true for other tumors that express high p63 levels including the ...... 102. 103. 104. 100. 101. 102. 103. 104. R4. CD24. CD44. R5 a. R4. R5. R5. R6. 100. 101. 102 ...... titis: Intraperitoneal (i.p.) administration of exces- .... residues (yellow regions) in each extracellular loop, ...... either capsule endoscopy, MR-Sellink procedure,.
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p/CIP
p16INK4a
▶ Amplified in Breast Cancer 1 ▶ Steroid Receptor Coactivators
▶ CDKN2A
p14ARF
p185neu
▶ ARF Tumor Suppressor Protein
▶ HER-2/neu
p16 ▶ CDKN2A
p19ARF ▶ ARF Tumor Suppressor Protein
p16INK4 ▶ CDKN2A
p16INK4A
p21 Axel H. Schönthal University of Southern California, Keck School of Medicine, Los Angeles, CA, USA
▶ INK4A
p16INK4A ▶ CDKN2A # Springer-Verlag Berlin Heidelberg 2017 M. Schwab (ed.), Encyclopedia of Cancer, DOI 10.1007/978-3-662-46875-3
Synonyms CAP20; CDKN1A; Cip1; Cyclin-dependent kinase inhibitor 1A; HRAS; mda-6; Sdi1; Waf1
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p21
Definition Traditionally, the p21 protein has been defined as an inhibitor of ▶ cyclin-dependent kinases (CDKs). Inhibition of this group of enzymes by p21 leads to the cessation of cell proliferation. Additionally, p21 binds to and inhibits the activity of PCNA (proliferating cell nuclear antigen, a subunit of DNA polymerase), which results in the termination of DNA replication. Because both of these functions lead to cell cycle arrest, p21 is also called an inhibitor of the cell cycle. Additional functions of p21 were discovered that are unrelated to its ability to inhibit cell proliferation. For example, p21 can regulate transcription; restrain ▶ apoptosis; impose on cytoskeletal dynamics and ▶ cell motility; interfere with cellular differentiation, ▶ senescence, and ▶ aging; act as cytoplasmic regulator of nuclear import; impinge on ▶ DNA repair; and affect the generation of induced pluripotent stem cells (Fig. 1). These multiple functions rely on different cellular localizations and different targets that are present either in the cytoplasm, in the nucleus, or on DNA. Because of this pleiotropic nature of p21 functions, its traditional definition as a “CDK inhibitor” or “cell cycle inhibitor” is incomplete; the well-informed investigator should not consider p21 merely as an inhibitor of the cell cycle. Rather, p21 presents itself as a multifunctional regulator of various cellular processes, with its subcellular localization as a prime determinant of this protein’s specific role.
Characteristics Because p21 has been discovered by diverse experimental approaches, it has received different suffixes, including wild-type p53-activated fragment 1 (Waf1), senescent-derived inhibitor-1 (Sdi1), CDK2-interacting protein (Cip1), CDK2associated 20K protein (CAP20), or melanoma differentiation-associated protein (mda-6). Most commonly, it is called p21Waf1 (p21Waf1) or p21Cip1 (p21Cip1). The p21 gene is called cyclin-dependent kinase inhibitor 1A (CDKN1A). It is composed of three
p21, Fig. 1 Central role of p21 in sensing and responding to a variety of stimuli. p21 responds to a variety of stimuli and exerts multiple functions. Some, but not all, of these functions are mediated via its inhibition of CDK activity. Inhibition of PCNA by p21 blocks DNA replication, but does not impair the ability of PCNA to repair DNA damage (This figure is based on a review article (Abbas and Dutta 2009), which comprehensively describes many of the p21-controlled processes shown here)
exons that are located on human chromosome 6 (6p21.2). Its 2.1 kb transcript encodes a 21 kD polypeptide (hence its name), which consists of 164 amino acids. The p21 protein is primarily located in the nucleus, although it may relocate to the cytosol where it exerts important functions as well. Orthologues of this gene have been cloned from mouse, rat, cat, and Xenopus and are likely to exist in organisms as diverse as insects, plants, and yeast. Cell Cycle Inhibition by p21 The activity of CDKs is required for cell cycle progression, while PCNA is essential for the execution of DNA replication. Therefore, the inhibition of CDK and PCNA activity by elevated levels
p21
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p21, Fig. 2 Cell cycle inhibitory function of p21. The p21 gene is a transcriptional target for multiple proliferation inhibitory signaling cascades, including p53 (which can be activated in response to DNA damage), transforming growth factor beta (TGF), tumor necrosis factor alpha (TNF), interferon gamma (IFN), or adenomatosis polyposis coli (APC). p21 binds to the DNA polymerase subunit PCNA and inactivates its DNA replicative function. As a consequence, DNA replication, but not DNA
repair, is blocked. p21 also binds to cyclin/cdk complexes and inhibits their enzymatic activity. As a result, the substrates of the cyclin/cdk complexes are not being phosphorylated, and the cells become arrested in the cell cycle. Thus, through the increase of p21 levels, the cells are able to stop proliferation. This process can be reversed: a reduction in the amount of p21 protein will release the inhibition of PCNA and of cyclin/cdk, and the cells resume growth
of p21 causes cell cycle arrest, in that way defining p21 as a negative regulator of cell proliferation (Fig. 2). In extension, p21 has been implicated in several other processes that are related to cell growth, such as differentiation, senescence, and carcinogenesis. The major targets of p21, the cyclin-dependent kinases, are considered the “engine of the cell cycle,” and they direct the events required for cellular proliferation. Each CDK consists of two parts: one part is a cyclin protein (which acts as a regulatory subunit) and the other part is the actual cyclin-dependent kinase (cdk, which constitutes the catalytic subunit). The complex, composed of one cyclin subunit plus one cdk subunit, is called CDK. Several different ▶ cyclin proteins (cyclin A,
cyclin B, etc.) as well as several different cdk (▶ cyclin-dependent kinases) subunits (cdk1, cdk2, etc.) exist and can combine to form different combinations of CDK heterodimers. The enzymatic activity of all CDK complexes, and thus their ability to drive cells through the cell cycle, is strictly regulated by posttranslational modifications (phosphorylation and dephosphorylation) and by their interaction with inhibitory proteins, the cyclin-dependent kinase inhibitors (abbreviated CKI or CDI). There are two families of CKIs: 1. The Cip/Kip (CDK inhibitory protein) family is constituted of p21 itself, p27Kip1, and p57Kip2.
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p21
p21, Fig. 3 Examples of inhibitory and stimulatory functions of p21 protein. Cytoplasmic p21 can bind to caspase 3 (and caspases 8 and 10) and ASK1 (apoptosis signal-regulating kinase 1) and thereby inhibit apoptosis. Moreover, cytoplasmic p21 functions as an assembly factor that promotes the association of cyclin D with cdk4 (or cdk6) and supports nuclear entry of this complex (without inhibiting its kinase activity), thereby stimulating cell cycle progression at low concentrations. Increased levels of nuclear p21 inhibit cyclin A/cdk2 and cyclin E/cdk2 complexes, leading to cell cycle arrest
2. The INK4 (inhibitor of kinase 4) family, of which the tumor suppressor p16INK4a is the best-studied member. While the INK4 proteins bind to and inhibit the activity of only two CDKs, namely, cdk4 and cdk6, members of the Cip/Kip family are rather indiscriminatory and affect all of the further abovementioned CDK complexes. p21 in particular has been labeled as a universal inhibitor of CDKs. Positive Regulation of Proliferation by p21 The major critical function of p21, in particular at elevated expression levels, is the inhibition of CDK activity, which subsequently causes cell cycle arrest and inhibition of cell division. However, at rather low concentrations, p21 does not inhibit all CDKs with the same efficiency. In the case of cyclin E/cdk2 and cyclin A/cdk2, a single
molecule of p21 is sufficient to completely inhibit the respective catalytic activity. Thus, in this instance, p21 fully conforms to its reputation as a potent CDK inhibitor. However, in the case of cyclin D/cdk4 and cyclin D/cdk6, two molecules of p21 are required for inhibition; at equimolar concentrations (one molecule of p21 plus one cyclin/cdk complex), p21 does not inhibit cyclin D/cdk4 or cyclin D/cdk6 activity (Fig. 3). Rather, under these conditions, p21 stimulates the assembly and nuclear targeting of these complexes. Moreover, the binding of p21 by cyclin D/cdk4 and cyclin D/cdk6 sequesters this inhibitor away from cyclin E/cdk2 and cyclin A/cdk2, thus indirectly promoting activation of these latter CDK complexes. As a consequence, the relatively low concentrations of p21, which are present in most cells, do not act cell growth inhibitory. For these reasons, the widely accepted definition of p21 as a general CDK inhibitor may have to be modified to
p21
accommodate its recognized role as a differential regulator of individual cyclin/cdk combinations. p21’s activity as an assembly factor for cyclin D/cdk4/6 is counterintuitive and in contrast to its function as a CDK inhibitor. In this context, the complete repression of p21 repression of p21 leads to impairment of cell cycle progression due to decreased cyclin D/cdk4/6 complex formation. This outcome clearly does not support the simplified expectation that repression of p21 should result in growth promotion, as predicted from its traditional role as a CDK inhibitor. The biological rationale behind this aspect of p21 function is not yet completely understood, and further studies are required to illuminate this conundrum. The p21 gene is a transcriptional target for multiple proliferation inhibitory signaling cascades, including p53 (which can be activated in response to DNA damage), transforming growth factor beta (TGFb), tumor necrosis factor alpha (TNFa), interferon gamma (IFNg), or adenomatosis polyposis coli (APC) (Fig. 1). p21 binds to the DNA polymerase subunit of PCNA and inactivates its DNA replicative function. As a consequence, DNA replication, but not DNA repair, is blocked. p21 also binds to cyclin/cdk complexes and inhibits their enzymatic activity. As a result, the substrates of the cyclin/cdk complexes are not being phosphorylated, and the cells become arrested in the cell cycle. Thus, through the increase of p21 levels, the cells are able to stop proliferation. This process can be reversed: a reduction in the amount of p21 protein will release the inhibition of PCNA and of cyclin/cdk, and the cells resume growth. Binding of p21 to CDK and PCNA The CDK and PCNA inhibitory activities of p21 are functionally independent and reside in separate parts of the protein. The amino-terminal region, which is strongly conserved among the Cip/Kip family members, contains distinct cyclinand cdk-interacting domains; amino acids 17–24 (the Cy1 site) constitute the binding site for cyclin, whereas residues 53–58 (K site) in conjunction with amino acids 74–79 (the 310 helix) contact the cdk. Thus, p21 simultaneously binds to both subunits of the CDK complex, whereby the
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interaction with the cyclin subunit serves to stabilize the ternary complex. As deduced from the crystal structure of the cyclinA/cdk2/p27Kip1 complex, the mechanism through which Cip/Kip proteins inhibit the kinase is through blocking the binding of ATP. This is achieved by a twofold process: first, the inhibitor inserts a small helix inside the catalytic cleft of the cdk subunit that directly blocks ATP binding; second, the inhibitor causes a conformational change within the aminoterminal region of the cdk subunit that results in the loss of many ATP-interacting elements. Together, these processes result in the efficient, yet reversible, inhibition of kinase activity. It remains to be established why binding of one molecule of p21 to complexes containing cdk4 or cdk6 does not result in a similar inhibition of kinase activity. In addition to modulating CDK activity, p21 protein is able to inhibit DNA synthesis through its ability to bind to and block the activity of PCNA, which is an accessory protein of the major replication enzymes DNA polymerase d and e. The interaction with PCNA is achieved via a high-affinity binding site that stretches from amino acids 143–160 in the carboxyterminal domain of p21. As a result, p21 masks contact sites on PCNA that are required for interaction with other proteins of the polymerase assembly. The interaction of PCNA with DNA, however, is not impeded by p21 binding. It is interesting to note that by binding PCNA, p21 inhibits DNA replication but not DNA repair. Rather, it appears that p21 actually stimulates PCNA-dependent nucleotide excision repair. Thus, by interacting with PCNA, p21 executes a twofold task: it blocks replicative DNA synthesis, which allows time for DNA repair to take place before the errors are duplicated, and at the same time, it helps the repair to be executed. Regulation of p21 Expression and Activity Low amounts of p21 protein are present in most cell types. However, the expression levels can be increased significantly in response to a wide spectrum of diverse external stimuli (Fig. 1) and through the activation of different intracellular signal transduction pathways. In general, p21
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expression can be regulated at multiple levels. The promoter of the p21 gene contains numerous binding sites for various transcription factors; most prominent among these is the tumor suppressor p53p53. At the posttranscriptional level, p21 expression is regulated via several miRNAs, as well as by a few proteins that bind to AU-rich elements in the 30 -UTR of p21 mRNA and thereby modulate its half-life. At the posttranslational level, the stability of p21 protein is regulated via phosphorylation by a number of different kinases, and it is degraded through the ubiquitin/ proteasome pathway and cleavage by caspases. Phosphorylation of p21 has been shown to modify the ability of this protein to interact with its respective targets and to direct its subcellular localization. Among the protein kinases known to phosphorylate p21 are Akt (also known as protein kinase B, PKB), glycogen synthase kinase 3 beta (GSK3 ), p38 (a stress-related MAP kinase), c-Jun N-terminal kinase (JNK), apoptosis signalregulating kinase (ASK1), protein kinase A (PKA), protein kinase C (PKC), Pim-1 (a proto-oncogenic serine/threonine protein kinase), Mirk (minibrain-related kinase), and cyclinE/cdk2. Although some details remain to be established, it is evident that phosphorylation of p21 has profound consequences for its function. For instance, phosphorylation of serine-130 (S130) prevents the interaction of p21 with cdk2, which in effect neutralizes its function as a CDK and cell cycle inhibitor. As well, phosphorylation of p21 might modulate its interaction with other proteins, and besides CDK complexes and PCNA, more than a dozen other proteins have been identified as binding partners for p21. Role of p21 in p53 Pathway The best understood regulation of p21 is in response to stress conditions that generate DNA damage, such as during cellular exposure to certain chemotherapeutic drugs, oxidants, or irradiation. These insults activate the tumor suppressor ▶ p53 (p53 protein, biological, and clinical aspects), which binds to the p21 promoter and increases p21 expression. Elevated levels of p21 cause cell cycle arrest and provide the cells with the opportunity to repair the DNA damage. In this
p21
sense, p21 exerts a protective function against stress, which relies on its ability to suppress cell proliferation. Cells that were engineered to lack the p21 gene have been found to inefficiently arrest in the cell cycle after DNA-damaging insults and are much more prone to cell death (apoptosis). Thus, p21 is a major executioner of p53 functions and contributes to cellular survival after exposure to harmful stimuli. In cells that are deficient in p53 function, as is the case in more than 50% of all human cancers, the transcriptional induction of p21 is not observed in response to many DNA-damaging agents. Role of p21 in Differentiation The expression of p21 is highly modulated during the course of cellular differentiation and development. At least in some tissues, the exit from the cell cycle during terminal differentiation is mediated by p21, and this function of p21 is regulated by pathways that are independent of p53. Thus, these differentiation-inducing signals act through pathways that target transcription factors other than p53. One such factor is MyoD, which is a central regulator of myogenesis that induces myocytes to exit the cell cycle and fuse to form myotubes. A binding site for MyoD has been identified in the p21 promoter and shown to mediate p21 induction during myogenesis. Furthermore, the ability of p21 to be induced in a p53-independent manner has been established for various differentiation models in vitro as well as during development in vivo. From these studies it has been determined that the induction of p21 is a primary mediator of differentiation. Moreover, in the case of hematopoietic stem cells, p21 has been shown to be the molecular switch that keeps these cells in relative quiescence; proliferative restriction is critical to the survival of these cells as increased cycling causes their premature depletion. Induced pluripotent stem (iPS) cell generation is the process of artificially reprogramming non-pluripotent (e.g., adult somatic) cells to become pluripotent stem cells. There are indications that the p53-p21 pathway acts as a barrier in iPS cell generation. For instance, silencing p53 or p21 expression has been shown to significantly
p21
increase the reprogramming efficiency of human somatic cells. Role of p21 in Senescence Most normal cells that are cultured in vitro cease to proliferate after a finite number of doublings, a process called ▶ senescence. It has been shown that p21 accumulates as cell cultures age and approach senescence. In parallel with the increase in p21, the enzymatic activity of cyclinE/cdk2 complexes is decreased and PCNA activity is inhibited. In addition, probably through indirect mechanisms, p21 has been implicated in the negative regulation of ▶ telomerase, which is an enzyme that is required for the extension of cellular life span. In this regard, it has been shown that the inactivation of the p21 gene delayed cellular senescence, i.e., the cells underwent more doublings before they reached senescence. In other studies, it was shown that the experimental inhibition of p21 function in early senescent cells stimulated the cells to reenter S-phase and replicate their DNA, although the cells did not undergo cell division. This finding is consistent with the fact that in early senescent cells, cyclinE/cdk2 complexes and PCNA are inhibited by p21. Upon removal of p21, both enzymes resume their function and drive cells through S-phase. Overall, p21 appears to be a critical initiator of the senescent process. However, once cells are fully senescent, other proteins, such as the CKI p16INK4a, take over for the long-term maintenance of this arrest. Role of p21 in Apoptosis The p21 protein protects cells from genotoxic and other cellular stresses. It was described above that after DNA damage, p53 transcriptionally activates the p21 gene, and elevated levels of p21 protein protect cells from apoptosis by arresting the cell cycle and permitting DNA repair. Moreover, cytoplasmic p21 can act as an anti-apoptotic component in a more direct manner (Fig. 3). It can bind to pro-caspase 3 (and caspases 8 and 10) and prevent its activation, thereby providing resistance to Fas-mediated apoptosis. Furthermore, p21 interacts with and inhibits the pro-apoptotic kinase ASK1 (apoptosis signal-regulating kinase 1),
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which phosphorylates p21 at serine-98 (S98). Both of these events are enhanced by the pro-survival kinase Akt/PKB (and possibly PKA), which phosphorylate p21 on T145, thereby stabilizing the protein and maintaining its location outside the nucleus. The pro-survival and anti-apoptotic function of p21, which is executed in the cytoplasm, seems to be in contrast to its primary nuclear function, which is inhibition of CDK and PCNA activity with ensuing cell cycle arrest and inhibition of cell growth. Thus, the tumor suppressor-like characteristics of nuclear p21 appear to conflict with its cytoplasmic purpose, which rather resembles typical oncoprotein function. Although this issue has not yet been completely clarified, it seems likely that exclusion of p21 from the nucleus results in the loss of tumor-suppressing function and selectively maintains its tumor-promoting activities. Role of p21 in Cell Migration Members of the Rho family of GTPases are best known for their role in the regulation of cytoskeletal dynamics (actin stress fiber formation, focal adhesion assembly, actin-myosin contractility) and cell migration. Cytoplasmic p21 protein has been shown to bind to and inhibit the Rho effector, Rho kinase (ROCK), which results in increased cell migration. As cells in general are required to choose between cell division and cell movement, high levels of p21 throughout the cell favor cell migration, because nuclear p21 inhibits proliferation and cytoplasmic p21 stimulates motility. This balance may be corrupted in tumor cells. For example, transformation of cells with the ras oncogene supports exclusion of p21 from the nucleus and stimulates its accumulation in the cytoplasm. As a result, such Ras-transformed cells not only are highly proliferative but also are prone to invasion and metastasis.
Clinical Relevance Tumor-Suppressing Function of p21 In theory, every cyclin-dependent kinase inhibitor should be a tumor suppressor. By virtue of their ability to arrest cells in the cell cycle and prevent
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their proliferation, CKIs are crucial negative regulators of cell growth. The elimination of CKI activity, for example, by mutation or deletion, should release CDK complexes from this form of inhibition and promote unrestricted, inappropriate cellular growth. Conversely, the artificially increased expression of CKIs in tumor cells, for example, through a gene therapy approach, should lead to the cessation of tumor cell proliferation. In reality, a tumor suppressor function of p21 has been difficult to prove. Unlike p16INK4a, which is a bona fide tumor suppressor and frequently found inactivated in human cancers, p21 has not yet convincingly revealed such a function. Efforts to find tumors that harbor mutations in the p21 gene yielded such events only at a very low rate. However, polymorphisms of p21 have been observed in various cancers and one of them (at position 149) was found to be associated with human esophageal cancer. Initially, the generation of mice with a homozygous deletion of the p21 gene yielded disappointing results; these mice did not exhibit an increased rate of spontaneous tumor formation. This was surprising because p21 is one of the major executioners of the tumor suppressor p53, and mice without the gene for p53 have a rapid rate of early tumor formation. Nonetheless, keratinocytes that were established from the p21-deficient mice exhibited increased susceptibility to transformation by the ▶ ras oncogene, and the resulting tumors grew much more aggressively as compared to ras-transformed keratinocytes from mice that contained the normal p21 gene. Thus, it appears that the lack of p21 function contributes to the promotion of malignant tumorigenesis and that the absence of p21 may become obvious and detrimental to cellular function only under conditions of stress, such as in response to genotoxins or aggravated growth stimuli by oncogenes.
Tumor-Promoting Function of p21 Early experiments with p21 primarily focused on its CDK inhibitory function, and for this reason potential tumor-promoting features of this protein
p21
were slow to be recognized. Some of the first clues came from the use of mice lacking both p21 and ATM (ataxia telangiectasia mutated; a kinase involved in DNA damage signaling pathways). Here, in the context of an atm-deficient mouse, loss of p21 resulted in slower development of spontaneous lymphomas, possibly due to increased apoptosis of tumor cells in the absence of p21. Similarly, growth factor-induced gliomagenesis was greatly reduced in mice lacking p21. In this latter case, the tumorpromoting effect of p21 was shown to require this protein’s cyclin-binding domain, and it is conceivable that p21’s ability (at low concentrations) to function as an assembly factor for CDK complexes that contain cyclin D (cyclinD/cdk4 and cyclinD/cdk6) (Fig. 3) may contribute to its pro-tumorigenic effects. Clinical studies could not yet clarify p21’s ambiguous role with regard to its tumorsuppressive versus pro-tumorigenic activities, as there are conflicting observations. For example, while loss of p21 (which is a rare event) in some cancer types is a negative prognostic marker, overexpression or cytoplasmic localization of p21 is a marker of poor prognosis and aggressiveness in several cancer types. In this context, it has been postulated that cytoplasmic localization of p21 protein favors its tumor-promoting functions, whereas primarily nuclear localization (especially at increased levels) is supportive of its tumorsuppressive activities.
Summary p21 is an important protein with multiple functions in diverse cellular processes. Although initially characterized as an inhibitor of CDK activity and cell cycle progression, several additional features have emerged, some of which are entirely independent of any CDK involvement. As well, it has been difficult to establish whether p21 displays primarily tumor-suppressive or rather tumor-promoting functions. In any case, it appears that its respective subcellular localization exerts decisive influence over this protein’s specific roles in the above-described events. Currently ongoing studies pursue the construction of
p27
separation-of-function mutants of p21 in order to reduce its multifunctional character, which should enable us to gain greater insight into selected functions of this protein.
Cross-References ▶ HRAS
References Abbas T, Dutta A (2009) p21 in cancer: intricate networks and multiple activities. Nat Rev Cancer 9:400–414 Besson A, Dowdy SF, Roberts JM (2008) CDK inhibitors: cell cycle regulators and beyond. Dev Cell 14:159–169 Child ES, Mann DJ (2006) The intricacies of p21 phosphorylation: protein/protein interactions, subcellular localization and stability. Cell Cycle 5:1313–1319 Jung YS, Qian Y, Chen X (2010) Examination of the expanding pathways for the regulation of p21 expression and activity. Cell Signal 22:1003–1012 Kreis NN, Louwen F, Yuan J (2015) Less understood issues: p21Cip1 in mitosis and its therapeutic potential. Oncogene 34:1758–1767 Sherr CJ, Roberts JM (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13:1501–1512
p21(RAS) ▶ HRAS
p27 Yasunobu Matsuda Department of Medical Technology, Niigata University Graduate of Health Sciences, Niigata, Japan
Synonyms CDKN4; Cyclin-dependent kinase inhibitor 1B (CDKI1B); KIP1; MEN1B; MEN4; p27KIP1
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Definition p27 is a 198-amino acid protein encoded by the cyclin-dependent kinase inhibitor 1B (CDKN1B) gene on the short arm of chromosome 12 and is a member of the CIP/KIP family of cyclindependent kinase (CDK) inhibitor proteins (p21, p27, and p57). Through its binding to cyclin E/CDK2 complexes, p27 acts as a cell cycle inhibitor at G1 phase. When quiescent cells begin to divide and enter cell division, p27 is phosphorylated by CDKs and rapidly degraded by ubiquitination. p27 has been examined as a tumor suppressor, because its decreased expression is often associated with poor survival in cancer patients. It has also been found that p27 is expressed in the cytoplasm of some sets of cancer cells and promotes tumor aggressiveness.
Characteristics The cell cycle is mainly controlled by several proteins termed cyclins and cyclin-dependent kinases (CDKs), which are activated in a strict order. The levels of cyclins/CDKs are stable in quiescent cells. When the cells are released from G1 phase, the levels of D cyclins are increased to assemble with their corresponding CDKs (mainly CDK4). Subsequently, the levels of cyclins A and E bound to CDK2 are increased to reach S phase. During DNA replication, the level of cyclin E is decreased while cyclin B bound to CDK1 is activated to start M phase. Importantly, their activities are controlled by CDK inhibitors (CDKIs). In mammals, there are two broad classes of CDKIs: the INK4 family members (p15, p16, and p19), which specifically form complexes with CDK4 and CDK6 to inhibit CDK4/cyclin D complexes, and the CIP/KIP family members (p21CIP1, p27KIP1, and p57KIP2), which can inhibit both CDK2/cyclin A and CDK2/cyclin E complex activities. The function of p27 is regulated in a cell cycledependent manner. When the cells are quiescent at G0 phase, p27 binds and inactivates cyclin E/CDK2 complexes. The interaction between p27 and CDK2 results in the hypophosphorylation of retinoblastoma protein (pRb).
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During G1 phase, p27 is progressively decreased, leading to the activation of cyclin E/CDK2 and cyclin A/CDK2 complexes. All these events enable cells to start the G1/S transition of the cell cycle. Overall, p27 plays a critical role in the maintenance of cell cycle regulation at G1 phase. Therefore, it is reasonable that the level of p27 is increased by extracellular stimuli that lead to cell growth arrest, such as the cytokine transforming growth factor (TGF)-b, serum starvation, and cellto-cell contact inhibition. p27 may also be involved in the regulation of cell size, because p27-knockout mice show an increased body size with organomegaly. p27 Is a Strong Tumor Suppressor In cancer cells, mutations of the p27 gene are rare. However, p27 has been regarded as a strong tumor suppressor in many types of human malignancies. Mice with null expression of p27 show spontaneous development of tumors in many tissues, and those with heterogeneous expression are susceptible to carcinogen-induced cancer development. In humans, reduced p27 levels are frequently observed in precancerous lesions as well as in aggressive cancer cells. Tight correlations have been reported between loss of nuclear p27 expression and poor survival in breast cancer, colon cancer, and hepatoma. Clinical studies have shown that immunohistochemical analysis of p27 is useful for predicting the prognosis of patients with epithelial, hematopoietic, and mesenchymal malignancies, suggesting that p27 can be regarded as an adverse prognostic indicator. p27 has been reported to act as an independent predictor of recurrence in hepatocellular carcinoma, among several types of G1/S cell cycle regulators (e.g., pRb, p21, p16, p53, cyclin D1, and cyclin E). Regulatory Mechanism for Degradation of p27 p27 protein is periodically expressed during the cell cycle. Specifically, it is expressed at high levels during G1 phase and decreases at S phase. Since the p27 mRNA levels remain unchanged throughout the cell cycle, it has been considered
p27
that the level of p27 protein is regulated by a posttranscriptional mechanism. The Skp1/Cullin/ F-box complex (SCF complex) has been identified as a main regulator of p27 degradation. SCF is a ubiquitin-ligase complex that consists of several subunits, including Cullin-1 (a major scaffold for the SCF complex), Skp1 (an essential component for recognizing F-box proteins), RBX1 (a component required for binding to ubiquitin conjugates), and variable F-box proteins that recognize and bind to specific target proteins to be processed for ubiquitin-mediated degradation. p27 is specifically targeted by S phase kinaseassociated protein 2 (Skp2) ubiquitin-ligasebound SCF. Skp2 is one of the most investigated F-proteins and has been found to degrade several CDKIs such as p27, p21, and p130. Skp2 can also interact with cyclin A/CDK2 complexes. The level of Skp2 expression is strictly correlated with the cell cycle. Specifically, it is expressed at the lowest level in G0 to early G1 phase and then increased during S phase, which is in contrast to the expression of p27. Taken together, Skp2 plays a positive role in cell cycle progression. Clinical studies have shown close relationships between increased Skp2 and decreased p27 in many types of human malignancies with a poor prognosis, suggesting that Skp2 plays an oncogenic role as a p27 inhibitor. Phosphorylation Modifies the Functions of p27 p27 protein has several phosphorylation sites, each of which acts independently as a modifier of different signal transduction pathways. (i) Serine 10 (Ser10) At early G1 phase of the cell cycle, p27 is phosphorylated at Ser10 by mitogenic stimuli. This enables export of nuclear p27 to the cytoplasm, which hampers the function of p27 as an inhibitor of cyclin E/CDK2 complexes. The biological significance of p27 Ser10 is supported by a previous report on mice carrying p27 with a Ser10 mutation, which were unable to export p27 from the nucleus and showed partial resistance to tumorigenesis.
p27
(ii) Threonine 157 and 198 (Thr157 and Thr198) When cells go from G1 to S phase, p27 is phosphorylated at Thr157 and Thr198 through Akt signaling, and nuclear import of p27 is inhibited. In this setting, p27 is sequestered from cyclin E/CDK2 to cyclin D1/CDK4 complexes, and its function changes from a CDK2 inhibitor to a CDK4 activator. (iii) Threonine 187 (Thr187) During late G1 and early S phase in the cell cycle, p27 in the cytoplasm is readily phosphorylated at Thr187 by cyclin E/CDK2 complexes. Skp2 recognizes this phosphorylated site and is recruited to SCF complexes. Therefore, Thr187 is a requisite for degradation of p27. (iv) Tyrosine 88 and 89 (Tyr88 and Tyr89) P27 is both inhibitory and non-inhibitory against cyclin D/CDK4 complex, and it has been reported that the phosphorylation status of Tyr88 and Tyr89 in p27 can determine the role of p27. These two tyrosine residues are phosphorylated in proliferating cells, resulting in the convergence of p27 from the inhibitor to non-inhibitor of CDK4. p27 and Nuclear Export Molecules In the nucleus, p27 interacts with nuclear poreassociated protein (mNPAP60) and the nuclear export protein chromosome region maintenance 1 protein (CRM1), causing export of nuclear p27 to the cytoplasm. Binding with p27 causes CRM1 to release cyclin D1 into the nucleus, which results in cell cycle progression. Cytoplasmic Displacement of p27 In many types of cancer cells, several oncogenic signaling molecules have been found to be implicated in abnormal cytoplasmic p27 expression. For example, Src family kinases phosphorylate p27 at tyrosine 74 (Tyr74) and Tyr88, while Akt phosphorylates p27 at Thr157 and Thr198, both of which enforce p27 accumulation in the cytoplasm. BCR-ABL, a fusion oncogene in chronic myeloid leukemia, phosphorylates p27 at Tyr74, Tyr88, and Tyr89 and increases the levels of cytoplasmic p27 by promoting Akt-mediated
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phosphorylation at Thr157. It has been shown that Thr157 and Thr198 of p27 are also phosphorylated by Pim family members (Pim1, Pim2, and Pim3), which are oncogenic serine/threonine kinases that promote cell cycle progression. These lines of evidence strongly indicate that excessive protein phosphorylation is closely correlated with cytoplasmic displacement of p27 in cancer cells and suggest that the localization of p27 determines whether it acts as an oncogene or a tumor suppressor. p27 and Cell Migration Aside from its cell cycle inhibitory function, p27 has unique activity to induce cell migration. For example, in hepatocyte growth factor-treated hepatoma cells, in which cell proliferation and motility are induced, p27 is exported from the nucleus to the cytoplasm via Ser10 phosphorylation. In this setting, p27 colocalizes with F-actin, causes actin rearrangement, and induces cell migration. Similarly, it has been found that p27 is required for cyclin D1-induced cell migration. In mammary epithelial cells, cyclin D1 maintains the level of p27 protein by decreasing the level of Skp2 (a ubiquitin ligase that specifically targets p27) and induces p27 phosphorylation at Ser10. Since breast cancer cells lacking the interaction of cyclin D1 with p27 are unable to migrate, p27 may play a role as a critical cell migration inducer. Cytoplasmic p27 has also been found to promote cell migration in hepatoma and glioma cells. Conclusion p27 was originally identified as a CDKI. Through its role in inhibiting the activity of cyclin E/CDK2 complexes, it plays a critical role in cell cycle inhibition. Thus far, p27 has been considered to be a tumor suppressor (Chu et al. 2008). However, accumulating evidence has indicated that p27 has a dual role during tumorigenesis. When abnormally phosphorylated and mislocated in the cytoplasm, p27 promotes cell proliferation via CDK4 activation or induces cell migration via interference with cytoskeletal actin (Susaki and Nakayama 2009; Wander et al. 2011). Since p27 is the only CDKI that shows an ambivalent effect on cell cycle progression, cautious clinical
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p27KIP1
p27, Fig. 1 Schematic representation of the mechanism of dual function of p27. In quiescent cells, p27 acts as a cyclin E/CDK2 inhibitor. With mitogenic stimuli, p27 is phosphorylated at Ser10 and exported into the cytoplasm. Following phosphorylation reactions convert p27 to promote cell cycle and cell migration and finally result in the protein degradation
application is required to ascertain its role in human malignancies (Fig. 1).
p300/CBP-Interacting Protein ▶ Amplified in Breast Cancer 1
References Chu IM, Hengst L, Slingerland JM (2008) The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy. Nat Rev Cancer 8:253–267 Susaki E, Nakayama KI (2009) Functional similarities and uniqueness of p27 and p57: insight from a knock-in mouse model. Cell Cycle 8:2497–2501 Wander SA, Zhao D, Slingerland JM (2011) p27: a barometer of signaling deregulation and potential predictor of response to targeted therapies. Clin Cancer Res 17:12–18
p40AIS
p27KIP1
p53
▶ p27
▶ TP53
▶ p53 Family
p51 ▶ p53 Family
p53 Family
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that each protein has unique roles in various biological and pathological processes from development to oncogenesis.
p53 Family Jacky K. H. Chung1 and Meredith S. Irwin2 1 Department of Medical Genetics and Microbiology, University of Toronto, Toronto, ON, Canada 2 Cell Biology Program and Division of Hematology-Oncology Hospital for Sick Children, University of Toronto, Toronto, ON, Canada
Synonyms Ket; p40AIS; p51; p63; p73; TP63; TP73; TP73L; Trp63; Trp73
Definition The p53 family consists of p53 and its two homologues, p63 and p73. All three proteins are transcription factors that bind specific DNA sequences to mediate gene expression involved in cell cycle arrest, apoptosis, and differentiation. p63 and p73 map to chromosomes 3q27 and 1p36, respectively. The three p53 family genes and their protein products share many characteristics, including some “p53-like activities.” However, some differences in structure and function suggest
p53
Characteristics Molecular Structure and Function In the late 1990s, two decades after the initial discovery of the p53 tumor suppressor protein, the p53 homologues p63 and p73 were identified. The p63 and p73 genes give rise to multiple mRNA, which are translated into several distinct isoforms (Fig. 1). Different isoforms result from the utilization of two promoters as well as alternative splicing. The three p53 family proteins have a common modular architecture with an N-terminal transactivation domain (TAD or TA), a DNA-binding domain (DBD), and an oligomerization domain (OD). The full-length p63 and p73 proteins that are translated from the first promoter are most similar to p53 and are referred to as TAp63 and TAp73, respectively. Like p53, TAp63 and TAp73 form oligomers and can induce apoptosis and cell cycle arrest. TAp63 and TAp73 regulate many of the same downstream target genes as p53 by binding to p53-responsive elements in genes such as ▶ p21, ▶ PUMA, and BAX. In addition, some unique target genes have been identified for p63 and p73, likely reflecting different promoter binding sequence preferences
TAD DBD
TAp63α TAp73α ΔNp63α
OD SAM TID
ΔNp73α
p53 Family, Fig. 1 The structure of the p53 family of proteins. Shown are the transactivation (TAD, orange), DNA-binding (DBD, blue), and oligomerization (OD, yellow) domains for p53, p63, and p73. These p63 and p73 domains show significant homology to p53 (TAD, 25%; DBD, 60%; and OD, 40%). Alternative splicing at the C-terminus of p63 and p73 generates multiple isoforms
(p63-a, p63-b, p63-g and p73-a, p73-b, p73-g, p73-d, p73-e, p73-F, p73-z, p73-Z) that are identical through the OD. The longest p63 and p73 isoforms (alpha) are shown in the figure and contain a SAM (green) and transinhibitory domain (gray) not found in most other C-terminal isoforms
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p53 Family
p53 Family, Table 1 Target genes of the p53 family Common p53-responsive p63 and p73 target genes
Unique p63 and p73 downstream genes
Puma Noxa p21 Mdm2 Bax CD95 Apaf-1 TNF TNF-R1 Perp AIP Redd1 Jagged 1/jagged 2 b4-integrin Dlx3 (p63) Ets-1 IKKa Gata-3 CyclinD1 Aquaporin (p73) P57 kip2
as well as unique tissue patterns of expression. Known TAp63 and p73 target genes are summarized in Table 1. p63 and p73 can induce many p53 target genes that are involved in cell cycle arrest and apoptosis (upper panel). However, both proteins can also upregulate novel target genes that cannot be activated by p53 (lower panel). Noted in parentheses are genes only regulated by p63 or p73, but not both. p63 and p73 also encode N-terminally truncated isoforms, DNp63 and DNp73, that lack the TAD. These DN variants are transcribed from the second promoter located within the third intron of both genes. Although the lack of TAD renders DN proteins transcriptionally inactive, they retain the ability to bind DNA and thus compete with transactivation-competent p53 family proteins for promoter binding sites. They can also form hetero-oligomers with TA proteins, preventing the formation of functional tetramers. Therefore, DNp63 and DNp73 are not only unable to induce downstream target genes, but they can act as dominant-negative proteins that block TAp63,
TAp73, and p53 activities and, thus, have antiapoptotic properties. Alternative splicing at the C-terminus adds further to the complexity of p63 and p73, resulting in unique coding sequences (Fig. 1). The longest p63 and p73 proteins (p63-a and p73-a) have a sterile a-motif (SAM) domain not found in p53. SAM domains are known to serve as proteinprotein interaction modules, raising the possibility that p63 and p73 splice variants with SAM domains are capable of recruiting isoform-specific binding proteins. A transcriptional inhibitory domain (TID) is also found in a subset of the different C-terminal isoforms. Thus, differential splicing of the C-termini influences the ability of these proteins to transactivate target genes. For example, in comparison to TAp63-a, TAp63-g more strongly induces downstream pro-apoptotic genes. Thus, the differences in coding sequences and isoform-specific binding partners result in different activities of the various isoforms. Various upstream pathways and stimuli, including DNA-damaging agents and oncogenes, such as E2F1 and Ras, regulate the expression and stability of p63 and p73 isoforms (summarized in Table 2). In addition, like p53, p63 and p73 are regulated by posttranslational modifications including phosphorylation, acetylation, and ubiquitination. Interactions with binding partners, such as p300 and ▶ MDM2, as well as heterotypic interactions with each other, further modulate the activities of the p53 family proteins. Roles in Development Unlike p53, which is ubiquitously expressed, p63 and p73 are expressed in a tissue-specific fashion, and each plays roles in critical developmental processes. In contrast to p53 null mice in which only a small percentage have developmental defects in neural tube closure, p63 and p73 mutant mice have significant developmental abnormalities. p63/ mice that lack all TA and DN isoforms have significant limb and craniofacial malformations as well as failed development of epithelial tissues including the skin. In humans, germline p63 mutations have been detected in patients with characteristics reminiscent of the p63 knockout mice. Heterozygous p63 mutations
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p53 Family, Table 2 Regulators of the p53 family Upstream regulators
Kinases
Binding proteins
Oncogenes: E2F-1 Ras DNA damage: Chemotherapeutic agents* g-radiation* BMP signaling Notch signaling c-Abl Chk Akt Itch Mdm2* MdmX* p300 Aspp family of proteins PML Yap Cyclins Pin1 PIAS p53 family proteins (heterooligomerization)
The p53 family is regulated by a number of proteins and signaling pathways. These upstream regulators can affect the p63 and/or p73 at the transcriptional or the posttranslational level. Some regulators have been shown to affect all three p53 family members (denoted with*).
have been found in six different human ectodermal dysplasia syndromes characterized by combinations of skin, hair, mammary gland, craniofacial, and limb abnormalities. Studies into these disorders have provided important clues as to which tissues express p63 and have revealed that p63 is essential in epithelial morphogenesis and involved in senescence. Studies of p73/ mice have demonstrated that DNp73 is crucial in the development of the nervous system. p73 knockout mice have significant neurologic abnormalities due to the absence and/or loss of specific populations of neurons. DNp73 promotes cell survival by the inactivation of the full-length p53 family proteins, and, thus, the loss of DNp73 leads to enhanced apoptosis in cortical and sympathetic ganglia neurons. The relative balance between the TA and DN forms of p63 and p73, as well as p53, is important in these
developmental processes and, similarly, in cancers that arise from these tissues. Role in Cancer: Tumor Suppressor or Oncogene? The high incidence of p53 mutations in human tumors coupled with the chromosomal locations of p63 and p73 led to the prediction that p63 and p73 were also tumor suppressor genes that are inactivated by mutations in certain human malignancies. Surprisingly, despite the functional similarities among the p53 family proteins, and specifically their ability to induce p53-responsive pro-apoptotic genes, only rare p63 and p73 mutations have been detected in both cell lines and primary tumors. Although the lack of mutations initially raised doubts as to whether these two genes have roles in human cancers, many studies have reported high levels of p73 and p63 expression in human cancers. However, many of these studies did not distinguish between the various isoforms. Evidence from mouse models and human tumors suggests that the relative expression levels of the different TA and DNp63 and p73 isoforms are important in tumor development and progression, as well as the response to treatment. Knockout mice lacking one or both copies of p53, as well as mice that express specific p53 point mutations, develop a wide range of tumors at a young age. In contrast, p63+/ and p73+/ mice develop premalignant and cancerous lesions only when aged. Furthermore, in p53 mutant mice, the additional loss of p63 or p73 leads to different tumor types and higher tumor burden with more metastases. Importantly, tumors from p63 or p73 heterozygous mice were shown to have loss of heterozygosity (LOH) of the remaining allele, suggesting that p63 and p73 have recessive properties of a classical tumor suppressor gene. However, the relative contribution of the different TA and DN isoforms is difficult to determine since these knockout mice lack the expression of all p63 or p73 isoforms. Loss of p63 and p73 has been described in several human tumor types. Loss of p63 expression is associated with bladder cancer progression and correlates with poor prognosis. TAp73
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silencing by methylation in leukemias and lymphomas has also been described. Furthermore, low expression of p63 and p73 has been reported in breast cancer due to a number of mechanisms including LOH and allele silencing. In addition to missense mutations, p53 is inactivated by binding to inhibitory proteins such as the papillomavirus E6 oncoprotein in cervical cancer and MDM2 in sarcomas. While TAp63 and p73 can likewise bind to MDM2, as well as other modulatory proteins such as MDMx, iASPP, YAP, and PML (see Table 2), the relative role of these complexes in human cancers has not been elucidated. However, there is growing evidence that inactivation of the putative tumor suppressor TA forms of p63 and p73 is mediated by hetero-oligomerization with a subset of p53 mutant proteins found commonly in tumors. These mutants include the p53 conformational mutants with “gain of function” properties. These mutant p53 proteins bind to and inhibit TAp63- and TAp73-dependent transactivation and apoptosis, leading to enhanced survival and growth. Furthermore, this binding affinity of mutant p53 for TAp73 is influenced by the status of a p53 polymorphism at codon 72. In certain tumors, such as head and neck squamous cell carcinoma, presence of arginine, instead of proline, at p53 codon 72 is associated with worse prognosis due, at least in part, to more potent binding to TAp73. Therefore, although cancerassociated missense changes are almost never found in the new p53 family members, other mechanisms exist to inactivate the tumor suppressor like forms TAp63 and TAp73. Ironically, despite the focus on p63 and p73 as tumor suppressors, there is perhaps more evidence supporting roles for DNp63 and p73 isoforms as oncogenes. Amplification of the genomic region encompassing p63 and overexpression of the DNp63 protein have been detected in several epithelial-derived tumors. In head and neck squamous cell carcinomas (HNSCCs) and breast cancers, DNp63 promotes the survival of transformed cells by blocking TAp73-dependent apoptosis. Thus, DNp63 behaves as a potent oncogene and highlights how perturbations in the equilibrium between DN and TA p63 and p73 proteins are
p53 Family
important in tumorigenesis. Whether this holds true for other tumors that express high p63 levels including the cervix, nasopharynx, and bladder remains to be determined. Much like p63, p73 is but highly expressed in several types of human malignancies. Overexpression of p73 has been reported in numerous cancer types including neuroblastoma; breast, lung, esophagus, bladder, ovarian, liver, and colon cancer; and certain types of leukemias. There is emerging evidence that when carefully studied, like p63, the upregulation of the DNp73 forms is relevant in the pathogenesis of these cancers. However, to date, specific overexpression of DNp73 transcripts and/or protein has only been shown in a few tumor types including mesenchymal or precursor-derived tumors such as rhabdomyosarcoma and neuroblastoma as well as breast, colon, and hepatocellular carcinomas. The tissue specificity of these findings is likely due to the fact that p73 has a restricted pattern of expression throughout development. The mechanisms responsible for p73 overexpression and specific mechanism(s) by which DNp73 promotes cancer development remain unknown. However, there is some evidence that DNp73 promotes immortalization of primary cells and can cooperate with oncogenes such as Ras to enhance transformation. Furthermore, the binding of DNp73 to p53 andTAp73 and TAp63 has been proposed to modulate differentiation in neuroblastoma and rhabdomyosarcoma. Clinical Relevance of p63 and p73: Prognosis and Chemosensitivity Evidence in cell and animal systems supporting a role for the different isoforms in tumorigenesis has led to the assessment of p73 and p63 expression as a marker for clinical prognosis. The relative ratio of TA versus DN isoforms as measured at the transcript level is linked to prognosis in several tumor types, including breast and colon cancers. High levels of DNp73 protein have also been correlated with poor prognosis in small series of patients with neuroblastoma and breast cancers. Immunohistochemistry for p63 has also begun to be used as a marker in several tumor types including prostate and breast. However,
p53 Family
large analyses of primary tumor samples will be required to determine if the relative expression of TA and DN isoforms of p63 and p73 can be linked to clinical prognosis. Nevertheless, roles for these two p53 family proteins in the response to DNA-damaging agents such as chemotherapies suggest that their expression has important therapeutic implications. Currently, the two major treatments for cancer, radiation and ▶ chemotherapy, both exert their cytotoxic effects by stabilizing and activating p53. However, these therapies are also capable of killing cancer cells harboring p53 mutations, suggesting that p53-independent mechanisms exist to initiate apoptosis. Many studies support a role for the p53 family proteins in chemosensitivity. TAp73 is induced by g-irradiation and many chemotherapeutic drugs and in a variety of cancer cell types, including those with mutant and wild-type p53. Studies using cells in which p73 or p63 is inactivated by genetic deletion, the use of dominant-negative proteins, and short interfering RNA (▶ siRNA) demonstrate that loss of p63 or p73 leads to chemoresistance. Similar to their effect on p53, many chemotherapies also induce p73 and p63 posttranslational modifications that stabilize and enhance their ability to induce apoptosis. These include c-abl-mediated phosphorylation in response to cisplatin and irradiation. p73 is also upregulated at the transcriptional level by some drugs. For example, doxorubicin treatment leads to E2F1-mediated induction of TAp73. Similar to p53, TAp73 and TAp63 mediate the expression of genes that control cell cycle arrest and apoptosis (Table 1). Thus, in response to anticancer treatments, TAp73, and perhaps TAp63, can substitute for and, in certain cases, cooperate with other p53 family proteins to eradicate transformed cells and promote tumor regression. The interactions among the p53 family proteins also modulate their ability to induce chemotherapy-induced apoptosis. The expression of various tumor-derived p53 mutant proteins leads to chemoresistance via the ability of these mutant proteins to bind to and inactivate TAp73 and p63. Experiments in cell lines demonstrate that manipulation of the levels of these p53 mutant proteins by overexpression or siRNA leads to
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chemoresistance or chemosensitivity, respectively. Some chemotherapies also affect the levels of DNp73 and p63. Furthermore, overproduction of DNp73 or DNp63 in cell lines also leads to chemoresistance, and this is likely due to heterooligomerization and inactivation of the full-length p53 family proteins. This paradigm has been best studied in head and neck squamous cancers where downregulation of DNp63 in response to cisplatin results in diminished DNp63-TAp73 complex formation and enhanced apoptosis. Given the importance of DN proteins in cancer cell survival, this represents a second potential mechanism through which chemotherapeutic drugs induce apoptosis. Although the mechanisms by which chemotherapies downregulate DNp63 and p73 are not known, it is clear the balance between DN and TA proteins, as well as mutant forms of p53, is an important determinant of chemosensitivity of tumor cells. Given the importance of p53 in tumor suppression, small-molecule inhibitors are actively being designed to activate p53. However, since p53 is nonfunctional in approximately half of all human cancers, there has been interest in developing therapeutic strategies to activate p73 and p63 to induce tumor cell death. Several different approaches might lead to enhanced p73 or p63 activity. Panels of drugs and small molecules can be screened for agents that either directly increase TAp73 or p63 or enhance their activation by upstream pathways (e.g., via E2F1 activation). Furthermore, in tumors with high levels of DNp73 or DNp63, strategies might be aimed at donwregulating these anti-apoptotic isoforms. Drugs that interfere with proteins that inactivate TAp73 and p63 can also be targeted. To this end, small peptides have been designed to modulate the interactions between the iASPP proteins and p63 and p73. Furthermore, therapeutic strategies using drugs and siRNA designed to interfere with the interaction between “gain of function” mutants of p53 and TAp73 have been successful in vitro. Finally, many drugs have been designed to interfere with p53 binding to its negative regulator MDM2. It is predicted that some of these drugs might also interfere with MDM2 binding to TAp73 and TAp63, raising the possibility that
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these small-molecule inhibitors may be useful for a broad spectrum of cancers. Concluding Remarks Since their discovery, studies of p63 and p73 have been focused on comparisons to p53. While the prospect of TAp63 and TAp73 compensating for the loss of p53 in tumor suppression and treatment is exciting, it is equally important to recognize very significant differences in their roles in both cancer and development. Not all tissues and cancers exhibit the same expression pattern of p63 and p73. The levels and balance between the various pro- and anti-apoptotic p53 family proteins appear to play important roles in both development and cancer. Given the importance of p63 and p73 in physiological apoptosis as well as in tumors and their response to chemotherapies, understanding how they function will likely lead to the development of better cancer treatments.
p63
p8 ▶ P8 Protein
P8 Protein Cédric Malicet and Juan Iovanna INSERM, Stress Cellulaire, Parc Scientifique et Technologique de Luminy, Marseille Cedex, France
Synonyms Candidate of Metastasis 1; Homo sapiens nuclear protein 1; NUPR1; p8
Definition
Irwin MS, Miller FD (2004) p73: regulator in cancer and neural development. Cell Death Differ 11(Suppl 1): S17–S22 McKeon F, Melino G (2007) Fog of war: the emerging p53 family. Cell Cycle 6(3):229–232 Mills AA (2006) p63: oncogene or tumor suppressor? Curr Opin Genet Dev 16(1):38–44 Moll UM, Slade N (2004) p63 and p73: roles in development and tumor formation. Mol Cancer Res 2(7):371–386 Stiewe T (2007) The p53 family in differentiation and tumorigenesis. Nat Rev Cancer 7(3):165–168
The p8 gene, first described as overexpressed in the pancreas during the acute phase of pancreatitis, encodes a ubiquitous nuclear and cytoplasmic stress protein. Expression of the p8 mRNA is rapid, strong, and transient in response to several stresses. The human p8 gene was assigned to chromosome 16, at position p11.2, and the gene is organized in three exons interrupted by two introns. The sizes of exons I, II, and III are 214, 150, and 329 nucleotides, respectively, and the complete mRNA sequence comprises 693 nucleotides (exclusive of the poly A tail) and has only one open reading frame.
p63
Characteristics
▶ p53 Family
The p8 gene was cloned from human, rat, mouse, and Xenopus laevis, conceptually translated from the Drosophila melanogaster genome or deduced from EST libraries (Bos taurus, Xenopus tropicalis, Zebrafish, Oryzias latipes, Bombyx mori, and Paralichthys olivaceus). p8 is a highly basic 82-aa polypeptide, with a theoretical molecular mass of about 8 kDa, containing a canonical
References
p73 ▶ p53 Family
P8 Protein
bipartite domain of positively charged amino acids typical of nuclear localization signals (NLS), and a nuclear/cytoplasmic location has been demonstrated for human p8. Importantly, the nuclear or cytoplasmic localization of p8 depends on growth conditions. When cells are growing, p8 is nuclear, whereas it is at the cytoplasm when cell growth is arrested. The transport to the nucleus is ATP dependent, and localization is also regulated by its p300-dependent acetylation. The p8 protein contains an N-terminal PEST region (Pro/Glu/Ser/Thr rich), suggesting a regulation of p8 expression by the ubiquitin (Ub)/ proteasome system. Homology searching in databases yielded no homology of p8 with other proteins of known function. Biochemical properties of the mammalian p8 proteins are a high isoelectric point (9.6–10.4), 14% of acidic amino acids, 20–24% of basic amino acids, 14–17% of phosphorylatable amino acids (serine, threonine, and tyrosine), and a high abundance of proline (6–9%) and glycine (5–6%). The negatively charged residues appear located at the aminoterminal region, and all the positive residues accumulate in the carboxy-terminal portion of the molecule. All these features are shared by some of the high-mobility-group (HMG) proteins, particularly by the HMG-I/Y, family. The overall identity of human p8 with human HMG-I/Y is only around 35%, but the molecular mass; the isoelectric point; the percentage of Arg + Lys, Glu + Asp, Ser + Thr, and Gly + Pro; the hydrophilicity plot, and the charge separation (despite a reverse orientation) are very similar. A characteristic property of these HMG proteins, also shared by p8, is that they are neither denatured by heating at 100 C nor precipitated by 2% trichloroacetic acid. NMR and CD analyses of p8 showed absence of stable secondary structure. The protein binds DNA weakly and is a substrate for protein kinase A (PKA). The phosphorylated p8 (PKAp8) has a higher content of secondary structure than the nonphosphorylated protein, and PKAp8 binds DNA strongly. Moreover, secondary structure prediction methods indicated that within the high homology region of other proteins, there is a basic helix-loop-helix secondary structure motif, characteristic of some classes of
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transcription factors. An architectural role in transcription is proposed, and several apparently unrelated functions have been ascribed to p8. p8 is described as a stage-specific component of the gonadotrope transcriptome that may play a functional role in the initiation of LHb gene expression during embryonic cellular differentiation. p8 is also a transcriptional regulator critical to two key cellular events in heart failure: cardiomyocyte hypertrophy and cardiac fibroblast matrix metalloprotease (MMP) expression. Furthermore, p8 is an important component of the defense program. For example, inactivation of the p8 gene increases liver sensitivity to CCl4 or increases sensitivity to systemic LPS treatment. Thus, p8 is an important element in the ▶ stress response. Finally, p8 and p53 are involved in an autoregulatory loop, p8 regulating p53 transactivation activity and p53 acting as a strong repressor of p8 expression. Also, p8 is involved in two major mechanisms, cell cycle regulation and ▶ apoptosis. To account for these various functions, it is suggested that the small size of the protein, its lack of specific tridimensional structure, and its nuclear-cytoplasm localization allow its interaction with several partners to target different signaling pathways. Several partners have been identified by yeast two-hybrid screening cDNA libraries. Tumor Establishment and Progression Tumor cells form ▶ metastasis to distant organs in a selective manner, and the organ specificity of the metastatic process is assumed to be governed, at least in part, by interactions between the malignant cells and local microenvironmental factors. It is currently admitted that development of metastases is primarily the result of the ability of disseminated tumor cells to initiate and continue growth in the target organ. In fact, cancer progression occurs in several steps; during transformation, some cancer cells are positively selected within the tumor on the basis of their growth capacity, low response to apoptotic signals, and ability to escape the immunological survey of the host. After leaving the primary tumor, transformed cells migrate through the body. Yet, metastasis will not develop in all tissues. Capacity
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for invading the target organ is a first limitation. But once within the organ, metastasis will develop only if transformed cells can cope with their new microenvironment. Therefore, invading cells are exposed to the stress induced by the new microenvironment, and their capacity to react by activating stress-associated genes should be determinant in metastasis formation. Supporting this hypothesis, several stress-associated genes were found overexpressed in tumors, and their expression level often correlated with aggressiveness. Therefore, the stress-associated genes might facilitate tumor progression and metastasis formation by helping cell adaptation to the microenvironment of the host tissue. p8 is overexpressed in many human cancers. Its expression is crucial for tumor development, and the stress-response mechanisms governed by p8 are required for tumor establishment and progression. Impossibility for p8-deficient cells to form colonies in softagar, to develop as subcutaneous tumors, or to generate intraperitoneal spreading strongly suggests that expression of p8 is required for the organization and development of tumors. Furthermore, p8 mediates the growth of tumor cells after metastatic establishment in a secondary organ, indicating that activated expression of p8 in metastatic cells is required for tumor progression. Some clinical data indicate that p8 expression in breast and pancreatic cancers correlates with aggressiveness and that metastatic cells express high levels of p8. However, the molecular mechanisms by which p8 allows tumor progression are still unknown, but its contributions to cell cycle regulation and apoptosis are probably involved. p8 and Cell Cycle Regulation In response to stress agents, cells activate various intracytoplasmic pathways, depending on cell type and on the nature of the agent that eventually reach the nucleus to modulate gene expression. By regulating genomic response, these stressassociated pathways will determine whether a cell reenters the cell cycle, undergoes cell cycle arrest, or enters a cell-death program. p8 is a stress gene, regulating cell cycle progression. It can act as a growth-promoting factor when it is overexpressed in pancreatic or HeLa cells or as a
P8 Protein
growth inhibitor when expressed in MEF (murine embryonic fibroblasts) or breast cancer-derived cells. These functions seem to involve regulation of the cyclin-dependent kinase inhibitor p27Kip1, in part through its interaction with JAB1. p8 and Apoptosis p8 expression has been inversely correlated to apoptosis in samples obtained from human pancreatic and breast cancers. According to this antiapoptotic effect, the interaction of p8/prothymosin alpha (ProTa), one of the molecular partners of p8, is very exciting. This natively unstructured protein was originally considered as a thymic hormone, but like p8, it was eventually attributed to several other functions. p8 and ProTa are two small proteins without stable secondary structure in solution, showing opposite electrostatic charges at neutral pH. They interact and promote mutual stabilization of their structures in a particular conformation, the resulting p8/ProTa complex becoming able to block staurosporine-induced apoptosis. In other words, two natively unfolded proteins, p8 and ProTa, which had been attributed an antiapoptotic function, are in fact inactive if alone and require interacting with each other to exert that function, probably because the active complex has acquired stable secondary structure. Moreover, p8 is involved in the effect of ▶ gemcitabine, the only chemotherapeutic treatment presently available for pancreatic cancer. It was demonstrated that in pancreatic cancer cells, a large part of gemcitabine-induced apoptosis results from the inhibition of the constitutive antiapoptotic activity of p8 (Fig. 1). p8 can also act as a proapoptotic protein. In fibroblast, the presence of p8 sensitizes cells to apoptosis induced by DNA damage. Moreover, recent works demonstrate a link between p8 and the antitumoral effects of ▶ cannabinoids. 9-tetrahydrocannabinol (THC) is the most abundant compound of Cannabis sativa, with potential therapeutic applications in patients with cancer. This antitumoral action of THC relies, at least in part, on its ability to induce p8 expression and subsequent p8-mediated apoptosis of tumor cells.
P8 Protein P8 Protein, Fig. 1 p8 and prothymosin alpha (ProTa) are required for caspase inhibition in stressed cells. In response to apoptogenic stimuli, Apaf1 oligomerizes with cytochrome c to form the apoptosome that recruits and activates caspase 9 that, in turn, activates effector caspase 3. Following a stress p8 and (ProTa) complex impedes apoptosome formation, which prevents caspase 9 activation and blocks the apoptotic cascade
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Stress
ProTα
p8
Apoptosome
Cytochrome c+
Pro-caspase 9 Caspase 9
Apaf 1
Pro-caspase 3 Caspase 3
Cell death
Conclusion In conclusion, p8 is a small, highly basic, and natively unfolded protein whose expression is induced by several stresses. p8 interacts with numerous partners to regulate transcription, cell cycle, and apoptosis. Functions of p8 depend on its molecular partner, its cellular location, the cell type, and its level of expression. Of particular interest is its role on tumor development. Finally, p8 might be a new drug-targetable gene whose blockade would prevent cancer progression and metastasis development.
Encinar JA, Mallo GV, Mizyrycki C et al (2001) Structural studies of human p8, an HMG-I/Y-like protein, with DNA binding activity modified by phosphorylation. J Biol Chem 276:2701–2707 Malicet C, Giroux V, Vasseur S et al (2006) Regulation of apoptosis by the p8/prothymosin alpha complex. Proc Natl Acad Sci U S A 103:2671–2676 Valacco MP, Varone C, Malicet C et al (2006) Cell growthdependent subcellular localization of p8. J Cell Biochem 97:1066–1079 Vasseur S, Hoffmeister A, Garcia S et al (2002) p8 is critical for tumour development induced by ras mutated protein and E1A oncogene. EMBO Rep 3:165–170
See Also
Cross-References ▶ Apoptosis ▶ Cancer ▶ Cannabinoids ▶ Gemcitabine ▶ JNK Subfamily ▶ Metastasis ▶ Stress Response
References Carracedo A, Gironella M, Lorente M et al (2006) Cannabinoids induce apoptosis of pancreatic tumor cells via reticulum endoplasmic stress-related genes. Cancer Res 66:6748–6755
(2012) ATP. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 302. doi:10.1007/978-3-642-16483-5_440 (2012) Cell Cycle. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 737. doi:10.1007/978-3-642-16483-5_994 (2012) High Mobility Group. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1694. doi:10.1007/978-3-642-164835_2729 (2012) Pancreas. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 2762–2763. doi:10.1007/978-3-642-16483-5_7055 (2012) PEST Sequence. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2828. doi:10.1007/978-3-642-16483-5_4478 (2012) Transcription Factor. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3752. doi:10.1007/978-3-642-164835_5901
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p94 ▶ BRaf-Signaling
PA2.26 ▶ Podoplanin
PA700 ▶ Proteasome
PACE ▶ Furin
Paclitaxel Keith Skubitz Division of Hematology, Oncology and Transplantation, University of Minnesota Medical School, Minneapolis, MN, USA
Definition Paclitaxel is an anticancer agent derived from the North America Pacific yew tree (Taxus brevifolia) that is used in the treatment of a number of common malignancies including lung, ovarian, and ▶ breast cancer.
Characteristics Paclitaxel is a cancer chemotherapeutic agent that is widely used for several types of malignancies (Fig. 1a, b). It is a natural product produced from the Pacific yew tree. While first isolated from the
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bark of the Pacific yew, it is now commonly synthesized from a compound obtained from yew needles or cell culture systems. Paclitaxel is a member of the taxane class of drugs and was the first taxane to enter clinical trials and receive FDA approval. Another commonly used taxane is taxotere, which is derived from the English yew tree. Although paclitaxel was identified in the 1970s, its development was limited by hypersensitivity reactions. It is poorly soluble in water, and it is therefore commonly administered in a vehicle known as Cremophor EL (a polyoxyethylated castor oil vehicle, and dehydrated ethanol (1:1, v/v), used to solublize some drugs that are poorly soluble in water), which may contribute to the hypersensitivity reactions observed when administered. Premedication with drugs such as antihistamines and glucocorticoids, and slower administration of the drug, allowed the practical use of paclitaxel in patients, but in some people, this reaction prevents its use. It is administered by intravenous infusion over times ranging from a few hours to several days. Paclitaxel is commonly used as first-line therapy in many common malignancies, including lung cancer, breast cancer, ovarian cancer, and head and neck cancer. It also has high activity in some specific uncommon malignancies such as angiosarcoma and Kaposi sarcoma. Paclitaxel is also used as a component of drug-eluting vascular stents, where slow local release of the drug inhibits restenosis. Mechanism of Action Paclitaxel binds the beta-subunit of tubulin and stabilizes tubulin polymerization (Fig. 2a, b). Microtubules are composed of polymerized alpha- and beta-tubulin monomers and play important roles in cell function. Tubulins are guanosine triphosphate (GTP)-binding proteins. Beta-tubulin has GTPase-activity. During the formation of microtubules, dimers of alpha- and beta-tubulin bound to GTP attach to the growing (+) end of the microtubule. After incorporation into the microtubule, the GTP is cleaved to GDP. The stability of the tubulin dimer in the microtubules is determined by whether it is bound to GDP or GTP, with GTP favoring assembly while GDP
Paclitaxel
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a Paclitaxel O CH3 O C
H C
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Paclitaxel, Fig. 1 Structure of paclitaxel and docetaxel. Molecular structure of paclitaxel (a) and docetaxel (b). Dissimilarities are marked in blue (a) and red (b)
(Reproduced from Andersen et al. (2006) BMC Clin Pharmacol 6(1):2, with permission per BMC Central policy)
favoring disassembly. Thus, microtubules are in a dynamic state. One important role of microtubules is the formation of the mitotic spindles, which pull the chromosomes apart during mitosis. Microtubules also contribute to cell shape as a critical component of the ▶ cytoskeleton. The cytoskeleton is composed of microtubules, actin, and intermediate filaments and plays an important role in the control of cell shape and locomotion. Microtubules are also involved in the movement of intracellular organelles. Paclitaxel binds polymerized tubulin and inhibits the dissociation rate of the tubulin subunits from the tubule to free monomer, thus stabilizing the microtubule. Paclitaxel administration to cells at micromolar concentrations results in the formation of microtubule bundles and asters and arrests cells in mitosis. However, much lower concentrations (1–20 nM) can be cytotoxic or cytostatic. In vitro studies suggest that the time a cell is exposed to paclitaxel above a threshold concentration may be an important determinant of biologic effect. While other mechanisms may also be involved, it is generally believed that the stabilization of tubulin
polymerization is the major mechanism by which paclitaxel exerts its antitumor activity. Mechanism of Resistance More than one mechanism of resistance to paclitaxel occurs. One clearly defined mechanism is mutations in the tubulin gene that result in altered paclitaxel binding. Other less well-defined mechanisms exist, at least one of which may include overexpression of transmembrane pumps, such as P-glycoprotein family members, that actively remove drug from the cell. Pharmacokinetics Paclitaxel is highly protein bound. Pharmacokinetic studies suggest a very large volume of distribution (~50–180 l/m2of body surface area), possibly reflecting binding to tubulin or another intracellular compartment. While renal elimination does not appear to be an important route of paclitaxel clearance, the liver plays a more important role in drug removal, and drug doses should be modified when liver function is abnormal.
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Paclitaxel
a
β-tubulin α-tubulin Tubulin heterodimer
Microtubule top end view Microtubule “Protofilament”
Microtubule axis
b
Microtubule “Plus end”
GTP-dependent “Treadmilling”
Microtubule “Minus end” Paclitaxel, Fig. 2 Schematic structure of a microtubule. The tubulin dimers associate to form a hollow tube known as a microtubule. The tubulin dimers are arranged in a helical manner with 13 dimers of tubulin per turn. When viewed from the side, the tubulin dimers form microtubule “protofilaments.” The arrangement of the dimers results in the exposure of an alpha-tubulin surface on one end of the
microtubule and a beta-tubulin surface on the other. The microtubules are dynamic structures, with tubulin dimers being added to the “+” end and dissociating from the “” end (Courtesy of Dr. Michael Blaber, Department of Biomedical Sciences, Florida State University, Tallahassee, Florida)
Common toxicities of paclitaxel include ▶ myelosuppression, hair loss, myalgias, fatigue, and peripheral neuropathy. The toxicities of paclitaxel depend on the dose and time of
administration. Paclitaxel is commonly given as a 1- or 3-h intravenous infusion, although a variety of infusion times ranging up to several weeks have been described. Different infusion times are
Palladium-Based Anti-Cancer Therapeutics
associated with different maximally tolerated doses. Longer infusions may be associated with less hair loss, neuropathy, myalgia, and hypersensitivity reaction. Newer formulations of paclitaxel have been developed that do not require Cremophor for solubility and have additional desirable properties. One such formulation is nanoparticle paclitaxel, in which paclitaxel is bound to albumin; this formulation has been FDA approved and is in clinical use. A second formulation is a polymer form in which paclitaxel is linked to poly-L-glutamic acid; this formulation is in late-stage clinical trials. Other formulations and analogs are also in development.
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Paired Basic Amino Acid-Cleaving Enzyme ▶ Furin
Palladium Antitumor Agents ▶ Palladium-Based Anti-Cancer Therapeutics
Palladium Antitumor Compounds ▶ Palladium-Based Anti-Cancer Therapeutics
References Hennenfent KL, Govindan R (2006) Novel formulations of taxanes: a review. Old wine in a new bottle? Ann Oncol 17:735–749 Huizing MT, Misser VH, Pieters RC et al (1995) Taxanes: a new class of antitumor agents. Cancer Invest 13:381–404 Monzo MR, Rosell R, Sanchez JJ et al (1999) Paclitaxel resistance in non-small-cell lung cancer associated with beta-tubulin gene mutations. J Clin Oncol 17:1786–1793 Rowinsky EK, Donehower RC (1995) Paclitaxel (taxol). N Engl J Med 332:1004–1014 Skubitz KM (1997) A phase I study of ambulatory continuous infusion paclitaxel. Anticancer Drugs 8:823–828
PADIs
Palladium Drugs ▶ Palladium-Based Anti-Cancer Therapeutics
Palladium-Based Anti-Cancer Therapeutics Sharon Prince1, Selwyn Mapolie2 and Angelique Blanckenberg2 1 Department of Human Biology, Health Science Faculty, Division of Cell Biology, University of Cape Town, Rondebosch, South Africa 2 Department of Chemistry and Polymer Science, Stellenbosch University, Matieland, South Africa
▶ Peptidylarginine Deiminase Enzymes
Synonyms
PADs ▶ Peptidylarginine Deiminase Enzymes
Palladium antitumor agents; Palladium antitumor compounds; Palladium drugs; Palladium-based drugs
Definition
PAF ▶ Platelet-Activating-Factor
Palladium-based anticancer drugs are a range of compounds containing the platinum group metal
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(PGM), palladium, in one of its various forms including metallic palladium (Pd(0)) and palladium ions in either the 2+ or 4+ oxidation states. In addition, radioactive 103Pd has also been used in cancer therapeutics. Examples include its use in fluoroscopy, a medical imaging technique which involves the real-time observation of internal organs, and in ▶ brachytherapy which uses medical implants of radioactive palladium-103 seeds.
Characteristics In this section, the advantages and disadvantages of palladium-based drugs over other metal-based drugs will be considered, specifically focusing on the inherent chemical and physical properties of palladium and how these influence drug–DNA interactions. Palladium Palladium is a platinum group metal and as such is very similar to platinum in both its chemical and physical properties. These similarities suggest that palladium and platinum could be used interchangeably to form analogous coordination compounds as antitumor agents. The ligand exchange kinetics for palladium is however 105 times faster than that of platinum which makes palladium significantly more reactive. Palladium complexes initially used were therefore found to react with various species in the physiological environment before reaching their target site. Studies have shown that the use of strongly coordinating or chelating ligands can be used to stabilize palladium ions thus reducing their lability in physiological media. Furthermore, palladium compounds are generally less toxic than their platinum counterparts. For example, while the median lethal dose (LD50) for platinum chloride administered orally in rats is 276 mg/kg, it is 2704 mg/kg for palladium, indicating that the palladium salt exhibits approximately tenfold lower toxicity than the platinum salt. Palladium Complexes The discovery of ▶ cisplatin as an antitumor agent has generated an enormous amount of interest
Palladium-Based Anti-Cancer Therapeutics
over the last few decades in the use of ▶ platinum complexes for the treatment of a range of cancers. A number of second-generation platinum compounds have been identified and are currently in clinical use along with cisplatin. Despite the success obtained with these drugs, their use has been associated with severe toxicity, acquired ▶ drug resistance, and low water solubility. This has led to the investigation of alternative metal complexes as antitumor agents, and based on the similarities between platinum and palladium, palladium has been regarded as a good alternative. Indeed, the last 30 years has seen an increase in the number of potential palladium anticancer drugs being synthesized and tested, and as early as the 1980s, palladium complexes with good water solubility and reduced toxicity were reported (Abu-Surrah and Kettunen 2006; Caires 2007; Abu-Surrah et al. 2008; Gao et al. 2009; Kapdi and Fairlamb 2014). There are two broad classes of palladium complexes, viz., mononuclear complexes which contain one palladium center and binuclear complexes which contain two palladium centers. Drug design for mononuclear palladium complexes incorporates several ligands which are biologically active and have known biological benefits. These include water-soluble ligands and ligands which are strongly coordinating to the metal ion. Planar ligands such as pyridine, quinoline, 1,10-phenanthroline, and their analogues are the most common ligand types employed as they allow for drug–DNA intercalation. An example is 2,6-dimethyl-4-nitro-pyridine (dmnp) which when complexed to palladium (Fig. 1) was found to be strongly active against four human cancer cell lines. Importantly, it was more active than cisplatin against three of the four cell lines including the platinum-resistant T47D ▶ breast cancer cell line (Gao et al. 2009). Palladium-based anticancer drugs have also been synthesized with sulfur donor ligands because sulfur-containing compounds have been shown to reduce nephrotoxicity. Both thiol and thiocarbonyl ligands complexed to palladium have shown promising results and have been found to be less toxic than their platinum analogues and cisplatin. Phosphine ligands and their metal complexes have been reported to have
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Palladium-Based AntiCancer Therapeutics, Fig. 1 Structure of [Pd (dmnp)2Cl2 (Gao et al. 2009)
Palladium-Based AntiCancer Therapeutics, Fig. 2 Structure of AJ-5 (Aliwaini et al. 2013, 2014)
anticancer activity, as well as antimicrobial and anti-HIV activity. As with the other ligands, phosphine ligands when complexed with palladium were also found to be more effective than their platinum counterparts. Amino acids are another class of ligands which have been employed because they do not dissociate easily in aqueous media and thus stabilize highly reactive palladium ions. A number of L-amino acid derivatives of 2,20 -bipyridine and 1,10-phenanthroline were used to synthesize both palladium and platinum complexes, and when they were tested in vitro, the palladium complexes were shown to have lower inhibitory doses (ID50) and to be superior nuclear transcription inhibitors compared to their platinum analogues (Gao et al. 2009). Binuclear palladium complexes can be synthesized using bidentate bridging ligands which, as for the mononuclear palladium complexes, are selected for their beneficial properties. Examples include bidentate phosphine ligands, and a series of phosphine-based dimeric complexes have been synthesized using 1,2-bis(diphenylphosphino)
ethane (dppe). Importantly, when tested in vitro and in vivo against mouse melanoma cell lines, the dimeric analogues display significantly higher anticancer activity than the mononuclear complexes (Abu-Surrah et al. 2008). One binuclear complex in particular was able to delay tumor growth in mice and was shown to both bind DNA and promote its degradation in a manner similar to apoptosis, although independent of ▶ caspases 1 and 3. It also appears to inhibit tumor cell metabolism. A novel binuclear palladacycle, [Pd(C^N)Cl (dppe)], more commonly referred to as AJ-5 (Fig. 2), was shown to be a promising metallodrug for treating advanced melanoma, notoriously chemotherapeutic resistant cancer, and breast cancer. AJ-5 was found to exhibit strong cytotoxic effects in metastatic melanoma cell lines as well as in melanoma-bearing mice at a 50-fold lower concentration than cisplatin (Aliwaini et al. 2013). Furthermore, it displayed strong cytotoxicity against breast cancer stem cells and ER-positive and ER-negative breast cancer cells (Aliwaini
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melanoma cell lines, respectively, and inhibited several pathways required for melanomagenesis. These pathways include activation of MAPK, AKT, STAT-3, and S6 kinase (Kapdi and Fairlamb 2014). The promising anticancer activity of palladium complexes, as described above, has been ascribed to their interaction with DNA via both covalent and non-covalent interactions. These will be discussed in the next section.
Palladium-Based Anti-Cancer Therapeutics, Fig. 3 Tris DBA palladium structure (Kapdi and Fairlamb 2014)
et al. 2014). The anticancer activity of AJ-5 in both melanoma and breast cancer cells was shown to involve the induction of double-strand DNA damage breaks and the p38 mitogenactivated protein kinase (MAPK) pathway which mediated cell cycle arrests and cell death by ▶ apoptosis and ▶ autophagy. Importantly, AJ-5 displayed no side effects. Several other dinuclear palladium complexes have been reported, including palladacyclopropanes which incorporate the E,E-dibenzylidene acetone (dba) ligand. These palladacyclopropanes constitute an unusual type of dimeric complex which contains palladium(0) with association between the metal and the ligand through the alkene functionalities of the ligand (Fig. 3). Pd2(dba)3 was the first of these complexes to be tested as an anticancer agent and was found to be a very promising drug candidate for treating ▶ melanoma. Indeed, it led to a 99% and a 96% decrease in cell count in the B16 mouse and A375 human
The Interaction of Palladium Complexes with DNA Metallodrugs can interact with DNA via covalent chemical bonds and/or non-covalent weaker interactions. The interaction between cisplatin and DNA has been extensively studied, and it is well known that its labile chloride ligands are exchanged for water molecules, thus forming an electrophilic complex. This leads to the platinum ion interacting with DNA at the N7 positions of two guanine or guanine and adenine bases which blocks DNA replication and can prevent transcription. A number of mononuclear, binuclear, and multinuclear palladium complexes have been synthesized which have lower toxicity and higher specificity and activity in cisplatin-resistant cell lines, and a major target of these complexes is DNA. Similar to platinum complexes, the interaction of palladium (Pd2+) salts with the oligonucleotide [d(CGCGAATTCGCG)]2 showed selective covalent binding to T8 imino and G4, N7 atoms with mononuclear and binuclear salts showing different selectivity. The mononuclear species interacted at the terminal regions of the oligonucleotides, preferentially at G2 and G10 bases, while the binuclear species selectively bound to N7 of G4 and the aquated species were more selective toward G2 and G4. Unlike platinum complexes, palladium complexes appear to have a good ▶ quantitative structure–activity relationship (Gao et al. 2009). Since palladium complexes show activity against cisplatin-resistant cancers, it was considered that they may also associate with DNA by non-covalent interactions such as groove binding, electrostatic binding, intercalation, and hydrogen bonding (Gao et al. 2009). Furthermore, the
Palladium-Based Anti-Cancer Therapeutics
activity of palladium complexes increases with increased lipophilicity which is a measure of how easily the complex can enter the cell through the phospholipid bilayer cell membrane (Abu-Surrah et al. 2008). The interaction of the mononuclear complex [Pd(bipy)(bmal)] 2H2O (where bipy = 2,20 -bipyridine and bmal = benzylmalonate) with fish sperm DNA was studied by ultraviolet–visible spectroscopy. The maximum absorbance of the complex was found to be 258 nm, and addition of DNA showed a concentration-dependent hypochromic red shift of this absorbance maximum. Related palladiumbipy complexes showed the same trend when reacted with calf thymus DNA (Trachtenberg et al. 2007). Together this suggests that there is a strong stacking interaction between the aromatic moieties of the complex and the base pairs of the DNA which are indicative of intercalative binding to the DNA. A similar trend was observed for the binuclear complex, [Pd2(m-bzta)4 1.5DMSO where bzta = benzothiazole-2-thiolate. The intrinsic binding constant (K) was calculated for this complex, and a value of 1.2 104 M1 was obtained which suggests a very strong interaction between the complex and DNA (Gao et al. 2007). Palladium Complexes in Clinical Trials Despite the reported advantages of palladiumbased drugs in anticancer regimens, there are only two in ▶ clinical trials, and both are used in ▶ photodynamic therapy. Below follows a short description of each of these drugs as well as their respective structures. WSTII is a novel negatively charged watersoluble bacteriochlorophyll derivative in which Cu2+ is replaced with Pd2+ bonded to 4 N donor sites. This complex is currently in an active phase I and II clinical trial to evaluate its therapeutic efficacy against renal tumors. It has also undergone three completed trials in which the efficacy in treating localized prostate cancer was evaluated but no results are as yet available (Mazor et al. 2005). WSTII is used as a sensitizing agent for vascular-targeted photodynamic therapy (VTP), and an in vitro study showed that the drug uptake, clearance, and phototoxicity are likely regulated by the serum albumin pathway.
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In vivo mouse studies showed rapid (60 min) clearance from the body; therefore, a short illumination period (5 min) was chosen. This protocol has been shown to shut down the tumor vasculature during the illumination period. TOOKAD is a palladium-bacteriopheophorbide photosensitizer similar to WSTII. Again, the palladium occurs in the Pd2+ oxidation state. TOOKAD has been shown to lead to vascular thrombosis and secondary tumor destruction. There are currently two closed phase III trials evaluating the efficacy of the drug against localized prostate cancer, and TOOKAD was found to be a safe and welltolerated VTP drug for salvage chemotherapy after radiation (Gao et al. 2006) (Fig. 4). Palladium-103 Brachytherapy Radioactive palladium has also been employed in radiation therapy to treat certain cancers, and an example of this is 103Pd brachytherapy which is currently used almost exclusively for treating prostate cancer. Brachytherapy involves radioactive seeds/sources being implanted in or near the tumor to provide a high radiation dose specifically to the tumor which ensures long-term biochemical and clinical tumor control. 103Pd is a stable radioisotope that is suitable for interstitial implantation usually in the form of a TheraSeed ®. Whereas brachytherapy has traditionally employed the 125 I radioisotope, 103Pd has an advantage over it because its half-life is significantly shorter (17 days vs. 60 days) which means that the initial dose rate of radiation from 103Pd is higher. Many short-term studies revealed no difference between the outcomes based on the two isotopes; however, a long-term study showed that 103Pd should be used in more severe cases due to this higher initial dose rate (Blasko et al. 2000). The Use of Palladium in Complementary Medicine Palladium has also found its way into the sphere of complementary medicine with the most frequently cited example being that of Poly-MVA, a composite of B complex vitamins, amino acids, and alpha-lipoic acid complexed to palladium ions (see Fig. 5). It is usually administered as a
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Palladium-Based Anti-Cancer Therapeutics, Fig. 4 Structure of WST11 and TOOKAD (Mazor et al. 2005; Gao et al. 2006)
Palladium-Based AntiCancer Therapeutics, Fig. 5 Palladium–lipoic acid complex (Ramachandran et al. 2010)
liquid dietary supplement and has been proposed as a nontoxic alternative to ▶ chemotherapy or in combination with other forms of anticancer therapy (combination chemotherapy). The incorporation of palladium into this mixture is claimed to
allow the lipoic acid to reach specific target sites, while the other components complement this action and provide necessary nutrients. The activity of Poly-MVA results from it interacting with and changing the electrical charge of DNA
Palladium-Based Anti-Cancer Therapeutics
molecules. It is also purported to boost the immune system, reduce pain, and help restore energy and appetite and is used for the treatment of medical conditions and diseases including several cancers, AIDS, and chronic fatigue. PolyMVA is classified as a radioprotective supplement which improves health by mitigating the reduction of tissue antioxidant levels during wholebody irradiation. There is currently only one closed phase 1 clinical trial for individuals with ▶ astrocytoma, but very little scientific evidence for the efficacy of this drug can be found (Ramachandran et al. 2010). Conclusion Palladium-based drugs provide a vast library of compounds with promising anticancer properties, and they have clear advantages over their platinum counterparts, including lower toxicity, lower cost, and better structure–activity relationships. There are however very few palladium compounds in use today. This is in part due to the limited knowledge available on the mechanism of action of these drugs which should be the focus of future research.
Cross-References ▶ Apoptosis ▶ Astrocytoma ▶ Autophagy ▶ Brachytherapy ▶ Breast Cancer ▶ Chemotherapy ▶ Cisplatin ▶ Drug Resistance ▶ Photodynamic Therapy ▶ Platinum Complexes ▶ Quantitative Structure Activity Relationship ▶ STAT3 ▶ Vascular Targeting Agents
References Abu-Surrah AS, Kettunen M (2006) Platinum Group Antitumor Chemistry: Design and development of New Anticancer Drugs Complementary to Cisplatin. Curr Med Chem 13:1337–1357
3377 Abu-Surrah AS, Al-Sa’doni HH, Abdalla MY (2008) Palladium-based chemotherapeutic agents: Routes toward complexes with good antitumor activity. Cancer Ther 6:1–10 Aliwaini S, Swarts AJ, Blanckenberg A, Mapolie SF, Prince S (2013) A novel binuclear palladacycle complex inhibits melanoma growth in vitro and in vivo through apoptosis and autophagy. Biochem Pharm 86:1650–1663 Aliwaini S, Peres J, Kröger WL, Blanckenberg A, de la Mare J, Edkins AL, Mapolie SF, Prince S (2014) The palladacycle, AJ-5, exhibits anti-tumour and anti-cancer stem cell activity in breast cancer cells. Cancer Lett 357:206–218 Blasko JC, Grimm PD, Sylvster JE, Badiozamani KR, Hoak D, Cavanagh W (2000) Palladium-103 brachytherapy for prostate carcinoma. Int J Radiat Oncol Biol Phys 46:839–850 Caires ACF (2007) Recent Advances Involving Palladium (II) Complexes for the Cancer Therapy. Anticancer Agents Med Chem 7:484–491 Gao E, Sun Y, Liu Q, Duan L (2006) An anticancer metallobenzylmalonate: crystal structure and anticancer activity of a palladium complex of 2,2'-bipyridine and benzylmalonate. J Coord Chem 59:1295–1300 Gao E-J, Wang K-H, Gu X-F, Yu Y, Sun Y-G, Zhang W-Z, Yin H-X, Wu Q, Zhu M-C, Yan X-M (2007) A novel binuclear palladium complex with benzothiazole-2thiolate: Synthesis, crystal structure and interaction with DNA. J Inorg Biochem 101:1404–1409 Gao E, Liu C, Zhu M, Lin H, Wu Q, Liu L (2009) Current Development of Pd (II) Complexes as Potential Antitumor Agents. Anticancer Agents Med Chem 9:356–368 Kapdi AR, Fairlamb IJS (2014) Anti-cancer palladium complexes: a focus on PdX2L2, palladacycles and related complexes. Chem Soc Rev 43:4751–4777 Mazor O, Brandis A, Plaks V, Neumark E, RosenbachBelkin V, Salomon Y, Scherz A (2005) WST11, A Novel Water-soluble Bacteriochlorophyll Derivative; Cellular Uptake, Pharmacokinetics, Biodistribution and Vascular-targeted Photodynamic Activity Using Melanoma Tumors as a Model. J Photochem Photobiol 81:342–351 Ramachandran L, Krishan CV, Nair CKK (2010) Radioprotection by a-Lipoic Acid Palladium Complex Formulation (POLY-MVA) in Mice. Cancer Biother Radiopharm 25:395–399 Trachtenberg J, Bogaards A, Weersink RA, Haider MA, Evans A, McCluskey SA, Scherz A, Gertner MR, Yue C, Appu S, Aprikian A, Savard J, Wilson BC, Elhilali M (2007) Vascular Targeted Photodynamic Therapy With Palladium-Bacteriopheophorbide Photosensitizer for Recurrent Prostate Cancer Following Definitive Radiation Therapy: Assessment of Safety and Treatment Response. J Urol 178:1974–1979
See Also (2012) AKT. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 115. doi:10.1007/978-3-642-16483-5_163
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3378 (2012) Antioxidant. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 216. doi:10.1007/978-3-642-16483-5_328 (2012) Cancer stem cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 626. doi:10.1007/978-3-642-16483-5_815 (2012) Caspase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 674–675. doi:10.1007/978-3-642-16483-5_873 (2012) Cell cycle. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 737. doi:10.1007/978-3-642-16483-5_994 (2012) Cell death. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 737. doi:10.1007/978-3-642-16483-5_6724 (2012) Cell lines. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 738. doi:10.1007/978-3-642-16483-5_1002 (2012) Clearance. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 880. doi:10.1007/978-3-642-16483-5_1211 (2012) Clinical trial. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 883–884. doi:10.1007/978-3-642-16483-5_1216 (2012) Combination chemotherapy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 952. doi:10.1007/978-3-64216483-5_6902 (2012) Cytotoxic. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1058. doi:10.1007/978-3-642-16483-5_1499 (2012) DNA. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1129. doi:10.1007/978-3-642-16483-5_1663 (2012) DNA replication. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1141. doi:10.1007/978-3-642-164835_1690 (2012) Dose rate. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1155. doi:10.1007/978-3-642-16483-5_1715 (2012) Fluoroscopy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1426–1427. doi:10.1007/978-3-642-16483-5_2215 (2012) Half-life. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1625. doi:10.1007/978-3-642-16483-5_2554 (2012) Isotope. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1921. doi:10.1007/978-3-642-16483-5_3164 (2012) Lipophilicity. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2058. doi:10.1007/978-3-642-16483-5_3384 (2012) Mitogen-activated protein kinase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2336. doi:10.1007/978-3-64216483-5_3770
Palladium-Based Drugs (2012) Oligonucleotide. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2606. doi:10.1007/978-3-642-16483-5_4207 (2012) Phase I and II clinical trials. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2852. doi:10.1007/978-3-64216483-5_4504 (2012) Phospholipid bilayer. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2869. doi:10.1007/978-3-64216483-5_4541 (2012) Photosensitizer. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2881–2882. doi:10.1007/978-3-642-16483-5_4559 (2012) Prostate cancer. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 3009–3010. doi:10.1007/978-3-642-16483-5_6576 (2012) Radiation therapy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3144. doi:10.1007/978-3-642-164835_4907 (2012) Salvage chemotherapy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3334. doi:10.1007/978-3-64216483-5_6947 (2012) STAT. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3502. doi:10.1007/978-3-642-16483-5_5481 (2012) Therapeutic efficacy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3667. doi:10.1007/978-3-64216483-5_5764 (2012) Thrombosis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3678. doi:10.1007/978-3-642-16483-5_5795 (2012) Toxicity. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3731. doi:10.1007/978-3-642-16483-5_5868 (2012) Transcription. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3752. doi:10.1007/978-3-642-16483-5_5899
Palladium-Based Drugs ▶ Palladium-Based Anti-Cancer Therapeutics
Palliative Care ▶ Supportive Care
Palliative Therapy
Palliative Therapy Michael MacManus Department of Radiation Oncology, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia
Definition The term “palliative” (from the Latin palliare, to cloak) is often applied to treatment that is given with the intention of reducing the severity of the symptoms of cancer or is intended to slow the progress of the disease. Palliative treatment is given without the intention of providing a cure. Much of the care that is provided to cancer patients is effectively palliative in nature and represents an enormous expenditure of resources by health agencies in developed countries.
Characteristics A decision to use palliative therapy in cancer implies that the patient has a malignant disease process that has been recognized as being impossible to cure. The use of “radical” or aggressive therapies with curative intent would be futile for such patients. Palliative therapies for cancer are therefore given without any real expectation of permanently eradicating the disease process causing the symptoms, although very rarely, long-term survival and even cure can be an unintended but welcome consequence. Palliative therapies for cancer work by inducing regression of all or part of the malignant disease process. This is in distinction to the disciplines of “palliative care” and “▶ supportive care,” which stress the skilful relief of pain and other physical and psychological symptoms in cancer and other chronic diseases, usually without direct treatment of the chronic illness responsible for the symptoms. In the course of the illness of a patient with cancer, the character of the treatment may become
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progressively more palliative in nature when initial treatment fails and the disease is gradually recognized as being incurable. In some diseases, such as ▶ notch/jagged signaling in neoplasia, the majority of patients present with disease that is already too advanced for cure and all therapy is palliative in nature at the outset. Palliative therapies are often, but not always, less intense or toxic than treatments given with curative intent. Palliative therapies often require that the patient experiences a temporary worsening of quality of life due to transient toxicity from the treatment, in exchange for the hope or expectation of a later improvement in the symptoms caused by the cancer. Alternatively the patient with incurable malignancy may have few symptoms at the outset but may still be offered palliative treatment in an effort to prevent an impending catastrophe, such as obstruction of a major airway, or to achieve longer survival due to a temporary reduction in the burden of disease. The range of symptoms that can be relived by palliative therapies is wide. This is a reflection of the enormous differences between different types of malignant disease and the endless possibilities for variation of patterns of disease progression between different patients with the same type of cancer. The best method of palliation varies widely between the different malignant disease types and the site of the disease responsible for the symptoms. The symptoms may reflect local anatomical problems, such as pain from bone destruction, or be systemic in nature, such as fevers or anemia. The most commonly used palliative therapies for cancer patients are radiotherapy (▶ ionizing radiation therapy), ▶ chemotherapy, hormonal therapy, and surgery, but a wide range of additional palliative anticancer therapies can have important roles in specific clinical situations. Examples of these other therapies include radionuclide therapy for bone metastases or thyroid cancer, immunotherapy and ▶ radioimmunotherapy for B-cell lymphoma, laser coagulation of bleeding bronchial carcinomas, radiofrequency ablation for liver tumors, and
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psoralen combined with ultraviolet light treatment (PUVA) in the treatment of some skin lymphomas. Surgical interventions can play a major role in the palliation of symptoms, such as placement of an esophageal stent to allow swallowing to resume in esophageal cancer, relief of intestinal obstruction by bowel diversion procedures, relief of spinal cord compression, or resection of a solitary or dominant brain metastasis. The most widely used palliative therapy modalities will be discussed in a little more detail below. Palliative Radiotherapy Palliative radiotherapy (also often known as radiation therapy) is a widely used and highly effective treatment for localized tumor lesions that are causing symptoms or are about to do so. Most palliative radiotherapy is now given using highenergy photons from linear accelerators or telecobalt machines. For more superficial tumors, electron beams or orthovoltage of superficial X-ray machines may be used. For some accessible tumors, radioactive sources may be placed directly into the tumor or adjacent to the tumor to provide local symptom control with a very low radiation dose to normal tissues more than a few centimeters from the source. Palliative radiotherapy differs from potentially curative radical or “definitive” radiotherapy, not only in the intent of the treatment but also in some technical aspects. Because cure is not the intention, lower total doses are often used than in radical radiotherapy, fraction sizes are usually larger, and fewer treatments are given so that overall treatment time is as short as possible to achieve the goal of timely palliation. Simpler and less costly beam arrangements may be used. It is not always necessary to include all of the disease in the treatment field to obtain a benefit, and the treatment intensity should ideally be just enough to provide relief of the symptoms for the duration of the patient’s remaining life span. For very ill patients with a very short anticipated life span, a single fraction of radiotherapy may be sufficient to relieve symptoms. However, most treatments are fractionated (which is given in separate small increments) on a daily basis. Most of the widely used fractionation schedules in common cancers involve the treatment of consecutive
Palliative Therapy
week days for approximately 1 or 2 weeks. There is a wide spectrum of radiosensitivity between different cancers, and this is considered when planning treatment. Some malignancies, such as follicular lymphoma, an often indolent type of ▶ malignant lymphoma, can be exquisitely radiosensitive and respond completely to doses as low as 4 Gy, without any toxicity. Others, such as recurrent rectal cancer, may be relatively radioresistant and require doses of radiation in excess of 40 Gy to obtain durable relief of pain, often with significant treatment-related side effects. Palliative radiotherapy is used across virtually the complete spectrum of cancers and plays a large role in all of the most common malignancies, including lung, breast, prostate, and bowel cancers, brain tumors, and ▶ multiple myeloma. Many of the most common consequences of these diseases are effectively treated with palliative radiotherapy, including pain caused by bone metastasis, breathlessness and hemoptysis caused by bronchial tumors, brain impairment caused by central nervous system metastases, or malignant spinal cord compression. Radiotherapy can reach any part of the body and is useful in situations where other treatments cannot gain access; for example, brain metastases may be relatively protected from chemotherapy by the blood-brain barrier but are often effectively treated with radiation. The toxicity of radiotherapy varies with the dose and fractionation and the region of the body treated. Because palliative doses are usually lower than radical doses, side effects are usually relatively mild and transient but commonly include fatigue, some skin redness, inflammation of mucous membranes in the treatment field, and localized hair loss. Palliative Chemotherapy The term chemotherapy in oncology usually means the delivery of cytotoxic drugs to kill cancer cells. Chemotherapy drugs are often very powerful agents targeted at proliferating cells. Most, but not all, have a relatively narrow therapeutic window, meaning that the difference between an effective dose and a dangerously toxic dose is relatively small. For this reason drugs with
Palliative Therapy
overlapping toxicities are often given together in combinations of two or more agents. Many of these drugs suppress bone marrow function and therefore need to be given in “cycles” some weeks apart, allowing bone marrow recovery to occur before the next cycle can be given. Malignant diseases show a very wide range of sensitivities to chemotherapy. In those diseases with the highest sensitivity to chemotherapy, including malignant lymphomas and germ cell tumors, chemotherapy is often given with curative intent at the outset. After relapse or after failure of salvage therapy, the patient may be recognized to have disease that is no longer curable. Chemotherapy may then play an important role in palliation. Sometimes the distinction between curable and incurable diseases may not be clearly made, and chemotherapy may serve both a palliative role, to relieve symptoms, and provide a small hope for long-term survival. In advanced follicular lymphoma, which is highly chemosensitive but is usually considered incurable, patients may survive for many years following palliative chemotherapy. Relapse or progression after initial palliative chemotherapy is usual and second-line chemotherapy may be considered. Subsequent courses of chemotherapy are usually more difficult to tolerate than early courses. In the late stages of an initially chemosensitive illness, the patient may be faced with the dilemma of choosing further chemotherapy associated with a high risk of toxicity and a low probability of response or concentrating solely on supportive care. There is a larger group of moderately chemotherapy-sensitive cancers where chemotherapy can play a significant role in palliation when the patient is known to have incurable disease. These diseases are not generally considered to be curable with chemotherapy alone. They include multiple myeloma and many epithelial cancers such as advanced non-small cell ▶ lung cancer, extensive-stage ▶ small cell lung cancer, ▶ ovarian cancer, ▶ breast cancer, and advanced head and neck cancers. Patients most likely to benefit from palliative chemotherapy are those with a good performance status and without serious concomitant illnesses. In advanced ▶ non-small cell lung cancer, for example,
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chemotherapy provides such patients with a significant but relatively short improvement in overall survival, perhaps 6 weeks on average. Some patients may consider the risk of toxicity excessive for a relatively short prolongation of their lives in this situation. However, quality of life is a major consideration when deciding whether or not to use palliative chemotherapy, and wellconducted studies have shown that quality of life is actually better in lung patients who receive chemotherapy than in those who do not. In other cancers such as breast cancer where response rates are usually higher and patients are often younger and fitter when palliative chemotherapy is needed, greater benefits may be obtained with chemotherapy and initial decision making about whether or not to have chemotherapy is easier. There is a further group of diseases that are relatively unresponsive to chemotherapy, in that the majority of patients do not have a response and randomized trials have shown that overall survival is not improved compared to patients who do not receive chemotherapy. Nevertheless, even in this group, excellent responses to chemotherapy are sometimes seen. This group includes diseases like malignant ▶ melanoma, renal cell carcinoma, and some soft tissue sarcomas. Like radiotherapy, chemotherapy can be a double-edged sword. In cancers with low chemosensitivity, it can often produce toxicity without any benefits whatsoever, unlike radiotherapy, which at least causes a response in most tumors that are treated even if they are chemoresistant. However, unlike radiotherapy chemotherapy is a whole-body treatment and often is the only available means for modifying the overall course of a widely disseminated cancer and improving overall survival. Hormonal Therapy Breast cancer and prostate cancer are very common malignancies that are often responsive to palliative hormonal therapies when patients are known to have incurable advanced disease. In both diseases, hormonal therapy can also play a role as adjuvant therapy for patients with potentially curable localized disease. Breast cancer cells commonly have receptors on the cell surface for
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Pancreas Cancer
estrogen and progesterone that indicate that the cells may behave more aggressively in the presence of estrogen in the bloodstream. In patients with advanced, receptor-positive breast cancer, the disease can often be made to regress by manipulating the patient’s hormonal environment. In the past this was commonly accomplished by inducing an artificial menopause by oophorectomy or radiation to the ovaries. In modern practice, estrogen receptor antagonists such as ▶ tamoxifen or ▶ aromatase inhibitors such as letrozole are most commonly used and are effective in both pre- and postmenopausal women. Compared to chemotherapy, these drugs have low toxicity. Response rates are high in receptor-positive cancers and may last several years, with regression of painful bony lesions or symptomatic soft tissue tumors. In advanced prostate cancer, often associated with widespread painful bone metastasis, tumor cells are usually dependent on the presence of circulating androgens which stimulate tumor growth by binding to ▶ androgen receptors on the cell surface. By inducing a reduction in the circulating level of androgens, as in bilateral orchidectomy, or by blocking the pituitary secretion of hormones that stimulate androgen production by the testis using LH-RH agonist drugs such as Zoladex or by using drugs that block androgen receptors on the tumor cells such as Bicalutamide, advanced prostate cancer can be made to regress in many cases. Such regressions last on average a little less than 2 years but in individual cases may last for many years and may be achieved with acceptable side effects.
and autonomy of the suffering person should be paramount.
Conclusion The palliative treatment of cancer is complex and should ideally be coordinated in a multidisciplinary team setting where a range of palliative therapies is available together with access to appropriate supportive care. Patients may benefit from the concurrent or sequential use of a range of different palliative treatments to achieve the best quality of life for the longest possible time. Treatments must be tailored both to the disease extent and to the wishes and personal philosophy of the patient, always bearing in mind that the dignity
Definition
References Bunn PA Jr (2002) Chemotherapy for advanced non-smallcell lung cancer: who, what, when, why? J Clin Oncol 20:23S–33S Hoskin PJ, Bownes P (2006) Innovative technologies in radiation therapy: brachytherapy. Semin Radiat Oncol 16:209–217 Lester JF, Macbeth FR, Toy E et al (2006) Palliative radiotherapy regimens for non-small cell lung cancer. Cochrane Database Syst Rev: CD002143 Martin CM (2006) Understanding palliative care. Consult Pharm 21:698–713
Pancreas Cancer ▶ Pancreatic Cancer
Pancreatic Cancer Daniel D. von Hoff Arizona Cancer Center, Tucson, AZ, USA
Synonyms Pancreas Cancer
Pancreatic cancer is an ▶ adenocarcinoma of the pancreas (carcinoma of the pancreas). Cancer of the pancreas is usually lethal. The pancreas is a gland that secretes both digestive enzymes and insulin (from special cells called islet cells). Estimated new cases and deaths from pancreatic cancer in the United States in 2009 are as follows: • New cases: 42,470 • Deaths: 35,240
Pancreatic Cancer
Characteristics Anatomy The pancreas is located on the upper abdomen located in the midst of many vital organs, including the liver, spleen, stomach, small bowel, and large bowel. Because of its central location, it is very problematic when cancer spreads from the pancreas directly into the adjacent organs. In addition, the head of the pancreas (the portion on the patient’s right side) covers the common bile duct. The common bile duct is the duct through which bile runs from the liver and gall bladder and is mixed with pancreatic juices and then emptied into the small bowel. Blockage of this common bile duct can lead to one of the early symptoms of pancreatic cancer. In addition, the location of the pancreas near the back (in an area called the retroperitoneum) is problematic as that is the area which contains important vessels, such as the superior mesenteric artery and veins and the celiac plexus. This means that a cancer in the pancreas will frequently (and very early in the course of the disease) invade these vessels, which enables the tumor to spread to distant sites by these vessels (such as to the liver or lung). Vessel invasion also makes the pancreas cancer inoperable. There are also many nerves that are located behind the pancreas, and those nerves are frequently affected by cancer of the pancreas. Etiology (Cause of the Disease) The cause of pancreatic cancer is unknown. In about 8% of patients, the disease appears to be inherited (familial pancreatic cancer). The primary risk factor for pancreatic cancer that is not inherited is smoking. Other risk factors include ▶ alcohol consumption, a history of surgical procedures for peptic ulcer disease, and a history of ▶ inflammation of the pancreas (pancreatitis). Diabetes is frequently associated with cancer of the pancreas. However, it is unclear if diabetes is a risk factor for the development of pancreatic cancer or whether it is just a result of the pancreatic cancer damaging the insulin-producing (islet cells) cells of the pancreas.
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Pathology It is very important to determine what type of cancer of the pancreas one is talking about. The types of cancer involving the pancreas are as follows: • Ductal adenocarcinoma of the pancreas: This is the most common (accounts for more than 90%) and the most lethal form of the disease. It is believed that this type of cancer arises in the cells that line the ducts of the pancreas. Ductal adenocarcinoma rapidly invades blood vessels, nerves, and other organs. It is frequently at an advanced stage when it is diagnosed and has a bad prognosis. • Mucinous cystadenocarcinoma: This pathologic type is an uncommon form of pancreas cancer. It tends to be less invasive and mainly causes problems because of its size. The usual treatment is surgical removal, unless other vital organs, blood vessels, or nerves are involved. It is not a benign condition. • Islet cell tumor: These are cancers that appear to arise from the small clusters of cells called islets, which are scattered throughout the normal pancreas. Islets have cells capable of making many different hormones including insulin and glucagon. Islet cell tumors can cause symptoms as the excessive hormones that they make (such as insulin) can cause severe physiologic problems (such as hypoglycemia or low blood sugar). In general, islet cell tumors have a far better prognosis than other types of cancer of the pancreas. The sections below will only deal with the most common type of pancreas cancers – ductal adenocarcinoma of the pancreas. Symptoms Unfortunately, the symptoms associated with ductal adenocarcinoma of the pancreas are fairly nonspecific and appear late. They include pain in the midepigastric (stomach area) or the back (usually due to nerve invasion by the tumor), nausea and/or vomiting, fatigue, loss of appetite, and weight loss. A change in bowel habits with
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light-colored stools is also a sign of the disease, as is a severe darkening of the urine. One rather drastic symptom is the appearance of jaundice (a yellowness of the whites of the eyes and skin), which is caused by the tumor closing off the bile drainage such that the bile (containing the pigment bilirubin) cannot be secreted, builds up in the blood, and is deposited in the skin. Making the Diagnosis The most effective way to determine whether or not there is a mass in the pancreas is via a special X-ray known as a spiral CT scan. Another, perhaps more sensitive method, is called an endoscopic ultrasound (or EUS) in which a tube (endoscope) is passed through the mouth into the stomach and an ultrasound device at the end of the tube sends out signals that are used to detect a mass in the pancreas. If a mass is detected, it is critical to obtain a histologic diagnosis to determine if the mass is cancer (or just a benign inflammation). A histologic diagnosis is obtained by inserting a needle into the mass to look for tumor cells, or the histologic diagnosis is obtained by performing an open surgical procedure and biopsy of the pancreas. Staging of the Disease Pancreatic cancer is staged as being localized, locally advanced, or metastatic (with distant spread). • Localized: This stage means the tumor is confined within the pancreas with no major blood vessel involvement or involvement of areas outside of the pancreas. • Locally advanced: Tumor involves major blood vessels or regional lymph nodes, but no cancer in other organs. In general, the tumor can be encompassed by a radiotherapy port. There can be no spread to distant organs. • Metastatic: The pancreatic cancer has spread beyond the pancreas (usually into the liver, other surrounding organs, or lung). Prognosis Pancreatic cancer has the worst survival of any cancer. The overall 1-year survival for all patients
Pancreatic Cancer
is about 18% with fewer than 2% of patients living 5 years. For patients with localized pancreatic cancer who have surgical resections of their disease and no evidence of tumor spread beyond the pancreas in their pathology resections, there may be as many as 20% who survive 2 years. However, it is rare to find that the pancreatic cancer is truly localized. For patients with locally advanced pancreatic cancer, the average survival is about 10 months if the patient is treated with radiation to the area in addition to chemotherapy. For patients with advanced metastatic pancreatic cancer, the average survival (with treatment) is about 6 months. Thus, the diagnosis of pancreatic cancer comes with a terrible prognosis. In order to make progress against the disease, it is important that new therapies be developed. Treatment • For patients with localized disease, the treatment is surgical resection. This gives the patient their only chance for prolonged survival (if the patient is fortunate to have truly localized disease). The surgical resection of the pancreas with surrounding organs and bowel reconstruction is frequently referred to as the Whipple procedure. Even with localized disease with no invasion noted on scans at the time of surgery, the pancreatic cancer frequently is found to have spread beyond the pancreas. Therefore, an important area of research is to use a ▶ neoadjuvant therapy approach. The idea with neoadjuvant therapy is to give therapy, such as radiation therapy, chemotherapy, or both, before surgery to try to “downstage” the disease. Hopefully, this approach will help make more patients truly operable with complete removal of their tumor. • For patients with locally advanced disease, there is controversy in terms of what constitutes the best treatment. The standard treatment is considered to be radiation plus chemotherapy (usually the drug 5-fluorouracil) to sensitize the tumor to the radiation. This treatment has been reported to increase the average survival for a patient with locally advanced disease from 5 up to 10 months. However, the
Pancreatic Cancer Basic and Clinical Parameters
radiation plus chemotherapy regimen is associated with substantial side effects. Therefore, a better approach (perhaps chemotherapy alone) for patients with locally advanced pancreatic cancer is being investigated. • For patients with metastatic pancreatic cancer, treatment is usually chemotherapy or supportive care (pain control and treatment of other medical problems) only. Until recently, there has been no chemotherapy that has improved patient survival. The anticancer agent, ▶ gemcitabine, has been shown to improve the survival of patients with advanced pancreatic cancer, plus improve the quality of life of the patient (decreased pain, improvement of performance status). When patients with advanced pancreatic cancer were treated with gemcitabine, their survival was improved from 2% (in the control arm treated with 5-fluorouracil) up to 18% for patients receiving gemcitabine. Currently, investigators are building on this modest advance against advanced disease by combining gemcitabine with other new cancer therapy approaches. Familial Pancreatic Cancer Only about 3–8% of pancreatic cancers are thought to be familial. It is an area of intense study to determine if you have a parent or brother and/or sister with the disease, what type of monitoring, and/or if treatment is necessary. It is clear that some type of monitoring, such as endoscopic ultrasound, should be performed at special centers.
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References Brentnall TA, Bronner MP, Byrd DR et al (1999) Early diagnosis and treatment of pancreatic dysplasia in patients with a family history of pancreatic cancer. Ann Intern Med 131:247–255 Burris HA III, Moore MJ, Andersen J et al (1997) Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol 15:2403–2413 Greenlee RT, Murray T, Bolden C et al (2000) Cancer statistics. CA Cancer J Clin 50:7–33 Moertel CG, Frytat S, Hahn RG et al (1981) Therapy of locally unresectable pancreatic carcinoma: a randomized comparison of high dose (6000 rads) radiation alone, moderate dose radiation (4000 rads + 5fluorouracil), and high dose radiation + 5-fluorouracil. Cancer 48:1705–1713
Pancreatic Cancer Basic and Clinical Parameters Jochen Gaedche and B. Michael Ghadimi Department of General, Visceral and Pediatric Surgery, University Medical Center, Göttingen, Germany
Definition Epithelial malignancies of the pancreatic gland that originate in more than 85% from ductal cells. In rare cases, tumors may also arise from acinar or endocrine cells.
Cross-References
Characteristics
▶ Alcoholic Pancreatitis ▶ Deleted in Pancreatic Carcinoma Locus 4 ▶ Neuroendocrine Tumors of the Pancreas ▶ Pancreatic Cancer ▶ Pancreatic Cancer Basic and Clinical Parameters ▶ Pancreatic Cancer Biology and Management ▶ Pancreatic Cancer Molecular Targets for Therapy ▶ Pancreatic Cancer Pathogenesis ▶ Pancreatic Cancer Stem Cells
Pancreatic adenocarcinoma is one of the most aggressive human malignancies reflected by a mortality rate which closely follows that of the incidence. Its incidence is steadily increasing, and today it is the fifth leading cause of cancer-related deaths in the western hemisphere. Most patients are diagnosed with pancreatic cancer in the late course of the disease with unspecific symptoms such as fatigue, weight loss, jaundice, and upper abdominal pain. At this stage ▶ pancreatic cancer frequently has invaded surrounding organs such
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Pancreatic Cancer Basic and Clinical Parameters
Pancreatic Cancer Basic and Clinical Parameters, Fig. 1 Abdominal magnetic resonance imaging (MRI) picture. The MRI depicts a huge mass in the upper abdomen that originates from the corpus of the pancreas
as the duodenum and stomach and the retroperitoneal tissue and blood vessels (Fig. 1). Lymph node metastases occur in most cases and reflect a high metastatic potential. Most patients present between the fifth and eighth decade of life with a male/female ratio of ~1.5:1. The etiology of pancreatic cancer remains unclear. However, environmental factors such as cigarette smoking and a high-fat diet may predispose patients to the development of pancreatic malignancies. Chronic inflammatory diseases such as chronic pancreatitis might also increase the risk for pancreatic adenocarcinoma. Genetic factors, such as p16 germline mutations and mutations in the mismatch-repair system, may also increase the probability to develop pancreatic adenocarcinoma. However, less than 10% of patients belong to a hereditary pancreatic cancer syndrome. Pathology The most frequent site of pancreatic cancer is the head of the pancreatic gland (60%). The remainder of cases arise in the body (15%) and the tail (5%) or disseminated throughout the pancreas (20%). The vast majority of pancreatic cancers are of ductal origin. Rarely, they originate from acinar or endocrine cells. Ductal adenocarcinoma of the pancreas are desmoplastic malignancies composed of mucin-producing glandular cells infiltrating a nonneoplastic stroma which accounts for more than 50% of the tumor tissue. In addition to the fibrocytic stroma, cancer cells
are also admixed with inflammatory cells, including lymphocytes. The histological progression from benign to malignant pancreatic disease starts from flat mucinous lesions to papillary lesions without atypia to lesions with atypia, to in situ carcinoma, and finally to infiltrating adenocarcinoma. At the time of diagnosis, pancreatic cancer generally has invaded the peripancreatic fat tissue and lymph nodes or adjacent organs such as the duodenum, stomach, peritoneum, and vessels. Cancers restricted to the pancreatic gland are rare. In particular, carcinomas located in the body and tail are diagnosed at a more advanced stage due to unspecific symptoms. Sites of hematogenous metastasis formation are primarily the liver and rarely the lung. However, local tumor recurrence due to remaining microscopic tumor foci even in smaller tumors seems to be the determining factor for patient’s survival. Staging
The UICC staging system is based on the size of the primary tumor (T), the extent of regional lymph node involvement (N), and the presence of metastases (M). Primary tumor (T): • TX Primary tumor cannot be assessed. • T0 No evidence of primary tumor. • Tis Carcinoma in situ. • T1 Tumor 2 cm. • T2 Tumor >2 cm.
Pancreatic Cancer Basic and Clinical Parameters
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Receptor tyrosin kinases (growth factor receptors)
Ligands
Autocrine loop
(growth factors)
p14 inactivation MDM2
No inhibition KRAS mutation
P53 degradation
Constitutive activation of different kinases
Loss of function No DNA repair no apoptosis
p53 mutation
Her2
Aneuploidy and genomic instability BRCA2 loss
P13K pathway Uncontrolled cell cycle progression
No homologous DNA repair
TβR I+II
HGF
AKT2 activation
Activation TGF
Antiapoptotic signal
SMAD 2+3
No growth inhibition no apoptosis RB inactivation
No heteromerization SMAD4 inactivation
CDK4 / CDK6 Activation
No suppression of angiogenesis
Chemo-and radioresistance No inhibition p16 inactivation
CyclinD1 Expression
EGFR EGF
Pancreatic Cancer Basic and Clinical Parameters, Fig. 2 Schematic diagram of major pathways in the development of pancreatic adenocarcinomas which finally lead to the loss of cell cycle control
• T3 Tumor beyond the pancreas but without involvement of the celiac axis or the superior misenteric artery. • T4 Tumor involves the celiac axis or the superior misenteric artery. Regional lymph nodes (N): • NX Regional lymph nodes cannot be assessed. • N0 No regional lymph node metastasis. • N1 Regional lymph node metastasis. Distant metastasis (M): • MX Presence of distant metastasis cannot be assessed. • M0 No distant metastasis. • M1 Distant metastasis. Genetics An array of technologies has been applied to investigate relevant chromosomal and genetic
changes in pancreatic cancer over the last couple of years. The detection of specific chromosomal changes and altered tumor suppressor genes and oncogenes has significantly improved our understanding of the development and progression of this dismal disease (Fig. 2). Cytogenetics
The application of modern fluorescence in situ hybridization (FISH) technologies such as comparative genomic hybridization (CGH) or spectral karyotyping (SKY) for the study of numerical and structural chromosomal aberrations has revealed new insights into pancreatic tumorigenesis. Pancreatic carcinoma cells have a surprisingly high degree of chromosomal instability but also have recurrent pattern of chromosomal alterations. Genetic losses usually involve chromosome arms and chromosomes 8p, 9p, 17p, 18q, 19p,
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and 21, whereas gains can be mapped to 3q, 5p, 7p, 8q, 12p, and 20q. These chromosomal regions are affected in up to 90% of pancreatic adenocarcinomas and correlate very well with those regions harboring oncogenes and tumor suppressor genes such as DPC4 at 18q, P16 at 9p, TP53 at 17p, and KRAS at 12p.
Oncogenes
The KRAS oncogene codes for a GTP-binding protein and plays a major role in pancreatic cancer. Activating point mutations of the KRAS oncogene occur in 70–90% of all ductal adenocarcinomas and are largely restricted to codons 12 and 13 of the KRAS gene at 12p12. Due to these mutations, the KRAS oncogene cannot be inactivated and the signal transduction pathway remains active stimulating proliferation and cellular transformation. KRAS mutations have been found in proliferative, noninvasive ductal lesions indicating that KRAS might play a role during early carcinogenesis. However, KRAS mutations have also been found in inflammatory and normal pancreatic tissue without neoplastic potential. This finding might limit the development of a gene-based test system using KRAS mutations as an indicator for neoplastic or malignant cells in pancreatic juice, blood, or stool of patients with pancreatic disease. Egfr (epidermal growth factor receptor) and Her-2/neu (heregulin, neuregulin, or glial growth factor receptor) belong to the Erbb2 protein family. In normal pancreatic tissue and chronic pancreatitis, Her-2 expression remains unaffected, whereas in patients with pancreatic cancer, overexpression of Her-2 can be found in early morphologic duct lesions. Using immunochemistry, overexpression of Her-2 ranges from 21% to 80%, and only 27% of the immunohistochemical-positive cases showed amplification of the HER-2 gene as detected by FISH. The EGFR gene is located on chromosome 7p12. Expression of Egfr protein can be detected in about 50% of pancreatic carcinomas. Egfr expression seems to play an important role in the metastatic potential.
Pancreatic Cancer Basic and Clinical Parameters
Tumor Suppressor Genes
The relevant tumor suppressor genes in pancreatic cancer are TP53, P16, and DPC4. Tp53 is a nuclear-binding protein that arrests cells at the G1/S checkpoint and also plays an important role in the induction of apoptosis after DNA damage. The TP53 gene resides on chromosome 17p13. It is inactivated by allelic loss and inactivating mutations in about 50% of pancreatic cancers. Therefore, loss of Tp53 protein function results in a disturbed cell cycle and loss of programmed cell death. P16 has been identified at 9p21 and is inactivated in about 90% of pancreatic cancers via allelic loss; inactivating mutations and/or hypermethylation of the promoter has been found. The P16 protein inhibits the promotion of the cell cycle by binding to the cyclin-Cdk4 complex and preventing Cdk4 activation of the Rb protein. Therefore, inactivation of p16 in pancreatic cancer dysregulates another relevant cell cycle checkpoint. The tumor suppressor gene DPC4 is biallelically inactivated in about 50% of pancreatic cancers. Located on chromosome 18q, DPC4 codes for a peptide which is closely related to the Mad family of proteins (sMAD). These molecules play an integral part in the signal transduction from Tgf-b superfamily cell surface receptors. Since it is known that Tgf-b inhibits cell growth and proliferation, inactivation of Dpc4 protein and loss of its inhibitory function may bestow a growth advantage upon cancer cells. Epigenetic Changes
Another cause of gene expression changes in the development of pancreatic cancer is DNA methylation. The methylation of the promoter regions of different genes (e.g., p16, RB, VHL, hMLH1, hMSH2) was also found in pancreatic carcinomas. Besides P16, the gene Preproenkephalin (ppEnk), which has growth-inhibiting function, was found methylated in 90% of the pancreatic cancers. Methylation of genes with a significant role in carcinogenesis occurs early in cancer development. The methylation of p16 can be observed in up to 50% of such cancers. The number of methylated loci increases with the size of the tumor and
Pancreatic Cancer Basic and Clinical Parameters
the age of the patients. Nonneoplastic epithelium is not methylated. Hereditary Pancreatic Cancer Several family studies have suggested that between 5% and 10% of pancreatic cancer may have a hereditary basis. A predisposition to the development of pancreatic cancer has been shown for several genetic syndromes including hereditary pancreatitis, hereditary nonpolyposis colorectal cancer (HNPCC), and the familial atypical mole-malignant melanoma (FAMMM) syndrome. Hereditary pancreatitis is an autosomal dominant disorder which is characterized by an early onset of age. Patients suffer from recurrent acute pancreatitis which leads subsequently to chronic pancreatitis carrying its significant risk factor for the development of pancreatic cancer. In the disorder, a mutation in the trypsinogen gene at 7q35 results in the inability to deactivate trypsin which results in autodigestion of pancreatic tissue. Hereditary nonpolyposis colorectal cancer (HNPCC) syndrome is another syndrome that predisposes individuals to pancreatic cancer. It is an autosomal transmitted disease, caused by germline mutations in the mismatch-repair system. Besides pancreatic cancer, patients also inherit a predisposition to other cancers, including colonic, breast, ovarian, and endometrial carcinomas. Patients with the FAMMM syndrome have an elevated risk for the development of multiple atypical nevi, malignant melanomas, and pancreatic cancer. In a subset of patients, a germline mutation in the p16 tumor suppressor gene is implicated. Diagnosis of Pancreatic Cancer
There are unfortunately no early signs or symptoms to identify pancreatic cancer as gastrointestinal obstruction and other compromising sequelae occur. Patients that are worked up for abdominal pain or jaundice suspicious for pancreatic cancer typically undergo the following diagnostic procedures. Abdominal Ultrasound Transabdominal pancreatic ultrasonography (US) is performed using
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high-resolution real-time linear array or sector scanners combined with Doppler examination. The sensitivity of US in pancreatic cancer is as high as ~85%. Lesions in the head are more visible than lesions in the body or tail of the gland, due to intestinal gas. Computer tomography (CT) is clearly superior in terms of sensitivity and specificity compared to ultrasonography. In summary, US is a reliable screening method for the detection of pancreatic masses and liver metastases but is not recommended for examining patients if a malignant disease is strongly suspected. Computed Tomography (CT) Current techniques including high-resolution spiral CT provide detailed images of the pancreas, the pancreatic and biliary duct system, the peripancreatic vessels, and the surrounding organs. Several studies have reported a sensitivity of 92% and a specificity of ~100% with spiral CT. However, only 65% of all tumors which were staged as resectable with spiral CT scans were actually candidates for resection. Therefore, laparotomy and surgical laparoscopy remain the only specific approaches to determine resectability. Magnetic Resonance Imaging (MRI) The importance of MRI in pancreatic cancer diagnostics, despite the advantage of avoiding ionizing radiation, is still to be determined. In most centers MRI is used to differentiate between malignant and benign disease when US or CT is equivocal or the administration of intravenous contrast agents is contraindicated. Magnetic Resonance Cholangiopancreatography (MRCP) Although the exact role of MRCP has not yet been determined, it has demonstrated its potential to display changes in the pancreatic duct pathology. MRCP and ERCP findings correlate with pathology in 80–90% of the cases. Endoscopic Retrograde Cholangiopancreatography (ERCP) Endoscopic techniques, such as ERCP, can be applied for diagnosis and interventional management for patients with
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Pancreatic Cancer Basic and Clinical Parameters
Pancreatic Cancer Basic and Clinical Parameters, Fig. 3 Pylorus-preserving pancreatectomy (PPPD). (a) Intra-abdominal situs before surgical resection – areas in
orange color mark the organs which will be removed. (b) Intra-abdominal situs after resection according to PPPD
pancreatic disorders with a sensitivity and specificity of 90%. Endoscopic pancreatographic features of malignant disease include stenosis or displacement of the pancreatic and bile duct (e.g., double duct sign), alteration of secondary branches, and extravasation of contrast dyes due to necrosis. An additional major role of ERCP is the opportunity to provide drainage of an obstructed common bile duct by facilitating the insertion of stents into the biliary system. Also tissue sampling can be performed during ERCP, using brush cytology, direct biopsy, endoscopic needle aspiration, and aspiration of pancreatic and bile fluid. The opportunity to obtain tumor material during endoscopic procedures might become an important step since molecular markers can be applied to these samples and might assist in the differential diagnosis of pancreatic disease.
Five-year survival rates increase to 30% when tumors smaller than 2 cm are resected. Surgical technique is dependent on location of the tumor and includes local excision in papillary tumors and left hemipancreatectomy in tumors of the pancreatic tail. However, the majority of pancreatic cancers are located in the pancreatic head on which two methods of resection are most commonly employed. The classical method, the WhippleKausch pancreatectomy, involves en bloc removal of (i) the distal third of the stomach and the right half of the greater omentum, (ii) the gall bladder including the distal bile duct system, (iii) the duodenum and the proximal 10 cm of the jejunum, (iv) the head and parts of the body of the pancreas, and (v) the peripancreatic and hepatoduodenal lymph nodes. The surgical therapy of choice and modern technique is the so-called the pyloruspreserving pancreatectomy (PPPD). Instead of resection of the distal stomach, the pylorus is attached directly to a jejunal loop in PPPD (see Fig. 3). It remains unclear if either method is superior, but advocates of PPPD claim better nutritional outcomes and shorter operative times compared to standard Whipple-Kausch pancreatectomy. Therefore, the PPPD is considered as state of the art in the major surgical centers in Europe.
Therapy of Pancreatic Cancer
Although there has been considerable progress in the biological understanding and diagnostic tools of pancreatic cancer, the neoplasms continue to have one of the poorest prognoses of all human cancers. Surgical resection of small tumors is the only option for curative treatment. The 5-year survival is below 5%, and due to advanced tumor stage at the time of diagnosis, therapies remain palliative for the vast majority of patients.
Pancreatic Cancer Biology and Management
In patients with advanced disease, multidisciplinary approaches are needed involving the surgeon, endoscopist, and radiologist to optimize palliative therapy in the setting of a limited life expectancy. Operative bypasses including choledochojejunostomies and gastroenterostomies have many advantages and remain as highly acceptable choices in the management of terminal disease. Nonoperative stenting should be reserved for elderly patients or patients with very advanced disease who are poor operative candidates. The resistance of pancreatic cancer to adjuvant, neoadjuvant, and palliative chemo- and/or radiotherapy has remained a consistent disappointment over the last decades. Trials with gemcitabine, a cytidine analogue, have shown a decrease of disease-related symptoms thus benefiting patient’s quality of life and have resulted in very modest prolongation in survival. Oral 5-FU (capecitabine) has been added to gemcitabine and some oncologists recommend additionally EGFR blockers. Overall, there are no convincing data about any drugs significantly increasing patient’s survival. Such results emphasize the importance of developing new diagnostic methods of pancreatic cancer to allow surgical resection at an earlier tumor stage.
Cross-References ▶ Pancreatic Cancer
References Baumgart M, Heinmöller E, Horstmann O et al (2005) The genetic basis of sporadic pancreatic cancer. Cell Oncol 27:3–13 Evans DB, Abbruzzese JL, Rich TA (1997) Cancer of the pancreas. In: DeVita VT, Hellmann S, Rosenberg S (eds) Cancer: principles and practice of oncology, 5th edn. Lippincott, Philadelphia Hruban RH, Yeo CJ, Kern SE (1998) Pancreatic cancer. In: Vogelstein B, Kinzler KW (eds) The genetic basis of human cancer. Mc Graw Hill, New York Klöppel G (1997) Pathology and classification of tumors of the exocrine pancreas. In: Trede M, Carter DC (eds) Surgery of the pancreas. Churchill Livingstone, Edinburgh
3391 Trede M, Carter JD (1997) The surgical options for pancreatic cancer. In: Trede M, Carter DC (eds) Surgery of the pancreas. Churchill Livingstone, Edinburgh
Pancreatic Cancer Biology and Management Narinder Kumar Sharma1, Sharmila Shankar2 and Rakesh Srivastava2 1 Department of Pharmacology, Toxicology and Therapeutics, and Medicine, The University of Kansas Medical Center, Kansas City, KS, USA 2 Department of Pathology and Laboratory Medicine, The University of Kansas Medical Center, Kansas City, KS, USA
Definition Pancreatic cancer is a type of cancer in which malignant (cancer) cells originate from the tissue of pancreas. It is one of the most lethal human cancers and continues to be a major challenging health disease at present. It is also known as “silent” disease because early pancreatic cancer usually does not cause symptoms. More than 95% of pancreatic cancers occur in the exocrine cells, and the vast majority of these tumors are adenocarcinomas. Less than 5% of all pancreatic tumors are neuroendocrine tumors, also known as endocrine or islet cell tumors. Pancreatic neuroendocrine tumors may be benign or malignant, and these usually grow slower than exocrine tumors.
Characteristics Human Pancreas The human pancreas is about 6 in.-long spongy organ located in the back of the abdomen surrounded by the stomach, intestines, liver, and other organs. The relatively broad head of the pancreas is toward the right side of the abdomen where the stomach is attached to the duodenum part of small intestine. The narrow tail of the
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pancreas extends to the left side of the abdomen next to the spleen. The pancreas has exocrine and endocrine glands that secrete pancreatic juices, enzymes, and hormones. Pancreatic juices, or enzymes, are released into the intestines by a series of ducts which assist in the digestion of fat, proteins, and carbohydrates. Exocrine glands and ducts form over 95% of the pancreas. Symptoms of Pancreatic Cancer Pancreatic cancer symptoms are quite vague and can easily be attributed to other less serious and more common diseases. Due to the lack of specific symptom, it spreads quickly and often goes undetected in its early stages. The various symptoms of pancreatic cancer include: • Pain in the abdomen from the tumor pushing against the nerves. • Yellowing of the skin and eyes and darkening of the urine, which is called jaundice, created when the cancer interferes with the bile duct and the liver. • Weight loss due to lack of appetite, nausea, and vomiting. • Blood clots in the deep veins of the arms and legs or other superficial veins. • Enlarged lymph nodes in the neck, when pancreatic cancer spreads (metastasis). • Neuroendocrine cancers (in the islet cell of pancreas) may cause the organ to produce large amount of insulin, which may cause dizzy feelings, chills, or diarrhea. Causes of Pancreatic Cancer Pancreatic cancer is primarily caused by damage to the DNA, i.e., mutations in DNA. These mutations can be inherited from parents or they can be acquired with age and environmental factors. There may be several risk factors that can contribute to the induction of pancreatic cancer. A risk factor is something that increases the likelihood that you will develop pancreatic cancer, but is not a guarantee. Various risk factors of pancreatic cancer include: • Smoking: Smokers are more likely to develop pancreatic cancer as compared to nonsmokers.
Pancreatic Cancer Biology and Management
• Even smokeless tobacco has been noted as a risk factor for pancreatic cancer. • Carcinogens: Carcinogens are a class of substances that are directly responsible for damaging DNA or promoting cancer. Many pesticides, dyes, and other chemicals used in metal refining are supposed to be carcinogenic. • Race: Pancreatic cancer is more frequently diagnosed in African-American than other races. • Age: The risk of pancreatic cancer increases after age 50. • Diabetes: Pancreatic cancer is diagnosed more often in people with diabetes. • Genetic factors: Pancreatic cancer is inherited in some families. Most cases are related to inherited genetic mutations. A number of mutated genes have been identified in pancreatic cancer, including K-ras, p53, DPC4, DCC, MCC, APC, c-erb-2, and RB1. • Age and cigarette smoking are consistently reported risk factors in pancreatic cancer. Cigarette smoking is estimated to contribute to more than 25% of pancreatic cancer incidence. • Alcohol consumption. Diagnosis of Pancreatic Cancer Different methods such as computed tomography (CT), magnetic resonance imaging (MRI), endoscopic ultrasound (EUS), endoscopic retrograde cholangiopancreatography (ERCP), and laparoscopy and biopsy are being used to diagnose pancreatic cancer. • CT scans are commonly used to diagnose pancreatic cancer and the extent of its spread. It can confirm the location of cancer in pancreas as well as in the organs near the pancreas, lymph nodes, and distant organs where cancer might have spread. Although doctors prefer to look at pancreas with CT scans, MRI scan can sometimes provide more information. • In EUS, an endoscope with ultrasound device mounted on its tip is passed through the patient’s mouth into the stomach to visualize cancer in the pancreas and adjacent large blood vessels or lymph glands. This device can give information of smaller tumors than those
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Pancreatic Cancer Biology and Management, Fig. 1 Pancreatic cancer. Pancreatic cancer also known as pancreatic carcinoma. It is an antagonistic type of cancer that assaults the pancreas, an important organ near the stomach and the liver. It is still not very well known what exactly causes pancreatic cancer, but it has been shown that some of the risk factors (i.e., cigarette smoking, alcohol,
high-fat diet, too much coffee intake, certain pesticides, dyes and chemicals, etc.) affect the DNA of the cells in the pancreas, which can result in abnormal cell growth and may cause pancreatic cancer. Pancreatic cancer can be treated by surgery, chemotherapy, radiation therapy, biologic therapy, and combinations of these therapies, based on the doctor’s commendation
detectable by CT scans. Similar to EUS, in ERCP, an endoscope equipped with a cannula is passed through the patient’s mouth. The cannula can take pancreatic cells for analysis. • In laparoscopy, a small camera (laparoscope) is inserted through one or more small incisions in the abdominal wall to examine abdominal cavity. • Fine needle biopsy is performed to confirm the diagnosis of pancreatic cancer. This can be performed by passing a long, thin needle through the abdomen then into the pancreatic tumor to remove tumor cells or by surgically removing a piece of pancreatic tissue.
Surgery is the best choice to treat pancreatic cancer that is considered curable and confined to the pancreases or can be done to alleviate the symptoms caused by the tumor(s). A very common surgical procedure called a Whipple is performed in people whose cancer may be curable. In this surgical procedure, a large portion of the pancreas, part of the stomach, gallbladder, small intestine, and bile duct are usually removed. Another less common surgical method includes the removal of the entire pancreas, known as total or distal pancreatectomy. Surgical removal of the entire pancreas or a part of the pancreas leads to less production of pancreatic juices or hormones, which can cause problems with digestion. Patients after surgery usually are suggested to take appropriate diet and medicine that will help relieve diarrhea or other problems such as cramping. Patients who do not have enough pancreatic hormones may develop other diseases, e.g., those who do not have enough insulin usually have
Treatment of Pancreatic Cancer The treatment methods for pancreatic cancer are removal by surgery, chemotherapy, and radiation (Fig. 1). These treatment methods depend on the stage of pancreatic cancer, type, and general health of the patient.
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diabetes. Hormones are given to these patients as part of their pancreatic cancer therapy to substitute those no longer produced by the pancreas. Chemotherapy and radiation therapy can be considered, depending upon the stage of the cancer and general health factors of the patient. Both of these therapies are useful but also have their own side effects. In chemotherapy various drugs are used to kill cancer cells. The patient may receive only one chemotherapy drug or a combination of chemotherapy drugs. These drug(s) can be injected into the vein or taken orally. A very common chemotherapy, drug gemcitabine, also known as Gemzar, is given intravenously to the patient. This drug is usually given alone for the treatment of metastatic pancreatic cancer and has direct effects on the killing of cancer cells. Another drug, fluorouracil, is also given intravenously and commonly used in combination with radiation therapy because it makes cancer cells more sensitive to the effects of radiation. Capecitabine also known as Xeloda is given orally and is converted in the body to a compound similar to fluorouracil. Capecitabine has similar effects on the cancer cells as fluorouracil and is also usually used along with radiation therapy. Chemotherapy drugs work by eliminating rapidly multiplying cancer cells, such as blood cells, which fight infection, help the blood to clot, or carry oxygen to all parts of the body. Therefore, patients receiving chemotherapy likely to get infections may bleed easily and may have less energy. Other rapidly dividing cells are hair follicle cells and digestive tract cells. Therefore, chemotherapy may result in many side effects including hair loss, poor appetite, nausea and vomiting, or diarrhea. Radiation therapy uses certain types of highenergy radiation, such as X-rays and protons, to reduce tumors or eliminate cancer cells. Radiation therapy usually comes from a machine that moves around the body of the patient and can be directed to specific points on the body. Radiation therapy may be delivered during surgery also known as intraoperative radiation. Radiation therapy
Pancreatic Cancer Biology and Management
damages cancer cell’s DNA, making it unable to multiply. Although cancer cells are highly sensitive to radiation and largely die, this therapy can also kill normal healthy cells. Usually, healthy cells damaged during radiation are resilient and can fully recover. Chemotherapy can also be used in combination with radiation therapy (chemoradiation). Chemoradiation is useful to treat cancer that has spread outside the pancreas, usually to nearby organs but not to distant parts of the body. Moreover, chemoradiation combination may also be used after the surgery to reduce the risk of pancreatic cancer recurrence. Unfortunately, cancer often comes back even when all of the tumors have been removed by surgery. But chemotherapy after surgery can delay the cancer’s return and help some patients to live longer. Biologic therapy is also known as immunotherapy. It helps to improve immune system in our body against cancer. In this therapy health experts use medicines/treatment that induces immunity in our body system against cancer. Although it is not clear how biologic therapy helps to induce immunity against cancer, it is believed that it may stop or slow the growth of cancer cells, make it easier for the immune system to destroy cancer cells, and stop the spreading of the cancer to the other parts of the body. Naturopathy as the treatment of disease is based on belief and ideology. Naturopathy was not very popular against cancer before the 1980s. Naturopathy has evolved as a naturopathy oncology which is based on the combination of belief, ideology, Ayurvedic treatment, homeopathic treatment, and acupuncture. In this therapy, health experts use combinations of different therapies in collaborative model against cancer. Naturopathy is becoming very popular among oncologists for the treatment of cancers. Self-Defense Against Cancer in Our Body Our body also has cells that can fight and kill cancer cells. When a normal cell is transformed
Pancreatic Cancer Biomarkers
into a cancer cell, some of the antigens on the cell surface change. These altered antigens are recognized by immune system cells that include macrophages, cytotoxic T cells, and natural killer cells. Expectation of Pancreatic Cancer Treatment (Prognosis) Although significant improvement has been made in improving survival rates of other cancer over the past few decades, very little has been achieved for the 5-year survival rate for pancreatic cancer, which increased from 3.0% in 1975 to 5.4% in 2005. Endocrine tumors, many of which are benign, have much better clinical outcomes, as compared to exocrine pancreatic cancer. Conclusion Pancreatic cancer remains a major unsolved health problem, with conventional cancer treatments having little impact on disease course. The major problem is the late diagnosis of this cancer. However, developments in early detection of patients will be expected to facilitate progress in improving patient prognosis. Overall, the best preventive measure is to maintain a healthy lifestyle such as exercise, more consumption of fruits, vegetables and whole grains, and quit smoking. Acknowledgments We would like to thank the members of our research group for their critical inputs in shaping this article.
References Greer JB, Brand RE (2011) New developments in pancreatic cancer. Curr Gastroenterol Rep 13(2): 131–139 Shi S, Yao W, Xu J, Long J, Liu C, Yu X (2012) Combinational therapy: new hope for pancreatic cancer? Cancer Lett 317(2):127–135 Tuveson DA, Neoptolemos JP (2012) Understanding metastasis in pancreatic cancer: a call for new clinical approaches. Cell 148(1–2):21–23
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Pancreatic Cancer Biomarkers Michael J. Baine1, Sukhwinder Kaur2, Aaron R. Sasson3 and Surinder K. Batra4 1 Department of Radiation Oncology, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA 2 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA 3 Department of Surgery, University of Nebraska Medical Center, Omaha, NE, USA 4 Eppley Institute for Research in Cancer and Allied Diseases and Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA
Definition Pancreatic cancer is the ninth leading cause of cancer diagnosis and fourth leading cause of cancer-associated death in the world. Pancreatic cancer biomarkers refer to cellular or biochemical mechanisms to aid in the detection, diagnosis, or prognostication of pancreatic cancer. Primarily, this term insinuates pancreatic exocrine tumors, specifically adenocarcinoma, but can be universally applied to all cancers arising within the pancreas. Additionally, the source and nature of pancreatic cancer biomarkers is not inherently defined and thus can denote any method to aid in its clinical detection or projection including but not limited to analysis of the proper tumor, tumorsurrounding tissues, implicated body secretions, and distant body fluids.
Characteristics The term pancreatic cancer (PC) biomarkers can be contextually applied to many entities for multiple functions. In general terms, it refers to any biochemical detection mechanism that can be
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applied in PC to offer clinical improvement. Considering physiologic aspects and clinical points of failure in highly lethal PC, this general term can have three distinct applications: improvement of diagnostic ability, providing prognostic information including likely patient time of survival and presence of therapeutic resistance mechanisms, and following of disease progression and treatment response. Clinically, each of these applications has meaningful clinical utility. Improved prognostic abilities, especially in the context of therapeutic efficacy, promise to aid physicians and patients in treatment planning, potentially altering treatment course for optimal patient outcome. Following of disease progression through biomarkers allows for rapid indication of treatment response, a therapeutic switch should progression be found during a treatment course, and a transition to palliative care at the earliest indicated time point, thereby preserving patient quality of life to the fullest extent possible. Currently, biomarker monitoring of PC patient progression is the only biomarker utility currently available clinically. This monitoring involves the analysis of plasma-based CA19-9, specifically the sialylated Lewis (a) carbohydrate antigen present on various mucin proteins, though this current method is plagued with non-specificity along with the fact that up to 15% of people lack to ability to produce sialylated Lewis (a) altogether. While each of these biomarker applications are doubtlessly important, the discovery of novel biomarkers to aid in PC diagnosis, especially early diagnosis, has the potential for the greatest clinical impact on patient overall survival and thus has been the subject of the majority of PC biomarker research. To date, the only treatment modality with proven ability to significantly and substantially alter PC patient outcome is resection, providing a 5-year survival rate of 24.6% and a median survival of 19.3 months as compared to 2.9% and 8.4 months in similar-stage non-resected patients. This point is further illustrated by studies indicating 5-year survival rates of 10–20%, 30–60%, and >75% in patients with resected pancreatic tumors 3 cm, 2 cm, and 1 cm in diameter, respectively. Unfortunately,
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few PC patients are diagnosed with resectable disease. Of the estimated 43,920 patients who will be diagnosed with PC in the United States in 2012, 80–85% will not be surgical candidates at the time of disease discovery. Of these, approximately 25–30% are diagnosed with borderline resectable disease, for whom chemoradiotherapy is utilized in an attempt to downstage the tumor to resectability but is met with only a 8–30% rate of success. Hence, for 70–75% of PC patients, their diagnosis is an unequivocal death sentence owing primarily to the lateness of disease discovery, a statistic that has remained virtually unchanged for the past 25 years. The principal cause attributing to the late discovery of PC is a general lack of specific or clinically worrisome symptoms until late in the disease course, mostly due to the anatomic and physiologic nature of the disease. Further, when PC does elicit symptoms, they are often general and thus lead neither the patient nor physician to suspect PC at the top of the differential diagnosis. As such PC can, for the majority of patients, be considered a symptomfree disease, resulting in a situation in which early diagnosis must come in the form of finding something that neither patient nor clinician knows they should be looking for. In such situations, the only method for disease detection is via screening mechanisms, such as are employed for prostate cancer (PSA), breast cancer (mammogram), and colorectal cancer (colonoscopy), putting stringent stipulations upon what such a test can entail for it to be clinically acceptable as discussed below. Despite decades of research into this field, however, no diagnostic marker has yet proven clinically viable for PC. Additionally, due to the anatomic location and physiologic functions of the pancreas, biomarker source can also be highly variable. Such sources include tumor and peri-tumoral tissue, pancreatic juice, peripheral plasma/serum, circulating cells, and other less directly implicated body fluids such as urine and bile. With each source comes a differing set of positive and negative properties and theoretical ties to the PC disease state, thus regulating specific targets and global approaches for study to discover utile PC biomarkers (Table 1).
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Pancreatic Cancer Biomarkers, Table 1 Theoretical properties of PC biomarker sources Biomarker source
Minimally invasive
High sensitivitya
Core biopsy
High specificityb
Screening applicability
Prognostic applicability
XXXX
XXXX
XXXX
X
XX
XXX
X
XXXX
XX
XX
Aspirate
X
X
XXX
Pancreatic juice
XX
X
XX
Serum/ plasma
XXX
XXX
CTCs
XXX
X
XXXX
XXXX
XXX
PBMCs
XXX
XXX
XXX
XXXX
XXX
Bile
X
X
Urine
XXXX
XX
Early diagnostic ability
X
X
XXX
X
X
X
XX
PC pancreatic cancer, CTCs circulating tumor cells, PBMCs peripheral blood mononuclear cells a Likelihood of tumor detection in a scenario of pancreatic tumor presence b Low risk of false-positive results in patients lacking a pancreatic tumor
Notes Only utile once a pancreatic lesion is discovered through other methods Risk of iatrogenic acute pancreatitis is low but not insignificant Lack of ductal cannulation reduces both invasiveness and specificity Specificity must come from choice of ideal marker as biomarker elevation can come from anywhere in the body Detection greatly increases the likelihood of distant disease spread Preliminary studies indicate early and specific changes in expression profiling May be best for ruling out hepatic and gallbladder pathology from differential diagnosis Specificity must come from choice of ideal marker as biomarker elevation can come from anywhere in the body
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Tumoral/Peri-tumoral Biomarkers Of the various areas from which to test for PC biomarkers, the most direct is in the tissue itself. Many studies have been conducted in the attempt to discern normal from neoplastic pancreata, being met with varying degrees of success. Two methods exist by which to obtain and analyze such tissue, each with its own advantages and disadvantages. Core Biopsy-Based Analysis Analysis of core biopsy specimens is one of the oldest methods for cancer detection and confirmation. Objectively, this method brings with it several distinct advantages not present with any other method, including obtainment of high-cellularity samples allowing for morphological and histological evaluation across multiple criteria, inclusion of both tumoral and peri-tumoral tissue as well as the junction between the two, and the presence of live cells in their respective surrounding microenvironment(s). Such properties offer an unparalleled look at the tissue of interest and provide the ability to directly link the presence of one or more cellular or morphotypic features to the existence of neoplastic disease and/or that disease’s therapeutic and prognostic phenotype. This method of pancreatic evaluation, however, does not come without multiple detracting attributes as well. Primarily, due to its depth and location within the abdomen, obtainment of core biopsies from the pancreas is no small order. As such, many medical centers have opted to forgo prooperative biopsies for PC altogether in favor of aspiration-based evaluation methods. Further, the size of the pancreas and invasiveness of the procedure preclude performing core biopsies without the presence of a previously identified pancreatic mass/lesion, thus preventing its application as a screening mechanism. Consequently, core biopsy-based PC biomarkers are segregated solely to functions of disease confirmation with further potential for prognostic utility. Aspiration-Based Analysis Methodological development for evaluation of free cells within an aspirate sample has sought to overcome multiple downfalls of the core
Pancreatic Cancer Biomarkers
biopsy-based evaluation methods. Accomplished either through transcutaneous aspiration of the main pancreatic duct with endoscopic ultrasound guidance or via direct aspiration following cannulation of the papilla of Vater through endoscopic retrograde cholangiopancreatography (ERCP), aspirate specimens are less invasive to obtain than their core biopsy counterparts and thus lend themselves better to patient screening. Further, aspiration of the main pancreatic duct allows for a theoretical evaluation of the entire ductal network, with potential of collecting cells and debris from ductal lesions throughout the pancreas. Aspirate evaluation also lends itself well to the analysis of both cytology and protein secretions, broadening the range of potential evaluable biomarkers. However, aspiration-based biomarkers carry with them many issues and potential complications. Presence of a cell or protein in an aspiration specimen is reliant on its being free within, or loosely adherent to the wall of a pancreatic duct or ductile, thus limiting the nature of evaluable entities. Obtainment of ample cellularity to allow for conclusive cytology or immunohistochemistry, especially if staining for multiple markers is desired, is also by no means guaranteed in pancreatic aspiration. Further, there is a small, though not insignificant, risk of pancreatic duct aspiration inducing acute pancreatitis, an acute inflammatory disease with significant morbidity and the realistic potential for patient mortality. Additionally, aspiration disallows the evaluation of cells within their natural environment(s) as well as the evaluation of considerable peri-tumoral tissue. As such, though its less invasive and more comprehensive nature makes aspiration superior to core biopsy evaluation for diagnostic means, it is still not a diagnostically viable mechanism and thus continues to be relegated to use in patients with suspicious lesions or symptomatology for confirmatory purposes. Pancreatic Juice A body fluid that has received substantial attention for PC diagnostics is pancreatic juice; the results of some such studies are reviewed in Chakraborty et al. Pancreatic juice, consisting of
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the secreted liquids, enzymes, chemicals, and sloughed cells from the pancreatic ductal system as it travels toward the duodenum, is endoscopically collected as it exits through the sphincter of Oddi and can be performed with or without the preceding step of secretin stimulation. Importantly, like aspirate evaluation, these methods have the ability to assess fluid, chemicals, and cells from throughout the entire acinar and ductal network of the pancreas, rendering a previously identified area of concern within the pancreas unnecessary. Further, pancreatic juice collection is less invasive than aspiration and does not pose the risk of iatrogenic pancreatitis. A myriad of downsides exist to pancreatic juice evaluation as well, though. For example, only a small volume is able to be collected without secretin stimulation, thereby limiting the tests which can be performed, but secretin stimulation itself serves to increase the volume of the pancreatic excretion and thus risks diluting relevant biomarkers to non-detectability. Additionally, collection of the juice from the duodenum risks sample contamination from both the small intestines and emptying stomach, potentially sacrificing both sensitivity (through dilution) and specificity of a given biomarker for PC. Lastly, the presence of a PC biomarker within pancreatic juice requires a communication of the tissue of origin for that biomarker, be it PC or peri-tumoral tissue, with the main pancreatic duct, a necessity which may not always be the case. Serum/Plasma Due to ease of accessibility, serum/plasma-based biomarkers have undergone the most extensive of study as a source of surrogate PC diagnostic or prognostic markers, the most famous of which is the currently FDA-approved CA19-9. An important advantage of these circulating biomarkers is the potential for detection of those derived from both tumor and stromal cells. The mildly invasive nature of their collection, accomplished via simple venipuncture, is also of significant benefit to their potential as a diagnostic screening mechanism. Unfortunately, the majority of such studies have been hampered by difficulty in isolating, identifying, and consistently detecting these
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low-abundance proteins. Additionally, as the search for PC biomarkers becomes farther removed from the pancreas, the greater likelihood of nonspecificity exists as biomarkers from the entire body would be thus detectable. This is of particular importance with regard to inflammatory or general neoplastic markers which may have utility if attributable to the pancreas specifically but otherwise can lead to clinical confusion and patient misdiagnosis. Consequently, choosing of proper biomarkers that are fully specific to pancreatic neoplasia in the quest for plasma/serumbased surrogate detection, prognostication, and/or following of progression of PC is of the utmost importance. Cellular Another tissue with high diagnostic potential and becoming of increasing interest is circulating cells derived from venipuncture. Regarding current biomarker interest, these cells come in two flavors: circulating tumor cells (CTCs) and peripheral blood mononuclear cells (PBMCs). CTCs are of interest due to the fact that their presence indicates not only the presence of cancer but also an increased likelihood of the presence of distant metastases, detectable or not, as well as the fact that their mutational profile can offer indications as to the prognosis and therapeutic responsiveness of both primary and metastatic lesions. Unfortunately, these benefits come at a steep price as the likelihood of detectable CTCs being present in patients with truly localized disease is low, thus making these methods of little benefit as an early diagnostic mechanism. Additionally, CTCs only make up a small portion of circulating cells, causing increased complications as these cells must be specifically isolated for analysis. The theory that PBMCs, consisting chiefly of monocytes, T cells, and B cells, may offer an early diagnostic test for PC with high utility is founded on two physiologically competing principles: (1) cancer cells are inherently antigenic, leading to a tumor-directed immune response and (2) as cancer develops, it gains the ability to evade the immune system through direct and indirect mechanisms. The effect that each of these principles instills upon the immune system as a whole results
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in both predictable and unpredictable alterations in immune cell populations as well as their transcriptional patterns, with the potential for monitoring to allow for a surrogate window into active immune modulation and its potential cause(s). Further, different cancers and immunogenic disease states utilize different combinations of methods for immune activation and suppression, providing a conceivable specificity to PBMC expression profiles as a biomarker mechanism. However, specific knowledge regarding cancer immune detection as well as immune evasion is currently in its infancy, making the discovery of cancer-specific and prognostic profiling a result of nondirected global approaches with obvious need for optimization prior to full assessment of clinical potential. Other Body Fluids Multiple other sources of surrogate markers have also been examined for the presence of PC biomarkers. PC patient urine, which benefits from a fully noninvasive nature, has been analyzed against that from healthy and CP controls by mass spectrometry, indicating a total of 60 proteins that were differentially expressed. Analysis of bile, obtainable through ERCP, has also determined that 127 protein fragments are elevated in PC patients. Such studies, however, suffer from a lack of intra-study validation and are currently singular in nature. Thus, while such fluids may represent mechanisms for future PC biomarker development, much future exploration is needed. Importance of an Optimal Testing Population Another point of importance in the discussion of PC biomarkers, particularly for biomarkers that are intended for diagnostic screening, is the statistical properties that such a marker must possess for clinical applicability. Primarily, screening tests must be mildly invasive and must have adequate performance with regard to sensitivity and specificity to elicit results that are ultimately beneficial to both clinician and patient. For the majority of screening tests (PSA, colonoscopy, mammogram, etc.), this stipulation skews diagnostic candidates toward increasing sensitivity, owing to the consideration that the purpose of the test is to add
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clinical suspicion to disease presence with the results of which being ultimately confirmed through more accurate, though often more expensive and invasive methods. Such confirmatory testing, however, does not exist in PC, especially in the case of small, localized tumors. Hence, a positive PC screening test has a high likelihood of resulting in invasive exploratory procedures such as EUS-FNAs and laparoscopies which also often fail to discern small tumors of the pancreas, thus raising the potential for pancreatectomies or pancreatoduodenectomies without concrete evidence of their necessity. These circumstances result in a situation in which PC diagnostic testing must be skewed toward increasing specificity under the consideration that it is better to give a portion of PC patients false-negative results than to lead a healthy patient to believe that they have this deadly disease, thus subjecting them to a barrage of tests and procedures causing significant financial, physical, and psychological burden that will, in many cases, generate only inconclusive results. Adding to this is the fact that successful screening for PC, due to its relative rarity, must, at its end, come down to the analysis and minimization of the false-positive (FP) to true-positive (TP) ratio (Table 2). The current annual incidence of PC in the United States is approximately 43,920 patients, a number that has been slowly creeping upward with time. As per the 2010 census, an approximated total of 93,555,000 people over the age of 50 (below which a diagnosis of PC is unlikely) are currently living in the United States, putting the per capita incidence of PC at about 1:2,000. Based on this, even a diagnostic test with perfect 100% sensitivity and a specificity of 99% will falsely diagnose 20 healthy people for every PC patient it correctly detects. With the lack of highly accurate confirmatory tests available for PC, especially in early disease, combined with their invasiveness and expense, even this optimal false-positive:truepositive (FP:TP) ratio cannot be considered acceptable. Hence, though improvement in the diagnostic performance of currently available surrogate diagnostic methods is obviously necessary for clinical applicability, it alone will fail to produce tests with true viability. Instead, equal focus
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Pancreatic Cancer Biomarkers, Table 2 False-positive to true-positive (FP:TP) ratios obtained by two theoretical tests (A and B) as well as CA19-9 when applied as a PC screening mechanism to various patient populations Test A Sensitivity 100% Screened population General population (50 years old) Chronic pancreatitis Family history (1+ relatives) Family history (2+ relatives) Family history (3+ relatives) Recent-onset type II diabetes
Pretest probability 1:2,000
Specificity 99%
Test B Sensitivity 90%
Specificity 90%
CA19-9 Sensitivity 70%
FP:TP ratioa
FP:TP ratioa
FP:TP ratioa
20.0
222.2
571.4
0.6–0.9
6.2–10.1
16.0–26.0
2.2–11.4
24.7–126.2
63.4–324.6
3.1–4.7
34.8–52.1
89.4–134.0
0.6
6.9
17.9
1.3–1.7
13.9–19.1
35.7–49.1
Specificity 80%
1:56–1:91 1:222–1:1,136
1:313–1:469
1:63
1:125–1:172
a
FP:TP Ratio indicates the number of PC-free patients who will test positive for every screened patient with PC correctly diagnosed per a test with the given diagnostic abilities
must be placed on optimal population(s) in which to administer such a test. Such populations must be well-defined as well as have a significant and substantial increase in risk of PC diagnosis over baseline. Based on current knowledge, three populations meet these criteria and may prove to provide enhancement of PC diagnostic test viability to allow for clinical applicability: chronic pancreatitis (CP), patients with a family history of PC, and patients with recent-onset type II diabetes mellitus (DM2). Both hereditary and nonhereditary CP is well established to be a significant risk factor for future PC diagnosis, a fact thought to primarily arise from the proneoplastic chronic inflammatory nature of the disease. While it is unclear what, if any, subsets of CP patients have the greatest PC risk, it appears that risk increases with time following CP diagnosis. For CP patients in general, however, it is estimated that the annual incidence of PC is 1.1–1.8% of the population. Such increase in risk would allow a biomarker with the ideal characteristics described above (100%
sensitivity and 99% specificity) to provide one to two TPs for each FP encountered, a potential that, though still not ideal, is a stark improvement over that allowed for in the general population. Further, this risk, and hence diagnostic biomarker utility, may be further heightened in specific CP population subsets, a potential which must be further explored. Patients with a family history significant for PC also represent an attractive population for diagnostic screening. Overall, it has been shown that patients with a PC-positive family history in any first-degree relative are at a 1.76–9.0-fold increased risk for PC diagnosis as compared to the general population. This risk further increases with the number of affected first-degree relatives, with patients having two or more affected relatives being at a 4.26–6.4-fold increased risk and a 32-fold increased risk observed in patients with 3 first-degree relatives with a PC diagnosis. As such, this population represents a realistic group in which to institute PC screening; however, it is noteworthy that only 5–10% of all PC patients are
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considered familial, and thus the overall impact on the disease will be low. Associations between new-onset DM2 and PC have indicated that this too may be a group in which diagnostic screening for PC may be viable. Newly diagnosed DM2 patients have a substantially increased risk of a PC diagnosis as compared to the general population, as it is reported that up to 0.8% of patients who are 50 years of age and diagnosed with DM2 will receive a PC diagnosis within the first 3 years, placing the overall risk at 1:125.
of relevant samples for study. This point highlights the utility of transgenic murine models in PC biomarker discovery, allowing the correlation of biomarker alteration with disease stage from preneoplastic through metastatic disease, though ample correlation across species to determine the generalizability of mouse-based results to human patients has yet to be undertaken and likely will require assessment for each biomarker study either individually or together in a global manner.
Conclusion PC biomarkers represent a wide array of entities derived from various locations within the body and designed for multiple potential purposes. Currently, a single biomarker, CA19-9, is approved for use in PC with proven, though suboptimal, utility for following patient progression but lacks the required specificity for diagnostic use. Biomarkers derived from the pancreas or peripancreatic tissue, such as tumoral and peritumoral tissues, pancreatic duct aspirate, and pancreatic juice, have the potential for the greatest specificity but are limited by their invasive natures. Peripherally derived biomarkers such as those from plasma/serum, CTCs, PBMCs, and urine are less invasive and thus are more conducive to screening for early PC diagnosis, though they may coincide with a significant loss of biomarker specificity or an increasingly complex testing design to reduce the potential for false positivity. Of these, CTCs and PBMCs have the greatest likelihood to offer reasonable specificity, though CTCs may have little ability in localized disease while PBMC profiling requires substantial optimization prior to realization of its full diagnostic utility. Additionally, in the study of PC biomarkers, consideration of the population in which to apply the test is of equal importance as optimization of the test itself and should be addressed as each series of studies move forward toward clinical testing. Of further note, the greatest current hindrance to establishment of an early diagnostic test for PC is the current lack of ability to discover the disease in its early stages, thus precluding the obtainment
Cross-References ▶ Pancreatic Cancer ▶ Pancreatic Cancer Regeneration
Development
and
References American Cancer Society (2009) Cancer facts and figures. American Cancer Society, Atlanta Baine MJ, Chakraborty S, Smith LM et al (2011) Transcriptional profiling of peripheral blood mononuclear cells in pancreatic cancer patients identifies novel genes with potential diagnostic utility. PLoS One 6(2):e17014 Bilimoria KY, Bentrem DJ, Ko CY, Stewart AK, Winchester DP, Talamonti MS (2007) National failure to operate on early stage pancreatic cancer. Ann Surg 246(2):173–180 Callery MP, Chang KJ, Fishman EK, Talamonti MS, William TL, Linehan DC (2009) Pretreatment assessment of resectable and borderline resectable pancreatic cancer: expert consensus statement. Ann Surg Oncol 16(7):1727–1733 Chakraborty S, Baine MJ, Sasson AR, Batra SK (2011) Current status of molecular markers for early detection of sporadic pancreatic cancer. Biochim Biophys Acta 1815(1):44–64 Chari ST, Kelly K, Hollingsworth MA et al (2015) Early detection of sporadic pancreatic cancer: summative review. Pancreas 44(5):693–712 Helm JF, Centeno BA, Coppola D et al (2008) Outcomes following resection of pancreatic adenocarcinoma: 20-year experience at a single institution. Cancer Control 15(4):288–294 Ishikawa O, Ohigashi H, Imaoka S et al (1999) Minute carcinoma of the pancreas measuring 1 cm or less in diameter – collective review of Japanese case reports. Hepatogastroenterology 46(25):8–15 Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D (2011) Global cancer statistics. CA Cancer J Clin 61:69–90
Pancreatic Cancer Desmoplastic Reaction and Metastasis Kenner BJ, Chari ST, Cleeter DF, Go VL (2015) Early detection of sporadic pancreatic cancer: strategic map for innovation-a white paper. Pancreas 44(5):686–692 Moniaux N, Escande F, Porchet N, Aubert JP, Batra SK (2001) Structural organization and classification of the human mucin genes. Front Biosci 6:D1192–D1206 Moniaux N, Andrianifahanana M, Brand RE, Batra SK (2004) Multiple roles of mucins in pancreatic cancer, a lethal and challenging malignancy. Br J Cancer 91:1633–1638 Mudan S, Giakoustidis A, Thillainayagam AV, Jacob J, Stebbing J (2010) Clinical utility of circulating tumor cell measurement in the diagnosis of indeterminate lesions of the pancreas. Future Oncol 6(1):177–179 Pannala R, Basu A, Petersen GM, Chari ST (2009) New-onset diabetes: a potential clue to the early diagnosis of pancreatic cancer. Lancet Oncol 10(1):88–95 Surveillance Epidemiology and End Results Cancer Statistics Review 1975–2006 (2011) National Cancer Institute. Available at http://seer.cancer.gov/csr/1975_ 2006/index.html Tsuchiya R, Noda T, Harada N et al (1986) Collective review of small carcinomas of the pancreas. Ann Surg 203(1):77–81 Weber C (2015) Biomarkers: the challenge to find biomarkers for the early detection of pancreatic cancer. Nat Rev Gastroenterol Hepatol. Epub ahead of print Wilkowski R, Thoma M, Schauer R, Wagner A, Heinemann V (2004) Effect of chemoradiotherapy with gemcitabine and cisplatin on locoregional control in patients with primary inoperable pancreatic cancer. World J Surg 28(10):1011–1018 Zhang SY, Zhang SQ, Nagaraju GP, El-Rayes BF (2015) Biomarkers for personalized medicine in GI cancers. Cancer Lett 365(2):141–148
Pancreatic Cancer Desmoplastic Reaction and Metastasis Rohini Gomathinayagam and Danny N. Dhanasekaran Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
Definition Desmoplastic reaction in pancreatic cancers is characterized by a marked increase in proliferation of stromal cells, accompanied by a synthesis and deposition of tumor-promoting extracellular matrix (ECM) proteins such as collagen, laminin,
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fibronectin, and vitronectin. Distinct cell types including endothelial cells, immune cells, fibroblasts, endocrine cells and, in particular, pancreatic cancer stellate cells (PSCs) that are embedded in the extracellular matrix constitute the desmoplasia – fibrotic sarcoma – of the pancreatic cancer. In pancreatic cancer, desmoplastic stroma can contribute to 90% of the tumor volume. Studies have shown that the desmoplastic reaction plays a greater role in metastatic tumor progression and drug resistance observed in pancreatic cancer.
Characteristics Histopathological studies have defined that the desmoplastic reaction is mediated by the copious secretion of different ECM proteins by both the stromal and cancer cells. These ECM proteins include stromal cell-derived biglycan, collagen I/III/IV, decorin, FAP-a, fibronectin, hyaluronic acid, laminin, lumican, MMP-2/9/11, osteopontin, periostin, SPARC/osteonectin, tenascin C, thrombospondin 1/2, as well as cancer cell derived ECM proteins, such as TIMP1/2, tPA, uPA, versican, and vitronectin. Of the different types of stromal cells, pancreatic stellate cells play a major role in this process. In the normal pancreas, PSCs are located in the periacinar space and account for approximately 4% of all pancreatic cells. These stellate cells in response to tumor cell derived growth factors, such as transforming growth factor-b (TGFb), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), Wnt-ligands, Hedgehog ligands, and/or oxidative stress, transdifferentiate into a-smooth muscle actin (a-SMA)-expressing “activated PSC.” ECM component of the desmoplastic reaction is primarily secreted by these activated PSCs. Therefore, the ratio of activated PSCs in relation to nondifferentiated stellate cells has been defined as Activated Stroma Index (ASI), and it has been used to define the extent of desmoplastic reaction in pancreatic cancer. In fact, higher ASI has been shown to predict poor prognosis in pancreatic cancer patients. This clinical correlation also
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underscores the tumor-promoting role of the associated desmoplastic stroma in pancreatic cancer. Desmoplastic Stroma and Pancreatic Cancer Progression In addition to ECM proteins, activated PSCs of the desmoplastic stroma secrete proliferative cytokines. Excess deposition of the desmoplastic constituents leads to a hypovascular and fibrotic pancreatic tumor microenvironment, which promotes epithelial to mesenchymal transition (EMT) of pancreatic cancer cells along with an increased drug resistance. The role of PSCs in such pancreatic cancer progression is evidenced by the studies in which the coculture of pancreatic cancer cells and PSCs leads to the expression of EMT markers and the stemness genes such as nestin, ABCG2, and LIN28 in the pancreatic cancer cells. The desmoplastic stroma also contributes to mechanisms by which invading tumor cells evade host immune surveillance. Galectin-1 and fibroblast activation protein-a synthesized by the activated PSCs bind to membrane glycoproteins of T-cells, thereby inducing apoptosis of tumortargeting T-cells. Desmoplastic Stroma and Metastasis of Pancreatic Cancer The desmoplastic reaction evoked by the PSCs form the major determinant of pancreatic cancer malignancy and metastasis. Interaction of PSCs with the pancreatic cancer cells leads to the production of migratory and invasive ECM proteins (Table 1). There seems to be bidirectional signaling between the tumor cell and the activated PSCs of the desmoplastic stroma. For example, extracellular matrix metalloproteinase inducer (EMMPRI) secreted by the pancreatic cancer cells induces the production of MMP-2 by PSCs, which facilitates the invasive migration of the cancer cells. Similarly, the invasive property of tumor cells via SERPINE2 is promoted only in the presence of ECM produced by the PSCs. In addition, PSCs have been shown to promote EMT in pancreatic cancer cells that contribute significantly towards the dissemination and metastasis of cancer cells. PSCs have also been observed to migrate to the
Pancreatic Cancer Desmoplastic Reaction and Metastasis Pancreatic Cancer Desmoplastic Reaction and Metastasis, Table 1 ECM proteins of desmoplastic stroma in tumor cell metastasis ECM protein Collagen I, III, IV Fibronectin Lumican MMP 2/9/11 Osteopontin C Osteonectin/ SPARC Tenascin Thromospondin 1/2 TIMP 1/2
Role in metastasis Invasion, metastasis, angiogenesis Migration Migration, invasion Invasion Invasion Invasion Invasion Invasion, angiogenesis Invasion
metastatic site where they appear to enhance metastasis through their physical interaction with pancreatic cancer cells. Owing to the abundance of ECM in desmoplastic stroma, integrins such as ITGA6, ITGB4, and ITGB5; and ephrins such as EPHA2 further contribute to the metastatic process in pancreatic cancer. Intriguingly, a distinctive subpopulation of PSCs expressing the surface marker CD10 is observed to promote tumor invasion and growth better than the CD10-negative subpopulations. Management of pancreatic cancer metastasis by targeting the desmoplastic reaction components using standard chemotherapeutic agents, such as gemcitabine and erlotinib, offer limited scope in pancreatic cancer management. It appears that the extensive ECM associated with the desmoplastic stroma of pancreatic cancer acts as a physical barrier that prevents the uptake of chemotherapeutic agents by the tumor cells. Therefore, there is an emerging interest in targeting the tumor microenvironment, particularly the desmoplastic/stromal components of pancreatic cancer. Several antifibrotic agents have shown promise in preclinical trials and include polyphenols, vitamin E, inhibitors of the rennin–angiotensin system, inhibitors of hedgehog signaling, enzymatic modes of elimination of hyaluran, and the depletion of tumor–associated fibroblasts. It has been shown that ellagic acid, a plant–derived phenol, has been
Pancreatic Cancer Desmoplastic Reaction and Metastasis
shown to inhibit the activation of PSCs. Different isomers of vitamin E have been shown to reduce desmoplastic reaction in animal models of pancreatic cancer. While a–tocopherol has been shown to reduce desmoplastic response, tocotrienols have been reported to exhibit a cytotoxic effect on PSCs by mediating autophagy and apoptosis. In addition, inhibitors of the renin–angiotensin system have been shown to inhibit the development of pancreatic fibrosis. Signaling pathways, which can revert the activated PSCs to quiescence, have been interrogated in the past decade. Retinoic acid-mediated inhibition of ERK, JNK, and p38MAPK has been demonstrated to induce such quiescence in PSCs. Overexpression or reintroduction of PPAR–g, C/EBP–a, and albumin has also been suggested to inactivate PSCs. Tumor-associated fibroblasts of the desmoplastic stroma produce several ECM proteins including fibroblast activation protein (FAP), a type II integral membrane glycoprotein that is associated in the turnover of the ECM. Therefore, subcutaneous administration of FAP-specific antibodies has been reported to attenuate tumor growth in mice models of pancreatic cancer. Within the desmoplastic stroma, mast cell-derived interleukin-13 and tryptase promote the proliferation of pancreatic stellate cells. Therefore, masitinib, a mast cell/stem cell growth factor inhibitor, in combination with gemcitabine was assessed for its efficacy in a phase III pancreatic cancer patient trial. The results indicated that masitinib conferred survival advantage to at least a subgroup of pancreatic cancer patients, thus validating mast cells as a therapeutic target to a certain extent. In addition, inhibition of candidate signaling pathways involved in the activation of PSCs has been tested for their ability to attenuate pancreatic cancer progression. Hedgehog signaling plays a crucial role in the epithelial to mesenchymal transition of the pancreatic cancer cells. Hedgehog ligands are secreted from the tumor epithelium and the activation of hedgehog signaling takes place in the adjacent stromal compartment. Administration of IPI-926, a hedgehog pathway inhibitor, was reported to improve the uptake of gemcitabine by the pancreatic tumor cells in the
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mouse models of pancreatic cancer. In such animal model, it has also been shown that the enzymatic depletion of hyaluronan/ hyaluronic acid using recombinant hyaluronidase (PEGPH20) can increase vascular permeability and drug delivery. Similarly, depletion of stroma by CD40activated macrophages has also been documented to improve survival in patients with PDAC. In a small cohort study with CD40-activated macrophages in patients with advanced pancreatic cancer, CD40-activated macrophages induced tumor attenuation, facilitated the depletion of desmoplastic stroma, and increased patient survival. Another strategy involves the differential expression of SPARC, an albumin transporter in cancer-associated fibroblasts. Since tumor cells do not express SPARC, nab-paclitaxel, an albumin bound form of paclitaxel, can be targeted to cancer-associated fibroblasts. Coadministration of the maximum tolerated dose of gemcitabine with nab-paclitaxel (MPACT trial) in patients with advanced pancreatic cancer resulted in an objective response rate of 48%, thus validating nab-paclitaxel as a stromal targeting agent. All of these results point to the desmoplastic stroma as a potential therapeutic target for the clinical management of pancreatic cancer. Conclusion Better prognosis and therapeutic management of pancreatic cancer requires identification of stromal targets and inhibition of stromal–pancreatic cancer cell communication. Understanding the intercellular communication among the different cellular populations within the tumor microenvironment and characterization of the critical factors involved in such communication could lead to better clinical management of pancreatic cancer.
References Apte MV, Park S, Phillips PA, Santucci N, Goldstein D, Kumar RK, Ramm GA, Buchler M, Friess H, McCarroll JA, Keogh G, Merrett N, Pirola R, Wilson JS (2004) Desmoplastic reaction in pancreatic cancer: role of pancreatic cancer cells. Pancreas 29:179–187
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Pancreatic Cancer Development and Regeneration Sonal Gupta Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Definition Understanding of progenitor cells and processes for embryonic development of the pancreas and regeneration holds the key for solving mysteries of ▶ pancreatic cancer development since many of these pathways are found to be activated in pancreatic tumors. It can also provide clues to regenerate the pancreas following injuries arising from diabetes, ▶ pancreatitis, and surgical insult.
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lumen which carries the pancreatic secretions to the common bile duct and intestine. Endocrine Component
Constituting about 1% of the adult human pancreas, more than a million of islets of Langerhans form the endocrine pancreas. Islets are comprised of four types of cells: Alpha cells (makes up 15–20% of islets) produce the hyperglycemic hormone glucagon in response to insulin, causing liver glycogenolysis and glucose transport toward the blood. Beta cells (makes up 50–80% of islets) produce hypoglycemic hormones insulin in response to blood glucose, which in turn, lowers blood glucose levels. Delta cells (makes up 3–10% of islets) contain somatostatin which modulates activity of alpha and beta cells by suppressing endocrine secretion of insulin and glucagon. F cells (1% of islets) make pancreatic polypeptide which inhibits the exocrine pancreas. Exocrine Component
Formed mainly of acinar cells which are round structures formed of many pyramidal cells around a central lumen, punctuated by centroacinar cells. They contain zymogen granules and produce the body’s main digestive enzymes like proteases, amylases, lipases, and nucleases. Stellate cells are elongated, periacinar, perivascular, and periductal myofibroblastic cells which have fat-storing capabilities. These cells can get activated during ▶ inflammation, associated with pancreatitis, causing morphological changes within the cells and also fibrosis associates with pancreatic cancer. Epithelial Duct Cells
Characteristics Functional Morphology of the Pancreas On average daily, the pancreas secretes 1,200 ml of pancreatic juice comprising of digestive enzymes necessary for digestion of food and hormones important for blood glucose homeostasis. The pancreas as a glandular organ comprises digestive enzyme-secreting exocrine cells (acinar cells), hormone-secreting endocrine cells (islets of Langerhans), and epithelial duct cells forming a
Acinar cells are connected to a network of epithelial, cubic duct cells that act as conduits to channel acinar contents into the gastrointestinal tract. The cells forming the pancreatic duct make up more than 10% of the total pancreas. Duct cells are surrounded by centroacinar cells which together form the intercalated ducts. Several intercalated ducts converge to form intralobular duct which in turn forms interlobular ducts. These ducts ultimately drain the pancreatic juices into the duodenum via the main pancreatic duct.
Pancreatic Cancer Development and Regeneration
Cellular Events During Embryonic Development of the Pancreas All the cells of the pancreas derive from common progenitor cells that emerge from the posterior foregut endoderm at the end of gastrulation. The two epithelial buds (dorsal and ventral), encased in mesenchyme, stratify and resolve into a branched epithelium of early pancreatic progenitors. The dorsal bud forms the head, body, and tail of the human pancreas, while the ventral bud forms the uncinate process and inferior part of the head. Thus, all epithelial tissues of the pancreas are derived from these two endodermal buds, which develop further via a dynamic signaling dialog between the epithelium and the overlying mesenchymes. The “primary transition” phase involves morphogenetic changes in the pancreatic epithelium, including dense branching, epithelial stratification, and microlumen formation along with appearance of first differentiated endocrine cells. The ensuing cell division creates a densely packed epithelium containing most of the progenitor cells for the islets, acini, and ducts whose number determines the eventual size of the mature pancreas. During the “secondary transition,” epithelium undergoes morphogenesis that converts the early progenitor cells to more developmentally restricted multipotent precursor cells that initiate the formation of islet, duct, acinar, and centroacinar cells. Centroacinar cells have been postulated to possess stem-cell-like qualities. Prenatal development ensures the expansion of ductal and acinar tissues and the maturation of islets; however, replication of differentiated endocrine cells is infrequent, with the increase of endocrine tissue during embryogenesis due almost exclusively to de novo formation from precursor cells in the tubules. Postnatal growth and maintenance of both exocrine and endocrine tissue depend primarily on proliferation of differentiated cells. Although proliferation of acinar cells and insulinexpressing beta cells decreases postnatally, it is seemingly sufficient to maintain the adult tissue mass. The common origin of various pancreatic cell types from embryonic duct-like epithelium seems to agree well with the postulated ductal origin of pancreatic adenocarcinoma (PDA), and thus a
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greater understanding of endocrine and exocrine development, their structural and physiologic relationships, and the principal intrinsic and extrinsic molecular regulators that drive their formation is critical to understanding the origin and nature of pancreatic cancer. Signaling Pathways During Development Pancreatic organogenesis is guided by a stepwise interplay between intercellular developmental signals from external growth factors acting as morphogens and their respective intracellular DNA-binding transcription factors (TF) which act as mediators of the developmental program. A signal from external growth factors gets relayed inside the cell through its binding to cellular transmembrane protein receptors which culminates into alteration of a specific intracellular transcriptional factor network and, thus, determines the developmental state of a cell by subsequent gene expression pattern. The following are few principal extracellular signaling pathways and the corresponding transcription factors they affect, which are known to shape the embryonic development of the pancreas: Hedgehog (Hh) Pathway: Gli TFs
Hedgehogs are a family of secreted peptides, comprising of sonic (SHh), Indian (IHh), and desert (DHh) hedgehogs, that bind to a transmembrane Patched 1 (PTCH1) receptor which in turn relieves Smoothened (Smo) repression and activates Gli family of TFs. During early specification of the pancreas, Hh produced throughout endoderm restricts the pancreatic domain to hedgehogfree islands. Hh inhibits early Pdx1 TF expression and the formation of the pancreas, until signals from notochord suppress Hh and cause endodermal acquisition of pancreatic cell fate. Transforming Growth Factor-beta (TGF-b) Pathway: Smad TFs
Secreted growth factors in this pathway include subfamilies of TGF-b, activins, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), etc. Ligand engagement-induced heterodimerization of types I and II receptors
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cause type II receptors to phosphorylate and activate type I receptor which then relays the signal inside the cell by phosphorylating one of the Smad TFs. Three types of Smad TFs involved are R-Smads (receptor-regulated), co-Smads (common mediator), and I-Smads (inhibitory). During early specification of the pancreas, TGF-b produced by notochord limits Hedgehog signaling within the pre-pancreatic domain of the early patterned endoderm and thus confers pancreatic identity on these cells. BMP and activin from the splanchnic mesoderm are required for early ventral pancreas specification. During late stages of pancreatic branching and growth, TGF-b signaling via ActRIIA/ActRIIB activin receptors causes proper endocrine and exocrine expansion and endocrine function.
Pancreatic Cancer Development and Regeneration
Receptor Tyrosine Kinase (RTK) Pathway: FGF, EGF, IGF, NEGF, PDGF, and Eph TFs
Numerous growth factors like ▶ fibroblast growth factor (FGF), epidermal growth factor (EGF), ▶ insulin-like growth factor (IGF), ▶ vascular endothelial growth factor (VEGF), ephrins, etc. bind to transmembrane receptors RTKs, which relay signaling inside the cell via ERK (extracellular signal-regulated kinase), or MAPK (mitogen-activated protein kinase) signaling cascades. RTKs are involved throughout the pancreatic development; for instance, Fgf10 is required for the proliferation and maintenance of proper beta-cell mass; VEGF expression is tightly regulated to prevent increased pancreatic vasculature which may lead to disruption in both endocrine and exocrine differentiation and abnormalities in insulin secretion.
Notch-Rbpj
This pathway mediates short-range signaling in adjacent cells since both its receptors (Notch1-4) and the ligands (Deltas and Jaggeds) are transmembrane proteins. Upon ligand binding, the receptor gets cleaved and migrates into the nucleus to bind and convert TF Rbpj into an activator resulting in the expression of Hes1 transcription factor. Hes1 binds and suppresses expression of pro-differentiation genes. During pancreatic development, Notch signaling promotes the expansion of progenitor cell population prior to “secondary transition” stage by suppressing differentiation. During the “secondary transition,” however, it determines the allocation of endocrine cells. Wnt Pathway: TCF/LEF TFs
Wingless/int (Wnt) is a family of secreted growth factors that transduce signaling in the cells by binding Frizzled receptors. This activates intracellular protein Disheveled (Dsh) which inhibits beta-catenin degradation, allowing its nuclear import and activation of target genes via binding to TFs Lefs and TCFs. Wnt signaling is inhibited in the early endoderm for pancreas specification but gets activated later for the development of acinar cell lineage.
Retinoic Acid (RA) Pathway and RAR TFs
▶ Retinoic acid is synthesized from vitamin A (retinol) via an enzymatic pathway which includes retinaldehyde dehydrogenases (Raldh). RA is carried to the nucleus with the help of adaptor proteins and two types of nuclear receptors, RARs (retinoic acid receptors), and RXRs (retinoid X receptors). RA bound to the RAR-RXR heterodimer enters the nucleus and activates expression of its target genes. During pancreatic development, RA produced from the dorsomedial splanchnic mesoderm promotes the outgrowth of the dorsal pancreatic bud and subsequently formation of the early endocrine cells. RA signaling is also critical during the early endodermal patterning. In addition to these intercellular signals leading to the activity of specific transcription factors, there are several other pancreas-dedicated transcription factors which are known to play important role at various stages of pancreatic development. While function of some is restricted to a particular stage, others are involved at more than one developmental stage. The latter includes chiefly homeodomain (HD)-containing Pdx1 (pancreas duodenal homeobox) and a basic helix-loop-helix (bHLH) TF Ptf1a (pancreas transcription factor 1a) which have critical roles
Pancreatic Cancer Development and Regeneration
during early, middle, and late stages of pancreatic development. Both are necessary for the growth, branching morphogenesis, and the transition to the protodifferentiated state. Pdx1 expression is first restricted to initial domains of peripancreatic buds and then expands to the proximal duodenum and distal stomach. It is required for the formation and growth of the protodifferentiated cells, during “secondary transition” for the formation of various cell lineages (acinar, ductal, and islet), and for differentiation and maintenance of beta cell at later stages. On the other hand, Ptf1a maintains pancreatic identity in the nascent pancreatic buds and maintains progenitor cell growth of early epithelium. It also defines the multipotent progenitor cell population that initiates the secondary transition while controlling the differentiation of acinar cells during later stages. There are other noteworthy transcription factors which play important roles at selected stages of pancreatic development. Forkhead factor FoxA3/Hnf3b controls the formation of anterior endoderm during gastrulation; HD transcription factor Hlxb9 participates in endoderm patterning and cell-lineage specification within the pancreatic domain by causing activation of Pdx1 in dorsal epithelium; bHLH TF neurogenin3 (Ngn3) is expressed specifically in the pre-pancreatic endoderm prior to budding and later specifies the pancreatic endocrine cell identity. Gata4 and Gata6 are expressed in early foregut endoderm and throughout the nascent dorsal and ventral pancreatic buds. They are required for development as they are potent inducers of endoderm and its pre-pancreatic regionalization by controlling expression of Pdx1 and Hlbx9 among other important TFs. The high-mobility group (HMG) TF Sry-box9 (Sox9) maintains the undifferentiated state of precursor cells during the primary and secondary transition. The basic leucine zipper (bZip) TFs MafA and MafB orchestrate the final stages of beta-cell differentiation. The pancreatic ductal network is comprised of larger main ducts and smaller intercalating ducts and centroacinar duct cells. Remarkably, both ductal systems are derived from distinct developmental programs. While Pdx1 and Ptf1a are
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required for the formation of small ducts but not large ducts, Hnf6 transcription factor is required for the development of the larger ducts but not small ducts. Regeneration in the Mature Pancreas Two major risk factors for pancreatic cancer development in humans are diabetes and chronic pancreatitis. Both these conditions cause extensive and almost irreversible damage to the pancreas resulting in loss of either endocrine or exocrine cells. At least in rodent experimental models, the mature pancreas has been shown to be fully regenerated following partial pancreatectomy and induced acute or chronic pancreatitis, wherein exocrine acinar cells are injured along with inflammation. There is debate, however, regarding the origin of the progenitor cells which leads to regeneration of the whole pancreas, including its exocrine, endocrine, and ductal components. Some reports suggest proliferation and transdifferentiation of duct cells to multiple pancreatic lineages, while others points to a like-begets-like model where preexisting endocrine and exocrine cells expand to reconstitute the pancreas. Regardless of the cellular origin, it is widely accepted that the process of regeneration involves dedifferentiation and subsequent redifferentiation of cells through reversible reactivation of various embryonic developmental signaling pathways, including Notch, Hedgehog, Wnt-b-catenin, etc. Aberrant regulation of these embryonic signaling pathways during regeneration is also believed to be one of the major events for the development of pancreatic cancer, as these pathways are well documented to be abnormally regulated during pancreatic carcinogenesis. Evidence is also emerging that regeneration that follows pancreatitis, in the presence of certain mutations (activating ▶ KRAS-G12D, inactivating ▶ TP53, etc.) and aberrant signaling processes (TGF-beta, Smad4, Ezh2, etc.), can cause pancreatic cancer precursor cells to get “locked” in the dedifferentiation stage which is evident by the activation of embryonic signaling pathways in pancreatic tumor cells.
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References Fendrich V, Esni F, Garay MV, Feldmann G, Habbe N, Jensen JN et al (2008) Hedgehog signaling is required for effective regeneration of exocrine pancreas. Gastroenterology 135:621–631 Jensen JN, Cameron E, Garay MV, Starkey TW, Gianani R, Jensen J (2005) Recapitulation of elements of embryonic development in adult mouse pancreatic regeneration. Gastroenterology 128:728–741 Jonsson J, Carlsson L, Edlund T, Edlund H (1994) Insulinpromoter-factor 1 is required for pancreas development in mice. Nature 371:606–609 Murtaugh LC, Melton DA (2003) Genes, signals, and lineages in pancreas development. Annu Rev Cell Dev Biol 19:71–89 Siveke JT, Lubeseder-Martellato C, Lee M, Mazur PK, Nakhai H, Radtke F et al (2008) Notch signaling is required for exocrine regeneration after acute pancreatitis. Gastroenterology 134:544–555
Pancreatic Cancer Metastases ▶ Pancreatic Cancer Metastasis
Pancreatic Cancer Metastasis Thilo Welsch Department of Visceral, Thoracic and Vascular Surgery, TU Dresden, Dresden, Germany
Synonyms Metastatic pancreatic cancer; Metastatic PDAC; Pancreatic cancer metastases; Pancreatic ductal adenocarcinoma metastasis
Definition Pancreatic cancer metastasis describes the process that leads to secondary malignant tumor cell growth at distant sites or organs from the parental cancer in the pancreas. Pancreatic cancer is often used as a synonym for pancreatic ductal
Pancreatic Cancer Metastases
adenocarcinoma (PDAC), which is the most frequent (>80%) and most lethal histologic type of cancer arising in the pancreas. Other histologic types are neuroendocrine tumors of the pancreas, acinar cell carcinoma, or adenosquamous carcinoma, which are not referred to in this entry.
Characteristics Pancreatic cancer is the fourth leading cause of cancer-related death in the Western world, and one main reason for the dismal prognosis is early metastasis to regional lymph nodes and distant organ sites (most frequently to the liver, peritoneum, and lung) (Iacobuzio-Donahue et al. 2009) (Fig. 1). Macroscopically, distant PDAC metastases appear as whitish, hard nodules in the surrounding tissue. At diagnosis, pancreatic cancer is already metastasized in over 80%, and only 1–2% of patients with distant metastases survive longer than 5 years (Siegel et al. 2013). Autopsy studies revealed that the vast majority of patients die from metastatic disease: 70% when all tumor stages are considered (Iacobuzio-Donahue et al. 2009). Furthermore, 75% of patients who had undergone curative resection of the primary tumor developed distant metastasis; others developed peritoneal carcinomatosis or local recurrence. Accordingly, the presence of lymph node or distant metastases negatively affects patient survival after curative resection, and several studies have underlined the prognostic value of the lymph node ratio, which is calculated by the number of tumor-infiltrated lymph nodes divided by the total number of surgically resected lymph nodes (Hartwig et al. 2011). Clinical Management Metastatic spread is the most critical determinant of resectability of pancreatic cancer. Crosssectional imaging using contrast-enhanced computed tomography (CT) or magnetic resonance imaging (MRI) is the mainstay for diagnosis and treatment allocation of pancreatic cancer patients; it allows identification of locally advanced tumor growth or distant metastasis (Fig. 2).
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Pancreatic Cancer Metastasis, Fig. 1 Immunohistological image of metastatic pancreatic ductal adenocarcinoma to a regional lymph node; brownish adenocarcinoma cells on the left infiltrate the lymphatic tissue (right)
Surgical Treatment
Surgical resection of PDAC is reserved for fit patients without distant metastasis or arterial infiltration and is only offered 15–20% of the patients with the initial diagnosis. The surgical resection (resection of the pancreatic head, tail, or total pancreatectomy) of the primary tumor includes a defined resection of regional lymph nodes. At the time of surgery for PDAC, 70–80% of the resected lymph nodes harbor metastatic tumor cells (N1 stage according to the TNM classification) (Hartwig et al. 2011). Meta-analyzed and randomized-controlled data failed to demonstrate a survival benefit when an extended (more radical) lymphadenectomy was performed. Synchronous liver or other distant metastasis (M1 stage according to the TNM classification) is usually considered a contraindication for surgical resection. Retrospective studies of selected cases (e.g., incidental intraoperative liver metastases or tumor-infiltrated interaortocaval lymph nodes) indicate that simultaneous resection of the primary tumor and single to few liver metastases, or resection of the interaortocaval lymph nodes, may improve patient outcome compared with palliative treatment. Likewise, surgical resection of metachronous recurrence or distant metastases is not standard. In most patients, recurrence is
observed within 2 years after initial tumor resection and accompanied by an elevation of the tumor marker carbohydrate antigen CA 19-9 (Welsch et al. 2009). Some retrospective data suggest an individual survival advantage in few selected patients with late recurrence (>9 months following primary tumor resection). If metastatic spread (e.g., to the liver) is observed during surgical exploration for PDAC of the pancreatic head, and consequently restricts curative resection, palliative surgery with hepaticojejunostomy and/or gastrojejunostomy should be considered in case of bile duct or duodenal obstruction, respectively. Palliative Treatment
The standard treatment for metastatic pancreatic cancer is palliative chemotherapy, and the currently applied regimen covers gemcitabine monotherapy, the FOLFIRINOX protocol, or a combination of gemcitabine and nab-paclitaxel. However, the median survival with palliative care is only 6–11 months (Von Hoff et al. 2013). Before initiation of palliative chemotherapy, histopathologic evidence of PDAC from either the primary tumor or distant metastases (e.g., obtained by percutaneous biopsy of liver metastases) is mandatory.
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Pancreatic Cancer Metastasis
Pancreatic Cancer Metastasis, Fig. 2 Computed tomography (CT) scan of a female patient with synchronous hepatic and pulmonary metastases of a pancreatic ductal adenocarcinoma showing a pancreatic ductal adenocarcinoma in the pancreatic head (a: abdominal CT scan;
arrow indicates the hypodense tumor mass in the pancreatic head), a (b) liver metastasis in segment 3 (arrow indicates the hypodense lesion), and (c) pulmonary metastases in the left lung (chest CT scan, arrow)
Genetic and Molecular Background Genome analysis of pancreatic cancer metastases provides evidence that acquisition of metastatic propensity arises in distinct subclones and classically occurs after the development of the parental nonmetastatic tumor cells. The process of gaining metastatic ability was approximated to last approximately 5 years (Yachida et al. 2010). Pancreatic cancer metastases display genetic heterogeneity, but the characteristic pancreatic cancer key mutations in the KRAS, TP53, and SMAD4 (DPC4) genes are concordantly identified in matched primary and metastatic cells. Today, it is still unknown which genetic or epigenetic aberrations are critical to trigger the metastatic process. Loss of Dpc4 expression, for example, was
correlated with metastatic stages, and Dpc4 immunolabeling can be used for differential diagnosis of PDAC metastases and organ-related lesions (e.g., lung metastasis and primary lung cancer) (Iacobuzio-Donahue et al. 2009). In a general, schematic perspective, metastasis requires detachment of cells from the parental tumor mass, invasion into adjacent lymph or blood vessels, and extravasation into distant organs with subsequent tumor cell seeding and proliferation. Thus, many research projects focus on in vitro analysis of tumor cell migration/adhesion and matrix degradation. Epithelial to mesenchymal transition (EMT) of tumor cells and its elementary, molecular set play a pivotal role in metastatic dissemination of PDAC. Furthermore,
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activation of the Wnt or the embryologic Hedgehog pathway is related to PDAC metastasis. Several experiments also point to the importance of the chemokine receptor CXCR4-CXCL12 axis for the metastatic process of PDAC. A growing field of research elucidates the role of pancreatic cancer stem cells (CSC) in the metastatic process. The subpopulation of pancreatic CSC is frequently characterized by the marker molecules CD133 and ALDH and the triplet CD44, CD24, and ESA.
Siegel R, Naishadham D, Jemal A (2013) Cancer statistics, 2013. CA Cancer J Clin 63:11–30 Von Hoff DD, Ervin T, Arena FP et al (2013) Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine. N Engl J Med 369:1691–1703 Welsch T, Keleg S, Bergmann F et al (2009) Comparative analysis of tumorbiology and CD133 positivity in primary and recurrent pancreatic ductal adenocarcinoma. Clin Exp Metastasis 26:701–711 Yachida S, Jones S, Bozic I et al (2010) Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Nature 467:1114–1117
Conclusion The poor prognosis of PDAC is largely determined by its propensity for metastasis that limits the success of available treatment options. Future major challenges are the development of diagnostic tests that enable early diagnosis of PDAC before metastasis and novel therapeutic agents blocking the metastatic process. The latter necessitates disclosure of the molecular key switch of the metastatic process. In surgery, randomized, controlled trials will have to determine whether synchronous resection of distant metastases can improve the quality of life and survival of PDAC patients.
Pancreatic Cancer Onset and Course
Cross-References ▶ Epithelial-to-Mesenchymal Transition ▶ KRAS ▶ Neuroendocrine Tumors of the Pancreas ▶ Pancreatic Cancer ▶ Pancreatic Cancer Molecular Targets for Therapy ▶ Pancreatic Cancer Stem Cells ▶ TP53 ▶ Trefoil Factors
References Hartwig W, Hackert T, Hinz U et al (2011) Pancreatic cancer surgery in the new millennium: better prediction of outcome. Ann Surg 254:311–319 Iacobuzio-Donahue CA, Fu B, Yachida S et al (2009) DPC4 gene status of the primary carcinoma correlates with patterns of failure in patients with pancreatic cancer. J Clin Oncol 27:1806–1813
▶ Pancreatic Cancer Pathogenesis
Pancreatic Cancer Pathogenesis Vinee Purohit1, Kamiya Mehla2 and Pankaj K. Singh3 1 The Eppley Institute for Research in Cancer and Allied Diseases, and Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA 2 The Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA 3 The Eppley Institute for Research in Cancer and Allied Diseases, and Department of Pathology and Microbiology, and Department of Biochemistry and Molecular Biology, and Department of Genetic Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA
Synonyms Pancreatic cancer pathogenesis
onset
and
course;
PC
Definition Pancreatic cancer pathogenesis refers to induction and progression of cancer originating from pancreatic cells. Over 95% of pancreatic tumors are
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ductal adenocarcinomas. Pancreatic cancer is the fourth leading cause of cancer-associated deaths in the United States (Siegel et al. 2012). The deepseated anatomical location of pancreas, a lack of clear early symptoms, and high invasiveness of the pancreatic tumor cells cause metastatic spread of the disease even before it is diagnosed. At diagnosis most pancreatic tumors are non-resectable (Hidalgo 2010). Patients undergoing surgical resection face tumor recurrence due to early metastasis. Metastasis often precedes tumor formation, and hence, the genetic alterations in the primary tumors are different from metastases (Rhim et al. 2012). The clinical response to the current therapies is poor, and the 5-year survival rate of the patients with pancreatic cancer is currently at 6% (Siegel et al. 2012).
Characteristics Pancreatic Cancer Epidemiology and Histological Classification Unlike other cancer types, pancreatic cancer has shown constantly increasing death rate in both sexes from the year 1930 (Siegel et al. 2012). Most patients are 60–70 years of age, but it frequently affects patients of much younger age group. The most common etiological causes of pancreatic cancer are chronic pancreatitis, diabetes mellitus, smoking, and obesity. Some hereditary factors such as cystic fibrosis, Peutz-Jeghers syndrome, and Lynch syndrome also lead to the development of pancreatic cancer (Hidalgo 2010). Histologically, pancreatic cancer can be classified into exocrine or endocrine cancer of the pancreas. About 95% the pancreatic tumors occur in the exocrine portion that constitutes 80% of the pancreas. Hence, pancreatic ductal adenocarcinoma is the most extensively studied cancer of the pancreas (Table 1). Progression of Pancreatic Cancer The metastatic spread of pancreatic cancer is dependent on the anatomical location of the tumor. Pancreatic tumors affecting the head of the pancreas most frequently obliterate the bile duct causing jaundice and spread of the disease
Pancreatic Cancer Pathogenesis
laterally to the liver and the gut. The tumors of the body and tail, in contrast, affect the spleen and the vertebral column and disseminate much faster leading to higher incidences of metastasis at the time of initial diagnosis. The onset of pancreatic cancer is marked by the occurrence of precursor lesions that may be mucinous cystic neoplasms (MCNs), intraepithelial papillary neoplasms (IPMNs), or pancreatic intraepithelial neoplasm (PanINs). The mucinous cystic neoplasms, as the name suggests, are mucin-producing and cyst-forming neoplasms of epithelial origin. MCNs most frequently occur in the body and the tail of the pancreas and demonstrate mild to moderate dysplasia. These lesions have a cyst-like appearance with the epithelial lining of columnar cells and an ovarian-type stroma. The MCNs demonstrate early changes including mutations in Kras gene, which increases in expression as MCN progresses to the carcinoma stage. However, the expression of Kras is not correlated to aggressiveness. Unlike Kras mutation, mutations in TP53 and SMAD4/ DPC4 genes occur relatively late in MCN. Studies have shown that TP53 expression is absent in mild to borderline tumors and is absent in the lesions lacking Kras mutations (Hruban et al. 2007). Like MCNs, the IPMNs are mucin-producing noninvasive papillary structures, which occur in the ductal portion, frequently in the head of the pancreas. Like MCN, the Kras mutation is increasingly observed from preneoplastic IPMN to the carcinoma stage. Overexpression of HER2/ Neu is also observed in IPMNs. Less frequently, a loss of TP53 and DPC4 has been reported in IPMNs. Inactivation of LKB1 and mutation of phophatidylinositide 3-kinase (PI3CA) are other important molecular changes. Claudin-4, CXCR4, S100A4, and mesothelin are upregulated in invasive IPMNs as compared to the noninvasive tumors with at least two of the proteins seen in 73% of the tumors under study. MUC5AC, claudin-18, and PKM2 are also upregulated in IPMNs. Invasive IPMNs also demonstrate increased expression of b-catenin, ATF4, ENO1, and SLC2A1 (Hruban et al. 2007). Unlike IPMNs and MCNs, the PanINs cannot be detected by imaging techniques currently
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Pancreatic Cancer Pathogenesis, Table 1 Subtypes of pancreatic tumors, their histological and underlying genetic alterations Tumor type Pancreatic ductal adenocarcinoma Mucinous noncystic carcinoma Signet ring cell carcinoma Undifferentiated anaplastic carcinoma Adenosquamous carcinoma Osteoclast-like giant cell tumor Serous cystadenoma
Mucinous cystadenocarcinoma Solid pseudopapillary carcinoma Acinar cell carcinoma Mixed acinarendocrine carcinoma Pancreatoblastoma
Histological alterations Invasive malignant epithelial neoplasms, abnormal glandular structures, moderately to poorly differentiated Gelatinous carcinoma with pooled mucin surrounded by epithelial cells Signet ring-shaped cells with intracytoplasmic mucins Epithelial neoplasm without differentiation to glandular or squamous. Highly proliferating tumors with perineural and lymphatic invasion Epithelial neoplasms with both squamous differentiation and glandular differentiation Pancreatic neoplasms having nonneoplastic giant cells in the vicinity of atypical cells Scar tissue surrounded by numerous cystic cells
Cystic tumors with mucinous epithelium Epithelial cells arranged in pseudopapillary pattern, expressing mesenchymal or endocrine markers
Underlying genetic alterations KRAS mutation, loss of function mutation in TP53, CDKN2A KRAS mutations, microsatellite instability KRAS mutations KRAS mutations
KRAS mutation and loss of DPC4, P53 KRAS mutation Loss of VHL, malignant potential detected by occurrence of KRAS mutation Relatively low incidence of mutations in KRAS, TP53 loss Mutations in b-catenin
Solid tumors with acinar patterns, lacks desmoplasia, but nodules of acinar-like cells are separated by fibrous bands Mixed tumors with ductal and endocrine components
KRAS mutation and alterations in APC-b-catenin, allelic loss on chromosome 11p Alterations in APC-b-catenin
Pediatric tumors with acinar portions and squamous cells separated by stroma
APC-b-catenin mutations, loss of MADH4, and overexpression of IGF4
available. The PanIN model is the most widely studied and well-established model of pancreatic tumor progression. On the basis of papillary architecture and cellular atypia, PanINs are classified into three main classes. With the advent of molecular analysis and study of tumor progression, it was observed that with the increasing histological grade of precursor PanIN lesions, the tumor-initiating cells accumulate mutations that pave the way for tumor formation. The progression of pancreatic cancer coincides with the accumulation of gene mutations causing activation of oncogenes, mainly KRAS, and inactivation of tumor suppressor genes, mainly p16, DPC4, and TP53. Germline mutations such as BRCA2, LKB1, and PRSS contribute to the familial onset of pancreatic cancer. These
alterations facilitate histopathological changes to facilitate pancreatic cancer progression from PanIN stage to pancreatic adenocarcinoma. Ductal lesions with minimal atypia demonstrate mutation in Kras and rarely transcriptional upregulation of HER2/neu. In addition to Kras mutation, there is a loss of p16 tumor suppressor gene on chromosome 9 that is found in early lesions. In contrast to these early mutations, the ductal lesions with advanced cytological and architectural atypia demonstrate the loss of heterozygosity for TP53, loss of DPC4 gene expression, and biallelic inactivation of BRCA2 (Hruban et al. 2000). Although the PanIN model provides a composite picture of molecular and histopathogenesis of pancreatic cancer, it is not necessarily the only route of carcinogenesis.
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According to an estimate, only 1 in 500 PanIN lesions does progress to pancreatic cancer (Kern et al. 2011). Furthermore, a study by Jones et al. has demonstrated that it is not individual mutations but signaling pathways that go awry in cancer. They demonstrate that pancreatic cancer has 12 core signaling pathways that are altered, and a given pancreatic tumor can bear up to 63 mutations on an average (Jones et al. 2008). It is evident that as the precursor lesions progress, they continue to accumulate mutations that can lead to higher chances of tumorigenesis. Here we have briefly summarized the pathways most frequently involved in the progression of pancreatic cancer. Oncogenes The most important gene that is mutated very early in pancreatic cancers is Kras. Kras mutations are found as early as in PanIN1A. In the genetically engineered mouse model, Kras mutation is sufficient to initiate pancreatic tumor formation (Hingorani et al. 2003). G12D mutation in Kras, a guanine nucleotide-binding protein, makes it constitutively active, resulting in increased growth factor receptor tyrosine kinase signaling (Lowy and Willumsen 1993). Pancreatic ductal adenocarcinoma is primarily ductal in phenotype. However, studies by Kopp et al. have demonstrated acinar to ductal metaplasia that facilitates PDAC from acinar cells. By tissue-specific induction of Kras in ductal/ centroacinar and acinar cell types, these authors demonstrated that PDAC originates and progresses to PanINs only in the acinar compartment (Kopp et al. 2012). Apart from Kras mutation, HER2/Neu overexpression at the mRNA level has also been suggested to cause pancreatic cancer. Though its occurrence is restricted to the early stages of pancreatic cancer and varies from 17% to 55%, HER2/Neu is an important gene to target pancreatic cancer oncogenesis (Dugan et al. 1997). Tumor Suppressors Other than activation of oncogenes, pancreatic cancer is initiated due to inactivation of tumor suppressors mainly p16, DPC4, and TP53. The TP53 tumor suppressor gene is frequently inactivated in about 40–80% pancreatic tumors
Pancreatic Cancer Pathogenesis
(Jean et al. 2002). TP53 is a transcription factor that regulates cell cycle by increasing the expression of p21 and thereby acting on G1/S checkpoint (Jean et al. 2002). Point mutations in this gene lead to inactivation of cell cycle checkpoint function and subsequent tumorigenesis. Furthermore, TP53 mutations provide gain of oncogenic function to facilitate tumorigenesis. P16, a member of INK4 family of cyclin-dependent kinase (CDK) inhibitors, is frequently deleted in pancreatic cancer. p16 inactivates CDKs and resultantly maintains retinoblastoma (Rb) protein in a dephosphorylated active state (Jean et al. 2002). This allows nuclear functions of Rb and its interaction with E2F transcription factor leading to uncontrolled cell division. Deleted in pancreatic cancer locus 4 (DPC4) or SMAD4 is another tumor suppressor that shows frequent biallelic or homozygous deletions in pancreatic cancer. Loss of DPC4 reduces transforming growth factor (TGF)-b-mediated transcription of p21 and p15 genes that are important cell cycle regulators. Additionally, mutations in BRCA2 and fragile histidine triad (FHIT) have also been reported in pancreatic cancer (Jean et al. 2002). Growth Factors and Other Signaling Pathways Pancreatic cancer cells are characterized by increased growth factor receptor signaling. Increased signaling through these growth factor receptors correlates with reduced survival in pancreatic cancer patients. The major growth factor pathways that are activated in pancreatic cancer are epidermal growth factor (EGF) (Friess et al. 1996; Jean et al. 2002), fibroblast growth factor (FGF) (Friess et al. 1996; Jean et al. 2002), hepatocyte growth factor (HGF) (Friess et al. 1996), transforming growth factor-b (TGF-b), and vascular endothelial growth factor (VEGF) (Friess et al. 1996). While EGF and FGF signaling pathways are involved in proliferation of pancreatic cancer cells, HGF and TGF-b pathways are involved in epithelial to mesenchymal transition, stemness, and metastasis. VEGF signaling is primarily responsible for angiogenesis in pancreatic tumors. In response to receptor tyrosine kinase activation, signaling activation of downstream PI3K/Akt and Erk pathways facilitates proliferation
Pancreatic Cancer Stem Cells
in pancreatic cancer cells (Friess et al. 1996). Activation of Hedgehog, Notch, SLIT/ROBO, and Wnt signaling pathways is responsible for stemness of pancreatic cancer cells and metastasis (Biankin et al. 2012; Quan et al. 2013).
References Biankin AV, Waddell N, Kassahn KS, Gingras MC, Muthuswamy LB, Johns AL, Miller DK, Wilson PJ, Patch AM, Wu J, Chang DK, Cowley MJ, Gardiner BB, Song S, Harliwong I, Idrisoglu S, Nourse C, Nourbakhsh E, Manning S, Wani S, Gongora M, Pajic M, Scarlett CJ, Gill AJ, Pinho AV, Rooman I, Anderson M, Holmes O, Leonard C, Taylor D, Wood S, Xu Q, Nones K, Fink JL, Christ A, Bruxner T, Cloonan N, Kolle G, Newell F, Pinese M, Mead RS, Humphris JL, Kaplan W, Jones MD, Colvin EK, Nagrial AM, Humphrey ES, Chou A, Chin VT, Chantrill LA, Mawson A, Samra JS, Kench JG, Lovell JA, Daly RJ, Merrett ND, Toon C, Epari K, Nguyen NQ, Barbour A, Zeps N, Australian Pancreatic Cancer Genome Initiative, Kakkar N, Zhao F, Wu YQ, Wang M, Muzny DM, Fisher WE, Brunicardi FC, Hodges SE, Reid JG, Drummond J, Chang K, Han Y, Lewis LR, Dinh H, Buhay CJ, Beck T, Timms L, Sam M, Begley K, Brown A, Pai D, Panchal A, Buchner N, De Borja R, Denroche RE, Yung CK, Serra S, Onetto N, Mukhopadhyay D, Tsao MS, Shaw PA, Petersen GM, Gallinger S, Hruban RH, Maitra A, Iacobuzio-Donahue CA, Schulick RD, Wolfgang CL, Morgan RA, Lawlor RT, Capelli P, Corbo V, Scardoni M, Tortora G, Tempero MA, Mann KM, Jenkins NA, Perez-Mancera PA, Adams DJ, Largaespada DA, Wessels LF, Rust AG, Stein LD, Tuveson DA, Copeland NG, Musgrove EA, Scarpa A, Eshleman JR, Hudson TJ, Sutherland RL, Wheeler DA, Pearson JV, McPherson JD, Gibbs RA, Grimmond SM (2012) Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature 491(7424):399–405 Dugan MC, Dergham ST, Kucway R, Singh K, Biernat L, Du W, Vaitkevicius VK, Crissman JD, Sarkar FH (1997) HER-2/neu expression in pancreatic adenocarcinoma: relation to tumor differentiation and survival. Pancreas 14(3):229–236 Friess H, Berberat P, Schilling M, Kunz J, Korc M, Büchler MW (1996) Pancreatic cancer: the potential clinical relevance of alterations in growth factors and their receptors. J Mol Med (Berl) 74(1):35–42 Hidalgo M (2010) Pancreatic cancer. N Engl J Med 362(17):1605–1617 Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, Jacobetz MA, Ross S, Conrads TP, Veenstra TD, Hitt BA, Kawaguchi Y, Johann D, Liotta LA, Crawford HC, Putt ME, Jacks T, Wright CV, Hruban RH, Lowy AM, Tuveson DA (2003) Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 4(6):437–450
3417 Hruban RH, Goggins M, Parsons J, Kern SE (2000) Progression model for pancreatic cancer. Clin Cancer Res 6(8):2969–2972 Hruban RH, Maitra A, Kern SE, Goggins M (2007) Precursors to pancreatic cancer. Gastroenterol Clin North Am 36(4):831–849, vi Jean ME, Lowy AM, Chiao PJ, Evans DB (2002) The molecular biology of pancreatic cancer. Pancreatic cancer: M. D. Anderson solid tumor oncology series. Springer, New York, pp 15–28 Jones S, Zhang X, Parsons DW, Lin JC, Leary RJ, Angenendt P, Mankoo P, Carter H, Kamiyama H, Jimeno A, Hong SM, Fu B, Lin MT, Calhoun ES, Kamiyama M, Walter K, Nikolskaya T, Nikolsky Y, Hartigan J, Smith DR, Hidalgo M, Leach SD, Klein AP, Jaffee EM, Goggins M, Maitra A, Iacobuzio-Donahue C, Eshleman JR, Kern SE, Hruban RH, Karchin R, Papadopoulos N, Parmigiani G, Vogelstein B, Velculescu VE, Kinzler KW (2008) Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 321(5897):1801–1806 Kern SE, Shi C, Hruban RH (2011) The complexity of pancreatic ductal cancers and multidimensional strategies for therapeutic targeting. J Pathol 223(2):295–306 Kopp JL, von Figura G, Mayes E, Liu FF, Dubois CL, Morris JP 4th, Pan FC, Akiyama H, Wright CV, Jensen K, Hebrok M, Sander M (2012) Identification of Sox9-dependent acinar-to-ductal reprogramming as the principal mechanism for initiation of pancreatic ductal adenocarcinoma. Cancer Cell 22(6):737–750 Lowy DR, Willumsen BM (1993) Function and regulation of ras. Annu Rev Biochem 62:851–891 Rhim AD, Mirek ET, Aiello NM, Maitra A, Bailey JM, McAllister F, Reichert M, Beatty GL, Rustgi AK, Vonderheide RH, Leach SD, Stanger BZ (2012) EMT and dissemination precede pancreatic tumor formation. Cell 148(1-2):349–361 Siegel R, Naishadham D, Jemal A (2012) Cancer statistics. CA Cancer J Clin 62(1):10–29 Quan M, Wang P, Cui J, Gao Y, Xie K (2013) The roles of FOXM1 in pancreatic stem cells and carcinogenesis. Molecular Cancer 12:159
Pancreatic Cancer Stem Cells Diane M. Simeone Department of Physiology, University of Michigan Medical Center, Ann Arbor, MI, USA
Definition A subpopulation of highly tumorigenic cells that possess the stem cell-like properties of the ability
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to self-renew and generate differentiated tumor cell types found in pancreatic cancer.
Characteristics ▶ Pancreatic cancer is an aggressive malignancy arising from the exocrine pancreas. Greater than 30,000 people per year are affected by the disease in the United States alone where it is the fourth leading cause of cancer death. Pancreatic cancer portends the worst prognosis of any solid tumor malignancy, with a 5-year survival rate of less than 3%. Conventional therapies, such as chemotherapy and ionizing radiation, often produce minimal clinical response in tumor shrinkage and cancer regression. While our understanding of the molecular mechanisms underlying the development and progression has significantly improved by examining genomic and proteomic profiling of human pancreatic cancers, these studies do not account for the heterogeneity of cancer cells within a particular tumor. There has been a paradigm shift in our understanding of how cancers develop and propagate termed the cancer stem cell theory. In the traditional model of how cancer develops and propagates, it is though that all cells within the cancer are able to proliferate extensively and form new tumors. In the cancer stem cell theory, tumors arise from a small population of cancer cells,
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usually less than 5% of cells, that have properties of adult stem cells; particularly the abilities to selfrenew and differentiate into the multiple cell types found in the tumor. Of clinical relevance, this model may explain why standard chemotherapy and ionizing radiation therapy may result in tumor shrinkage, but do not prevent cancer recurrence, since cancer stem cells are resistant to standard types of therapies. Conventional treatments may be able to target the bulk of the tumor, but not necessarily these tumor-initiating cells, subsequently leading to recurrence or disease progression following standard treatment. The existence of cancer stem cells was originally demonstrated in acute myelogenous leukemia and now has been well established in several types of solid tumors, including breast cancer, brain cancer, prostate cancer, colon cancer, head and neck cancer, lung cancer, and melanoma. Identification of cancer stem cells within human tumors has been facilitated by the development of xenografts (or implants) of human tumors into immunodeficient mice for study. Using this model system, either primary tumors derived directly from patients or implanted and expanded in mice can be dissociated into single cell suspensions. These cells then undergo ▶ flow cytometry and are isolated based on differential cell surface marker expression and implanted into mice with monitoring of tumor growth (Fig. 1).
Pancreatic Cancer Stem Cells, Fig. 1 Tumor formation in NOD/SCID mice injected with 500 CD44+ CD24+ ESA+ cells, with no tumor formation seen at the injection site of 500 marker negative CD44 CD24 ESA cells
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Study of human pancreatic cancers has identified a subpopulation of cells that express cell surface markers ▶ CD44 CD24 ESA as putative pancreatic cancer stem cells. In pancreatic cancer, less than 1% of the cancer cells are CD44+ CD24+ ESA+. As few as 100 CD44+ CD24+ ESA+ cells injected into immuneincompetent NOD/SCID mice form tumors (Table 1), while 10,000 unsorted primary bulk pancreatic cancer cells were required to initiate tumor growth, revealing a greater than 100-fold enhanced tumorigenic potential in the triple surface marker positive cell population compared to bulk pancreatic cancer cells. The CD44+ CD24+ ESA+ pancreatic cancer cells possess the stem cell properties of selfrenewal and the ability to produce differentiated progeny that recapitulate the phenotype of the parent tumor. Tumors formed in NOD/SCID mice as xenografts derived from pancreatic cancer Pancreatic Cancer Stem Cells, Table 1 Tumor formation ability of sorted pancreatic cancer cells using cell surface markers Cell number Unsorted CD24+ ESA+ CD24 ESA CD44+/CD24+/ESA+ CD44/CD24/ ESA
10 K 4/6 6/8 2/8 10/12 1/12
1K 0/6 5/8 1/8 10/12 0/12
500 0/3 5/8 0/8 7/12 0/12
100 0/3 2/8 0/8 6/12 0/12
stem cells are histologically identical to patient’s primary tumor from which the cells were derived (Fig. 2). While some different types of tumors may have common cell surface makers that best define the cancer stem cell population, it is likely that different tumor types each have their own “best” set of makers that identify cancer stem cells. In addition, the secondary tumors that form from implantation of the primary tumor’s CD44+ CD24+ ESA+ cells reproduce the surface expression pattern of commonly expressed proteins seen in the original tumor (Fig. 3). The capacity of these highly, tumorigenic CD44+ CD24+ ESA+ pancreatic cancer cells to both self-renew and produce differentiated progeny highlights their stem cell properties. It is not known at present whether these pancreatic cancer stem cells arise from a mutated stem cell, or a differentiated cell that has regained stem cell-like properties because of genetic alterations. Several studies examining other cancers have supported the concept that cancer stem cells likely arise from self-renewing adult stem cells which are transformed by dysregulation of a self-renewal pathway. Characterizing normal pancreatic stem cells will greatly enhance our understanding of pancreatic cancer stem cells. Unfortunately, despite extensive effort, isolation of normal pancreatic stem cells has not yet been achieved to date. A critical consequence of the identification of pancreatic stem cells is the potential impact this
Pancreatic Cancer Stem Cells, Fig. 2 H&E staining of the tumor generated from CD44+ CD24+ ESA+ cells (right) has similar histologic features to the corresponding patient’s primary pancreatic tumor (left). Magnification, 200
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Pancreatic Cancer Stem Cells, Fig. 3 Highly tumorigenic pancreatic cancer cells display stem cell-like properties. (a–f) Phenotypic diversity in tumors arising from CD44+ CD24+ ESA+ cells. Plots are representative of the CD44, CD24, and ESA staining patterns of human pancreatic cancer cells. (a and d) Staining pattern from a patient tumor. CD44+ CD24+ ESA+ tumorigenic cells from the
tumor were then isolated (b and e) and injected into the flank of NOD/SCID mice. (c and f) The staining pattern of the resultant tumor that arose from the CD44+ CD24+ ESA+ cells. The tumorigenic cells formed tumors that contained phenotypically diverse cells (c and f) similar to those seen in the original tumor (a and d)
may have on approaches to treatment of pancreatic cancer. It is likely that therapies specifically targeting this stem cell population will be needed to improve the likelihood of long-term cure for patients with pancreatic cancer. Approaches targeting markers expressed specifically on the cell surface of pancreatic cancer stem cells (i.e., CD44) or drugs that target signaling pathways that are specifically altered in these pancreatic cancer stem cells, such as sonic hedgehog, are likely to lead to significant improvements in outcome for pancreatic cancer patients. In short, a better understanding of pancreatic cancer stem cells will aid in refining current anticancer therapies and ultimately help to identify novel diagnostic markers and therapeutic targets.
References Al-Hajj M, Wicha M, Morrison SJ et al (2003) Prospective identification and characterization of a tumorigenic breast cancer cell. Proc Natl Acad Sci U S A 7:3983–3988 Bao S, Wu Q, Sathornsumetee S et al (2006) Stem cell-like glioblastoma cells promote tumor angiogenesis through vascular endothelial growth factor. Cancer Res 66:7843–7848 Bonnet D, Dick JE (1997) Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3:730 Li C, Heidt DG, Dalerba P et al (2007) Identification of pancreatic cancer stem cells. Cancer Res 67:1030–1037 Reya T, Morrison SJ, Clarke MF et al (2001) Stem cells, cancer, and cancer stem cells. Nature 414:105–111
Pancreatic Cancer Molecular Targets for Therapy
Pancreatic Cancer Molecular Targets for Therapy Steven N. Hochwald, William G. Cance and Elena Kurenova Departments of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY, USA
Definition ▶ Pancreatic cancer, synonym pancreas cancer, is a virulent disease with no effective therapy besides surgical resection. The possibility for surgical tumor removal is limited by early metastatic spread of tumor to sites outside the pancreas. The survival for patients with metastatic pancreatic cancer is less than 1 year following diagnosis. Clinicians must translate the available knowledge of the molecular basis of this disease into rationale and effective therapeutic strategies for treatment. Pancreas cancer is one of the tumors with the highest number of genetic mutations of any solid malignancy. These include oncogenes, tumor suppressor genes, and DNA-stability genes. The stability genes include mismatch repair (MMR) and base excision repair (BER) genes, which control the mutation rate of other genes. A number of genetic alterations have been, and still are being, tested as molecular target for antipancreatic cancer therapy. It is clear that novel molecular targets and strategies need to be developed for the treatment of pancreatic cancer.
Characteristics Pancreatic cancer ranks 13th in incidence but eighth as a cause of cancer death worldwide. In the United States and Europe, pancreatic cancer is the fourth leading cause of cancer death in both men and women. Chemotherapy and radiation therapy have had little impact on survival, prompting the National Cancer Institute to declare that survival for pancreatic cancer has remained
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unchanged for three decades and its treatment has consistently been identified as an area of unmet medical need. Oncogenes KRAS Oncogene
The KRAS proto-oncogene is the most commonly mutated gene in pancreatic cancer. KRAS is a member of the RAS family GTP-binding proteins and has intrinsic GTPase activity. The Ras proteins mediate a wide variety of cellular functions including proliferation, differentiation, and survival by transduction of growth-promoting signals from the cell-surface tyrosine kinase receptors to intracellular pathways. Activating KRAS point mutations at codon 12 result in substitution of amino acids and are early genetic alterations, occurring in 30% of early pancreatic neoplasms and with a frequency approaching 100% in advanced pancreatic adenocarcinoma. Ras has been considered an important therapeutic target in pancreatic cancer due to its frequency of mutation and possible role in the initiation of this malignancy. Unfortunately, Ras inhibitors to date have not been clinically effective. Most studies have focused on the efficacy of farnesyltransferase inhibitors (FTIs) which inhibit posttranslational modification of Ras by interfering with lipid modification of the C-terminus of Ras. Despite showing promise in vitro and in xenograft models, FTIs have failed to significantly improve survival of patients with pancreatic cancer, possibly due to compensatory geranyltransferase activity preserving Ras function. A phase II trial of FTI alone on 53 patients with locally advanced or metastatic pancreatic cancer showed a median survival of 2.6 months and a 6-month survival rate of 19%. Another phase II trial on 20 patients with metastatic disease showed a median survival of 19.7 weeks and a 6-month survival rate of 25%. A phase III trial combining the FTI tipifarnib with gemcitabine chemotherapy did not lead to any improvement in median survival compared with gemcitabine chemotherapy alone. Therefore, at the present
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time, the role of FTIs in the treatment of pancreatic cancer remains uncertain. Activated KRAS signals to multiple critical pathways including the Raf–mitogen-activated protein kinase (MAPK), phosphoinositide-3kinase, and RALGDS pathways. These downstream targets are logical alternative points of study for therapeutic intervention. Signaling Pathways of Ras Raf–MAPK
The Raf serine/threonine kinases lead to MAP kinase activation resulting in proliferative behavior in a variety of cell types. Activating B-Raf mutations have been demonstrated in a variety of malignancies including melanoma and colorectal, thyroid, and ovarian cancers. However, B-Raf and Ras mutations appear to be mutually exclusive in these cancers. Pancreatic cancers rarely have B-Raf mutations, except for a low incidence in the histologic subtype of pancreatic medullary carcinoma. Nevertheless, inhibition of MAP kinase leads to pancreatic cancer cell cycle arrest and decreased proliferation. This arrest may be mediated through increased expression of p27KIP1. Phosphoinositide-3-Kinase (PI3K) Pathway
The PI3K pathway regulates cell size, survival, and proliferation via several downstream effectors including AKT. Activating mutations in the catalytic subunit of PI3K and loss-of-function mutants of the PTEN tumor suppressor have been frequently seen in some cancers but not in human pancreatic adenocarcinoma cells. However, the PI3K pathway appears to have general importance in pancreatic adenocarcinoma. Decreased PTEN expression in pancreatic adenocarcinoma, possibly due to promoter hypermethylation, has been reported. In addition, activation of the PI3K pathways appears necessary to maintain Ras-transformed tumor growth in nude mice after Ras expression is abolished. Finally, inhibition of PI3K appears to increase the sensitivity of pancreatic adenocarcinoma cells to chemotherapy. These observations have made the PI3K pathway an interesting potential target for drug
Pancreatic Cancer Molecular Targets for Therapy
development, but no successful clinical trials have been reported to date. Activation of the serine/threonine kinase AKT is common in pancreatic cancer; inhibition of which sensitizes cells to the apoptotic effect of chemotherapy. Inhibition of either phosphatidylinositol-3 kinase or AKT led to a decreased protein level of the antiapoptotic gene BCL-2 and an increased protein level of the proapoptotic gene BAX. Furthermore, inhibition of AKT decreased the function of nuclear factor kB (NF-kB), which is capable of transcriptional regulation of the BCL-2 gene. Inhibiting this pathway had little effect on the basal level of apoptosis in pancreatic cancer cells but increased the apoptotic effect of chemotherapy. The antiapoptotic effect of AKT activation in pancreatic cancer cells may involve transcriptional induction of a profile of BCL-2 proteins that confer resistance to apoptosis; alteration of this balance allows sensitization to the apoptotic effect of chemotherapy. Nuclear Factor kB
The NF-kB transcription factor is an important mediator of mutated KRAS signaling in pancreatic adenocarcinoma cells. This pathway is activated by a variety of cellular stresses through stimulation by cytokines and growth factors and subsequently aids in the control of apoptosis and immune responses. Most primary pancreatic cancers and cell lines demonstrate constitutive NF-kB activity. NF-kB induction has been shown to be necessary for Ras transformation of several cell types. NF-kB helps to regulate cell survival genes, VEGF, urokinase, and other proinvasive and angiogenic factors. The NF-kB pathway may also contribute to chemoresistance which is widespread among pancreatic adenocarcinomas possibly due to the ability of NF-kB to upregulate BCL-2 and BCL-XL. The diverse roles of NF-kB have led to the development of inhibitors that have been studied in patients with advanced pancreatic cancer. The phosphorylated form of I-kB is degraded by the 26S proteasome, and inhibition of the proteasome prevents I-kB degradation, thereby preventing the activation of NF-kB. Bortezomib (PS-341, Velcade) is a 26S proteasome inhibitor
Pancreatic Cancer Molecular Targets for Therapy
that causes cell cycle arrest of pancreatic cancer cells via accumulation of cyclin-dependent kinase inhibitor p21cip1/waf1 and apoptosis via downregulation of BCL-2. In vivo experiments have shown that PS-341 increases the cytotoxicity of gemcitabine. However, despite encouraging preclinical data, neither PS-341 alone nor combined with gemcitabine is considered of any benefit to patients with metastatic pancreatic cancer. This is due to findings from a randomized phase II trial involving 87 patients with metastatic pancreatic adenocarcinoma, where the use of PS-341 alone or in combination with gemcitabine resulted in a median survival of only 2.5 and 4.8 months, respectively. Ras Family GTPases
The Ras family of GTPases has more than 150 members and is divided into five groups: Ras, Rab, Arf, Rho, and Ran. Some of the best characterized proteins include the Rho family. Activating mutations of Rho family members have not been demonstrated in human pancreatic tumors; however, overexpression of Rho has been shown to correlate with pancreatic cancer metastasis. Ral GTPases are downstream of Ras and are members of the Ras subfamily. Ral A has been shown to be activated in several pancreatic cancer cell lines, and RAL A inhibition suppressed tumorigenicity of Ras-transformed human cells. Much evidence supports KRAS and many of its downstream effectors in both the initiation and maintenance of pancreatic cancer. While agents that target FTIs have not shown to be of benefit in pancreatic cancer, ongoing studies are investigating the possibility of targeting Ras-directed downstream signaling mediators. Tumor Suppressor Genes CDKN2A/INK4A/p16
Chromosomal alterations in the 9p21 region that contains the gene for CDKN2A/INK4A/p16 are frequently observed in pancreatic cancer. These alterations typically result in cellular inactivation through deletion or mutation, although gene silencing by hypermethylation does also occur.
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The CDKN2A gene encodes a cyclin-dependent kinase inhibitor that induces cell cycle arrest at G1 in cooperation with normal retinoblastoma function. In animal models, loss of CDKN2A is an early event in pancreatic cancer that functions to enhance the oncogenic effect of KRAS. SMAD4 and TGF-b
The SMAD4 gene plays an important role in signaling pathways involving transforming growth factor beta (TGF-b), bone morphogenetic protein, and activin. Inactivation of SMAD4 is noted in approximately 50% of pancreatic cancers. SMAD4 has been termed a progression allele for pancreatic adenocarcinoma as it is lost in later stages of pancreatic intraductal neoplasms. Reports suggest that wild-type SMAD4 restricts activated KRAS-initiated neoplasms that would otherwise progress to frank invasive disease in the context of SMAD4 deficiency. TGF-b is the prototypic member of a superfamily of proteins that upon ligand binding phosphorylate receptor-regulated SMAD proteins regulating a variety of cellular functions including proliferation, migration, differentiation, and apoptosis. In numerous cell lines, TGF-b exerts a growth inhibitory effect that involves cell cycle regulators, repression of c-Myc, and induction of apoptosis. Elevation in TGF-b signaling inhibits epithelial cancer initiation in vivo, and defects in this pathway promote pancreatic tumorigenesis. Evidence suggests that SMAD4 deficiency inhibits TGF-b-induced cell cycle arrest and cell migration, thereby altering TGF-b signaling from one of tumor suppression to tumor promotion. p53
Loss of activity of the transcription factor p53, which plays a critical role in cell cycle arrest and apoptosis, has been reported in pancreatic tumors. Missense mutations in part of the p53 gene encoding the DNA-binding domain of this tumor suppressor protein result in the production of a protein that is able to translocate to the nucleus but is unable to function in DNA binding and therefore accumulates in the nucleus. This accumulation has been described in high-grade tumor and late intraepithelial lesions, suggesting that these
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mutations contribute to the later stages of pancreatic tumorigenesis. As with SMAD4 deletions, loss of p53 activity induces further chromosomal instability in the context of mutated, activated KRAS. Growth Factors Epidermal Growth Factor Receptor (EGFR)
The EGFR is a family of transmembrane glycoproteins that has four members. Upon phosphorylation, the activated receptor subsequently signals to a variety of intracellular adaptor molecules and mediators. These allow EGFR to play a role in a range of cellular functions including proliferation, survival, and motility. Two major downstream pathways in normal EGFR signaling are the Ras–Raf–MAPK and the PI3K/Akt pathways. Constitutive activation of EGFR promotes oncogenic cellular processes including uncontrolled proliferation with migration, invasion, angiogenesis, and resistance to apoptosis. Overexpression of EGFR has been observed in many pancreatic tumors and appears to correlate with poor prognosis and disease progression. Preclinical studies have shown that antagonizing EGFR signaling inhibits growth and metastasis of pancreatic tumors in xenograft animal studies. Two therapeutic approaches have been utilized to interfere with EGFR signaling. The first involves blocking ligand binding with monoclonal antibodies specific to epitopes within the ligandbinding domain. Once bound to the domain, these antibodies prevent receptor dimerization and subsequent tyrosine phosphorylation. The second approach to inhibit EGFR signaling uses small-molecule tyrosine kinase inhibitors (TKIs) that function inside the cell. These TKIs inhibit the kinase activity of the receptor independent of ligand binding through competition with ATP binding, thereby preventing receptor activation. The advantage of kinase inhibitors over receptor antibodies is that cross-reactivity with the ATP-binding domain of other EGFR family members as well as the ability to inhibit truncated receptor mutants that lack a functional extracellular domain may allow for more thorough receptor inhibition with TKIs. The disadvantage of TKIs is
Pancreatic Cancer Molecular Targets for Therapy
that other kinases can be inhibited leading to increased toxicity or death of normal cells. A small-molecule EGFR TKI, erlotinib, has been evaluated in randomized studies of patients with advanced pancreatic cancer. Patients were randomized to gemcitabine chemotherapy with placebo or gemcitabine with erlotinib. The latter treatment was well tolerated and resulted in a small but significant improvement in survival. This study validated EGFR as a target in pancreatic cancer and provides further rationale for ongoing studies of targeted molecular agents in this disease. Vascular Endothelial Growth Factor Receptor (VEGFR)
Angiogenesis refers to the process by which new blood vessels develop from existing vasculature. Tumor blood vessels are highly disorganized and structurally abnormal resulting in irregular blood flow, increased permeability, poor nutrient and drug delivery, and areas of hypoxia. VEGFR is one of the central mediators of angiogenesis and is overexpressed in pancreatic cancer. Preclinical data suggest that this growth factor has mitogenic activity on endothelial as well as tumor cells, making it a target for dual cancer therapy intended to produce both antiangiogenic and antitumor effects. Angiogenesis has prognostic importance in pancreatic cancer, a disease in which VEGF expression correlates positively with local recurrence, metastatic potential, and overall survival. The VEGF pathway has been most commonly targeted with monoclonal antibodies that bind the ligand (e.g., bevacizumab) or small-molecule VEGFR TKIs. Bevacizumab has been shown to suppress pancreatic tumor growth in preclinical models. Subsequently, a phase II study evaluated the combination treatment of bevacizumab with gemcitabine in 52 patients with metastatic pancreatic cancer. Results showed a 21% partial response rate with a median survival of 8.8 months. Overall, the treatment was well tolerated, but plasma VEGF levels did not correlate with outcomes. The response rates and survival results from this phase II study were considered encouraging, and this treatment regimen was
Pancreatic Cancer Molecular Targets for Therapy
evaluated in a phase III study. In this doubleblinded randomized trial in 602 patients with pancreatic cancer, bevacizumab plus gemcitabine treatment was compared with gemcitabine plus placebo therapy. Unfortunately, bevacizumab plus gemcitabine treatment failed to show a survival benefit compared with gemcitabine monotherapy. An alternative approach to targeting the VEGF pathway involves the use of TKIs that operate on an intracellular level. Such agents have already been approved for use in other cancers and include sunitinib in renal cell and gastrointestinal stromal tumors and sorafenib in advanced renal cell carcinoma. The knowledge obtained with the use of targeted anticancer molecular agents suggests that less selective inhibitors with multiple targets may be more efficacious than inhibitors of single targets. This is likely due to the cross talk and redundancy in molecular pathways that promote essential functions such as proliferation and survival. In addition, a single inhibitor with multiple targets may promote fewer drug interactions, potentially inducing less toxicity than combinations of more specifically targeted agents. Sorafenib is a multitargeted TKI of Raf-1, VEGR-2, and the platelet-derived growth factor receptor. Proposed antitumor activity of sorafenib targets both antiangiogenic and antiproliferative mechanisms. A phase I study in 23 patients with advanced pancreatic cancer demonstrated encouraging results with a 57% incidence of disease stability. Subsequently, a phase II study of gemcitabine with sorafenib was performed, and preliminary results have been reported. Unfortunately, there were no objective responses to this combination with a median survival of 4 months. Cytoplasmic Tyrosine Kinases
Several agents targeting a variety of pathways are in early clinical testing in pancreatic cancer and include those directed at Src and focal adhesion kinase (FAK). Src was the first reported protooncogene and encodes a nonreceptor tyrosine kinase with involvement in the regulation of proliferation, differentiation, survival, motility, angiogenesis, and cell–cell interactions. Malignant Src activity is related to the overexpression
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of the wild-type protein rather than expression of a mutated genotype. Src activity may contribute to both constitutive and EGF-induced VEGF expression and, therefore, to angiogenic potential. This has been shown in pancreatic cancer cells in vitro and in animal models growing human pancreatic tumors. Src inhibitors are in phase I and II clinical testing, and clinical trials combining an oral Src inhibitor with gemcitabine are in process. Unfortunately, combination did not improve efficacy over what would be expected from gemcitabine alone. FAK is a cytoplasmic protein tyrosine kinase that, as its name suggests, is localized to focal adhesions, which are contact points between a cell and its extracellular matrix (ECM). Tyrosine phosphorylation of FAK occurs in response to clustering of integrins, during formation of focal adhesions and cell spreading, and upon adhesion to fibronectin. FAK does not function as a classic oncogene, affecting cell transformation. Rather, it promotes a more invasive and metastatic phenotype of an established malignancy. FAK appears to have many functions in cells, linking integrin signaling to downstream targets, acting as part of a survival signal pathway, and having a connection with cell motility. FAK is phosphorylated following activation of a number of transmembrane receptors. FAK functions not only as a kinase, but also as a scaffolding protein for the assembly of a number of cellular signaling molecules, suggesting that FAK is a critical mediator of cell–ECM signaling events. Our laboratory group has played an important role in defining the biology of FAK in human tumors, including pancreatic cancer. We have demonstrated in pancreatic cancer cells, as well as in vivo models, that FAK inhibition is an effective antineoplastic strategy by inducing apoptosis and sensitizing tumor cells to chemotherapy. FAK inhibitors are currently in phase I testing. TKI FAK inhibitor in combination with PD-1 inhibitor and gemcitabine will be tested in patients with pancreatic cancer. Matrix Metalloproteinases Tissue invasion and the ability to metastasize are among the hallmarks of cancer. Disruption of
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cellular junctions and the interactions between cells and the ECM are essential prerequisites for cell detachment from the primary tumor, invasion of the blood stream, and growth at distant sites. Several families of proteases are implicated in these processes. The largest and best characterized is the family of matrix metalloproteinases (MMPs), which are zinc-dependent proteolytic enzymes with different substrate specificities for ECM molecules (e.g., collagen, fibronectin, laminin, and elastin). Among the various family members, MMP-2 and MMP-9 are most commonly implicated in tumor angiogenesis. The activity of MMPs may be inhibited by members of the family of proteins known as tissue inhibitors of metalloproteinases (TIMPs). In pancreatic cancer, overexpression of several MMPs has been demonstrated in several studies. It has been suggested that an imbalance between MMPs and TIMPs plays a role in pancreatic tumor progression and that MMP-2 activation correlates with more advanced pathologic stages and with early recurrence after resection. Synthetic MMP inhibitors (MMPIs) have been developed and tested against a variety of tumors. One such MMPI is marimastat, an orally bioavailable drug that is active against several MMPs. In preclinical models, marimastat and other MMPIs delay tumor growth and prolong animal survival, but no cytotoxic effect is observed. Phase I studies found an inflammatory polyarthritis to be the dose-limiting toxicity of marimastat. Pharmacokinetic analyses showed that marimastat is rapidly absorbed and eliminated, with time to maximum concentration of 1–2 h and an elimination half-life of 4–5 h. In a dose-finding study among patients with pancreatic cancer, marimastat doses of 5, 10, and 25 mg twice daily were considered adequate. CA19-9 changes in that study suggested evidence of activity, which was also noted in a phase II trial in which patients who had CA19-9 stabilization or responses (30% of cases) lived longer than patients with a rising tumor marker. Based on these findings, randomized trials of marimastat were performed. Bramhall et al. randomized 414 patients with advanced pancreatic cancer to receive either
Pancreatic Cancer Molecular Targets for Therapy
gemcitabine or marimastat. Although the median survival in the 25-mg marimastat group was lower than that in the gemcitabine group (125 days vs. 167 days), the log-rank analysis showed no difference between the survival curves of the two groups. Survival of patients treated with the other doses of marimastat (5 mg b.i.d. and 10 mg b.i.d.) was much lower than that of patients given gemcitabine. A subgroup analysis of patients with metastatic disease (approximately 65% of the cases) suggested an interaction between marimastat and disease stage; the median survival of patients with nonmetastatic disease was longer than that of patients with metastatic disease. In the gemcitabine group, there was no difference in median survival between patients with and without metastases. Consistent with the mode of action of these two drugs, the median progressionfree survival among patients treated with marimastat, which has no cytotoxic effect, was approximately half that of patients treated with gemcitabine. Likewise, the reported response rates were lower in the marimastat arms of the trial (3% vs. 26% for gemcitabine). Musculoskeletal toxicity was reported in 39–55% of patients across different dose levels of marimastat, but this toxicity was grade 3 or 4 in only 7–12% of cases. A different approach was taken in a second phase III trial, in which 239 patients were treated with gemcitabine and were concurrently randomized to receive marimastat (10 mg b.i.d.) or placebo. The median and 1-year survival was nearly identical in the two groups. Likewise, the log-rank comparison between the two survival curves yielded no differences in overall or progressionfree survival. In this trial, there was no interaction between marimastat and disease stage. The results of these trials indicate that marimastat, alone or in combination with gemcitabine, has no role in the treatment of advanced pancreatic cancer. Furthermore, another MMPI, BAY12-9566 proved inferior to gemcitabine in a randomized trial of 277 patients with advanced pancreatic cancer. Therefore, the currently available studies of MMPIs show that these agents have no role in the treatment of advanced pancreatic cancer. However, due to
Pancreatitis
their cytostatic effect, it is conceivable that the administration of marimastat or other MMPIs to patients with tumors in earlier stages could improve on the existing therapies. The suggested interaction between marimastat and disease stage in one of the trials is in line with this hypothesis.
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Pancreatitis Sonal Gupta Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA
References Aho U, Zhao X, Lohr M et al (2007) Molecular mechanisms of pancreatic cancer and potential targets of treatment. Scand J Gastroenterol 42:279–296 Bramhall SR, Schulz J, Nemunaitis J, Brown PD, Baillet M, Buckels JA (2002) A double-blind placebo-controlled, randomised study comparing gemcitabine and marimastat with gemcitabine and placebo as first line therapy in patients with advanced pancreatic cancer. Br J Cancer 87:161–167 Cohenuram M, Said M (2007) Epidermal growth factor receptor inhibition strategies in pancreatic cancer: past, present and future. JOP 8:4–15 Di Marco M, Grassi E, Durante S, Vecchiarelli S, Palloni A, Macchini M, … Biasco G (2016) State of the art biological therapies in pancreatic cancer. World J Gastrointest Oncol 8(1), 55–66. doi:10.4251/wjgo.v8. i1.55 Hansel DE, Kern SE, Hruban RH (2003) Molecular pathogenesis of pancreatic cancer. Annu Rev Genomics Hum Genet 4:237–256 Kindler HL, Friberg G, Siingh DA et al (2005) Phase II trial of bevacizumab plus gemcitabine in patients with advanced pancreatic cancer. J Clin Oncol 23:8033–8040 Matsuoka T, Yashiro M (2016) Molecular targets for the treatment of pancreatic cancer: clinical and experimental studies. World J Gastroenterol 22(2):776–789. doi:10.3748/wjg.v22.i2.776 Sulzmaier FJ, Jean C, Schlaepfer DD (2014) FAK in cancer: mechanistic findings and clinical applications. Nat Rev Cancer 14:598–610. doi:10.1038/nrc3792 Wallace JA, Locker G, Nattam S et al (2007) Sorafenib (S) plus gemcitabine (G) for advanced pancreatic cancer (PC): a phase II trial of the University of Chicago Phase II Consortium. Program and abstracts of the 2007 American Society of Clinical Oncology Gastrointestinal Cancers Symposium, 17–21 Jan Orlando (Abstract 127)
Pancreatic Ductal Adenocarcinoma Metastasis ▶ Pancreatic Cancer Metastasis
Definition Pancreatitis is a disease characterized by the ▶ inflammation of exocrine pancreas that is initiated by the premature activation and intracellular release of the digestive enzymes produced in pancreatic acinar cells. The release of these enzymes causes destruction of acinar cells and a subsequent robust inflammatory response, resulting in a significant injury to the pancreas.
Characteristics Pancreatitis was first described as a disease of autodigestion in 1896 by pathologist Hans Chiari. Common symptoms include searing upper abdominal pain that radiates into the back; nausea and vomiting, especially after eating; fever; rapid heartbeat; and jaundice (the yellowing of the skin, the whites of the eyes (conjunctiva), and mucous membranes caused by increased levels of bilirubin in the blood that may be due to liver damage as well as other diseases) – discoloration is the result of increased levels of bilirubin in the blood due to obstruction of the biliary drainage from the liver through the biliary tree into the small intestine. Over the period of time, indigestion due to lack of sufficient pancreatic enzymes also causes weight loss. Causes and Diagnosis Among various factors known to cause pancreatitis, the most common factors are alcohol abuse and presence of gallstones (80%). Pancreatitis associated with prolonged alcohol abuse is known as alcoholic pancreatitis. Recognized for well over 100 years, it remains one of the least
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understood alcohol-associated human diseases. Other triggers include trauma, steroids (such as prednisolone), mumps, autoimmune pancreatitis, scorpion sting, hyperlipidemia (elevated blood triglycerides), hypothermia, high blood calcium, ERCP (endoscopic retrograde cholangiopancreatography), and drugs (azathioprine, didanosine, pentamidine, valproic acid, statins, metformin, gliptins, etc.). These causative factors are also referred by a mnemonic GETSMASHED. Other causes are type 2 diabetes, pancreatic cancer, pancreatic duct stones, microbial infections, etc. Diagnosis is largely centered on the characteristic of abdominal pain, abdominal ultrasound or endoscopic ultrasound (EUS) accompanied by biopsy, CT scan (computed tomography scan), and ERCP. Pancreatic function test and glucose tolerance tests are also used to assess the normal functionality of pancreas. Elevated blood amylase and lipase are also considered indicators for pancreatitis although controversial. Types of Pancreatitis Pancreatitis is generally classified as either acute (sudden occurrence and lasting few days) or chronic (persisting for a long time or constantly recurring). Acute Pancreatitis
Acute pancreatitis is a condition that develops when the pancreas is damaged by an inflammation that leads to swelling and sometimes to necrosis (death) of parts of the pancreas. The digestive enzymes from damaged pancreas cause breakdown of fatty tissue in the abdomen. The most common risk factor for acute pancreatitis is the presence of gallstones. The pathophysiology of gallstone-induced pancreatitis was first described by pathologist Eugene Opie in 1901. In about 85% of patients, acute pancreatitis is a mild disease and is associated with a rapid recovery within a few days of onset of the illness. In about 15–20% of patients, acute pancreatitis can lead to severe damage of the pancreas, often leading to a severe illness associated with extended hospital stay, multiple surgical procedures, and
Pancreatitis
occasionally death in some patients. Patients with severe acute pancreatitis are at risk for developing the following complications: • Parts of the pancreas get destroyed causing the release of digestive enzymes and subsequent death in the pancreas (pancreatic necrosis) and the surrounding abdominal areas (peripancreatic necrosis). Treatment involves surgical removal of the dead tissue in extreme cases. • Failure of multiple body organ systems such as the heart, lungs, liver, and kidney due to damage from toxins released in the bloodstream from the dead pancreatic tissue. • More than 80% of death among pancreatitis patients is due to infected pancreatic necrosis. Treatment involves urgent and often multiple surgeries to remove all the dead tissue. • Pseudocysts: a collection of fluid around the pancreas. The fluid in the cyst is usually pancreatic juice that has leaked out of a damaged pancreatic duct. Characterized by abdominal pain and bloating due to indigestion, it is usually diagnosed by a CT scan. Although not commonly required, treatment includes surgical, endoscopic, or radiological intervention. • Pancreatic abscess occurs in around 3% of acute pancreatitis patients, after more than a month of initial attack. It often develops from infected pancreatic pseudocysts and primarily treated through surgical or endoscopic drainage. Left untreated, it can lead to sepsis, fistula formation, or chronic pancreatitis. • Pancreatic fistula: leakage of pancreatic juice in the abdomen from an injury to the pancreas. As a common occurrence after surgery, fistula forms from the portion of the pancreas left behind after removal of the dead pancreas. Although it heals in the vast majority of patients without any further surgery, some patients require additional surgery to remove the part of the pancreas that is contributing to the fistula. • Damage to surrounding organs such as the small bowel, colon, and duodenum due to the inflammation.
Pancreatitis
Intracellular trypsinogen activation and NF [kappa]B activation are two important and independent molecular events consistently observed during the early stages of pancreatitis and capable of inducing acute pancreatitis in experimental models. Mouse models shows that intra-acinar NF[kappa]B activation is required as well as sufficient to induce inflammatory response of pancreatitis including local inflammation and systemic inflammatory response while acinar death due to pathologic trypsinogen activation leads to about half of all pancreatic cell necrosis during pancreatitis. Chronic Pancreatitis
Chronic pancreatitis (CP) is characterized by a widespread scarring and inflammatory destruction of pancreatic tissue over many years. Although the most common etiological factor in chronic pancreatitis is prolonged alcohol abuse (70%), in many patients, this condition may develop without any apparent cause (20%). It also occurs most commonly after an episode of acute pancreatitis, as the result of ongoing inflammation of the pancreas. Smoking increases the risk of non-gallstone acute pancreatitis twofold in non-alcoholics and fourfold in alcoholics. Smoking is also the most important factor influencing progression of acute to chronic pancreatitis. Other minor causes which constitute 10% of cases include tropical pancreatitis, hereditary pancreatitis, cystic fibrosis, pancreas divisum, and hyperparathyroidism. Risks posed by CP include: • Diabetes mellitus due to damaged insulinsecreting endocrine cells of the pancreas. The overall prevalence of diabetes is 47% in CP patients. The incidence of diabetes increases over time, ranging from 0% to 22% at the onset of symptoms and more than 80% 25 years after the onset of CP. The independent predictors of diabetes are distal pancreatectomy, smoking in patients with prior surgery, pancreatic calcifications, age at the onset of CP, smoking, and chronic pain in patients without surgery.
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• ▶ Pancreatic cancer. • Severe chronic pain. • Malsorption of nutrients from the digestive tract leading to weight loss. • Pseudocyst. • Stomach bleeding. Treatment often involves pain medications and supplementation with pancreatic enzymes or insulin with meals to aid in nutrient absorption. The pancreatic enzyme product (PEP) approved by the US Food and Drug Administration (FDA) contains pancrelipase, a mixture of porcine lipase, amylase, and proteases. Around 50% of patients require surgical intervention which produces superior results to conservative and endoscopic treatment approaches. Management includes a low-fat diet and alcohol abstinence. Autoimmune Pancreatitis
It is an idiopathic chronic inflammatory disease that produces pancreatic masses and ductal strictures. Although considered a type of chronic pancreatitis, autoimmune pancreatitis is distinct from conventional chronic pancreatitis but bears more resemblance to ▶ pancreatic cancer, both clinically and radiographically. Most patients are more than 50 years old at diagnosis and twice as many men as women are affected. The most common clinical features are painless obstructive jaundice, new-onset diabetes mellitus (endocrine dysfunction in 67% of cases), and weight loss from impaired digestion and decreased appetite (pancreatic exocrine insufficiency in 88% of cases). Most patients have no history of alcohol abuse or other traditional risk factors for chronic pancreatitis. Diagnosis of autoimmune pancreatitis involves combination of serological, radiographic, and histopathological analyses. Gamma globulin, total IgG, and IgG4 are usually elevated in autoimmune pancreatitis with serum IgG4 considered the most sensitive and specific marker, elevated in 63–94% of patients. Antilactoferrin and anti-carbonic anhydrase II autoantibodies are also elevated in autoimmune pancreatitis. The
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most common radiographic feature is diffuse enlargement of the entire pancreas. The “sausagelike” appearance of the gland is a feature best seen with CT and magnetic resonance imaging (MRI). Histological evaluation is the gold standard for diagnosis with the following characteristic features: • Parenchymal and often periductal lymphoplasmacytic infiltration (CD4+ and CD8+ lymphocytes, IgG4-positive plasmacytes), which is typically florid in intensity. • Storiform fibrosis: characterized by ductal luminal destruction, acinar atrophy, and arrangement of myofibroblasts in a storiform pattern resembling an inflammatory pseudotumor. • Obliterative phlebitis: Venulitis is seen mainly in small and medium-size pancreatic and peripancreatic veins. The inflammatory response and fibrosis disrupt the venous endothelium and often result in obliterative phlebitis. Corticosteroids (prednisone) are very effective in treating autoimmune pancreatitis. In fact, a poor response to steroid therapy raises suspicion of pancreatic cancer, necessitating reevaluation of the diagnosis. Genetic Basis of Pancreatitis Pancreatitis results from a complex interaction of multiple genetic and environmental factors. Genetic studies for hereditary, idiopathic, and tropical forms of pancreatitis have established at least the following five pancreatitis susceptibility genes: PRSS1
Expressed in acinar cells, it encodes cationic trypsinogen, which is the most abundant isoform of trypsinogen in human pancreatic juice. Most of the bona fide disease-causing missense mutations in the PRSS1 gene led to enhanced trypsinogen autoactivation and/or increased trypsin stability resulting in autosomal dominant or hereditary pancreatitis.
Pancreatitis
SPNIK1
Expressed in acinar cells, the serum protease inhibitor, Kazal type 1 gene (SPINK1), encodes pancreatic secretory trypsin inhibitor (PSTI), one of the key defensive mechanisms against prematurely activated trypsin within the pancreatic acinar cells during ongoing inflammation. Multiple loss-of-function variations in the SPINK1 gene have been found in patients with idiopathic chronic pancreatitis. CFTR
It encodes cystic fibrosis transmembrane conductance regulator and is expressed in the ductal and centroacinar cells. As an anion channel, it allows movement of chloride or bicarbonate across the duct cell luminal membrane into the duct, increasing the pH, as well as initiating and driving pancreatic juice flow. By diluting and alkalinizing the protein-rich acinar secretions, CFTR prevents the formation of protein plugs that predispose to pancreatic injury. Various loss-of-function mutations have been reported to be associated with idiopathic and alcoholic chronic pancreatitis. CTRC
Expressed in acinar cells, it encodes the chymotrypsinogen C gene. Multiple mutations associated with chronic pancreatitis are believed to cause impaired trypsinogen, trypsin degradation, and induction of endoplasmic reticulum stress. CASR
Expressed in both acinar and duct cells of the pancreas, it encodes a plasma membrane-bound G-protein-coupled calcium-sensing receptor. As a monitor and regulator of pancreatic juice calcium concentration, it triggers ductal electrolyte and fluid secretion when Ca2+ levels are elevated, which otherwise could increase the risk of trypsinogen activation and trypsin stabilization causing acute pancreatitis. Multiple mutations and SNPs have been associated with hereditary pancreatitis. Experimentally Induced Pancreatitis To better understand the pathophysiology of pancreatitis for the development of targeted treatment
Pancreatitis
options, various experimental animal models have been developed which resemble the human pancreatitis to various degrees. It includes the noninvasive models of acute pancreatitis such as hormone-induced, alcohol-induced, immunemediated, diet-induced, gene knockout, and L-arginine and invasive models including closed duodenal loop, antegrade pancreatic duct perfusion, biliopancreatic duct injection, combination of secretory hyperstimulation with minimal intraductal bile acid exposure, vascular-induced, ischemia/reperfusion, and duct ligation. Caerulein, a cholecystokinin-pancreozymin analogue, has been widely used for the induction of acute pancreatitis in laboratory animals. Caerulein administration causes intracellular proteolytic enzyme activation resulting in pancreatic acinar autolysis. Increased concentrations of pancreatic enzymes in blood plasma, acinar cell necrosis, and pancreatic inflammation are the common observations. A relatively simple and inexpensive model, it has been frequently used to study the cell biology and pathophysiology of acute pancreatitis and regeneration of damaged tissue, as its effects are reversed after being discontinued. In a diet-induced model of acute pancreatitis, young female mice are fed with a cholinedeficient diet containing ethionine (CDE diet) which induces severe acute necrotizing pancreatitis. It is the simplest system to establish for the investigation of acute hemorrhagic pancreatitis. L-arginine-induced necrotizing acute pancreatitis: Intraperitoneal (i.p.) administration of excessive doses of L-arginine (500 mg/100 g body weight) in rats selectively damages pancreatic acinar cells without any morphological change in islets of Langerhans or other organs. Although the exact mechanism of L-arginine pancreatitis is not clear, it is proposed that its metabolites, oxidative stress, and metabolic acidosis contribute to the pancreatic injury in this model. Pancreatic Cancer and Pancreatitis Meta-analysis of various epidemiological studies revealed the association of pancreatic cancer with several risk factors among which chronic pancreatitis is the most considerate risk factor. It has been
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reported that among patients with hereditary pancreatitis, 40% develop pancreatic cancer in their lifetime. The chronic inflammation incurred during pancreatitis results in pancreatic cancer development over a period of time increasing the risk by 10- to 20-folds. Chronic pancreatitis is accompanied by a series of events including fibrosis, tumor-stromal interaction, loss of immune surveillance, and DNA repair response, leading to the development of pancreatic tumor. It is known that numerous inflammatory mediators induced due to smoking, obesity, diabetes, alcohol abuse, and chronic pancreatitis are capable of causing genomic damage, altered gene expression, and induction of oncogenic signaling pathways leading to the development of precancerous precursor lesions called pancreatic intraepithelial neoplasias (PanINs) and further growth and progression of pancreatic cancer. In adult mice models, brief bouts of experimentally induced pancreatitis lead to PanINs and pancreatic cancer when acinar cells express mutant KRAS oncogene.
Cross-References ▶ Inflammation ▶ Pancreatic cancer
References DiMagno MJ, DiMagno EP (2012) Chronic pancreatitis. Curr Opin Gastroenterol 28:523–531 LaRusch J, Whitcomb DC (2011) Genetics of pancreatitis. Curr Opin Gastroenterol 27:467–474 Momi N, Kaur S, Krishn SR, Batra SK (2012) Discovering the route from inflammation to pancreatic cancer. Minerva Gastroenterol Dietol 58:283–297 Sah RP, Garg P, Saluja AK (2012) Pathogenic mechanisms of acute pancreatitis. Curr Opin Gastroenterol 28:507–515 Su KH, Cuthbertson C, Christophi C (2006) Review of experimental animal models of acute pancreatitis. HPB (Oxford) 8:264–286
See Also (2012) Diet. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1109. doi:10.1007/978-3-642-16483-5_1610 Gooch JW (2011) Discoloration. In: Gooch JW (ed) Encyclopedic Dictionary of Polymers, 2nd edn. Springer
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Pannexins
New York, p 234. doi:10.1007/978-1-4419-62478_3821 Gooch JW (2011) Yellowing. In: Gooch JW (ed) Encyclopedic Dictionary of Polymers, 2nd edn. Springer New York, p 821. doi:10.1007/978-1-4419-6247-8_12958
Pannexins Charles P. K. Lai, John F. Bechberger and Christian C. Naus Department of Cellular and Physiological Sciences, The University of British Columbia, Vancouver, BC, Canada
Definition Pannexins are a second family of ▶ gap junction genes, distinct from ▶ connexins. However, in function they are similar to connexins, forming hemichannels, proteinaceous cylinders spanning the plasma membrane and encompassing a hydrophilic channel.
Characteristics Overview. Only one mammalian family of gap junction genes had been identified, namely, the connexins. However, a novel family of mammalian gap junction genes with low sequence similarity to the invertebrate gap junctions, innexins (Inxs), has been discovered in chordates and termed pannexins (Panxs). Currently, three pannexin members (Papanicolaou test, Panx2, and Panx3) have been identified in vertebrates. Previous studies on Inx mutants in Drosophila have demonstrated Inx-specific functions including synaptogenesis in the giant fiber system, epithelial organization and morphogenesis, and germ cell differentiation processes. The role of pannexins in mammalian cells and tissues has yet to be fully explored. Similarities Between Connexins and Pannexins. Despite sequence dissimilarity between connexins and pannexins, the two
Connexin
Pannexin
Pannexins, Fig. 1 Comparison of the topology of connexin versus pannexin proteins. They are both tetraspan transmembrane proteins with two extracellular loops, one intracellular loop, and intracellular amino and carboxy-termini. While connexins possess three cysteine residues (yellow regions) in each extracellular loop, pannexins only have two
protein families share structural resemblance. Similar to connexins, pannexins have a predicted topology of four membrane-spanning domains, two extracellular loops, a cytoplasmic loop, and cytoplasmic amino- and carboxy-termini. Intriguingly, whereas connexins contain three regularly spaced cysteine residues in the two extracellular loops, pannexins, like innexins, only have two such residues. Numerous studies have suggested the importance of the cysteine residues in facilitating functional connexin-based gap junctions and hemichannels, and therefore the variation in the number of cysteine residues may underlie functional differences between pannexin and connexin functions. Analogous to connexins, pannexin hemichannel and intercellular channel formation are pannexin specific. Although the two channels have similar characteristics, their differences imply that they could exhibit unique functions via different mechanisms (Fig. 1). Pannexins as ▶ tumor suppressors. Gap junctions, and their constituent connexin proteins, have long been considered to play a role in the control of cell proliferation, with disruptions in expression and gap junctional coupling correlating with cell transformation. However, pannexins
Papanicolaou Test
have just been identified and their implication in cancer is only now being examined. Indeed, like connexins, pannexins are now emerging as being ▶ tumor suppressors. Panx1 is normally expressed in brain astrocytes and has been shown to be reduced in brain tumors, specifically gliomas. Panx2 has also been suggested as a tumor suppressor gene in glial cells. Thus transfection of either Panx1 or Panx2 suppresses the tumorigenic phenotype of glioma cells. Highthroughput microarray analysis of human brain tumor samples has also shown an overall reduction of Panx2 gene expression in gliomas. Furthermore, a correlation between Panx2 upregulation and postdiagnosis survival in patients with glial tumors was found using a brain cancer gene expression database (REMBRANDT). In addition, the Panx2 gene is located within chromosomal region 22q13.3 where deletion was often found in human astrocytomas and ependymomas. Summary. Targeting some of the cellular mechanisms controlling proliferation, migration, and invasion would constitute effective therapeutic strategies. One such mechanism involves intercellular communication through gap junctions. Gap junctions and their constituent proteins, connexins and pannexins, have been shown to play a role in controlling proliferation in many cell types. This has been exploited for possible therapeutic potential by directly targeting cell proliferation or by utilizing properties of connexins/pannexins and/or gap junction channels to augment ▶ suicide gene therapy. While the clinical application of therapies targeting Cx/Panx pathways has not yet been realized, valuable approaches to such therapy remain fertile territory.
References Lai CP, Bechberger JF, Thompson RJ et al (2007) Tumorsuppressive effects of pannexin 1 in c6 glioma cells. Cancer Res 67:1545–1554 Litvin O, Tiunova A, Connell-Alberts Y et al (2006) What is hidden in the pannexin treasure trove: the sneak peek and the guesswork. J Cell Mol Med 10:613–634
3433 Naus CC, Goldberg GS, Sin WC (2005) Connexin in growth control and cancer. In: Winterhager E (ed) Gap junctions in development and disease. Springer, Berlin/Heidelberg/New York, pp 253–273 Oskam NT, Bijleveld EH, Hulsebos TJ (2000) A region of common deletion in 22q13.3 in human glioma associated with astrocytoma progression. Int J Cancer 85:336–339 Panchin YV (2005) Evolution of gap junction proteins – the pannexin alternative. J Exp Biol 208:1415–1419
Panzem ▶ Methoxyestradiol
Papanicolaou Test Definition A polychrome staining method originally developed by George N. Papanicolaou. Routinely used in all cytology labs for staining cells so that they can be seen microscopically. Is excellent for evaluating nuclear and cytoplasmic structure. Is the microscopic examination of exfoliated cells obtained by swabs from the uterine cervix (cytology). Abnormal cells showing morphological signs of papillomavirus infection and/or malignant changes can be identified, in which case usually a direct inspection of the cervix (colposcopy) is performed, eventually followed by biopsy and histological diagnosis. Cytology is currently used as the method in cervical cancer screening programs. Because of the relatively high false-negative rate of cytology, it is being evaluated if detection of papillomavirus infection may become a supplementary method in cancer screening.
Cross-References ▶ Fine Needle Aspiration Biopsy
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Papillary Thyroid Carcinoma
Papillary Thyroid Carcinoma
PAR1
Definition
▶ Protease-Activated Receptors
Papillary thyroid carcinoma (PTC) derives from the follicular thyroid cells. It is the most common type of thyroid cancer. PTC usually occurs sporadically. However, familial occurrence has been noted. Patients with familial adenomatosis polyposis (FAP), which is caused by mutations in the APC gene, seem to have an increased risk of developing PTC. Patients with ▶ Cowden syndrome, an inherited hamartoma syndrome caused by mutations in the tumor-suppressor gene PTEN, also have an increased risk of developing thyroid cancer, specifically follicular thyroid carcinoma, but PTC has also been reported. Somatic rearrangements in RET have most often been found in about 10–40% of PTC. In transgenic mice, these rearrangements are capable of causing PTC. A somatic point mutation in BRAF (V600E; previously designated as V599F) has been identified as the most common (35–70%) genetic change is PTCs.
PAR2 ▶ Proteinase-Activated Receptor-2
PAR3 ▶ Proteinase-Activated Receptor-3
PARAHOX Isabelle Gross INSERM U1113, Université de Strasbourg, Strasbourg, France
Cross-References ▶ APC Gene in Familial Adenomatous Polyposis ▶ Cowden Syndrome ▶ Follicular Thyroid Tumors ▶ RET ▶ Thyroid Carcinogenesis
See Also (2012) APC. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 234. doi:10.1007/978-3-642-16483-5_347
Definition The ParaHox gene cluster is an array of homeobox genes that, similarly to Hox genes, encode transcription factors involved in specifying regional identity during embryonic development. The ParaHox gene cluster includes the CDX genes (CDX1, CDX2, CDX4) and GSX genes (GSX1, GSX2). The ParaHox gene cluster is considered as evolutionary related to the Hox gene cluster: the two gene clusters are thought to arise by duplication of a primitive ProtoHox gene cluster.
PAR ▶ Proteinase-Activated Receptor-1 ▶ Proteinase-Activated Receptor-4
Parallel Gene Expression Analysis ▶ Microarray (cDNA) Technology
Parathyroid Hormone-Related Protein
Parathyroid Hormone-Related Protein Jürgen Dittmer Klinik für Gynäkologie, Universität Halle-Wittenberg, Halle (Saale), Germany
Synonyms PLP; PTHLH; PTHrP
Definition PTHrP was discovered as a humoral factor that induces hypercalcemia of malignancy. PTHrP shares with parathyroid hormone (PTH), a protein that regulates calcium homeostasis, the ability to interact with the parathyroid hormone receptor 1 (PTH1R), a ▶ G-protein-coupled receptor. Activation of PTH1R in the bone, kidney, and intestine leads to increased calcium blood levels.
Characteristics Protein Structure Three isoforms of the PTHrP protein exist, containing 139, 141, or 173 amino acid, each of which is coupled to a prepropeptide of 36 amino acid on its N-terminus. The mature PTHrP consists of a least three functional domains, the N-terminus, the mid-region domain, and the C-terminal domain. In the 173 amino acid isoform, the last 34 amino acids may form a fourth functional domain. Following posttranscriptional cleavage of PTHrP, these domains are able to act separately. The N-terminus shows similarities to the N-terminus of PTH and is a secretory peptide that interacts with PTH1R. This interaction gives rise to an increase in cellular cAMP levels, but can also lead to activation of calcium (▶ calciumbinding proteins) or protein kinase Cdependent pathways. The mid-region domain translocates into the nucleus and is able to
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accumulate in the nucleolus. Nuclear translocation is blocked upon phosphorylation of this domain by CDK1/2 (▶ cyclin-dependent kinases). The import of the mid-region domain into the nucleus is therefore thought to be regulated in a cell cycle-dependent manner. The C-terminal domain (osteostatin) antagonizes the bone-absorbing action of the N-terminal domain of PTHrP, probably by interfering with PTH1R internalization. The C-terminal domain contains four serine phosphorylation sites that are required for its mitogenic activity on vascular smooth muscle cells. The last 34 amino acid of the 173 amino acid isoform has been shown to interfere with nuclear localization of PTHrP and to raise the cellular cAMP level. PTHrP mRNA undergoes extensive splicing. N-terminal splicing of PTHrP mRNA varies depending on the promoter that initiated transcription. C-terminal splicing of the PTHrP mRNA that give rise to the different protein isoforms seems to be independent of promoter usage. All PTHrP transcripts contain the coding exons for the three functional domains (Fig. 1). Regulation of PTHrP Expression PTHrP expression is mainly regulated on the transcriptional level. In humans, three different PTHrP promoters (P1, P2, and P3) have been identified. The best characterized promoter is the proximal P3 promoter. It contains functional binding sites for ▶ Ets transcription factors, Sp1, and for the TGFb (▶ transforming growth factor-b)regulated Smads. The Ets binding site is required for the activation of PTHrP by the ▶ Ras/Raf/ MEK-1/ERK1/2 pathway. This signaling cascade is used by tyrosine receptor kinases, such as EGFR (▶ epidermal growth factor receptor ligands), and ▶ G-protein-coupled receptors, such as the calcium-sensing receptor, to activate PTHrP transcription. TGFb upregulates PTHrP gene transcription by recruiting Smad proteins to the Smad site of the PTHrP P3 promoter where the Smad proteins cooperate with Ets- and Sp-like proteins. A number of cytokines, including IL-1b, IL-2, IL-4, and IL-6, also activate PTHrP expression on the transcriptional level. In
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Parathyroid Hormone-Related Protein Bipartite NLS Importin β
PTH1R –36 Prepropeptide
1
34 PTH-like
66
β-arrestin 94
Mid-region T 85
122
139141
173
Osteostatin S ST S 119 130/2 138
Parathyroid Hormone-Related Protein, Fig. 1 The human parathyroid hormone-related protein. The three initial translational products (36/139, 36/141, and 36/ 173) of human PTHrP contain a prepropeptide of 36 amino acid at their N-termini. The mature protein consists of three functional domains, the PTH-like N-terminal domain, the mid-region domain, and the C-terminal domain (osteostatin). The PTH-like domain interacts with the PTH1 receptor. The mid-region domain harbors two
sequences that mediate its translocation into the nucleus. The first sequence interacts with importin-b; the other is a bipartite nuclear localization sequence (NLS). T and S denote threonine- and serine-based phosphorylation sites, respectively. Thr85 can be phosphorylated by CDK1/2. The b-arrestin interaction domain of osteostatin is indicated. Arrestins are involved in internalization and inactivation of ligand-stimulated G-protein-coupled receptors, such as PTH1R
addition, the interaction of cells with particular ▶ extracellular matrix proteins can trigger PTHrP expression. Inhibition of PTHrP transcription is achieved by treatment with nuclear receptor ligands, such as vitamin D, retinoic acid, and glucocorticoids. Vitamin D represses PTHrP transcription through a vitamin D response element in the P1 promoter.
role in skin development. Proliferative or antiproliferative effects on regeneration processes, such as wound healing, have been attributed to PTHrP. An important role of PTHrP in controlling the vascular tone is emerging. As a vasorelaxant PTHrP regulates the contractility of many different vascular beds, including kidney microvessels and the arteries and veins of the mammary gland. During pregnancy, PTHrP is essential for the fetal–placental calcium transport, an activity that seems to be dependent upon the mid-regional part of PTHrP. Furthermore, a role of PTHrP in the survival of neurons is discussed (Fig. 2).
Normal Functions The normal function of PTHrP is to regulate many biological processes critical in embryonal development and adult physiology. PTHrP usually acts locally as a paracrine or autocrine factor. As a paracrine factor, PTHrP plays a crucial role in bone development where it is expressed by the periarticular perichondrium to activate proliferation of PTH1R-expressing chondrocytes, thereby preventing premature ossification. PTHrP knockout mice die shortly after birth because of fatal skeletal dysplasia. Of similar importance is PTHrP for mammary gland development. PTHrP is required for ductal morphogenesis and sexual dimorphism as it triggers the formation of mammary-specific mesenchyme. A number of other processes have been linked to PTHrP function, such as lactation and tooth development. In tooth development, PTHrP stimulates bone resorption of the alveolar bone, a process needed for tooth eruption. By interfering with keratinocyte differentiation, PTHrP likely plays a
PTHrP and Cancer PTHrP is expressed by many tumors, including carcinomas of the breast, kidney, prostate, colon, stomach, and lung. In hematopoietic malignancy, it is predominantly found in HTLV-I (▶ human T-lymphotropic virus)-induced adult T-cell leukemia, probably as a result of the ability of the HTLV-1 protein Tax to strongly induce PTHrP transcription. Cancers can secrete PTHrP at levels that are sufficient to raise PTHrP blood levels and to induce hypercalcemia of malignancy, a disease that complicates cancer. Besides this endocrine cancer-related function, PTHrP displays autocrine and paracrine activities that seem to influence tumor progression and metastasis. One of the best characterized examples for the importance
Parathyroid Hormone-Related Protein
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PTHrP
Tumor cell TGbR
Ca
TGF b
R
Ca 2
+
Ost
Osteoclast precursor
at
tur
Ma
ion
RANK
eoc
RANKL
Osteoblast
last
Bone matrix
Parathyroid Hormone-Related Protein, Fig. 2 Involvement of PTHrP in skeletal metastasis. PTHrP produced by breast cancer cells that metastasized to bone may fuel a vicious cycle by stimulating osteoblasts to interact with osteoclast precursors via RANK-RANKL (▶ RANK-RANKL signaling). As a result, the osteoclast precursors will mature to osteolytic osteoclasts. Following
its destruction, the bone matrix releases TGFb and calcium which then activate their corresponding receptors on the tumor cells (TGbR and CaR). The activated receptors further stimulate PTHrP production and more osteoclasts are recruited for bone degradation. This positive feedback loop is thought to facilitate metastasis growth by providing space for tumor expansion
of PTHrP in cancer is its involvement in breast cancer growth in the bone. By secreting PTHrP, breast cancer cells that metastasized to bone induce osteoclastic bone absorption leading to bone destruction and release of TGFb which then acts back on the cancer cells and further boosts PTHrP expression in these cells. This positive feedback fuels a vicious cycle that accelerates osteolysis and facilitates expansion of the metastatic cells in the bone environment. It is likely that calcium is supporting this process. Calcium is also released upon bone degradation and stimulates PTHrP expression in the tumor cells by activating the calcium-sensing receptor CaR. PTHrP secreted into the bone may also interfere with hematopoiesis by stimulating the activity of hematopoietic stem cells. Other studies show that PTHrP protects cancer cells from apoptosis, stimulate their proliferation, and trigger their ability to migrate and invade extracellular matrix. A number of genes that are targeted by PTHrP in cancer cells have been identified. Among them are
genes encoding integrin a6 (▶ integrin signaling), PAI-1 (▶ plasminogen activating system), CDK1 (▶ cyclin-dependent kinases), and ▶ KiSS-1. Activation of the PI3K/AKT pathway (▶ AKT signal transduction pathway in oncogenesis) by PTHrP has also been shown. The PTHrP domain that mediates these effects varies with the targeted gene. For example, integrin a6 is activated via the mid-region domain, whereas the N-terminal domain seems to be important for the repression of CDK1. Supporting the notion that PTHrP is a tumor-promoting factor, many survival analyses of cancer patients revealed that PTHrP expression in the tumor increases the risk of a relapse and is linked with unfavorable prognosis. In breast cancer, coexpression of PTH1R along with PTHrP further reduced disease-free survival. It should be noted, however, that the value of PTHrP as a prognostic factor of breast cancer is still a matter of debate, as also data have been published that show that PTHrP expression in breast cancer correlates with a more favorable prognosis.
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PARP Inhibitors
References
Particle-Induced Cancer Dittmer J (2004) The importance of PTHrP for cancer development. Gene Ther Mol Biol 8:451–464 Dittmer J (2007) Mechanisms involved in the regulation of PTHrP expression. In: Luparello C (ed) Novel aspects of PTHrP physiopathology. Nova Science Publisher, Hauppauge Liao JL, McCauley LK (2006) Skeletal metastasis: established and emerging roles of parathyroid hormone related protein (PTHrP). Cancer Metastasis Rev 25:559–571 Strewler GJ (2000) The physiology of parathyroid hormone-related protein. N Engl J Med 342:177–185
Anne Thoustrup Saber1, Ulla Vogel1, Steffen Loft2 and Håkan Wallin1 1 National Institute of Occupational Health, Copenhagen, Denmark 2 Department of Environmental Health, University of Copenhagen, Copenhagen, Denmark
Definition Particle-induced cancer refers to cancer arising after exposure to particles.
PARP Inhibitors Definition
Characteristics
Poly(ADP-ribose) polymerase inhibitors are a large number of low molecular weight compounds (NAD+ analogues) that competitively inhibit PARP activity. Among the first to be described were nicotinamide, benzamide, and 3-aminobenzamide. However, their potency and specificity is rather low. Subsequently, more sophisticated new compounds have been developed such as 4-amino-1,8naphthalimide, 3,4-dihydro-5-methoxyisoquinolin1(2H)-one (PD 128763), 8-hydroxy-2methylquinazolin-4(3H)-one (NU1025), or 2-methylbenzimidazole-4-carboxamide (NU1064), to name but a few. These are much more potent than the first-generation inhibitors and possess an improved pharmacokinetic profile. With respect to specificity, while all inhibitors inhibit PARP-1 (by definition), at least some of the first-generation inhibitors also interfered with other ADP-ribosyl transfer reactions, such as mono-ADP-ribosylation of proteins or NAD+ glycohydrolases, albeit at different IC50 levels. Furthermore, the novel poly(ADP-ribose) polymerases are likely to be inhibited as well, perhaps at similar IC50 levels as PARP-1.
Particles are here defined as solid spherical or fibrous materials suspended in air. It has been documented that particle-induced cancer occurs in animals and humans after inhalation of solidinsoluble particles. The International Agency for Research on Cancer (IARC) has classified a number of particles according to their carcinogenicity. Group 1 agents are carcinogenic to humans. Group 2A agents are probably carcinogenic to humans, while group 2B agents are possibly carcinogenic to humans. In the outdoor environment, ultrafine particles are formed by the combustion processes, e.g., of diesel (group 2A) and biomass (group 2A). Indoor sources of particle pollution include smoke from tobacco (group 1), cooking fires, wood stoves, fireplaces, and candles (smoky coal is classified as group 1 carcinogen). Some occupational exposures to aerosols of solid particles have been associated with ▶ cancer in the respiratory tract. Examples are quartz (group 1), asbestos (group 1), soot (group 1), welding fumes (group 2B), and wood dust (group 1). The effects of particles deposited in the lung may be related to carcinogenic effects that have been noted in animals after surgical implantation of some stable solid materials such as thin films of polymeric or metallic materials or solid metallic bodies of specific compositions.
Cross-References ▶ Poly(ADP-Ribosyl)ation
Particle-Induced Cancer
In the hitherto largest study, in which 500,000 persons from 51 urban areas in the USA were followed for over 16 years, the incidence of lung cancer correlated with long-term exposure to fine particulate matter: For every 10 mg/m3 increase in fine particles (PM2.5), lung cancer mortality increased by approximately 8% each year. The study provides the strongest evidence yet for linking ▶ lung cancer to air pollution in urban areas. Particle Size and Form Particles in urban air tend to have a multimodal distribution, partly reflecting the sources. Particles are traditionally divided in three different size fractions: the ultrafine (90th percentile for men 14 days after surgery to avoid postsurgical contamination by tumor cells. IF MRI of spine is planned at this time, the LP should be performed after the MRI of the spine to avoid misinterpretation of the MRI due to post-LP changes. Non-lumbar CSF that is frequently obtained at time of surgical resection is not considered adequate for staging. • Optic pathway tumors require baseline vision evaluation. They may also require endocrine evaluation. • Suprasellar tumors require baseline endocrine evaluation. Special Considerations
• Patients with suspected CNS germ cell tumors should have serum and CSF tumor markers (beta HCG and alpha fetoprotein) obtained at diagnosis. • For gliomas and craniopharyngiomas, which do not usually spread, MRI of spine and LP are not typically required. • For patients presenting with either bone pain or low blood counts, one also needs to consider the rare possibility of bone or bone marrow metastasis that may occur in disseminated medulloblastomas or PNET. After confirmation of histological diagnosis, such patients should have a bone scan and bilateral bone marrow aspirates and biopsies. • Young patients with AT/RT should be evaluated for synchronous renal and liver second primary rhabdoid tumors. Therapeutic Approaches Therapy usually comprises a combination of surgical resection, with or without addition of chemotherapy and/or irradiation. Clinical trials are beginning to explore use of targeted therapy and biological modifiers, but these are not yet proven effective. Surgical Resection
Surgical resection depends on the location and type of tumor. Maximal safe surgical resection is usually recommended except in germ cell tumors, diffuse intrinsic pontine gliomas, and some others. Complete resection of localized medulloblastomas,
Pediatric Brain Tumors
PNET, gliomas, and ependymomas improves prognosis significantly and should therefore be attempted if safely feasible. Where only biopsy is feasible or needed, it yields important information on the histological type of the tumor, which may be important in determining the therapeutic approach. Radiation Therapy
Irradiation is an important part of therapy of many brain tumors in older children. Its extent and dose depend on tumor type. In infants and young children, however, irradiation to the whole brain or large areas in the brain causes severe neurocognitive long-term sequelae. Therefore, current therapeutic approaches attempt to minimize, delay, or avoid radiation therapy for most brain tumor types in infants and young children. Chemotherapy
Multi-agent chemotherapy is an effective part of treatment for most types of malignant childhood brain tumors. Tumors that usually are not treated with chemotherapy include completely resected craniopharyngiomas, WHO grade I astrocytomas, and completely resected ependymomas. Approach to Therapy in Specific Tumor Types Malignant Brain Tumors in Infants and Young Children
In order to avoid or delay irradiation in treatment of infants and young children, current approaches to treatment of malignant brain tumors utilize more intensive chemotherapy regimens. These frequently include high-dose chemotherapy followed by myeloablative chemotherapy with autologous hematopoietic stem cell rescue. Gliomas
Low-grade gliomas may be cured by surgery only if they are completely resected. Low-grade gliomas that are incompletely resected and are progressive, recurrent, or symptomatic but not amenable to surgery will require additional intervention. Presently, in North America, chemotherapy is the first line of treatment for children less than 10 years of age and may result
Pediatric Brain Tumors
in event-free survival in the range of 30–40% and overall survival in the range of 70–80%. Approaches for older children differ depending on institution and usually include irradiation with or without chemotherapy. Targeted therapy for select situations is currently in clinical trials. High-grade gliomas, even if completely resected, require additional therapy. This comprises radiation and chemotherapy. However, for infants and young children, many institutions prefer approaches with high-dose chemotherapy so that irradiation can be avoided, or at least minimized or delayed. For diffuse intrinsic brain stem gliomas, irradiation prolongs survival and is the current standard treatment. However, prognosis remains dismal. Clinical trials are ongoing to investigate additional strategies. Ependymomas
Treatment of ependymomas is dictated by the histological subtype, extent of disease (localized vs. disseminated), and location in the brain (posterior fossa vs. supratentorial region). Surgery is the mainstay of therapy for localized ependymoma. Gross totally resected localized ependymomas located within the posterior fossa, regardless of histology, are treated by conformal radiotherapy to the tumor bed following surgery and achieve cure rates in excess of 70%. However, treatment of localized completely resected supratentorial ependymoma depends upon histology. Completely resected cellular ependymomas are observed following complete resection, whereas gross totally resected anaplastic ependymomas in the supratentorial compartment are treated by adjuvant conformal irradiation. Patients with less than complete resection, regardless of histology, receive neoadjuvant chemotherapy followed by second-look surgery for residual disease followed by conformal focal irradiation. Needless to say, patients with incompletely resected and disseminated ependymomas carry a dismal prognosis. Medulloblastomas
Medulloblastoma is the most common malignant brain tumor in children and accounts for
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approximately 20% of all pediatric CNS tumors and 40% of all posterior fossa tumors. The peak incidence of medulloblastoma is between 5 and 7 years of age with 10% of cases diagnosed within the first year of life and the incidence decreasing with age. Approximately 30% of all medulloblastomas occur in children older than 10 years of age. Medulloblastomas account for less than 1% of all brain tumors in adults. The treatment of children with medulloblastoma can be divided into three main categories: • High-risk medulloblastoma (patients with disseminated disease, less than gross total resection, and anaplastic medulloblastoma) in patients older than 3 years of age • Standard-risk medulloblastoma (nondisseminated disease, gross total resection, and non-anaplastic histology) in patients older than 3 years of age • Medulloblastoma in infants and young children Patients older than 3 years of age with highrisk medulloblastoma are usually treated with craniospinal irradiation along with posterior fossa irradiation boost followed by intensive chemotherapy resulting in long-term survival for >70% of patients. The standard of care for patients older than 3 years of age with average-risk medulloblastoma is reduced-dose craniospinal irradiation along with a boost to the posterior fossa followed by chemotherapy, resulting in long-term survival for >80% of patients. The present North American cooperative group, Children’s Oncology Group (COG) trial, is trying to reduce the irradiation in patients between 3 and 8 years of age. Future clinical trials intend to use histology and molecular factors as prognostic features when designing new treatments. Medulloblastoma in infants and young children is typically treated using high-dose chemotherapy and autologous hematopoietic stem cell rescue (AuHCR) with an intention of avoiding radiation therapy, or at least minimizing and/or delaying it. Many cooperative group trials have identified desmoplasia as a good prognostic
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factor, opening the possibility of further reducing the intensity of treatment for this particular group. PNETs
Although PNETs have been shown to have a different gene expression profile than medulloblastomas, they are treated similar to high-risk medulloblastoma patients. Infants and young children with CNS PNET are treated with intensive induction chemotherapy followed by myeloablative chemotherapy and AuHCR. Patients older than 3 years of age receive craniospinal irradiation and boost to the tumor bed followed by intensive chemotherapy. Pineal PNETs behave very differently than PNETs in other supratentorial locations. Infants and children T haplotype is of clinical importance. Reports as well as the NCBI dbSNP database show a tri-allelic polymorphism of ABCB1
(2677G > T/A) and one rare mutation (2677G > C). Functionally evaluation showed that nonsynonymous polymorphisms (2677G > T, A, or C) at amino acid position 893 (Ala > Ser, Thr, or Pro) have a great impact on both the activity and the substrate specificity of ABCB1. The polymorphisms of 2677G > T (893 Ala > Ser) are reportedly associated with high risk of lung cancer.
P ABCC1 (MRP1/GS-X Pump) and ABCC2 (MRP2/ cMOAT) Human ABCC1 (MRP1) gene is located on chromosome 16p13.1 and spans at least 200 kb consisting of 31 exons. The ABCC1 gene encodes a 1531 amino acid protein which has a molecular weight of 190 kDa in its mature glycosylated form. In the promoter region of the ABCC1 gene, there are a number of putative transcription factor motifs, such as the activator proteins AP1 and AP2 (Sp1), glucocorticoid response element (GRE), and also estrogen response element (ERE) and cAMP response element (CRE). Human ABCC2 (MRP2 or cMOAT) is a 1545 amino acid protein whose exon gene is located in chromosomal region 10q23–24. Although there is limited sequence similarity between ABCC1 and ABCC2 (49%), the primary structure and
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Pharmacogenomics in Multidrug Resistance
Pharmacogenomics in Multidrug Resistance, Fig. 2 Nonsynonymous polymorphisms of human ABCG2. Schematic illustration of the structure of ABCG2 protein and the locations of amino acid changes. R482G and R482T are acquired mutations
N590Y R575stop F571I F431L S441N
F208S I206L
D620N
OUT
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G51C
F489L
V12M H2N
IN COOH
R482G R482T
Q126stop
E334stop S248P
Q141K T153M
Q166E R160Q
membrane topology of the two proteins are similar. In addition, the two transporters also have similar substrate characteristics. Both ABCC1 and ABCC2 transport a wide range of organic anions, including glutathione disulfide (GSSG), glutathione-metal complexes, glutathione conjugates, as well as glucuronate and sulfate conjugates. Elevated expression of ABCC1 mRNA and/or protein levels has been observed in many multidrug-resistant cancer cells. Transfection of ABCC1 cDNA in cultured cells resulted in enhanced resistance to many cytotoxic agents including doxorubicin, vincristine, and VP-16. ATP-dependent transport of these anticancer drugs can be enhanced by the presence of glutathione (GSH) in membrane vesicles prepared from ABCC1-overexpressing cells, suggesting that ABCC1 cotransports anticancer drugs and GSH. In patients, human colorectal cancers frequently overexpress ABCC1 and g-glutamylcysteine synthetase (g-GCS), a ratelimiting enzyme of GSH biosynthesis, as compared to the surrounding normal tissue. The frequency of ABCC1 expression in carcinoma was higher than that in adenoma (p < 0.0001). The ABCC1 upregulation and the p53 status were significantly correlated.
ABCG2 (BCRP/MXR1/ABCP) Human ABCG2 (BCRP/MXR1/ABCP) is another member of the ABC transporter gene family, and its overexpression in cancer cells is related with cellular resistance to anticancer drugs, such as mitoxantrone, topotecan, and SN-38, an active metabolite of ▶ irinotecan (Fig. 2). SNPs of ABCG2 have been documented to be a significant factor in the patients’ responses to medication and/or the risk of diseases. Sequencing of the ABCG2 gene from human samples has revealed over 80 different, naturally occurring sequence variations. Drug resistance profiles of fluorescence diagnosis expressing two major SNP variants, i.e., V12M and Q141K, toward SN-38 demonstrated that the IC50 value for Q141K was about 50% of that for the wild type (WT). The contributions of the minor SNP variants, i.e., F208S, S248P, F431L, S441N, and F489L, to drug resistance toward SN-38, mitoxantrone, doxorubicin, daunorubicin, or etoposide were significantly lower than that of WT. The Q141K polymorphism located in exon 5 (c.421C > A) leads to the replacement of the negatively charged glutamic acid residue with a positively charged lysine residue. This polymorphism affects the ATP-binding domain, between the Walker A motif (amino acid residues 83–89) and the signature region (amino acid residues
Pharmacokinetics and Pharmacodynamics in Drug Development
186–189). The Q141K variant was also detected in all ethnic groups tested: the allele frequency ranged between 0% and 35% (the Africans in North of Sahara, the Africans sub-Saharan, and AfricanAmerican subjects with low allele frequencies; the Japanese and Chinese populations with high allele frequencies). The SNP (Q141K) was postulated to cause increased sensitivity of normal cells to anticancer agents that are ABCG2 substrates such as topotecan, diflomotecan, and SN-38. Perspectives Drug transporters as well as drug metabolism play pivotal roles in determining the pharmacokinetic profiles of drugs and, by extension, their overall pharmacological effects. There are an increasing number of reports addressing genetic polymorphisms of drug transporters. Information is still limited, however, regarding the functional impact of genetic polymorphisms in drug transporter genes. Detailed functional analysis in vitro is critically important to provide clear insight into the biochemical and therapeutic significance of genetic polymorphisms. Functional validation of SNPs and their linkage with clinical data would provide a new approach to individualized pharmacotherapy in the twenty-first century.
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See Also (2012) Fluorescence. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1426–1427. doi:10.1007/978-3-642-16483-5_2215 (2012) Gamma-glutamylcysteine synthetase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1494. doi:10.1007/978-3-64216483-5_2314 (2012) Genetic polymorphism. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1528. doi:10.1007/978-3-64216483-5_2382 (2012) Genotype. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1540. doi:10.1007/978-3-642-16483-5_2396 (2012) Glutathione. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1559. doi:10.1007/978-3-642-16483-5_2438 (2012) Multidrug resistance. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2393. doi:10.1007/978-3-642-16483-5_3887 (2012) Personalized medicine. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/ Heidelberg, p 2828. doi:10.1007/978-3-642-164835_4476 (2012) SN-38. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3456. doi:10.1007/978-3-642-16483-5_5386 (2012) Topotecan. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3730. doi:10.1007/978-3-642-16483-5_6948 (2012) Walker A motif. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3941. doi:10.1007/978-3-642-16483-5_6228
P Cross-References ▶ Blood-Brain Barrier ▶ Chemotherapy ▶ Irinotecan
References Borst P, Elferink RO (2002) Mammalian ABC transporters in health and disease. Annu Rev Biochem 71:537–592 Ishikawa T (2003) Multidrug resistance: genetics of ABC transporters. In: Cooper DN (ed) Nature encyclopedia of the human genome, vol 4. Nature Publishing Group, London, pp 154–160 Kalow W, Meyer UA, Tyndale RF (eds) (2001) Pharmacogenomics. Marcel Dekker, New York Sakurai A, Tamura A, Onishi Y et al (2005) Genetic polymorphisms of ATP-binding cassette transporters ABCB1 and ABCG2: therapeutic implications. Expert Opin Pharmacother 6:2455–2473
Pharmacokinetics and Pharmacodynamics in Drug Development Simon Pacey, Debashis Sarker and Paul Workman Cancer Research UK Center for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, UK
Definition Pharmacokinetics ▶ Pharmacokinetics (PK) is the study of how a drug is absorbed, distributed, metabolized, and excreted over time.
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Pharmacokinetics and Pharmacodynamics in Drug Development
Pharmacodynamics ▶ Pharmacodynamics (PD) is the study of how a drug affects its target(s) in a dose- and timedependent fashion.
Characteristics To maximize the chance of successful drug development, a comprehensive knowledge of the compound under investigation is required. Assuming the drug target has been correctly selected, drug efficacy requires delivery into the patient such that adequate drug concentration is achieved within the plasma and tumor (measured by PK studies) to effect target modulation (measured by PD studies) resulting in anticancer effect(s). These principles have been incorporated into a pharmacological audit trail consisting of a series of questions that should be addressed during drug design and development. These can be summarized as: • Is the drug target expressed in the tumor of interest? • Are adequate plasma and tumor drug levels achieved? • Does this level of drug exposure result in target modulation? • Can effects be demonstrated on the biochemical pathway downstream of the target? • Are the desired biological effects achieved, e.g., cell cycle arrest, induction of apoptosis, or inhibition of angiogenesis? • What are the therapeutic consequences of drug exposure and target modulation? Related to these questions, it is valuable to determine if biomarkers of sensitivity or resistance can be identified. From these questions, it is obvious that PK and PD studies form an important part of the knowledge base required for modern drug development. The advantage of using PK/PD biomarkers, and of implementing the pharmacological audit trail, is that decisions in drug development can be made on a rational basis, therefore making the process more effective.
Pharmacokinetics It is usual to assume that the therapeutic and toxic effects of a drug are related to its concentration at the site of action. Previously, it was estimated that around 40% of drugs failed clinical development due to poor PK properties. Inappropriate PK can lead to inadequate or variable drug exposure and hence to lack of therapeutic activity or undesirable toxicity. This figure has fallen to around one in ten, due mainly to more thorough preclinical PK modeling. Early in drug discovery, physicochemical properties need to be addressed, in order to maximize the chance of the final molecule possessing appropriate drug-like properties. The Lipinski rule of five provides a useful guide. This is based on the fact that most marketed drugs have a molecular weight of less than 500 Da, partition coefficient cLog P < 5 (a measure of lipophilicity), lomefloxacin, on the one hand and thymine on the other hand. This leads to the formation of thymine-containing CPDs with the predominance of thymine cyclobutane dimer. Other relevant photosensitization reactions that do not involve the participation of O2 are triggered by bifunctional furocoumarins such as 5-methoxypsoralen (5-MOP) and 8methoxypsoralen (8-MOP) used as photogenotoxic agents in the so-called “psoralen plus UVA” (PUVA) therapy of hyperproliferative skin diseases, photopheresis of T cell lymphoma, and pathogen inactivation in plasma and platelet components. UVA-excited psoralens are able to undergo [2 + 2] photocycloaddition through either their pyrone or furan moiety to the 5,6-bond of thymine giving rise to mono-adducts and highly deleterious interstrand cross-links. The second main category of UVA-sensitized degradation reactions of DNA is oxygen dependent giving rise to the so-called “photodynamic effects”. Most of the exogenous photosensitizers are able to generate 1 O2 through the type II mechanism. However, several photoactive agents including riboflavin and methylene blue are able to abstract one-electron mostly from guanine according to the type I photosensitization mechanism. Subsequently, hydration of the resulting guanine radical gives rise to 8-hydroxy-7,8-dihydroguanyl radical that
Photo-Induced DNA Damage
is then converted into 8-oxoGua and 2,6-diamino4-hydroxy-5-formamidopyrimidine (FapyGua) upon one-electron oxidation and one-electron reduction, respectively (Fig. 3). Indirect evidence has been provided for the formation of complex DNA photoproducts when cells are incubated with anticancer and immunosuppressor thiopurines including azathioprine, 6-mercaptopurine, and 6thioguanine prior to UVA exposure. A reasonable mechanism for the observed generation of DNA-protein adducts and interstrand DNA crosslinks involves nucleophilic addition of amino residues of amino acids or nucleobase to the guanine radical cation produced by one-electron abstraction from the guanine base by UVA-excited 6thioguanine that has been metabolized and inserted into DNA as an internal photosensitizer. Detection of DNA Photodamage in Cells and Skin Various methods have been developed for measuring DNA photoproducts in isolated cells and skin. Therefore, several polyclonal and monoclonal antibodies raised against either CPDs or 64PPs are available to detect in a semiquantitative manner the formation of bipyrimidine photoproducts in fibroblast, keratinocytes, and skin using immunoassays and immunohistochemical methods. Other global methods involved the use of DNA repair enzymes that cleave damaged DNA at the sites of CPDs with subsequent detection of the nicks by either the alkaline comet assay or the alkaline elution technique. In addition, bipyrimidine photoproducts can be mapped at the sequence level after suitable chemical or enzymatic conversion of the lesions into strand breaks using the ligation-mediated polymerase chain reaction (LM-PCR) method that, however, suffers from a lack of sensitivity. High-performance liquid chromatography associated with electrospray ionization tandem mass spectrometry (HPLCESI-MS/MS) appears to be a relevant and quantitative method for measuring each of the possible main position isomers of CPDs, 6-4PPs, and DEWs due to its high specificity and accuracy. It may be also mentioned that HPLC-ESI-MS/MS could be used for monitoring the formation of oxidized purine and pyrimidine nucleosides
Photo-Induced DNA Damage
when the extent of modification is high. The association of electrochemical detection with HPLC constitutes a suitable alternative for measuring 8-oxo-7,8-dihydro-20 -deoxyguanosine (8-oxodGuo) that is characterized by a low ionization potential. However, modified comet assay or alkaline elution technique that requires preincubation of released DNA with DNA repair glycosylases remain methods of choice for the sensitive detection classes of oxidation products, mostly due to the lack of adventitious oxidation reactions during damage analysis. Repair of DNA Photodamage It is well documented that the bulky bipyrimidine photoproducts are removed from cellular DNA by the nucleotide excision repair (NER) pathway that implicates several proteins involved in the recognition of the damage, unwinding of DNA, excision of the lesion as oligonucleotides, DNA polymerization, and oligonucleotide ligation as the main successive steps. It was found mostly by using immunoassays that 6-4PPs are more rapidly excised than CPDs. HPLC-ESI-MS/MS measurements of each of the bipyrimidine photoproducts showed that CPDs at CT and CC sites that are more mutagenic than the other cyclobutane pyrimidine dimers are repaired more efficiently in the global NER pathway than TT and TC CPDs, although less rapidly than 6-4PPs and DEWs. Another interesting information deals with the slower removal of UVA-induced TT cyclobutane dimers with respect to those generated by UVB irradiation for similar amounts of DNA photoproducts. This could be due to either epigenetic regulations or modification of the DNA repair pool, possibilities which remain to be further investigated. As a final remark, repair of single-strand breaks and oxidized bases is usually faster than that of bipyrimidine photoproducts. Thus, the half-life of 8-oxoGua that typically is less than 1 h in human cells has to be compared with that of TT CPDs (~24 h) and 6-4PPs (~3–4 h). Skin Carcinogenesis The causative relationship between UV exposure and nonmelanoma skin cancer is well documented,
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and important molecular targets have been identified. These consist of patched protein in the hedgehog pathway for basal cell carcinoma and p53 for squamous cell carcinoma. In contrast, the role of UV exposure in melanoma pathogenesis is more complex, and the site distribution of melanomas is different from that of BCC and SCC. In most cases, this does not correspond to the most sun-exposed areas of the body. It has been proposed that melanoma is more closely associated with intermittent intense sun exposure rather than long-term and constant exposure as it is established for SCC. Induction of damage to DNA is a key initiating event in the induction of skin cancer by solar radiation, and emphasis has been placed on UVB radiation that overlaps with DNA absorption and triggers the formation of bipyrimidine photoproducts (vide supra). Yet, involvement of UVA in skin carcinogenesis is a growing concern. In that respect, the decision of the International Agency for Research on Cancer to classify artificial UV devices as carcinogens further emphasizes the carcinogenic risk associated with UVA. Cutaneous carcinogenesis is a multiple-step process that is initiated by formation of UVB-induced mutations that mostly occur at bipyrimidine sites including C to T or CC to TT transitions, known as UV-signature mutations or UV fingerprints. Functional loss of tumor suppressor genes is a fundamental cause of cancer progression. This is illustrated by the observation of high frequency of the above-specific mutations in the p53 gene in SCC and in BCC patients. Moreover, 90% of sporadic BCCs also exhibit mutations in the Patched 1 (PTCH1) gene whereas for the 10% remaining cases, mutations were observed in cellmembrane associated smoothened homologue (SMO). As a consequence, upregulation of the hedgehog (Hh) signaling pathway is noted while such activation is known to be a key factor in the development of a number of human malignancies. A substantial proportion of mutations observed in PCTH1 and SMO are UV point mutations. In contrast, p53 is rarely mutated in melanomas whereas UV-type base substitution mutations in the tumor suppressor gene PTEN (phosphatase and tensin homologue) are very frequent in melanomas of both xeroderma pigmentosum patients and general
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population. It was shown that melanoma induction by UVA requires the presence of melanin pigment being associated with oxidatively generated DNA damage within melanocytes. On the other hand, UVB exposure gives rise to melanoma in a pigment-independent manner that is associated with direct-induced DNA damage. The next steps of carcinogenesis consist in tumoral promotion and progression that involve inflammation and immunosuppression. UVA radiation may be involved in this process through the occurrence of oxidation reactions triggered by photosensitized processes leading to the production of 1O2 and to a lesser extent of ●OH.
Photothermal Ablation
Photothermal Therapy Klaramari Gellci and Mohammad Mehrmohammadi Department of Biomedical Engineering, Wayne State University, Detroit, MI, USA
Synonyms Laser-induced hyperthermia; Photothermal ablation; Photothermal treatment
Definition Cross-References ▶ Photocarcinogenesis ▶ Skin Cancer ▶ Skin Carcinogenesis ▶ Squamous Cell Carcinoma ▶ Uveal Melanoma ▶ Xeroderma Pigmentosum
References Cadet J, Wagner JR (2013) DNA base damage by reactive oxygen species, oxidizing agents and UV radiation, Cold Spring Harb Perspect Biol 5:a012559 Cadet J, Douki T, Ravanat J-L (2015) Oxidatively generated damage to cellular DNA by UVB and UVA radiation, Photochem Photobiol 91:140–155 Cadet J, Grand A, Douki T (2015) Solar UV radiationinduced DNA bipyrimidine photoproducts: formation and mechanistic insights. Top Curr Chem 571:3–17 Mouret S, Charveron M, Favier A, Cadet J, Douki T (2008) Differential repair of UVB-induced cyclobutane pyrimidine dimers in cultured human skin cells and whole human skin. DNA Repair (Amst) 7:704–712 Premi S, Wallisch S, Mano CM, Weiner AB, Bacchiocchi A, Wakamatsu K, Bechara EJ, Halaban R, Douki T, Brash DE (2015) Chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure. Science 347:842–847
Photothermal Ablation ▶ Photothermal Therapy
Photothermal therapy (PTT) is a method of heat ablation in which light energy is utilized to induce localized heat within the tissue and destroy the pathologic cells. Often in practice and in order to not harm surrounding healthy tissues, the targeted diseased cells are labeled with light-absorbing nano-sized particles, which significantly increases the light energy absorption and also allows for utilizing laser lights in near-infrared (NIR) wavelengths to achieve larger penetration depths. With the aid of these nano-sized agents (i.e., nanoparticles), PTT is capable of destroying pathologic cells selectively, at larger penetration depth and with no damage to surrounding non-labeled healthy tissue.
Characteristics ▶ Hyperthermia is a noninvasive approach for ▶ cancer therapy, whereby biological tissues are exposed to higher than normal temperatures to promote destruction of abnormal cells. PTT is a specific form of hyperthermia that uses heat generated from light (laser) to cause damage to abnormal (diseased) cells. PTT would be particularly useful for individuals with cancer because of its ability to be selective through the assistance of biocompatible nano-sized particles. Unlike traditional techniques of cancer therapy, such as chemotherapy and radiation, PTT has the ability to not
Photothermal Therapy
harm surrounding, healthy cells. Selective thermal destruction of cancer offers a unique mechanism of treatment to the patients who have difficulties with these conventional therapy methods. PTT can also be combined with other cancer treatments such as chemotherapy to enhance the performance of the treatment. PTT can operate in three basic modes: (a) light only, (b) light with endogenous molecules for photothermal conversion, and (c) light with exogenous metal nanostructures for enhanced and selective photothermal therapy. These nanoparticles are engineered to generate heat after the absorption of a laser light, usually in the near-infrared range (650–1,064 nm), and localize hyperthermia in malignant cells. Because of the high photothermal conversion efficiency of metal, the third approach is the most effective in terms of heat generation. However, it must be noted that utilizing nanoparticles as enhancing PTT agents involves issues such as delivery to target cancer sites and potential toxicity and side effects of metallic nanostructures. Using strategies such as antibody-antigen or ligand-receptor interaction, one can actively target and deliver the metal nanostructures to specific cancer tissues. Metals such as gold and silver are considered as biocompatible and low in toxicity and do have good potential for PTT applications. Principles of Photothermal Therapy Heat Generation in a Laser-Tissue Interaction
The major heat generation mechanism in PTT is through interaction of the laser light with the tissue. The interaction between incident light and the tissue can be studied in a microscopic or a macroscopic level. This process partially converts the laser energy (high-frequency electromagnetic waves) into thermal energy. Microscopic Model: When a photon with energy of hv (v = frequency) hits a molecule (M) within the tissue, the light energy could be absorbed, and the molecule M is excited from
the normal state to an excited state M ðhvþ
M ) M Þ . The molecule M* suffers an inelastic collision (equal energy loss) with the neighbor
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M1 (an electron, an atom, or a molecule), transferring part of its energy and decay to an energy lower (stable) state: M þ M1 ðecin Þ ) M þ M1 ðecin þ DeÞ. The amount ðecin þ DeÞ represents the increase of the thermal vibration of the particle M1, and, as a consequence, there is a microscopic temperature increment by the energy absorbed from the photon. The macroscopic effect of the temperature increase is observed due to the high number of M-type molecules, which absorbed the energy from a high number of photons presented in a laser beam and transformed this energy in thermal vibration and heat. Macroscopic Model: In a macroscopic level, the assumption is that the heat generated is directly related with the laser propagation in the tissue. Therefore, the light propagation in the tissue plays an important role in macroscopic model of laser-tissue interaction and heat generation. When a laser beam irradiates a sample, a part of the beam is reflected and the other part penetrates through the tissue. The penetrated light is subject to absorption and scattering events. The absorption and the scattering are characterized for absorption coefficient (ma) and scattering coefficient (ms), which represents, respectively, the rate of radiation energy loss per penetration length unit, due the absorption and the photons scattering. These two coefficients are specific to each tissue and depend on the laser wavelength. To simplify, initially consider an absorber and not scattering sample. In this case, the beam attenuation is described by Beer’s Law: I Z ¼ I 0 ema Z where Iz is the beam intensity that depends on the penetration length z and I0 is the intensity for depth (z) = 0 (i.e., the surface of the tissue). The generated heat per unit and per time unit, in a very small thickness Dz, is given by: ðr, ZþDZ Þ r, ZÞ Qðr, zÞ ¼ Iðr, ZÞIDZ ¼ @Ið@Z ¼ ma I ðZÞ. This equation expresses that the generated heat in the tissue is equal to the absorbed energy and can be described as the absorption coefficient multiplied by the local light intensity (fluence). In most cases, the light is both absorbed and scattered into the sample simultaneously. The beam attenuation continues to be described by a similar law
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from Beer’s Law, but the attenuation coefficient is the sum of the absorption and scattering coefficients, which is called total attenuation coefficient ðmtotal ¼ ma þ ms Þ. In this case, the total deposited heat can be expressed as: Qðr, zÞ ¼ ðma þ ms ÞI ðZ Þ.
Cell Death Mechanisms in Photothermal Therapy
Melamed et al. described the causes of cell death in PTT. The major causes of cell death in PTT process include necrosis and apoptosis. Necrosis is characterized by loss of plasma membrane integrity and subsequent release of intracellular contents including damage-associated molecular patterns (DAMPs) into the extracellular milieu. This abnormal release can trigger detrimental inflammatory and immunogenic responses, making necrosis an undesirable pathway for cell death. In apoptosis on the other hand, cells maintain their membrane integrity and produce “eat me” signals to mark the cells for phagocytosis without incurring inflammation. ▶ Apoptosis may lead to secondary necrosis in which the cell experiences loss of membrane integrity and release of damageassociated molecular patterns (DAMPs) but without activating phagocytosis. To date, the most commonly reported in vitro cellular response to PTT is necrosis, although a few studies have suggested that apoptosis is the primary mechanism of cell death under certain light exposure conditions.
Selective Photothermal Therapy with the Aid of Nano-sized Particles PTT, without the assistance of any external agents, has the limitation of being nonspecific between malignant and normal cells. In other words, without utilizing light-absorbing agents to label pathologic cells, both normal and diseased (such as cancer) cells are subject to death when they are exposed to the laser light. Such damage to healthy tissue could be even more severe since often the diseased tissue is deep inside and the superficial normal tissue is exposed to a larger light fluence (i.e., larger energy). To avoid such side effect, it is necessary to deploy mechanisms to vary the response of diseased (cancerous) cells
Photothermal Therapy
to the incident light by increasing its energy absorption. Such distinction between normal and diseased cells is vital to maintain the integrity of the healthy cells while at the same time destroying the cancerous cells. Nanotechnology (nano-sized agents) has shown great promises to overcome limitation of heat therapy and allows for a range of unique properties that make the various nanomaterials desirable agents for photothermal therapy. The utility of nanomaterials increases the selectivity of photothermal therapy and allows for a more targeted heat response from the cells. In selective PTT by the aid of nano-sized agents, nanomaterials are delivered to tumors either intravenously based on their enhanced permeability and retention (EPR) effect or intratumorally. Subsequent exposure of the tumor to light at the nanoparticle (NP) resonant energy causes synchronized oscillation of the NP conduction-band electrons that results in heat production. This heat can increase the tumor temperature sufficiently to cause irreversible cellular damage and subsequent tumor regression. The temperature rise in the tumor depends on the photothermal conversion efficiency of the NPs, the concentration of NPs in the tumor, and the dosage of light delivered. To maximize the probability of successful thermal ablation, NPs are designed to absorb near-infrared (NIR) wavelengths of light (l 650–1,064 nm), which penetrate more deeply into biological tissues than visible wavelengths due to minimal absorbance by water and hemoglobin in this regime. Many nanomaterials of interest are synthesized out of noble metals such as gold (Au). Au nanomaterials strongly scatter light, and their maximum absorption wavelength could vary based on their various morphologies and sizes. Au is one of the desired nanomaterials for PTT due to its low cytotoxicity and its ability to clear out of the system, both important characteristics in allowing the clinical expansion of PTT. Au nanomaterials currently being invested for their application in PTT include Au nanorods, Au nano-popcorns, silicacoated Au/Fe2O3 nanoaggregates, Au nanoroses, luminescent Au-speckled silica NPs, thermoresponsive polymer-encapsulated Au nanorods, amphiphilic mixed polymers grafted Au NPs, and
Photothermal Therapy
Au nanomatryoshkas. Although Au has been the most commonly researched PTT agent, there are a variety of other agents that may be used such as carbon nanomaterials, palladium nanosheets, and copper sulfide nanoparticles. For combination therapies, an antitumor drug may be combined with a PTT agent in order to prompt a photothermalinduced drug delivery. Applications of Photothermal Therapy in Cancer Treatment PTT has been investigated as a minimally invasive approach to cancer therapy in various cancer types. Colon cancer has been studied in animal models using the intravenous delivery of gold nanorods by Goodrich et al. In their study, the control group, which consisted of laser-only, nanorod-only, and untreated tumored animals, had a shorter survival length than the animals that received the nanorod-based PTT at 800 nm laser irradiation. Au nanoshelled microcapsules were used to test their effectiveness as PTT agents in a rodent model of ▶ breast cancer by Wang et al. The tumors were irradiated at 808 nm for 10 min. The volume of the tumors gradually decreased, and some of the mice even had their tumors completely eliminated by 17 days after treatment. Au nanoshells also demonstrated their ability to produce a therapeutic response in a canine brain model. Nanoshells accumulated in the tumor and then were thermally ablated at 808 nm. Schwartz et al. demonstrated that the damage from the laser irradiation had only been done to the tumor while successfully keeping the normal brain tissue intact. The utility of Au nanoshells in treatment of prostate cancer is studied by Stern et al. When exposed to an 810 nm infrared laser for 3 min, Au nanoshells intravenously injected into mice were able to regress prostate tumors up to 93%. There was also no thermo-harm done on non-tumorous sites. Nanomaterials can also be combined with antibodies in order to exclusively target cancer cells. In a study by Chen et al., nanoparticles targeted with Mucin 7 were able to cure urothelial cancer cells at a laser wavelength of 532 nm. A lower amount of energy was required to harm the
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malignant bladder cells, which contributes to the preservation of the surrounding healthy cells. Nanomaterials were shown to be beneficial for combination therapies in cancer such as photothermal and chemotherapy. Mild heating by photothermal promotes the cellular uptake of drugs and makes the cell weaker, therefore making the malignant cells more susceptible to the effects of the treatment. Studies have shown that combination treatment for breast cancer could destroy the tumor completely. While nano-sized particles made out of Au demonstrate a great performance in PTT, it must be noted that there are less known about some Au nanomaterials, such as hollow gold nanospheres. Although these nanoparticles have been shown to successfully ablate different types of cancer by using a near-infrared laser irradiation, questions still remain about the clearance and safety of such nanomaterials. Conclusion In conclusion, photothermal therapy, with assistance of nano-sized particles, offers a unique mechanism for selective therapy of pathologies such as cancer, without damaging normal surrounding tissues and at clinically relevant depths. Large number of studies demonstrated the utility of selective PTT in treatment of various types of cancer and by using different types of nano-sized particles, often made out of Au.
Cross-References ▶ Apoptosis ▶ Breast Cancer ▶ Cancer ▶ Chemotherapy ▶ Hyperthermia ▶ Urothelial Carcinoma
References Chen F, Cai W (2015) Nanomedicine for targeted photothermal cancer therapy: where are we now? Nanomedicine 10:1–3
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3570 Chen CH, Wu Y-J, Chen J-J (2015) Gold nanotheranostics: photothermal therapy and imaging of Mucin 7 conjugated antibody nanoparticles for urothelial cancer. Biomed Research International, Article ID 813632 Goodrich GP, Payne JD, Sharp K, Bao L, Sang KL (2009) Efficacy of photothermal ablation using intravenously delivered NIR-absorbing nanorods in colon cancer. SPIE BiOS: Biomed Opt 7181:71810Q–71810Q-5 Huang X, Jain PK, El-Sayed IH, El-Sayed MA (2008) Plasmonic photothermal therapy (PPTT) using gold nanoparticles. Lasers Med Sci 23:217–228 Jabeen F, Najam-ul-Haq M, Javeed R, Huck CW, Bonn GK (2014) Au-Nanomaterials as a Superior Choice for Near-Infrared Photothermal Therapy. Molecules 19 (12):20580–20593 Melamed JR, Edelstein RS, Day ES (2015) Elucidating the fundamental mechanisms of cell death triggered by photothermal therapy. ACS Nano 9:6–11 Nadejda R, JinZhong Z (2009) Photothermal ablation therapy for cancer based on metal nanostructures. Science in China Series B: Chemistry 52:1559–1575 Schwartz JA, Shetty AM, Price RE, Stafford RJ, Wang JC, Uthamanthil RK, Pham K, McNichols RJ Coleman CL, Payne JD (2009) Feasibility study of particle-assisted laser ablation of brain tumors in orthotopic. Cancer Res. 69(4):1659–1667 Wang S, Dai Z, Ke H, Qu E, Qi X, Zhang K et al (2013) Contrast ultrasound-guided photothermal therapy using gold nanoshelled microcapsules in breast cancer. Eur J Radiol 177–122 Welch AJ (1984) The thermal response of laser irradiated tissue. IEEE J Quantum Electron 20:1471–1481 Zezell DM, Ana PA (2015) High power lasers and their interaction with biological tissues. Lasers in dentistry: guide for clinical practice. Joh Wiely & Sons, Inc, Hobken, NJ, pp 11–18
Photothermal Treatment Berlin/Heidelberg, pp 126–131. doi:10.1007/978-3540-85516-3_72 Kreidberg J (2009) Animal models. In: Avner E, Harmon W, Niaudet P, Yoshikawa N (eds) Pediatric nephrology. Springer, Berlin/Heidelberg, pp 397–417. doi:10.1007/978-3-540-76341-3_16 Moll R (2006) Breast cancer. In: Ganten D (ed) Encyclopedic reference of genomics and proteomics in molecular medicine. Springer, Berlin/Heidelberg, pp 174–180. doi:10.1007/3-54029623-9_4360 Schleicher Pliska E (2015) Cancer. In: Loue S, Sajatovic M (eds) Encyclopedia of immigrant health. Springer, New York, pp 340–344. doi:10.1007/978-1-44195659-0_112 Sheriff A, Herrmann M, Voll RE, Gaipl US, Kalden JR (2006) Inflammatory response. In: Encyclopedic reference of genomics and proteomics in molecular medicine. Springer, Berlin/Heidelberg, pp 877–882. doi:10.1007/3-540-29623-9_4580 Tadros T (2013) Nanoparticle. In: Tadros T (ed) Encyclopedia of colloid and interface science. Springer, Berlin/Heidelberg, pp 747–748. doi:10.1007/978-3-642-20665-8_121 Weiss C, Roedel C (2013) Bladder. In: Brady LW, Yaeger TE (eds) Encyclopedia of radiation oncology. Springer, Berlin/Heidelberg, pp 29–40. doi:10.1007/978-3-54085516-3_108 Wong E (2011) Ablation. In: Kreutzer JS, DeLuca J, Caplan B (eds) Encyclopedia of clinical neuropsychology. Springer, New York, p 6. doi:10.1007/978-0-38779948-3_3 Yu S, Sousa N, Almeida OFX (2014) Apoptosis. In: Stolerman IP, Price LH (eds) Encyclopedia of psychopharmacology. Springer, Berlin/Heidelberg, pp 1–12. doi:10.1007/978-3-642-27772-6_385-2
Further Reading (2008) Tumor. In: Rédei GP (ed) Encyclopedia of genetics, genomics, proteomics and informatics, 3rd edn. Springer, The Netherlands, p 2045. doi:10.1007/9781-4020-6754-9_17595 (2012) Plasma membrane. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2900. doi:10.1007/978-3-642-16483-5_4599 Bechmann I (2006) Brain. In: Ganten D (ed) Encyclopedic reference of genomics and proteomics in molecular medicine. Springer, Berlin/Heidelberg, pp 166–173. doi:10.1007/3-540-29623-9_5620 Computer Science and Communications Dictionary (2001) Near infrared. Springer-Verlag US, New York, p 1076. doi:10.1007/1-4020-0613-6_12129 Goldstein B (2011) Chemotherapy. In: Kreutzer JS, DeLuca J, Caplan B (eds) Encyclopedia of clinical neuropsychology. Springer, New York, pp 544–546. doi:10.1007/978-0-387-79948-3_98 Huth BJ (2013) Colon cancer. In: Brady LW, Yaeger TE (eds) Encyclopedia of radiation oncology. Springer,
Photothermal Treatment ▶ Photothermal Therapy
Ph-positive Chronic Leukemia ▶ Chronic Myeloid Leukemia
Phyto-cannabinoids ▶ Cannabinoids
Phytochemicals in Cancer Prevention
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of a particular cancer, who move to an area with a
Phytochemicals in Cancer Prevention high prevalence of that cancer, rapidly adopt the Andreas J. Gescher1, Karen Brown2 and William P. Steward2 1 Department of Cancer Studies, Cancer Biomarkers and Prevention Group, University of Leicester, Leicester, Leicester, UK 2 Department of Cancer Studies, University of Leicester, Leicester, UK
Synonyms Nutraceuticals; Plant-derived agents
Definition Phytochemicals are molecules which occur in the plant kingdom; the prefix phyto- is used in words related to botany. Strictly speaking, the term phytochemical encompasses compounds belonging to a large range of chemical classes, from essential minerals such as selenium to large organic molecules such as plant proteins, fats, and carbohydrates, which serve as nutrients. Most people would probably associate the term phytochemical with small non-nutrient organic molecules contained in edible fruits and vegetables. Representative examples of potentially cancer chemopreventive phytochemicals (with their dietary precursors in brackets) are genistein (soya), vitamin c (fruits, vegetables), b-carotene (fruits, vegetables), curcumin (curry spice turmeric), lycopene (tomatoes), and tea catechins such as epigallocatechin gallate (green tea), resveratrol (red grapes, other fruits), and apigenin (leafy vegetables) (see Fig. 1).
Characteristics Epidemiology There is epidemiological evidence which links the incidence of cancer in people in certain areas of the world to the geography of their origin. Often emigrants from areas characterized by a low incidence
high risk of the indigenous population of their chosen immigrant country. For example, Japanese women, who traditionally have a low risk of developing breast cancer, adopt the high breast cancer risk pattern prevalent in the USA within a generation. The low risk of breast cancer in Japan has been tentatively linked to the ubiquitous consumption of plentiful amounts of soya. During the last two decades, many attempts have focused on the chemical isolation and pharmacological characterization of dietary constituents, exemplified by those shown in Fig. 1, which may be responsible for the cancer-preventive properties of the parent foodstuff. In the case of soya, constituent chemicals which have been hypothesized to cause, or contribute to, the cancer-preventive properties are the isoflavones genistein and daidzein. Cancer Chemopreventive Mechanisms Research on chemopreventive mechanisms of phytochemicals has primarily focused on their ability to modulate the following biochemical processes: activation/detoxification of chemical carcinogens by xenobiotic enzymes, DNA repair, cell cycle progression, cell proliferation, differentiation, ▶ apoptosis, expression and functional activation of ▶ oncogenes or tumor suppressor genes, ▶ angiogenesis, ▶ metastasis, and hormonal and growth-factor activity. It is important to note that phytochemicals engage pharmacological mechanisms in a species- and tissue-specific manner. Table 1 shows the spectrum of mechanisms in cells in vitro activated by curcumin as a paradigm of polyphenolic phytochemicals. Cancer Chemopreventive Activity in Rodents Phytochemicals have been shown to possess preclinical cancer chemopreventive efficacy in two types of rodent model. Within weeks of continued exposure to carcinogens such as azoxymethane or benzanthracene, rodents develop cancer in a variety of tissues depending on dose and route of administration. These are the so-called carcinogen-induced models of carcinogenesis. They contrast with “genetic models,” rodents (usually mice) which harbor a gene defect,
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Phytochemicals in Cancer Prevention, Fig. 1 Examples of chemopreventive phytochemicals
O
O OCH 3
H3 CO Curcumin
HO
OH
OH
OH OH
O
O
HO
Apigenin OH
HO
Genistein
O
O OH
HO
O
HO
O
OH HO
HO
OH Resveratrol
OH
Ascorbic acid
OH O
HO
OH O OH
OH O OH OH
Epigallocatechin gallate
Lycopene
β-Carotene
deletion or mutation, which predisposes them to the formation of malignancies. Particularly interesting for cancer chemoprevention research are rodent models with the propensity to develop cancer caused by a genetic defect, which resembles the genetic fault underlying the corresponding disease in humans. Two examples of genetic mouse models are the Apc Min and the TRAMP (transgenic adenocarcinoma of the mouse prostate) mice. Apc Min mice harbor a functionally inactive mutated Apc tumor suppressor gene. All mice with the min genotype develop tumors in the small intestine. The Apc Min mouse is a model of an inherited human disease called familial adenomatous polyposis coli (FAP)
characterized by an APC gene mutation not unlike that seen in Apc Min mice. FAP patients present with colorectal adenomas early in life, which ultimately progress to colorectal carcinomas. In TRAMP mice, expression of the SV40 transforming sequences is targeted to the prostate, and all male TRAMP mice develop prostate tumors. In chemoprevention efficacy testing experiments, carcinogen-induced or genetic models receive the phytochemical under study usually added to the diet, and the number and size of tumors which develop are compared with those in animals which received control diet only. The chemopreventive phytochemicals shown in Fig. 1 have been demonstrated to possess cancer chemopreventive activity
Phytochemicals in Cancer Prevention
in a variety of carcinogen-induced and genetic preclinical models. Clinical Trials Definitive phase III clinical trials which show without doubt that a specific isolated phytochemical (rather than a dietary mixture) can prevent cancer in humans have yet to be published. Two randomized trials with a lung cancer endpoint, the a-tocopherol, b-carotene (ATBC) prevention study and the b-carotene and retinol efficacy trial (CARET), were conducted in the early 1980s. Their outcome suggests that b-carotene was not only devoid of imparting any health benefit, but that it might have even exacerbated the risk of lung cancer. A survey of good–quality trials conducted up to 2013 in which vitamins and mineral supplements were studied for ability to prevent cancer failed to find convincing evidence for efficacy. Results like these intimate that ultimate incontrovertible clinical proof of the ability of isolated phytochemicals to prevent a particular malignancy will be exceedingly difficult to obtain. In order to gather indirect evidence for anticarcinogenic efficacy of phytochemicals, pharmacodynamic markers reflecting chemopreventive efficacy are under intense exploration. Clinical evidence for the potential role of specific phytochemicals in the prevention of certain malignancies has emanated from secondary findings of trials and from small pilot studies. However, there have also been reports suggesting detrimental effects of such agents, and consumption of some phytochemicals has been associated with an increase in certain malignancies. Selenium and folate are examples for agents for which initial clinical evidence of benefit as to their effect on incidence of prostate cancer has subsequently been contested. One reason for such discrepancies may be that phytochemicals are now realised to exert different - sometimes contrary - pharmacological effects at different doses. Whether individuals experience preventive - or indeed detrimental - effects of phytochemicals seems also to be influenced by nutritional status and pre-existing levels of these substances in the
3573 Phytochemicals in Cancer Prevention, Table 1 Chemopreventive mechanisms of curcumin in cells in vitro Pharmacological effect Inhibition of carcinogen activation/DNA binding Stimulation of carcinogen detoxification Control of cell cycle and proliferation
Inhibition of oncogene product activity Induction of apoptosis/ differentiation
Inhibition of angiogenesis, metastasis, invasion
Molecular target #CYP1A1 activity #CarcinogenDNA adduct formation "Detoxifying enzymes (e.g., GST, QR) #Cyclin D1 expression
#Rb phosphorylation #NF-kB activation #c-Jun, c-Fos expression #c-Myc, c-Jun, c-Fos expression #NF-kB, IKK activation
#BCl-2; "p53 and BAX expression; "caspase activity; "BID cleavage; "cytochrome C release #VEGF; #MMP-2 mRNA expression; "TIMP-1 mRNA expression
Model system Human breast cancer cells
Rodent liver; human hepatoma cells Prostate/breast/ squamous carcinoma cells, lymphoma cells, murine epidermal cells
Mouse epidermis Human myeloma/ breast cancer/ leukemia cells
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Human breast carcinoma cells
Abbreviations: CYP cytochrome P450, GST glutathione S-transferase, QR quinone reductase, Rb retinoblastoma tumor suppressor, NFkB nuclear factor kB transcription factor, IKK I kB kinase, VEGF vascular endothelial growth factor, MMP matrix metalloproteinase, TIMP tissue inhibitor of metalloproteinase
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biophase. The Continuous Update Project of the World Cancer Research Fund International (WCRF) analyses emerging scientific research for links between diet and cancer, and the WCRF internet site provides regular updates concerning potential benefit of diet constituents.
Cross-References ▶ Angiogenesis ▶ Apoptosis ▶ Metastasis ▶ Oncogene
References Fortmann SP, Burda BU, Senger CA et al (2013) Vitamin and mineral supplements in the primary prevention of cardiovascular disease and cancer: an updated systematic evidence review for the US Preventive Services Task Force. Ann Intern Med 159:824–834 Omenn GS (1998) Chemoprevention of lung cancer: the rise and demise of beta-carotene. Annu Rev Public Health 19:73–99 Surh YJ (2003) Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer 3:768–780 World Cancer Research Fund International (2015) http://www.wcrf-uk.org/uk/our-research/our-continuousupdate-project
Phytoestrogens Valerie Speirs Leeds Institute of Molecular Medicine, University of Leeds, Leeds, UK
Definition Phytoestrogens are natural chemicals of plant origin which have similar action to the mammalian ▶ hormone estrogen.
Characteristics Phytoestrogens are nonsteroidal polyphenolic compounds. They are found naturally in many
Phytoestrogens
edible plants and have structural similarity to the mammalian steroid hormone 17b-estradiol (Fig. 1). This permits binding to ▶ estrogen receptors (ERs), but they have a much higher affinity for ERb than ERa. Based on their chemical structure, phytoestrogens can be broadly divided into four distinct categories: the ▶ isoflavones (▶ genistein, daidzein, biochanin A), the lignans (enterolactone, enterodiol), the coumestans (coumestrol), and the stilbenes (▶ resveratrol). Phytoestrogens are found in a range of plant products consumed in the diet although their concentrations depend largely on the food type. Isoflavones are found mainly in soy-based products including soybeans, tofu, soy milk, textured soy protein, and miso. Of these, the best studied are genistein and daidzein. The coumestans are found in plants less frequently consumed in the human diet, e.g., alfalfa sprouts, clover, and pinto beans. Lignans are present in flaxseed and are ingested as plant lignan precursors from most fruits and vegetables. These are then converted by gut microflora and into mammalian lignans. Stilbenes are found mainly in peanuts but also in the skin of grapes and as a component of red wine. Potential Benefits of Phytoestrogens Many putative health benefits of phytoestrogens have been proposed, the most interesting of which include their potential role as anticancer agents (▶ phytochemicals in cancer prevention), especially in hormonally regulated ▶ cancer (▶ estrogenic hormones). This stems from epidemiological (▶ Epidemiology of cancer) evidence revealing a much higher incidence of breast cancer in Asian populations who consume a soy-rich diet and where the average daily intake of phytoestrogens is 30 mg/day compared to 10,000 m/ml in choriocarcinoma). • Most (70–80%) PSTTs behave in a benign fashion in which the tumor is confined to the uterus and simple hysterectomy or tumor resection is the treatment of choice. For the malignant PSTTs in which the tumor has locally invaded or distantly metastasized, chemotherapy may be required in conjunction with surgery. Metastases may develop several years after the initial diagnosis. • The behavior of PSTT is difficult to predict since it has been demonstrated that no correlation exists between clinical outcome and molecular or morphological markers.
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• With the use of dose-intensive chemotherapy, imaging techniques to define disease spread, surgery for localized disease, and close surveillance with serologic measurement of b-hCG levels, most patients with PSTT are curable. Pathological Features On gross inspection, most PSTTs are well circumscribed or polypoid, projecting into the uterine cavity or predominantly involving the myometrium. Invasion frequently extends to the uterine serosa but rarely to the adnexal structures. Under a microscope, PSTT is composed of neoplastic intermediate (extravillous) trophoblastic cells with morphology similar to trophoblastic cells in a normal placental site. The large and polygonal tumor cells infiltrate deep into the myometrium and insinuate themselves between smooth muscle fibers. The cells may be present singly, in nests or masses. PSTT is characterized by extensive deposition of fibrinoid material and a unique pattern of vascular invasion – replacement of the muscular wall of an artery by trophoblastic cells and fibrinoid material deposition. Molecular Etiology The molecular etiology of PSTT is largely unknown because of its rarity and lack of animal models for studies. • The trophoblastic nature (fetal origin) of PSTT has been demonstrated by molecular genetic studies showing that the tumor contains new (paternal) alleles not present in adjacent normal uterine tissue. • PSTT is associated with abnormal expression of cell cycle regulatory genes including cyclins, cyclin-dependent kinases, and p53. • As compared to the normal extravillous (intermediate) trophoblastic cells in the implantation site, the majority of PSTTs overexpress the activated (phosphorylated) form of mitogen-activated protein kinase (MAPK). The RAS/RAF/MEK/MAPK signaling pathway participates in various cellular activities including proliferation, differentiation, apoptosis,
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angiogenesis, and migration. It is likely that activation of MAPK contributes to the local invasion and distant metastasis in PSTTs.
References Chang YL, Chang TC, Hsueh S et al (1999) Prognostic factors and treatment for placental site trophoblastic tumor – report of 3 cases and analysis of 88 cases. Gynecol Oncol 73:216–222 Ichikawa N, Zhai YL, Shiozawa T et al (1998) Immunohistochemical analysis of cell cycle regulatory gene products in normal trophoblast and placental site trophoblastic tumor. Int J Gynecol Pathol 17:235–240 Kobel M, Pohl G, Schmitt WD et al (2005) Activation of mitogen-activated protein kinase is required for migration and invasion of placental site trophoblastic tumor. Am J Pathol 167:879–885 Shih IM (2007) Gestational trophoblastic neoplasmspathogenesis and target-based therapy. Lancet Oncol 8:642–650
Plant-Derived Agents ▶ Phytochemicals in Cancer Prevention
Plasma Cell Disorder ▶ Multiple Myeloma
Plasmacytoma Siegfried Janz Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
Definition Plasmacytoma (PCT) refers to a solitary, localized, malignant, monoclonal plasma cell neoplasm that grows either in the bone (solitary bone plasmacytoma: SBP) or soft tissue
Plant-Derived Agents
(solitary extraosseous or extramedullary plasmacytoma: SEP). PCT is a rare, curable cancer. Most patients are treated with moderate-dose radiotherapy. A subset of patients requires surgical intervention. Adjuvant chemotherapy is not indicated. The likelihood of local control after treatment is high. The most common pattern of relapse is systemic, indicating progression of PCT to multiple myeloma (MM). This is more likely to occur after treatment for SBP compared to SEP. The importance of the diagnosis PCT rests in large measure with the potential for this malignancy to progress to MM. A comprehensive review of PCT is available (Kilciksiz et al. 2012).
Characteristics Epidemiology PCT is a rare form of cancer that affects fewer than 10% of patients with plasma cell neoplasms. Plasma cell neoplasms account for 1–2% of malignancies and occur at an overall rate of ~4.5 per 100,000 people per year. PCT is more common in males, with a male-to-female ratio of ~2:1 in case of SBP and ~3:1 in case of SEP. The median age of patients with PCT is 55 years, approximately 15 years younger than patients with MM. SBP develops into MM in 50–60% of patients. Progression of SEP to MM is less frequent (~20% at 10 years after diagnosis), leading to a higher survival rate compared to SBP. Etiology Although no definitive cause for PCT has been identified, it is likely that the genetic and environmental factors associated with increased risk of MM also play a role in the etiology of PCT. Genetic risk factors for MM include gender (male preponderance), race (e.g., African Americans in the USA exhibit twice the incidence of US whites), and age (median age at diagnosis is ~70 years). Clustering of MM in certain families points to a hereditary predisposition consistent with an autosomal dominant mode of inheritance. Some of the underlying tumor susceptibility alleles or “MM genes” have been identified. Associations of MM with occupational exposure to
Plasmacytoma
various metals (nickel), chemical compounds (aromatic hydrocarbons, silicone, petrochemical agents), pesticides, and animal viruses (farming) – protracted infections that can lead to sustained immune stimulation of B lymphocytes with antigen (Helicobacter pylori, human herpesvirus-8); acquired immunodeficiency syndromes, such as HIV/AIDS, that can result in reduced immune surveillance by T lymphocytes; and autoimmune diseases, such as rheumatoid arthritis – have all been proposed. However, the evidence is not strong. Pathogenesis The natural history of PCT is poorly defined. SBP and SEP share many histopathologic, genetic, and biologic features, including accumulations of neoplastic plasma cells that produce monoclonal immunoglobulin; recurrent chromosomal translocations that involve the immunoglobulin heavy-chain locus, IGH, at 14q32; dependence on the plasma cell growth, differentiation, and survival factor, interleukin 6 (IL-6); and a common global gene expression profile. Nevertheless, there are also major differences between SBP and SEP that may point to a different tumor precursor (cell of origin) and pathway of neoplastic development. The abovementioned epidemiological differences of SBP and SEP in terms of tumor progression and overall survival support this view. Just like MM, SBP is thought to be derived from an antigen-experienced isotype-switched post-germinal center B lymphocyte that has undergone somatic hypermutation of the expressed immunoglobulin heavy- and lightchain genes. The clinical observation that SBP can be an intermediate step in the evolution from monoclonal gammopathy of undetermined significance (MGUS) to MM underlines the view that SBP and MM share a common pathway of neoplastic cell transformation. Pathogenetic factors implicated in MM are thus likely to be also important for SBP. Factors of this sort include cytogenetic and molecular genetic alterations that result in the deregulated expression of oncogenes. The interaction of tumor cells with the bone marrow microenvironment, which leads to
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the production of cytokines and growth factors, is also of crucial importance. Unlike MM, SEP is believed to arise from a B lymphocyte or plasma cell that has performed isotype switching and resides in the mucosaassociated lymphoid tissue of the aerodigestive tract. Most SEP express IgA. It is possible that isotype switching occurred outside germinal centers, without T-cell help, under the influence of microbial factors from the aerodigestive tract. But this has not been demonstrated. The molecular genetics of SEP, including the cellular signaling pathways that govern growth, proliferation, and survival of tumor cells, is less well defined than the molecular genetics of SBP, its counterpart in the bone. Likewise, the interaction of SEP with the special microenvironment of the subepithelial mucosa is less well understood than the interaction of SBP/MM with the bone marrow. As mentioned above, SEP can progress to MM, albeit less frequently than SBP. The genetic and/or epigenetic changes underlying the switch from bone-avoiding SEP to bone-seeking MM are unknown. Clinical Features PCT can arise in any body part. Tissue biopsy shows monoclonal plasma cell histology. Bone marrow plasma cell infiltration does not exceed 5% of nucleated cells. Peripheral blood cell count, renal function, and calcium are within the reference range. Serum or urine monoclonal protein (M protein) is absent in more than half of patients. In the majority of patients in which M protein can be detected, it is minimal. Levels of immunoglobulins not involved in the M spike are preserved. A rare occurrence is multiple solitary PCT, which, at first glance, sounds like an oxymoron. Nonetheless, this form of PCT is believed to exist, without evidence of MM, in up to 5% of patients with SBP or SEP. • SBP: The most common symptom is pain at the site of the skeletal lesion, which shows predisposition for the red marrow-containing axial skeleton. Compression fractures of the thoracic and lumbar vertebral bodies (spinal disease, observed in ~50% of cases) result in
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severe spasms, back pain, and nerve root or spinal cord compression. The latter represents an emergency requiring immediate attention to avoid permanent neurologic damage, including paraplegia, bowel and bladder dysfunction, and chronic pain. Occasionally, patients may present with peripheral polyneuropathy or features consistent with the syndrome of POEMS: polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes. • SEP: Although extraosseous tumors occur in any tissue site, 80% of SEPs develop as headand-neck tumors in the upper aerodigestive tract, especially in the paranasal sinuses, pharynx, nasal cavity, gums, and oral mucosa. Patients with tumors involving the base of the skull may present with cranial nerve palsies. In one third of the cases, local lymph nodes are involved at presentation. Imaging Studies Radiographic skeletal survey is preferred over bone scan, which is less sensitive for detecting bone lesions. Approximately 25–50% of bone trabeculae must be destroyed for a bone defect to be visible on a plain radiograph, where SBP classically exhibits a lytic appearance with clear margins and a narrow zone of transition to healthy surrounding bone. Rare occurrences of a cyst, a trabeculated lesion resembling a giant cell tumor or aneurysmal bone cyst, and sclerotic lesions associated with POEMS syndrome have been described. Computed tomography (CT) is more sensitive for evaluating the extent of bone destruction. For SPB, magnetic resonance imaging (MRI) is helpful to monitor the treatment response. In most cases of SEP with nasal cavity or maxillary sinus involvement, radiographic assessment shows local bone destruction. Treatment The treatment of choice for SBP is moderate-dose radiotherapy (40 Gy for spinal lesions and 45–50 Gy for other bone lesions) administered once daily at 1.8–2.0 Gy per fraction in a continuous course. Virtually all patients have major symptom relief. Local tumor recurrence is
Plasmacytoma
~10%. Surgery is contraindicated in the absence of structural instability or neurologic compromise. Chemotherapy using regimens proven in MM may be considered for patients not responding to radiation therapy. No role exists for adjuvant chemotherapy. In the case of SEP, the accepted treatment is radiotherapy, surgery when a lesion can be completely resected, or combined surgery and radiotherapy, which may provide the best results. Chemotherapy may be considered for patients with refractory or relapsed disease. As an exception to the general rule that adjuvant chemotherapy is not indicated in PCT, this therapy may be considered for the subset of patients with positive surgical margins after resection or tumors larger than 5 cm and exhibiting high-grade histology. Follow-Up Outpatient Care and Progression to MM For both SBP and SEP, periodic evaluation for progression and development of MM is recommended every 6 weeks for the first 6 months, with less frequent clinic appointments thereafter. Besides a complete physical examination, the following tests are recommended: complete blood cell count; complete metabolic panel with lactic dehydrogenase, calcium, phosphorus, C-reactive protein (CRP), and b2 microglobulin; sedimentation rate; serum protein electrophoresis with immunofixation; serum immunoglobulin quantification; urinary protein electrophoresis with immunofixation; and skeletal bone survey. Orthopedic and/or ear-nose-throat follow-up is recommended for SBP and head-and-neck SEP, respectively. SBP progresses to MM in the majority of patients, with a median onset of conversion between 2 and 5 years. In SEP, the rate of progression is lower, ranging from 10% to 30% at 10 years after diagnosis. The prognosis for patients with SEP is better than for those with SBP. Medical and Legal Pitfalls Failure to diagnose spinal cord compression syndrome or impending pathologic fractures can cause irreversible neurologic damage.
Plasmacytoma
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cases fatal malignancy. These complications include generalized bone destruction, hypercalcemia, anemia, hyperviscosity, infections, and amyloid. Basic Research PCT occurs not only in human beings but also in many other mammals, including dogs, cats, cattle, rats, and mice (Fig. 1). The laboratory mouse is the premier experimental model organism for research on the biology and pathogenesis of PCT. Laboratory mice are prolific, easy to maintain, and available worldwide. Additionally, the mouse genome can be readily manipulated and modified, using gene targeting and other methods. This affords good opportunities for recapitulating genetic changes of human PCT in transgenic mice. Several mouse models of human PCT have been developed, including a myeloma-like SBP model on the genetic background of C57BL/ 6 (Vanderkerken et al. 2003) and an SEP model in strains BALB/c (Potter 2003).
Cross-References Plasmacytoma, Fig. 1 Histopathology and cytomorphology of plasmacytoma (PCT). (a) Photomicrograph of a histological section of a PCT that developed in the gut-associated lymphoid tissue of a BALB/c mouse that harbors a widely expressed human interleukin-6 (IL-6) transgene. Neoplastic plasma cells are rich in cytoplasm and have a single, eccentric nucleus with clumped chromatin, which forms in some cases the characteristic wheel spoke-like pattern (arrow). A large, binucleated tumor cell is located in the center (arrowhead). (b) Electron micrograph of a neoplastic plasma cell from the same tumor. The cell nucleus (arrow) contains euchromatin in the center (light gray) and heterochromatin in the periphery (black) – tightly attached to the inside of the nuclear membrane. The abundance of rough endoplasmic reticulum (arrowhead) in the cytoplasm indicates that PCT cells produce copious amounts of protein: immunoglobulin (Ig). Typically, this leads to an M spike (extragradient, paraprotein, M protein) in the tumor-bearing mouse, just like the M spike seen in myeloma patients
Inappropriate follow-up or failure to evaluate progression to MM delays institution of appropriate therapy to control the well-known systemic complications of this difficult-to-treat and in most
▶ Monoclonal Gammopathy of Undetermined Significance (MGUS) ▶ Multiple Myeloma
References Kilciksiz S, Karakoyun-Celik O, Agaoglu FY, Haydaroglu A (2012) A review for solitary plasmacytoma of bone and extramedullary plasmacytoma. Scientific World Journal 2012:895765 Potter M (2003) Neoplastic development in plasma cells. Immunol Rev 194:177–195 Vanderkerken K, Asosingh K, Croucher P, Van Camp B (2003) Multiple myeloma biology: lessons from the 5TMM models. Immunol Rev 194:196–206
See Also (2012) POEMS syndrome. In: Schwab M (ed) Encyclopedia of cancer. Springer, Berlin/Heidelberg, p 2934. doi:10.1007/978-3-642-16483-5_4652
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Plasminogen-Activating System Salvatore Ulisse, Enke Baldini, Sarah Bocchini and Massimino D’Armiento Department of Experimental Medicine, University of Rome “Sapienza”, Rome, Italy
Definition The plasminogen-activating system (PAS) consists of two immunologically distinct serine proteases, the urokinase plasminogen activator (uPA) and the tissue-type plasminogen activator (tPA); their specific inhibitors, the plasminogen activator inhibitor 1 (PAI-1) and 2 (PAI-2) and the protease nexin-1 (PN-1), which belong to the serine protease inhibitor superfamily of serpins; and the glycolipid-anchored cell membrane receptor for the uPA. The two plasminogen activators convert, in the extracellular environment, the proenzyme plasminogen into the serine protease plasmin. The latter is involved in fibrin degradation during clot lysis and in a number of physiological and pathological processes requiring basement membrane (BM) and/or ▶ extracellular matrix (ECM) remodeling, such as wound healing, mammary gland development and its postlactational involution, tissue regeneration, ▶ angiogenesis, tumor ▶ progression, and metastasization (Fig. 1). Besides fibrin, in fact, plasmin may directly degrade several ECM and BM components, including laminin, vitronectin, type IV collagen, and proteoglycans, and may also activate latent ▶ matrix metalloproteinases (MMPs). The physiological switch-off of the PAS may occur either through plasmin inhibition, by a2-antiplasmin, or tPA and uPA inhibition, by PAI-1, PAI-2, and PN-1 (Fig. 1).
Characteristics Plasminogen conversion to plasmin into the blood circulation during clot lysis is mostly due to the action of tPA, while uPA plays a major role in ECM remodeling at the tissue level. The uPA is
Plasminogen-Activating System
secreted from the cells as a single-chain proenzyme (pro-uPA or sc-uPA) which binds to its specific uPAR (Fig. 2). The membrane-bound pro-uPA is then converted into active uPA, a two-chain molecule held together by a single disulfide bridge (tc-uPA), by the action of different enzymes, including plasmin, cathepsin B, and kallikrein. In view of the fact that also plasminogen may bind to plasma membrane receptors, the occurrence in the same cells of both uPAR and plasminogen receptors may result in the formation of cell membrane-associated plasmin, leading to a localized pericellular ECM degradation. Beyond plasmin-mediated effects, the uPA/uPAR complex has a number of non-proteolytic effects at both extra- and intracellular level. In particular, it activates other receptors such as the formyl-peptide receptor 2 (FPR2) and the epidermal growth factor receptor (EGFR), which in turn regulate migration, chemotaxis, and cytokine production. The uPA/uPAR also interacts with several integrins enhancing their binding to ligands and therefore activating outside-in integrin signaling. Furthermore, evidence suggests that binding of uPA to its cognate receptor increases its affinity for the ECM component vitronectin (Vn). A direct interaction of uPAR/Vn was shown to be both required and sufficient to initiate downstream changes in cell morphology, migration, and signal transduction. As mentioned, uPA activity is inhibited by PAI-1, PAI-2, and PN-1, which interact with active uPA either free or bound to the uPAR. In the latter case, the complex is rapidly internalized by the cells, leading to the degradation of both uPA and its inhibitor, while the uPAR is recycled to the cell membrane (Fig. 2). The Plasminogen-Activating System in Cancer Progression An efficient ECM and BM degradation, achieved through the action of different proteolytic enzymes, is required for cancer invasion and dissemination. Several studies, which employed different experimental models, indicated that PAS, and especially the uPA/uPAR complexes, plays a relevant role in this process. For example, it has been demonstrated that lung ▶ metastasis of different ▶ melanoma-derived cell lines inoculated
Plasminogen-Activating System Plasminogen-Activating System, Fig. 1 Schematic representation of the major biological processes involving the plasminogenactivating system. MMP matrix metalloproteinases, tPA tissue plasminogen activator, uPA urokinase plasminogen activator, PAI plasminogen activator inhibitor, PN-1 protease nexin-1
3599 α2-antiplasmin
PAI-1, PAI-2, PN-1
tPA, uPA
Fibrin degradation
Plasmin
Plasminogen
Pro-MMPs
Clot lyses
MMPs
Growth factor activation
Extracellular matrix remodeling
Cell adhesion and migration
Cell proliferation
Wound healing, tissue regeneration, angiogenesis, tumor progression and metastatization
Plasminogen-Activating System, Fig. 2 Schematic representation of the urokinase plasminogen activator (uPA), its cognate receptor (uPAR), and the plasminogen activator inhibitor (PAI-1) interaction and function. ECM extracellular matrix
ECM degradation PAI-1
Plasminogen Plasmin
Pro-uPA +
uPA Plasma membrane
uPAR Complex internalization Recycling
in nude mice, positively correlated with cancer cell uPA expression, and that overexpression of uPA in ▶ prostate cancer cells, following transfection with uPA cDNA, increased skeletal metastasis in vivo. Also, inhibition of uPA activity by low-molecular-weight inhibitors or antibodies, as well as inhibition of uPA or uPAR expression by antisense oligonucleotides, induced a decreased tumor growth and metastasis. The main function of PAS, along with members of the MMPs family, was originally thought to be limited to the degradation of ECM and BM,
Degradation
required for local diffusion and spread to distant sites of malignant cells as well as for tumor angiogenesis. However, it has become increasingly clear that PAS affects also multiple aspects of the neoplastic evolution, including tumor cell proliferation, ▶ adhesion and ▶ migration, intravasation, and growth at the metastatic site (Fig. 1). In particular, uPA, directly or through the generation of plasmin, promotes cell proliferation and tumor neoangiogenesis by activation or release of several growth factors, including epidermal growth factor (EGF), ▶ insulin-like growth factor (IGF),
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▶ transforming growth factor-b (TGF-b), basic ▶ fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and ▶ vascular endothelial growth factor (VEGF). Moreover, uPAR, following uPA binding, may affect cell proliferation and other cellular functions by triggering intracellular signaling, leading to the activation of tyrosine and serine protein kinases (i.e., lymphocyte protein tyrosine kinase, lck; hematopoietic cell kinase, Hck; focal adhesion kinase, FAK; and mitogenactivated protein kinase, MAPK). Since uPAR lacks transmembrane and cytosolic domains, the activation of the intracellular signaling is realized through its interaction with at least three different transmembrane proteins, which include members of the integrins adhesion receptor superfamily, G-protein coupled receptor (GPCR), and caveolin. The occurrence of metastasis implies the ability of malignant cells to migrate from the primary site to distant places. Cell migration initiates with the extension of the plasma membrane at the leading edge of the cell, to which corresponds an intracellular reorganization and polymerization of new actin filaments. The latter are then stabilized by the formation of new adhesions to ECM components. At the same time, at the rear or trailing edge of the cells, plasma membrane releases its binding to ECM. Thus, consecutive cycles of attachment and detachment from ECM substrates take place during cancer cell migration. Several reports demonstrated the ability of PAS to affect this process, and the involved molecular mechanisms include (i) ECM proteolysis at the leading edge of the cell allowing plasma membrane extension; (ii) ECM-cell adhesion receptor degradation causing the release of the trailing edge of the cell; (iii) activation of motogenic growth factors such as HGF, bFGF, and TGF-b; and (iv) modulation of new cell adhesion to ECM. Regarding the latter, it has been documented that expression of uPAR on plasma membranes dramatically increases the contacts between cell and ECM. This is likely due to the uPAR/Vn interaction, which brings all surface receptors closer to their matrix ligands. Moreover, the engagement of Vn by uPAR occurs preferentially within membrane structures known as lipid rafts, enriched in signal transduction molecules, thus promoting the
Plasminogen-Activating System
activation of the signal transduction machinery. These effects, however, occur only in the presence of low levels of PAI-1, since when the concentration of PAI-1 exceeds that of uPA, the inhibitor displaces uPAR binding to Vn causing the release of the cell from the ECM. In addition, PAI-1 inhibits the binding of Vn to integrins receptors and reduces cell migration. A further mode by which the uPA/uPAR complex may contribute to cancer progression emerged from studies employing the human breast cancer MDA-MB468 cell line, where uPAR was found to promote epithelial-mesenchymal transition (EMT) under hypoxic condition through the activation of different signaling molecules, including ERK, PI3K/ Akt, Src, and Rac1. The EMT phenotype could be reverted by reoxygenation and inhibition of the uPA/uPAR interaction or of downstream effectors. Clinical Significance of PAS Components Expression in Cancer Consistent with their role in cancer progression and metastasis, a marked increase in the expression of uPA, uPAR, and PAI-1, with respect to normal counterpart tissues, has been observed in malignant tumors. For example, in breast cancers the expression of uPA and uPAR was shown to be upregulated approximately of 5- and 19-fold, respectively, compared with normal or benign tumor tissues. Similarly, in thyroid papillary carcinomas, the upregulation of uPA and uPAR expression by more than fourfold, with respect to normal matched thyroid tissues, was documented. Different clinical studies demonstrated that high tumor tissue levels of uPA and its cognate receptor uPAR correlate with a poor prognosis in several malignancies including leukemia and breast, lung, brain, esophageal, gastric, pancreatic, colorectal, hepatocellular, endometrial, ovarian, kidney, and bladder cancers. However, paradoxically, high levels of PAI-1 expression, considered the primary inhibitor of uPA, are also associated with more aggressive diseases and poor prognosis in several types of cancer. Different hypotheses have been proposed to explain these observations, including an essential role of PAI-1 in tumor angiogenesis, cancer cells adhesion, migration, and inhibition of apoptosis.
Plasminogen-Activating System
The clinical relevance of PAS component expression is particularly evident in ▶ breast cancer, in which the prognostic value of uPA, uPAR, and PAI-1 expression in cancer tissue has been exhaustively investigated. The first information about the prognostic value of uPA expression in breast tumors dates from 1988, when it was demonstrated that patients with breast tumors containing a high level of uPA activity had a significantly shorter disease-free interval, compared to patients with low level of tumorassociated enzyme activity. This initial observation was then confirmed in several large case studies, and nowadays uPA is considered the most potent independent prognostic factor, in terms of overall survival and/or disease-free interval, with a predictive value stronger than those of patient age, tumor size, estrogen and progesterone receptors, and ▶ HER-2/neu or p53 expression. Also high levels of PAI-1 associate with adverse outcome in breast cancer patients and it has been demonstrated that it represents an independent prognostic factor. Moreover, the concomitant assay of both uPA and PAI-1 possess a greater prognostic value with respect to that of each marker alone. Therefore, both uPA and PAI-1 are now candidate molecular markers for routine clinical use in patients with breast cancer. PAS as Target for Anticancer Therapy The involvement of PAS in cancer progression and the observation that its inhibition is devoid of toxicity, as verified in uPA- or uPAR-deficient mice, identify the PAS inhibition as a suitable target for anticancer therapies. The first evidence showing that uPA inhibition could be effective in preventing cancer metastasis was reported in 1983. In this study, it was demonstrated that antibodies inhibiting uPA activity were capable of preventing metastasis from the human epidermoid carcinoma cell line Hep3 in the chick chorioallantoic membrane system. Since then, different therapeutic strategies have been designed to inhibit the uPA/uPAR function, including selective inhibitors of uPA activity, antagonist peptides, and monoclonal antibodies able to prevent uPA binding to uPAR, antisense, or gene therapy reducing
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uPAR expression and peptides that inhibit uPAR/ integrins interaction, thus preventing uPARmediated cell signaling and adhesion. These approaches have demonstrated, in xenograft models, antitumor effects, including both reduction in local tumor growth and cancer dissemination. However, these new anticancer therapeutic strategies, although promising, need definitive confirmation in humans and, up to this day, only one uPA inhibitor, the WX-UK1/MESUPRON ® (Wilex AG, Munich, Germany), entered clinical trial. After successful completion of phase I studies, phase II studies were carried out on breast and pancreatic tumors requiring long-term treatment. Patients with locally advanced, inoperable, nonmetastatic pancreatic cancer were treated with a combination of MESUPRON ® and the chemotherapeutic agent gemcitabine. Coadministration of these drugs led to an increase of the tumor response rate and improvement of the progression-free survival (PFS), compared to the gemcitabine alone. Patients affected by breast cancer received MESUPRON ® in combination with the chemotherapeutic agent capecitabine. The obtained results showed again an increment of the objective tumor response rate and a modest increase in PFS in patients, compared to capecitabine monotherapy (http://www.wilex.de/ portfolio-english/mesupron/phase-i-ii-with-mesu/).
Cross-References ▶ Adhesion ▶ Angiogenesis ▶ Breast Cancer ▶ Extracellular Matrix Remodeling ▶ Fibroblast Growth Factors ▶ HER-2/neu ▶ Insulin-Like Growth Factors ▶ Matrix Metalloproteinases ▶ Metastasis ▶ Migration ▶ Progression ▶ Prostate Cancer ▶ Transforming Growth Factor-Beta ▶ Vascular Endothelial Growth Factor
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References Blasi F, Carmeliet P (2002) uPAR: a versatile signalling orchestrator. Nat Rev Mol Cell Biol 3:932–943 Choong PFM, Nadesapillai APW (2003) Urokinase plasminogen activator system: a multifunctional role in tumor progression and metastasis. Clin Orthop Relat Res 415S:S46–S58 de Bock CE, Wang Y (2003) Clinical significance of urokinase-type plasminogen activator receptor (uPAR) expression in cancer. Med Res Rev 24:13–39 Duffy MJ (2004) The urokinase plasminogen activator system: role in malignancy. Curr Pharm Des 10:39–49 Madsen CD, Ferraris GM, Andolfo A, Cunningham O, Sidenius N (2007) uPAR-induced cell adhesion and migration: vitronectin provides the key. J Cell Biol 177:927–939 Noh H, Hong S, Huang S (2013) Role of urokinase receptor in tumor progression and development. Theranostics 3:487–495 Rockway TW, Nienaber V, Giranda VL (2002) Inhibitors of protease domain of urokinase-type plasminogen activator. Curr Pharm Des 8:2541–2558 Schuliga M, Westall G, Xia Y, Stewart AG (2013) The plasminogen activation system: new targets in lung inflammation and remodeling. Curr Opin Pharmacol 13:386–393 Smith HW, Marshall CJ (2010) Regulation of cell signalling by uPAR. Nat Rev Mol Cell Biol 11:23–36 Ulisse S, Baldini E, Sorrenti S, D’Armiento M (2009) The urokinase plasminogen activator system: a target for anti-cancer therapy. Curr Cancer Drug Targets 9:32–71
Platelet
formation. They contain fibrinogen, factor V, vitronectin, thrombospondin, and von Willebrand factor, which are released upon platelet activation. Platelets are produced in the bone marrow. The progenitor cell for platelets is the megakaryocyte which sheds platelets into the circulation.
See Also (2012) Von Willebrand Factor. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3937. doi:10.1007/978-3-642-164835_6220. Rattenholl A, Steinhoff M, Schneider SW, Görge T (2012) Proteinase-Activated Receptors. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 3095-3096. doi:10.1007/978-3-642-164835_4810
Platelet-Activating-Factor Synonyms PAF
See Also (2012) Extracellular Matrix. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin Heidelberg, p 1362. doi:10.1007/978-3-642-16483-5_2067 (2012) MAPK. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin Heidelberg, p 2167. doi:10.1007/978-3-642-16483-5_3532 (2012) P53. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer, Berlin Heidelberg, p 2747. doi:10.1007/978-3-642-16483-5_4331
Platelet Synonyms
Definition Is a potent inflammatory mediator; it is an etherlinked analogue of phosphatidylcholine, bearing an ester linked acetyl moiety at the sn-2 position of glycerol.
Cross-References ▶ Lipid Mediators
Thrombocyte
Definition Cells without nuclei which circulate in the blood. They are involved in thrombus (blood clot)
Platelet-Derived Endothelial Cell Growth Factor ▶ Thymidine Phosphorylase
Platelet-Derived Growth Factor
Platelet-Derived Growth Factor Carl-Henrik Heldin Ludwig Institute for Cancer Research, Uppsala University, Uppsala, Sweden
Definition Platelet-derived growth factor (PDGF) is a family of growth factors with mitogenic activity for connective tissue cells, such as fibroblasts and smooth muscle cells, as well as for certain other cell types.
Characteristics PDGF isoforms are disulfide-bonded homodimers of homologous A-, B-, C- and D-polypeptide chains, as well as a heterodimer PDGF-AB. PDGF A- and B-chains are proteolytically processed during secretion from the producer cell, whereas PDGF C- and D-chains are secreted as inactive precursors containing N-terminal CUB domains that need to be cleaved off before these isoforms can bind to receptors. The mature growth factor domain has about 100 amino acid residues with a perfect conservation of 8 cysteine residues. The two subunits in the dimers are arranged antiparallel. In addition to the two interchain disulfide bonds, each subunit contains six additional cysteine residues that are arranged in a characteristic cystine knot structure. The amino acid sequences of PDGF chains are homologous to those of vascular endothelial cell growth factors (▶ vascular endothelial growth factor). PDGF isoforms exert their cellular effects via binding to two structurally related protein tyrosine kinase receptors (▶ Receptor tyrosine kinases), denoted a- and b-receptors. The A-, B- and C-polypeptide chains bind with high affinity to a-receptors, whereas B- and D-polypeptide chains bind to b-receptors (Fig. 1). The dimeric PDGF isoforms bind two receptor molecules simultaneously; receptor dimerization leads to juxtaposition of the kinase domains of the receptors, whereby they phosphorylate each other in trans.
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The “autophosphorylation” activates the receptor kinases and initiates intracellular ▶ signal transduction by recruitment of SH domain (▶ SH2/SH3 domains) containing signal transduction molecules. Thereby, several signal transduction pathways are initiated, ultimately leading to cell proliferation, cell migration, changes in cell morphology, and inhibition of cell death (Fig. 2). Intracellular cell signaling is characterized by extensive cross-talk between different signaling pathways. Moreover, stimulatory and inhibitory pathways are often induced in parallel. Genes The PDGF A-, B-, C- and D-chain genes are localized on chromosomes 7, 22, 4, and 11, respectively. In all genes, the first exon encodes untranslated sequences and the signal sequence. In the A- and B-chain genes, the second and third exons encode N-terminal sequences that are removed during proteolytic processing, the fourth and fifth exons encode most of the mature parts of the proteins, and exon 7 is mainly noncoding. Exon 6 encodes C-terminal sequences that in the case of the B-chain may be removed during processing. The A-chain occurs as two different splice forms, with and without the exon 6 sequence. Since the exon 6 sequence contains a stretch of basic amino acid residues that bind to extracellular matrix molecules, the absence or presence of the exon 6 sequence affects the localization of PDGF. In the C- and D-genes, exons 2 and 3 encode the CUB domain, exon 4 (exon 4 and 5 for the D-chain gene) encodes a linker region, and exon 5 and 6 (6 and 7 for the D-chain gene) encode the growth factor domain. Bioactivity Sequencing of PDGF revealed that the B-chain is almost identical to the v-Sis ▶ oncogene product, the transforming protein of simian sarcoma virus. Subsequent studies have shown that transformation of cells by the simian sarcoma virus occurs via autocrine stimulation involving a PDGF-like growth factor. The demonstration that overactivity of PDGF had transforming effects prompted studies on the expression of PDGF in human tumors. PDGF was
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Platelet-Derived Growth Factor
Platelet-Derived Growth Factor, Fig. 1 Binding of PDGF isoforms to a- and b-tyrosine kinase receptors
found to be commonly expressed in glioblastomas and sarcomas; tumors derived from cell types normally expressing receptors, suggesting autocrine stimulation of growth. PDGF is expressed also by several cancers that are derived from cell types that do not express PDGF receptors. Such tumor-derived PDGF has been shown to be involved in a paracrine manner in the formation of supporting connective tissue cells that surrounds the tumor cells. PDGF stimulation of connective tissue cells in the stroma also contributes to the increased interstitial fluid pressure of tumors, which causes a decreased uptake of drugs into the tumor and precludes efficient chemotherapy of tumors. PDGF has also been shown to have an angiogenic activity (▶ angiogenesis), through direct effects on capillary endothelial cells, as well as indirectly through the recruitment of supporting pericytes that reinforce the walls of the newly formed vessels. Clinical Relevance The normal function of PDGF is to promote the development of different kinds of connective tissue cells during embryonal development. Often
PDGF is produced by different epithelial cell types and acts on neighboring connective tissue cells expressing PDGF receptors. In the adult, PDGF stimulates wound healing, and topically applied PDGF-BB (becaplermin) has been shown in clinical trials to increase the rate of healing of different types of wounds. PDGF also regulates the interstitial fluid pressure in connective tissue. PDGF released from platelets may also exert a negative feedback control of platelet aggregation; binding of PDGF to a-receptors on platelets inhibits platelet aggregation. The important function of PDGF in the development and homeostasis of connective tissue suggests that overactivity of PDGF may result in fibrotic reactions. In support of this notion, overexpression of PDGF has been shown to be involved in glomerulonephritis, liver cirrhosis, myelofibrosis, and lung fibrosis. The stimulating effect of PDGF, released from platelets or secreted from macrophages, on smooth muscle cells at sites of injury to the endothelial cell layer of arteries, also contributes to the intimal hyperplasia seen in atherosclerotic reactions. In malignancies, PDGF may be involved in autocrine stimulation
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Platelet-Derived Growth Factor, Fig. 2 Ligandinduced dimerization of the PDGF b-receptor, followed by autophosphorylation and docking of SH2-domaincontaining signaling molecules. The importance of
individual signaling pathways for the effects of PDGF in cell growth, survival, migration, and actin reorganization is depicted
of tumor cell growth as well as in paracrine stimulation of cells in blood vessels and fibroblasts in the stromal compartment. Certain tumor types show specific perturbations in PDGF signaling pathways through mutations of genes for PDGF isoforms or PDGF receptors, including dermatofibrosarcoma protuberans (fusion of the genes for PDGF B-chain and collagen 1a1), chronic myelomonocytic leukemia and hypereosinophilic syndrome (fusion of genes for PDGF a- or b-receptor with different other genes), ▶ gastrointenstinal stromal tumor (point mutation in the PDGF a-receptor gene), and glioblastoma (▶ amplification of the PDGF a-receptor gene) (▶ Brain Tumors). The involvement of PDGF in several serious disorders including malignancies makes PDGF
antagonists highly warranted. Several types of such antagonists have been developed, including molecules that bind PDGF and prevent it from binding to its receptors, e.g., antibodies, soluble extracellular domains of receptors (e.g., ▶ STI-571 or ▶ Imatinib) or DNA ▶ aptamers, as well as low molecular weight inhibitors of the receptor kinase.
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Cross-References ▶ Angiogenesis ▶ Autocrine Signaling ▶ Brain Tumors ▶ Endocytosis ▶ Fibrosarcoma
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Platelet-Derived Growth Factor Receptor (PDGFR) Inhibitors
▶ Gastrointestinal Stromal Tumor ▶ Imatinib ▶ Platelet ▶ Receptors ▶ Signal Transduction ▶ STI-571
References Fredriksson L, Li H, Eriksson U (2004) The PDGF family: four gene products form five dimeric isoforms. Cytokine Growth Factor Rev 15:197–204 Heldin C-H, Westermark B (1999) Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 79:1283–1316 Heldin C-H, Östman A, Rönnstrand L (1998) Signal transduction via platelet-derived growth factor receptors. Biochim Biophys Acta 1378:F79–F113 Pietras K, Sjöblom T, Rubin K et al (2003) PDGF receptors as cancer drug targets. Cancer Cell 3:439–443 Rosenkranz S, Kazlauskas A (1999) Evidence for distinct signaling properties and biological responses induced by the PDGF receptor a and b subtypes. Growth Factors 16:201–216
Platelet-Derived Growth Factor Receptor (PDGFR) Inhibitors ▶ Receptor Tyrosine Kinase Inhibitors
Platinating Agents ▶ Platinum Complexes
Platinum Antitumor Agents ▶ Platinum Complexes
Platinum Antitumor Compounds ▶ Platinum Complexes
Platinum Complexes Irina Bosman and Ganna V. Kalayda Institute of Pharmacy, University of Bonn, Bonn, Germany
Synonyms Platinating agents; Platinum antitumor agents; Platinum antitumor compounds; Platinum drugs
Definition In 1844, Michele Peyrone synthesized cisplatin (cis-platinum(II)-diammine-dichloride, CDDP) without being aware of its tumor-inhibiting effect. Not until the mid-1960s, Barnett Rosenberg discovered the inhibition of cell division in the presence of cisplatin. Due to its outstanding effectiveness in treating numerous tumors, cisplatin became the prototype for a new class of antineoplastic substances. Meanwhile, it has become one of the most frequently used cytotoxic drugs in tumor therapy. However, its use is limited due to its side effects and the development of resistance which has led to the search for new platinum complexes. Most of the platinum complexes are uncharged cis configurated square-planar platinum(II) complexes that can be described by the general formula cis-[PtA2 X2], with A2 as either two monodentate or one bidentate stable amine ligand(s) and X2 as two monodentate or one bidentate anionic leaving ligand(s). Octahedral platinum(IV) complexes, which are now under development, can be described by the general formula cis-[PtA2 X2 Y2] with Y2 as monodentate anionic leaving ligands. The advantage of platinum(IV) complexes is the possibility of oral administration due to their
Platinum Complexes
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increased stability and solubility in the gastrointestinal tract.
imidazole ring. Besides monoadducts, cisplatin forms bifunctional 1,2- or 1,3-intrastrand crosslinks and interstrand cross-links (Fig. 1).
Characteristics
Cellular Response
In the following paragraphs, the processes taking place after the administration of ▶ cisplatin are described. Different characteristics of other platinum complexes are outlined in the section “Approved Platinum Complexes.” Mode of Action Bioactivation
After intravenous administration, a high amount of cisplatin is bound to plasma proteins and thereby inactivated. Because of the high extracellular chloride ion concentration (100 mM), free cisplatin exhibits relatively low reactivity in the plasma. After entering the cell containing low chloride ion concentrations (~4 mM), reactive monoaqua and diaqua complexes are formed by exchange of the chloride ligands. Upon activation, cisplatin is able to form complexes with a variety of macromolecules present in the cell. Formation of Platinum–DNA Adducts
The cytotoxic effect is mainly a consequence of the formation of platinum–DNA adducts. The primary target is the N7 position of the purine bases due to the high nucleophilicity of the Platinum Complexes, Fig. 1 Formation of platinum–DNA adducts
1. Inhibition of DNA synthesis As a result of the structural change of the DNA helix, binding of DNA polymerases is hampered inhibiting replication and transcription. 2. DNA repair mechanisms Platinum–DNA adducts are mainly repaired by ▶ nucleotide excision repair (NER). Due to the small substrate specificity of this repair system, it is unlikely that platinum–DNA adducts of structurally different platinum complexes are differentiated. A complete removal of the DNA damage is not possible in this way because of the limited capacity of the NER. Furthermore, adducts can be bypassed by some DNA polymerases. This translesion DNA synthesis – the so-called replicative bypass (post replicative repair) – allows the cells to progress through the S-phase of the cell cycle. The mismatch repair (MMR) system is an important prerequisite for the cytotoxic activity of cisplatin. Base-pair mismatches are recognized and repaired by MMR proteins. The DNA strand previously synthesized is cut out and the DNA is again synthesized beyond the damaged sites entering in a vicious cycle. In the long term, these futile repair attempts lead
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Platinum Complexes
Platinum Complexes, Fig. 2 Cell death pathways activated in response to cisplatin (Modified from Wang and Lippard 2005). TCR transcription-coupled repair
Cisplatin
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to an induction of apoptosis. Thus, deficiency of MMR is associated with cisplatin resistance (see below). 3. Binding of HMG proteins and transcription factors The disturbance of the DNA structure results in the binding of different proteins. Among them high mobility group box proteins – such as HMGB1 – protect platinum–DNA adducts against repair proteins by building stable bonds. Many HMG box proteins are transcription factors and partly exhibit a higher affinity for the platinated DNA than for their natural substrate. The transcription factors are intercepted by the platinum–DNA adducts; therefore, the transcription is inhibited. This phenomenon is called “transcription factor hijacking.” 4. Induction of ▶ apoptosis and ▶ necrosis The DNA damage caused by cisplatin triggers apoptosis and necrosis (Fig. 2). Mechanisms of Resistance Resistance can be intrinsic or acquired by chronic drug exposure. The resistance to cisplatin is multifactorial; different resistance mechanisms may
develop in parallel. Four main resistance mechanisms can be distinguished. Reduced Accumulation
After exposure to platinum complexes, a decreased influx, an increased efflux, or a combination of both phenomena was observed. Previously, it has been assumed that platinum complexes enter the cell primarily by passive diffusion (Fig. 3). During the last decade, a link between copper transporters (CTR1, ATP7A, ATP7B) and platinum influx and efflux has been discovered in numerous studies. Furthermore, results indicate an involvement of organic cation transporters (OCT). Platinum resistance may develop by upregulation of efflux or downregulation of influx transporters. Increased Inactivation
Platinum complexes react to a great extent with intracellular molecules that contain thiol groups, e.g., glutathione (GSH) or metallothionein. Only a relatively small fraction of the intracellular cisplatin binds to the genomic DNA; more intracellular platinum is bound to GSH and other peptides and proteins. The conjugate with GSH is
Platinum Complexes Platinum Complexes, Fig. 3 Mechanisms of cisplatin influx, efflux, and detoxification. CTR1 copper transporter 1, ATP7A adenosine triphosphate alphapolypeptide, ATP7B adenosine triphosphate beta-polypeptide, MRP2 multidrug resistance protein 2
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biologically inactive. The platinum–GSH complex can be removed from the cell by the transporter MRP2 (Fig. 3). In addition, the formation of cross-links between platinum–DNA monoadducts and the opposite DNA strand can be prevented by reaction of the thiol-containing molecules and the monoadducts. An increased intracellular GSH production can contribute to the development of resistance. Increased Adduct Tolerance and Failure of Apoptotic Pathways
An increase of the replicative bypass leads to an increased adduct tolerance as well as to a high mutation rate, whereby changes, which lead to subsequent development of resistance, are favored. Defects in the MMR system prevent the beginning of futile repair cycles, hence contributing to resistance. The complex cascades of apoptosis offer a further starting point for the development of resistance. Many factors can be changed: examples are the loss of p53 function, reduced activity of caspases, or a deregulated MAPK pathway. Increased Repair
A general resistance mechanism is an increased repair by the NER system. The capacity of this kind of resistance mechanism seems, however, to be limited.
ATP7A ATP7B
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Approved Platinum Complexes The chemical structures of approved platinum complexes are shown in Fig. 4. Cisplatin
Indications. FDA-labeled indications: metastatic malignant tumor of testis, metastatic ovarian tumor, and advanced transitional cell carcinoma of bladder. Furthermore, one finds a broad range of non-FDA-labeled indications. Adverse effects. Relevant and dose-limiting side effects are nephrotoxicity, ototoxicity, emesis, and neurotoxicity. Carboplatin
Characteristics. By the exchange of the labile chloro-ligands for cyclobutan-1,1dicarboxylate, water solubility and stability were increased compared to cisplatin; the toxicity was decreased. However, cisplatin and carboplatin show crossresistance, which points at a similar mechanism of action. Indications. FDA-labeled indication: advanced ovarian cancer. Carboplatin is approved worldwide for the treatment of many other tumors. Adverse effects. The only dose-limiting side effect is myelosuppression. Among the serious side effects are an imbalance of electrolytes, a peripheral neuropathy, and a less-common visual disturbance.
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platination is substantially lower after treatment with oxaliplatin. However, the rate of single-strand breaks is higher. The replicative bypass is probably disturbed by the bulky DACH ligand leading to an increased singlestrand break rate and enhanced cytotoxicity. An intact MMR system does not seem to be essential for the cytotoxic effect of oxaliplatin, because the DACH ligand prevents the attachment of the MMR proteins. These changes in the repair mechanisms are a possible explanation for the absence of crossresistance to cisplatin. Oxaliplatin is effective in the treatment of metastatic colon carcinoma, which exhibits intrinsic resistance to cisplatin and carboplatin. Indications. FDA-labeled indications: colon cancer and advanced colorectal cancer. Adverse effects. The crucial and dose-limiting toxicity of oxaliplatin is peripheral sensory neuropathy. Myelosuppression is low. Neither ototoxicity nor nephrotoxicity has been observed.
6 O
Platinum Complexes, Fig. 4 Approved platinum complexes: cisplatin (1), carboplatin (2), oxaliplatin (3), nedaplatin (4), lobaplatin (5), heptaplatin (6)
Oxaliplatin
Characteristics. Oxaliplatin shows another spectrum of efficacy and resistance. The oxalate ligand as the leaving group leads to a slightly reduced reactivity compared to cisplatin. The bulky 1,2-diaminocyclohexane (DACH) ligand is probably responsible for the improved water solubility as well as the changed profile of activity. Using equimolar and equitoxic concentrations of cisplatin and oxaliplatin, it was noted that the extent of DNA
Characteristics. The complex contains glycolic acid as a leaving group. Nedaplatin exhibits a lower reactivity than cisplatin, but a higher one than carboplatin. It has a better side effect profile than cisplatin, but does not, however, show genuine advantages regarding treatment response and survival rate. Studies comparing nedaplatin with carboplatin are missing. Indications. No FDA approval. Approved in Japan for the treatment of several tumors. Adverse effects. Myelosuppression, including severe thrombocytopenia, is dose limiting. Nonhematological toxicity is reduced compared to cisplatin – except ototoxicity. Lobaplatin
Characteristics. Lobaplatin was developed to extend the spectrum of activity of the platinum complexes. It is as active as cisplatin with absence of crossresistance. Its efficacy in cisplatin-resistant tumors has to be proven in larger phase III studies.
Platinum Complexes
Indications. No FDA approval. The complex has received approval in China for the treatment of different kinds of tumors. Adverse effects. Thrombocytopenia represents the dose-limiting toxicity.
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cellular thiols. Although picoplatin showed promising activity at the early stages of clinical development, no advantage over the control group could be observed in the Phase III clinical trials. Multinuclear Platinum Complexes
Heptaplatin
Characteristics. Heptaplatin exhibits a more favorable adverse effect profile and a slightly reduced resistance compared to cisplatin. Indications. No FDA approval. Heptaplatin gained approval in South Korea for the treatment of advanced gastrointestinal tumors. Adverse effects. Nephrotoxicity is dose limiting. New Platinum Complexes Under Development
Two to four platinum centers with cis- or trans-configuration are connected by polyamine chains of different lengths. This approach has led to the development of highly antitumor active complex BBR3464. BBR3464 was able to overcome resistance through formation of long-chain interstrand cross-links, because this type of crosslinks is not recognized by the NER system and HMG proteins. Unfortunately, clinical development of BBR3464 had to be discontinued due to dose-limiting gastrointestinal and hematological toxicity observed in Phase II clinical trials.
Platinum(IV) Complexes
These octahedral complexes exhibit some advantages compared to the square-planar Pt(II) complexes. They can be administered orally due to their increased stability and solubility in the gastrointestinal tract. Adverse effects are reduced due to their lower reactivity. New opportunities of synthesis and more possibilities of structural variations are opened. In the body, they are reduced to more reactive Pt(II) complexes which form DNA adducts. In addition, there is evidence that the Pt (IV) complexes may interact directly with the DNA. Satraplatin is the most promising Pt(IV) compound in clinical evaluation. In a Phase III clinical trial in prostate cancer, satraplatin reduced the risk of disease progression and improved progressionfree survival. However, FDA approval did not follow due to the lack of convincing benefit for overall survival. Sterically Hindered Platinum Complexes
The introduction of bulky substituents was supposed to lead to the reduction of detoxification and repair mechanisms. Picoplatin (formerly AMD473, ZD0473), cis-ammine-dichloro(2-methylpyridine) platinum (II), was designed in order to reduce the inactivation of the complex by glutathione and other
Drug Delivery Conjugates with Platinum Complexes
Targeting of the complexes to the disease site can be achieved by the use of selective carriers or through retention in the tumor tissue. The enhanced permeability and retention (EPR) effect can be used when platinum complexes are coupled to macromolecules, which tend to accumulate in tumor tissue due to its leaky vasculature. A liposomal formulation of cisplatin, lipoplatin, has obtained an orphan drug status from the European Medicines Agency (EMA) for the treatment of pancreatic cancer. Phase III clinical trials are ongoing. Polymer micelles containing cisplatin and oxaliplatin are under clinical investigation.
References Rabik CA, Dolan ME (2007) Molecular mechanisms of resistance and toxicity associated with platinating agents. Cancer Treat Rev 33:9–23 Wang D, Lippard SJ (2005) Cellular processing of platinum anticancer drugs. Nat Rev Drug Discov 4:307–320 Galluzzi L, Senovilla L, Vitale I, Michels J, Martins I, Kepp O, Kastedo M, Kroemer G (2012) Molecular mechanisms of cisplatin resistance. Oncogene 31:1869–1883 Dilruba S, Kalayda GV (2016) Platinum-based drugs: past, present and future. Cancer Chemother Pharmacol Epub ahead of print
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Platinum Drugs ▶ Platinum Complexes
Platinum-Refractory Testicular Germ Cell Tumors Christian Kollmannsberger Division of Medical Oncology, British Columbia Cancer Agency, Vancouver Cancer Centre, University of British Columbia, Vancouver, BC, Canada
Synonyms Cisplatin-refractory germ cell tumors; Cisplatinresistant germ cell tumors; Treatment-refractory germ cell tumors
Definition Platinum-refractory germ cell tumors (GCTs) are GCTs which no longer respond to cisplatin-based chemotherapy. Cisplatinum-refractory disease is defined as response or disease stabilization during, but disease progression within 4 weeks after cisplatin-based chemotherapy, whereas absolutely cisplatin-refractory disease refers to patients with progression during cisplatin-based treatment. In a broader sense, multiply relapsed (3 recurrences) GCTs are also often referred to as treatment-refractory GCTs.
Characteristics Today, approximately 70–80% of all patients with metastatic germ cell cancer will achieve long-term cure after standard-dose, cisplatin-based combination chemotherapy, such as PEB (cisplatin, etoposide, bleomycin) or VIP (etoposide, ifosfamide, cisplatin). Patients relapsing after
Platinum Drugs
cisplatin-based first-line chemotherapy have a less favorable prognosis, although 20–25% of patients are still cured with conventional secondline cisplatin-based chemotherapy. However, patients relapsing again after conventional or even high-dose salvage chemotherapy have a very poor prognosis, particularly those with cisplatin-refractory or absolutely cisplatinrefractory disease and less than 5% of these patients are long-term survivors. The molecular basis for the exquisite chemotherapy sensitivity and mechanisms of cisplatin resistance of malignant GCTs is poorly understood. Mechanisms of Platinum Resistance Despite various studies, no uniform hypothesis has been developed to explain the exquisite chemosensitivity of most GCTs as well as the chemoresistance of the minority of malignant GCT. Multiple factors on different cellular levels seem to play a role in the induction of cell death following cisplatin-based chemotherapy, but the exact mechanisms of cisplatin resistance are still unknown. Cisplatin is believed to kill cells through interaction with the DNA, mainly by the formation of various DNA adducts, which lead to the initiation of apoptosis. The tumor cell can escape the initiation of apoptosis on several levels: First, cisplatin can be inactivated by changes in the level of thiolcontaining cell compounds such as glutathione or metallothionein or can be exported out of the cell by several export pumps, even before it reaches the DNA. Second, cisplatin-induced DNA damage can be repaired, predominantly by the so-called nuclear excision repair (NER) pathway prior to the activation of the apoptotic cascade; third, the recognition of the critical DNA damage by mechanisms, which initiate apoptosis, can fail; and fourth, the execution of apoptotic cell death may be prevented by antiapoptotic signals or by defects of apoptosis effectors. A number of studies predominantly performed in cell lines and xenograft models have suggested a correlation between glutathione and metallothionein levels and cisplatin resistance as well as between various export pumps, such as the
Platinum-Refractory Testicular Germ Cell Tumors
ABC transporters and the lung resistance protein, and cisplatin resistance. However, validation on clinical tumor samples is generally lacking. Investigations of the role of the NER, which is thought to be the most important DNA repair mechanism for cisplatin-induced damage, indicate a low capacity of GCT cells for NER. The low intrinsic capacity of the NER demonstrated in GCT cell lines has been attributed to low levels of xeroderma pigmentosum complementation group A protein (XPA) and the NER protein ERCC1. Alternatively, it has been proposed that the DNA adducts could be concealed by testisspecific high mobility group (HMG)-box proteins preventing damage detection and repair by NER factors. The finding of a low NER capacity itself and its potential clinical relevance have not been confirmed in samples from patients with GCTs, yet it is conceivable that a low NER activity contributes to the overall chemosensitivity of GCTs. A high level of wild-type p53 in GCTs has commonly been regarded as the biological explanation for GCT chemosensitivity. Two studies investigating the role of p53 in refractory GCT have demonstrated that p53 mutations are rare even after treatment, that these mutations are unlikely to be the cause for chemotherapy resistance, and that the inactivation of p53 does not lead to a sensitivity change to chemotherapy in vitro. These findings suggest that induction of apoptosis in testis cancer cells can be executed even independent from p53, which questions the previously suggested important role of p53 in cisplatin resistance. A high incidence of microsatellite instability (MSI) was found in samples of patients with refractory GCT. In most refractory cases, MSI was found in several loci, whereas in unselected GCT, only very few patients had a MSI and most of them just in one locus. MSI in refractory GCT indicates that defects in DNA mismatch repair pathway may represent a clinically relevant resistance mechanism. Other proteins involved in the regulation of apoptosis, such as BAX, BCL-2, BCL-XL, and others, have also been investigated, but no single factor seemed to correlate with treatment response. More research is necessary to conclusively define the role for antiapoptotic regulators
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downstream of the initiation of apoptosis in chemotherapy resistance of GCT. Unfortunately, most models described earlier are based predominantly on in vitro analyses of cell lines and largely lack confirmation of their relevance in clinical material. Treatment of Patients with CisplatinRefractory GCTs The treatment of patients with cisplatin-refractory disease remains a challenge due to the limited number of effective treatment options. Treatment for these patients remains largely noncurative and palliative. Drugs with No or Minor Activity in Refractory GCTs
A large number of different agents have been evaluated in patients with refractory testicular cancer, mostly based on a promising preclinical activity in cell lines or xenograft models (Table 1). However, the vast majority of these agents could not demonstrate any meaningful clinical activity, despite a well-received preclinical rationale and results (Table 1). Active Agents
Only four chemotherapy agents, oral etoposide, paclitaxel, gemcitabine and oxaliplatin, have thus far demonstrated clinical activity in these patients (Table 2). These agents were tested based on preclinical results indicating activity in germ cell cancer cell lines or tumor models as well as based on the lack of cross-resistance with cisplatin. Toxicity with all these single-agent therapies is acceptable. Given as single-agent therapy, all of these treatments yield response rates of approximately 20%. In all these studies, single patients were long-term survivors. Over the past years, these agents have been combined in combination chemotherapy regimens. The main objective of these studies was proof of feasibility of combination chemotherapy in these intensively pretreated patients as well as to improve the outcome for this particular patient population. Response rate and survival served as outcome measures in these trials. Achieving a high response rate in refractory patients is important since the induction of a remission may
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Platinum-Refractory Testicular Germ Cell Tumors
Platinum-Refractory Testicular Germ Cell Tumors, Table 1 Agents without or minor clinical activity in patients with refractory germ cell cancer (Modified according to Kollmannsberger et al. 2006) Author, year Williams et al. (1983) Williams et al. (1985) Drasga et al. (1987) Harstrick et al. (1990) Hoskins et al. (1990) Murphy et al. (1992) Stoter et al. (1992) Bokemeyer et al. (1993) Motzer et al. (1993) Moasser et al. (1995) Puc et al. (1995) Kollmannsberger et al. (2000) Kollmannsberger et al. (2002) Kondagunta et al. (2004) Rick et al. (2006)
Agent Amsacrine Mitoxantrone Iproplatin Epirubicin Mitomycin C Iproplatin Epirubicin Vinorelbine Suramin All-trans retinoic acid Topotecan Bendamustine Irinotecan Temozolomide Thalidomide
No. Patients 6 14 14 16 7 15 18 7 14 16 15 19 15 14 15
Responses 0/6 0/14 1/14 1/16 2/7 0/14 0/18 0/7 0/14 0/14 0/14 1/19 0/15 0/14 0/15
Platinum-Refractory Testicular Germ Cell Tumors, Table 2 Results of chemotherapy in selected studies for patients with refractory or multiple relapsed GCTS (Modified according to Kollmannsberger et al. 2006)
Author, year Motzer (1994) Bokemeyer (1996) Bokemeyer (1999) Einhorn (1999) Kollmannsberger (2002) Hinton (2002) Miki (2002) Kollmannsberger (2004) Pectasides (2004) Theodore (2004) Pectasides (2004) Einhorn et al. (2007)
Treatment Paclitaxel Paclitaxel
No. Patients 31 24
Patients pretreated with HD-CT 16% 50%
Cisplatinrefractory disease 76% 75%
Response rate (confidence interval) 26% [95%-CI: 3–29] 25% [95%-CI: 10–47%]
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All patients “resistant” defined as failure to achieve a durable complete remission to a cisplatin-based regimen and one or more of the following unfavorable prognostic features: relapse after CR or progression within 4 weeks after first-line therapy; poor or no response to prior conventional dose cisplatin/ifosfamide therapy; extragonadal primary site Refractory being defined as a disease progression during or within 2 months after cisplatin-based chemotherapy n.s. not stated
Platyfish-Swordtail Melanoma
subsequently allow the resection of residual masses and may thus be a chance to still achieve long-term survival in selected patients. The first study which gave proof of the feasibility and activity of combination chemotherapy in these heavily pretreated patients was a phase II study investigating the combination of paclitaxel and gemcitabine. Twenty-one percent of patients responded and single long-term survivors were observed. A subsequent study which used paclitaxel/gemcitabine as treatment regimen after failure of salvage high-dose chemotherapy with autologous stem cell support found a response rate of 31%. Again, some long-term survivors were seen. Two phase II studies examined the activity and toxicity of a combination consisting of oxaliplatin and gemcitabine in refractory germ cell cancer patients. These studies demonstrated again the feasibility of combination of chemotherapy in this heavily pretreated patient population. Both studies reported high response rates of 46% and 32% with a number of patients surviving long term. These response rates are the highest published to date. Other tested combinations include paclitaxel/oxaliplatin and irinotecanbased combinations such as irinotecan/oxaliplatin or irinotecan/cisplatin. Hematological toxicity is usually the main side effect in these extensively pretreated patients. Some trials have investigated the use of biological agents for the treatment of refractory or relapsed GCTs. None of the tested agents including suramin, an antitrypanosomal drug, all-trans-retinoic acid (ATRA), or thalidomide have shown activity. Novel molecular targets are also being explored now in germ cell cancer, including agents targeting the VEGF and EGF pathway. Preclinical experiments have suggested an important role for these pathways in germ cell cancers.
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markers after cisplatin-based chemotherapy or patients relapsing after salvage chemotherapy (sometimes referred to as “desperation surgery”). Approximately, 20–25% of patients may remain disease-free after a complete resection of all tumor manifestations. The complete resection of residual masses is the most crucial factor for success in this setting. The complete resection of residual masses also appears to be an important step toward long-term disease-free survival in cisplatin-refractory patients who achieve a remission on salvage chemotherapy. Most long-term survivors in trials investigating novel agents had surgery subsequent to a chemotherapy response as part of their salvage treatment for refractory disease. Surgery alone or surgery after salvage chemotherapy should therefore always be considered for selected patients with cisplatin-refractory disease, particularly in patients presenting with localized and potentially completely resectable recurrences or with completely resectable masses after response to salvage chemotherapy.
References Albers P, Ganz A, Hannig E et al (2000) Salvage surgery of chemorefractory germ cell tumors with elevated tumor markers. J Urol 164:381–384 Kollmannsberger C, Nichols C, Bokemeyer C (2006) Recent advances in management of patients with platinum-refractory testicular germ cell tumors. Cancer 106:1217–1226 Looijenga LH, Oosterhuis JW (2002) Pathobiology of testicular germ cell tumors: views and news. Anal Quant Cytol Histol 24:263–279 Mayer F, Honecker F, Looijenga LH et al (2003) Towards an understanding of the biological basis of response to cisplatin-based chemotherapy in germ-cell tumors. Ann Oncol 14:825–832 Porcu P, Bhatia S, Einhorn LH (2000) Results of treatment after relapse from high-dose chemotherapy in germ cell tumors. J Clin Oncol 18:1181–1186
Salvage Surgery
Salvage surgery remains an important treatment option for chemotherapy-refractory patients, in particular for patients with limited and potentially respectable metastases. A number of studies have investigated the role of surgery for patients with persistently elevated
Platyfish-Swordtail Melanoma ▶ Xiphophorus
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Pleiotrophin Evangelia Papadimitriou Laboratory of Molecular Pharmacology, Department of Pharmacy, School of Health Sciences, University of Patras, Patras, Greece
Synonyms HARP; HBBM; HB-GAM; HBGF-8; HBNF; HBNPF; Heparin-affin regulatory peptide; Heparin-binding brain mitogen; Heparin-binding growth factor 8; Heparin-binding growthassociated molecule; Heparin-binding neuritepromoting factor; Heparin-binding neurotropic factor; OSF-1; Osteoblast-specific factor; PTN
Definition Pleiotrophin (PTN) is an 18 kDa growth factor that has high affinity for heparin and together with midkine forms a family of structurally related heparin-binding growth factors. The two proteins share 45% homology in their amino acid sequence and ten perfectly conserved cysteine residues. They also use the same receptors and share many biological activities, the best characterized being neural development and tumor growth.
Characteristics PTN consists of 168 amino acids that are highly conserved across different species, such as human, mouse, rat, bovine, fish, chicken, frog, and insects. The cleavage of the 32 amino acids signal peptide leads to a secreted protein, which consists of 24% of cationic residues, mainly lysines, organized in two clusters at the NH2and COOH-terminal regions. The tertiary structure of PTN is arranged in two beta sheet domains connected with a flexible linker. Each of the domains possibly contains three antiparallel beta strands and possesses one thrombospondin type 1 (TSR-1) homology motif. Although the two
Pleiotrophin
TSR-1 motifs have been implicated in its binding to heparin, data suggest that the carboxyl terminal TSR-1 domain is the main heparin-binding site of PTN. The NH2- and COOH-terminal lysine-rich regions lack a detectable structure and appear to form random coils. PTN interacts with heparan sulfate proteoglycans (HSPGs) and other glycosaminoglycans (GAGs), such as dermatan sulfate and chondroitin sulfate A, and can thus be found located onto the extracellular matrix and the surface of different cell types. GAGs induce PTN dimerization and enhance the mitogenic but not the neurite outgrowth activity of PTN. PTN binds N-syndecan, a HSPG implicated in the neurite outgrowth activity of PTN. Another receptor for PTN may be ▶ anaplastic lymphoma kinase (ALK), which is a transmembrane ▶ tyrosine kinase receptor of a 200 kDa molecular mass, expressed by many types of cancer cells. There are numerous data questioning a direct interaction of PTN with ALK and favouring a possible indirect activation. The most studied PTN receptor is receptor protein-tyrosine phosphatase beta/zeta (RPTPbeta/ zeta), which seems to be responsible for the migratory responses of neuronal, endothelial, and ▶ cancer cells to PTN. RPTPbeta/zeta directly interacts with alpha v beta 3 integrin on the cell surface and alpha v beta 3 is required for the stimulatory effect of PTN on cell migration through RPTPbeta/zeta. The same pathway is also implicated in both PTN- and vascular endothelial growth factor 165 (VEGF165)-induced cell surface localization of ▶ nucleolin, which is also a PTN receptor over-expressed in activated endothelial and many cancer cells. The signaling pathways activated by PTN are not fully elucidated; up to date PTN is known to activate ▶ c-src, ▶ focal adhesion kinase, ▶ phosphatidylinositol 3-kinase, Akt, and ▶ extracellular signal-regulated kinases ½. Little is still known on the regulation of PTN expression. PTN transcription is directly stimulated by the transcription factors HOXA5 and activator protein-1. PTN protein levels are also increased by cAMP, ▶ platelet-derived growth factor BB, ▶ hypoxia, serum, hydrogen peroxide, members of the fibroblast growth factor family,
Pleiotrophin
tumor necrosis factor alpha, epidermal growth factor, endothelial nitric oxide synthase and interferon gamma and down-regulated by VEGF165. Numerous biological activities have been attributed to PTN. The first and most prominent is stimulation of neurite outgrowth and a role in the growth and maturation of brain. PTN also induces proliferation of several types of cells, is involved in bone remodelling and, participates in normal spermatogenesis, and regulates the expansion and regeneration of hematopoietic stem cells. A role of PTN in human ▶ cancers was first suggested after purification of the growth factor from conditioned media of the highly malignant ▶ breast cancer cell line MDA-MB-231. Screening of various human tumor cell lines and tumor specimens of different origin revealed that PTN is expressed in many types of cancer, such as ▶ astrocytomas, ▶ melanomas, meningiomas, ▶ neuroblastomas, choriocarcinomas, leukemias, and cancers of ▶ pancreas, ▶ prostate, stomach, colon, ovary, and ▶ lung. PTN receptors are also upregulated in a plethora of tumors and are being tested as targets for anticancer therapy. Regarding the biological activity of PTN in cancer, there is ample evidence that it is a tumorpromoting factor. This is supported by data showing that overexpression of PTN in human embryonic kidney cell line 293 and human adrenal carcinoma cell line SW-13 leads to autonomous growth in soft agar and formation of tumors in athymic nude mice. Moreover, the secreted PTN stimulates proliferation of fibroblast, endothelial, and epithelial cells. PTN purified from lung cancer cell lines also stimulates the growth of fibroblast and endothelial and SW-13 epithelial cells. In the same line, overexpression of the bovine PTN cDNA in NIH 3 T3 cells results in a transformed phenotype, as judged by increased cell numbers at confluence, anchorageindependent growth, and tumor formation in nude mice. In the same line, deletion of the tumor suppressor gene PTEN leads to upregulation of PTN, which seems to participate in tumorigenesis caused by PTEN loss. Inhibition of PTN expression in several types of cancer cells inhibits colony formation of the cells, decreases angiogenicity, and prevents tumor
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growth in mice. For example, human breast cancer MDA-MB-231 cells transfected with a mutant cDNA that encodes a truncated form of PTN fail to form plaques or colonies in soft agar and are unable to form tumors in athymic nude mice. Stable transfection of human melanoma WM852 cells that express high levels of PTN mRNA, with PTN-targeted ribozymes, quenches production of PTN, inhibits colony formation of the cells, and prevents tumor growth in mice. In another highly metastatic human melanoma cell line 1205Lu, transfection with PTN-targeted ribozymes decreases tumor growth and ▶ angiogenesis in nude mice. A replication-deficient recombinant adenovirus generated to express antisense PTN at high efficiency induces transcripts that completely inhibit PTN protein production and decrease melanoma cell growth in soft agar and SCID mice. The downregulation of PTN in these cells coincides with the downregulation of the cell cycle regulator cyclin E and the upregulation of the cell cycle inhibitor p21WAF1/Cip1. PTN-targeted ribozymes have been used to deplete PTN mRNA from Colo357 ▶ pancreatic cancer, choriocarcinoma, and glioma cells. Reduction of PTN results in a decrease in the proliferation rate, soft agar colony formation, and tumor growth in nude mice. In human prostate LNCaP cells, antisense PTN expression decreases cell ▶ migration, as well as anchorage-dependent and anchorage-independent growth, and abrogates the stimulatory effects of ▶ hydrogen peroxide and fibroblast growth factor 2 on LNCaP cell proliferation and migration. In the same line, antisense PTN expression in human prostate cancer PC3 cells induces apoptosis and decreases cell migration and tumor growth and metastasis in vivo. Besides a direct effect of PTN on tumor cells, there is also a plethora of reports indicating a positive correlation between PTN and in vivo or in vitro angiogenesis, a key step in the progress of many tumors. Numerous in vitro studies demonstrate that PTN is involved in the control of endothelial cell migration, proliferation, and differentiation into tube-like structures. The angiogenic potential of PTN in vivo has been shown in the chicken embryo chorioallantoic
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membrane assay and in mice using matrigel implants. Moreover, PTN expression by monocytes/macrophages leads to downregulation of their monocytic cell markers and upregulation of endothelial cell characteristics, thus inducing the transdifferentiation of monocytes into functional endothelial cells. PTN is found to colocalize with capillaries of human atherosclerotic plaques and may serve to recruit stromal tissue and blood supply to the expanding tumor. Culture supernatants derived from a PTN-transfected human adrenal carcinoma cell line (SW-13) were shown to possess mitogenic activities for fetal bovine heart endothelial cells and human umbilical vein endothelial cells (HUVEC), making PTN a candidate tumor angiogenesis factor. In the same line, PTN purified from lung cancer cell lines stimulates the proliferation of HUVEC in vitro, and ribozyme targeting of PTN mRNA, which is constitutively expressed in the human melanoma cell line 1205Lu, reduces the number of vessels in the primary tumor and the metastatic spread of these cells in athymic nude mice. PTN-transfected MCF-7 human breast carcinoma cells are mitogenic for HUVEC in vitro and strongly angiogenic in vivo, in mice xenografts, and in the rabbit corneal assay. Endothelial cell functions in vitro and angiogenesis in the chicken embryo chorioallantoic membrane in vivo induced by culture medium of human ▶ prostate cancer LNCaP cells were also inhibited when PTN expression was diminished and angiogenesis in prostate cancer PC3 xenografts in vivo was diminished when PTN expression was down-regulated. Finally, in MCH66 murine mammary tumor cells, which metastasize depending only on tumor angiogenesis, PTN could be regarded as the only plausible candidate for this activity. In contrast to a stimulating effect of PTN on tumor growth and angiogenesis, it has also been suggested that overexpression of PTN in NIH 3 T3 cells may be implicated in cellular quiescence rather than an oncogenic phenotype. In several cases, PTN has been identified as a confluence-specific protein, secreted by normal cells but not cells transformed by ▶ ras or other ▶ oncogenes. High PTN expression is associated with poor vasculature in neuroblastomas, and
Pleiotrophin
HOXA5 that induces apoptosis of breast cancer cells and inhibits angiogenesis directly activates PTN transcription. PTN directly binds VEGF165 leading to inhibition of VEGF165-induced endothelial cell proliferation, migration, and tube formation on matrigel. This negative regulatory effect is attributed to both thrombospondin type I repeats of PTN, which are located in the b-sheet domains of the molecule and are responsible for the binding to VEGF165. PTN and VEGF165 also compete for binding to RPTP beta/zeta and this may be another mechanism through which PTN regulates the angiogenic effects of VEGF165. The identification of PTN domains responsible for its angiogenic and transforming activities is considered important, and the data existing so far suggest distinct or even opposite effects for different PTN regions. The last 25 amino acids of the C-terminal region are considered important for the binding to ALK, RPTPbeta/zeta, and integrin alpha v beta 3 and for the angiogenic effect of PTN. The last 43 amino acids of the same region enhance plasminogen activator activity and decrease the plasminogen activator inhibitor levels. An angiogenic role for the residues 65–136 as well as peptides that correspond to both the NH2 and COOH domains of the molecule has also been suggested. Proteolysis of PTN by plasmin results in the production of five peptides with distinct activities on endothelial cell activation in vitro or angiogenesis in vivo. PTN is also a substrate for trypsin or chymotrypsin and matric metalloproteinase 2, and the hypothesis that PTN is a possible substrate for several proteolytic enzymes, which may control its angiogenic or/and tumorigenic potential, is reinforced by the presence of proteolytic forms of PTN in media from endothelial or tumor cells. In addition to being a therapeutic target, PTN may also be an attractive circulating growth factor for prognostic monitoring. In mice, serum PTN levels increase as a function of tumor size. In humans, elevated serum PTN levels are measured in patients with pancreatic or colon cancer, but not in patients with stomach cancer. In both mice and humans, serum PTN levels drop after successful tumor removal. Elevated PTN serum levels are also measured in 30% of patients with chronic
Pleomorphic Xanthoastrocytoma
pancreatitis, a disease that frequently precedes pancreatic cancer. Moreover, PTN expression increases from 7% observed in normal tissue to 34% in inflammatory and 67% in pancreatic cancer tissues. Finally, PTN may represent a promising new diagnostic marker for ▶ testicular cancer with high sensitivity in early-stage disease. In summary, PTN exhibits important biological activities in cancer and its expression or/and expression of PTN receptors at high levels may play a crucial regulatory role in many tumors of diverse origin. Clarifying the exact role of PTN in diverse tumor types could lead to the development of therapeutic tools that can be used alternatively or in addition to existing therapies to control tumor growth, metastasis and/or resistance.
Cross-References ▶ Anaplastic Lymphoma Kinase ▶ Angiogenesis ▶ Astrocytoma ▶ AP-1 ▶ Breast Cancer ▶ Cancer ▶ Colorectal Cancer Clinical Oncology ▶ Extracellular Signal-Regulated Kinases 1 and 2 ▶ Fibroblast Growth Factors ▶ Focal Adhesion Kinase ▶ Hydrogen Peroxide ▶ Hypoxia ▶ Lung Cancer ▶ Migration ▶ Neuroblastoma ▶ Nucleolin ▶ Oncogene ▶ RAS Genes ▶ Receptor Tyrosine Kinases ▶ Pancreatic Cancer ▶ PI3K Signaling ▶ Platelet-Derived Growth Factor ▶ Prostate Cancer ▶ Prostate Cancer Clinical Oncology ▶ SRC ▶ Testicular Cancer ▶ Vascular Endothelial Growth Factor
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References Mikelis C, Koutsioumpa M, Papadimitriou E (2007) Pleiotrophin as a possible new target for angiogenesis-related diseases and cancer. Recent Pat Anticancer Drug Discov 2:175–186 Papadimitriou E, Mikelis C, Lampropoulou E, Koutsioumpa M, Theochari K, Tsirmoula S, Theodoropoulou C, Lamprou M, Sfaelou E, Vourtsis D, Boudouris P (2009) Roles of pleiotrophin in tumor growth and angiogenesis. Eur Cytokine Netw 20:180–190 Pantazaka E, Papadimitriou E (2012) PTN (pleiotrophin). Atlas Genet Cytogenet Oncol Haematol 16:821–837 Pantazaka E, Papadimitriou E (2014) Chondroitin Sulfatecell membrane effectors as regulators of growth factormediated vascular and cancer cell migration. BBA General Subjects, 1840:2643–2650
See Also (2012) AKT. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 115. doi:10.1007/978-3-642-16483-5_163 (2012) Chondroitin sulfate. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 821–822. doi:10.1007/978-3-642-16483-5_1118 (2012) Glycosaminoglycans. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1570. doi:10.1007/978-3-642-16483-5_2453 (2012) Heparan sulfate proteoglycans. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1647. doi:10.1007/978-3-642-16483-5_2638 (2012) HOXA5 gene. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1739. doi:10.1007/978-3-642-16483-5_2820 (2012) Leukemia. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2005. doi:10.1007/978-3-642-16483-5_3322 (2012) Midkine. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2313. doi:10.1007/978-3-642-16483-5_3743 (2012) N-Syndecan. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2567. doi:10.1007/978-3-642-16483-5_5622 (2012) Pancreas. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 2762– 2763. doi:10.1007/978-3-642-16483-5_7055 (2012) Receptor protein tyrosine phosphatase beta/zeta. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3198. doi:10.1007/9783-642-16483-5_4979
Pleomorphic Xanthoastrocytoma ▶ Astrocytoma
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Pleural Effusion
Pleural Effusion Juliana Guarize1 and Lorenzo Spaggiari2 1 Department of Thoracic Surgery, European Institute of Oncology, Milan, Italy 2 University of Milan School of Medicine, Milan, Italy
Synonyms Malignant pleural Thoracentesis
effusion;
Pleurodesis;
Definition Pleural effusion is the presence of excessive fluid in the pleural cavity. The pleura is a two-sheet, serous membrane that covers the chest wall, mediastinum (parietal pleura), and lungs (visceral pleura). Between the parietal and visceral pleura, there is a virtual space filled with a small liquid pellicle with a high daily turnover which permits sliding. Daily fluid production is about 0.01–0.02 mL kg1 h1 being continuously absorbed in a way that remaining the pleural fluid is about 0.1–0.2 mL kg1. Fluid movement and absorption are supported by a balance between plasmatic and pleural pressures (hydrostatic and oncotic pressures) and thoracic lymphatic drainage. Pleural effusions occur because of an upset in these mechanisms that reabsorb the fluid normally present in the pleural space.
Characteristics Pleural effusion is a common clinical problem seen at the advanced stage of several malignant diseases and is called ▶ malignant pleural effusion (MPE). The physiopathology of a malignant pleural effusion includes vascular and lymphatic alterations due to a direct invasion of the pleura by a primary tumor or a metastasis. These pleural
changes affect the normal reabsorptive flow of fluid from parietal to visceral pleura and can even cause an increased capillary leaking and increased fluid production. A malignant effusion can be present whatever the direct neoplastic involvement of the pleura as a clinical consequence of a malignancy or its treatment (radio or chemotherapy, cachexia, thromboembolism, pneumonia, and others), and in these cases it is called paramalignant effusion. An indirect increased capillary permeability, increased hydrostatic pressure, a decreased oncotic pressure, increased negative intrapleural pressure (as in cases of atelectasis), and decreased or blocked lymphatic drainage are the final organic alterations responsible by fluid overload. The main causes of MPE are reported with different incidence rates between the literature series. Widely, lung cancer is the most common cause of malignant pleural effusion in men. In women, breast cancer seems to be the first cause, followed by lung cancer and genital tract cancer. Other related causes of MPE are lymphoma, gastric cancer, and mesothelioma. In about 10% of malignant effusions, no primary tumor is identified. Table 1 shows main neoplasms related to pleural effusion. Clinical Manifestations and Diagnosis The most common related symptom is dyspnea (shortness of breath or difficult or labored breathing). Other symptoms are cough, chest discomfort, and pain. Many patients with pleural
Pleural Effusion, Table 1 Causes of malignant pleural effusion Male Lung cancer Lymphoma Gastrointestinal tract Genitourinary tract Melanoma Less common tumors Primary unknown
Female Breast cancer Female genital tract Lung cancer Lymphoma Gastrointestinal tract Melanoma Less common tumors Primary unknown
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Pleural Effusion, Fig. 2 CT scan disclosing massive pleural effusion with mediastinal shift and lung atelectasis Pleural Effusion, Fig. 1 Chest radiogram showing a large right pleural effusion due to breast cancer
effusions are asymptomatic, especially if a small pleural effusion is present. Physical examination is poor in cases of small effusions or should reveal decreased chest expansion, dullness to percussion, and reduced breath sounds in cases of a larger effusion. A simple chest radiography is the first diagnostic approach. It can confirm the presence of an effusion and other parenchymal alterations. Small pleural effusions could not be seen in a simple chest radiogram, but larger pleural effusions can cause a complete opacification of the hemithorax. Figure 1 shows a chest radiogram with a large right pleural effusion. When a massive pleural effusion is present there are symptoms of a tension hydrothorax with mediastinal shift, severe dyspnea, and hemodynamic instability (Fig. 2). Loculated effusions are difficult to diagnose in a simple chest radiogram because it is difficult to distinguish from other pulmonary parenchymal process such as atelectasis and consolidation. Ultrasound and CT scan are useful diagnostic means, especially in cases of small or loculated effusions and can determine the precise site for thoracentesis. Thoracentesis Thoracentesis is useful as a preliminary “looking for” diagnosis and can play a therapeutic role.
The fluid analysis permits distinguishing an exudative from a transudative effusion. Transudative effusion is a plasma filtrate with the same characteristics and results of an elevated hydrostatic pressure or increased capillary permeability. Exudative effusion is a rich protein fluid resulting from a local pleural inflammation or obstructed lymphatic drainage or both, and it is common in neoplasm. The fluid analysis includes the cell count (total and differential), total protein count, lactate dehydrogenase (LDH), glucose level, pH, amylase, and cytology. Usually MPE is an exudative effusion that is characterized by serum ratio of protein greater than 0.5 (relation between pleural and plasmatic protein level), LDH ratio greater than 0.6 (relation between pleural and plasmatic LDH level), or the absolute LDH pleural level greater than 2/3 of the upper normal limit for serum level and low glucose level. Other analyses include cholesterol, triglycerides (when a chylothorax is suspected), and ADA (adenosine deaminase), helpful to differentiate neoplastic from tuberculous effusion. A hemorrhagic fluid is highly correlated to a malignant effusion. The fluid analysis also permits to identify neoplastic cells and some direct and indirect tumor markers.
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Pleural Effusion
Pleural Effusion, Fig. 3 Thoracentesis of hemorrhagic pleural effusion
Some tumor markers such as ▶ CEA and CA 15-3 may improve the diagnostic value of cytology. High fluid levels of CEA are seen in pulmonary squamous cell carcinoma and pulmonary adenocarcinoma. Differently, high levels of CA 15-3 are observed in breast cancer. Chromosomal and tumor growth factors analyses are still being investigated as a worth diagnostic mean. When an exudative effusion is present and the etiology is not clear, thoracoscopy and pleural biopsy have been described as the most rational diagnostic approaches. Treatment The goal of the treatment is the resolution of the effusion and its recurrence prevention. Secondary treatment endpoints are minimizing patient’s symptoms and reducing hospital stay. Therapeutic Thoracentesis
Therapeutic thoracentesis can immediately provide relief from dyspnea and other acute symptoms, but it has an elevated recurrence rate (as high as 98–100% Fig. 3). Thoracentesis is indicated in symptomatic patients. There is no absolute contraindication for thoracentesis. Relative contraindications are bleeding disorders, anticoagulation, and mechanical ventilation. Complications related to thoracentesis include pneumothorax, hemothorax, pain, and vasovagal
reaction (with bradycardia, low systemic blood pressure, and reduced conscious level) during the procedure. Reexpansion pulmonary edema is a rare complication during a thoracentesis. In MPE, repeated thoracentesis can be indicated in patients with a slow reaccumulation rate (more than one month) and a poor expected survival. For patients with a rapid reaccumulation rate, an indwelling catheter (as known as tunneled pleural catheter) seems to be a better treatment option. Tunneled Pleural Catheter
The placement of tunneled pleural catheter is indicated in patients with recurrent and symptomatic MPE, particularly in cases of trapped lung with poor response to chemical pleurodesis. Tunneled catheters allow patients to lead an independent life outside the hospital with intermittent drainage of the fluid. Complications are not frequent and include: reaccumulation of the effusion, loculations within the pleural space, empyema, and cellulitis at the catheter insertion site. Pleurodesis
Instillation of sclerosing agents in the pleural space causes an inflammatory process with a fusion between parietal and visceral pleura. A successful pleurodesis provides complete lung expansion and stops the fluid collection. Pleurodesis can be performed with chemical agents or mechanical abrasion of the parietal pleura.
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Pleural Effusion, Fig. 4 One-port videoassisted thoracoscopy (VATS) for pleural biopsy and talc poudrage
Asbestos-free talc has been the most established and diffused method for the management of MPE. It can be directly instilled in the pleural space with a chest tube or via a thoracoscopic procedure. Chemical pleurodesis with a chest tube is less effective than pleurodesis with a video-assisted thoracoscopic surgery (VATS). Pleurodesis with VATS (Fig. 4) has a very high efficacy in terms of effusion control, and it is the preferred approach in patients with a prolonged survival (more than 6 months) and a good performance status. The advantage of this method is that it offers a possibility of a uniform distribution of the talc in a direct view and high accuracy pleural biopsy. Talc is usually well tolerated with few side effects. Most common ones are fever and pleuritic pain. Other described complications are acute respiratory distress syndrome (ARDS) and talc
pneumonitis. These complications are rare and appear to be related with larger doses of talc (>5 g). Other chemical agents have been described as sclerosing agents. Bleomycin sulfate is the most used antineoplastic drug for pleurodesis. Described side effects are fever, chest pain, and nausea. The drug has a limited systemic absorption, and the efficacy is about 70–80% with a high cost. Tetracycline was frequently used in the past as a sclerosing agent with widely different results between patients’ series. Other agents as interferons (alpha and beta) and interleukins (IL2) have showed controversial results in the literature. Mechanical Pleurodesis
Thoracoscopic mechanical pleurodesis has a similar effusion control rate when compared with talc
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pleurodesis. It has, however, more complications. The most common complications are prolonged lung deflation, bleeding, subcutaneous emphysema, and wound inflammation. Other Treatment Options
Surgical pleurectomy and decortication are options with very high morbidity and mortality. It requires thoracotomy with frequent complications and prolonged hospital stay. It is usually reserved in cases of MPE related to malignant pleural mesothelioma. Pleuroperitoneal shunting is an alternative for patients who have intractable symptomatic pleural effusions that have failed chemical pleurodesis or are not candidates for surgery. Shunting insertion is well tolerated in minithoracotomy or thoracoscopic procedure. Complications related to this technique are shunt occlusion, infection, and tumor implant. Chemotherapy The treatment of the underlying malignancy is not effective in the control of the MPE mostly of the cases. Exceptions include pleural effusions related to lymphoma, breast cancer, small cell carcinoma of the lung, germ cell tumors, prostate cancer, and ovarian cancer.
Cross-References ▶ Carcinoembryonic Antigen
References Gasparri R, Leo F, Veronesi G et al (2006) Video-assisted management of malignant pleural effusion in breast carcinoma. Cancer 106:271–276 Rusch VW (2002) Pleural effusion: benign and malignant. In: Thoracic surgery, 2nd edn. Churchill Livingstone, New York, pp 1157–1170 Terracciano D, Di Carlo A, Papa P et al (2004) New approaches in the diagnostic procedure of malignant pleural effusions. Oncol Rep 12:79–83 Villena V, Lopez-Encuentra A, Echave-Sustaeta J et al (2003) Diagnostic value of CA 549 in pleural fluid. Comparison with CEA, CA 15–3 and CA 72–4. Lung Cancer 40:289–294
Pleurodesis
See Also (2012) Malignant pleural effusion. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2149. doi:10.1007/978-3-642-164835_3516 (2012) Pleural biopsy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2923. doi:10.1007/978-3-642-16483-5_4624 (2012) Pleuroperitoneal shunting. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2927. doi:10.1007/978-3-642-164835_4628 (2012) Pneumothorax. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2932. doi:10.1007/978-3-642-16483-5_4649 (2012) Thoracentesis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3672. doi:10.1007/978-3-642-16483-5_5782
Pleurodesis ▶ Pleural Effusion
Plexin Ligands ▶ Semaphorin
Plexins William P. J. Leenders Department of Pathology, Radboud University Medical Center Nijmegen, Nijmegen, The Netherlands
Synonyms PLXN; Semaphorin receptors
Definition Plexins are large, transmembrane receptors with structural homology in their extracellular domains to the ▶ scatter factor receptor, c-MET. The
Plexins
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Out
Cell membrane Rac GTP In GTP Plexin A
B
C
Rho
D
Repulsion
Attraction
Plexins, Fig. 1 Structural domains of plexin family members: four subfamilies have been identified, plexin A–D. Blue boxes indicate Sema domains; yellow boxes represent Met-related sequence (MRS) motifs. The red box indicates an atypical MRS motif of plexin D1 which distinguishes it from the other plexin family members. Plexin B subfamily members have a potential ▶ furin-like
proteolytic site, marked by a gray ribbon. Two intracellular conserved domains are shown as ovals. In the family of B plexins, RhoA and Rac small GTPase binding sites are activated upon semaphorin binding, resulting in repulsion (growth cone collapse) or attraction (filopodia formation and extension), respectively
family of plexins consists of four groups, plexin A, B, C, and D, each with several subgroups. The plexins differ mostly from each other in their extracellular domains. These ectodomains are typically composed of a ▶ semaphorin-like or Sema domain, followed by two or three Met-related sequences (MRS). The Met-related sequences of class B plexins show the highest similarity to c-Met. The intracellular parts of the plexins are highly conserved during evolution and are characterized by two conserved regions, known as the SEX-plexin (SP) domain (Fig. 1).
PLXND1-Sema3E. Interactions of plexins with the class 3 semaphorins are mostly mediated by the neuropilins, but exceptions to this rule exist. For example, Sema3E binds directly to PLXND1 in the absence of neuropilins. The neuropilins are also receptors for the larger variants of the angiogenic factor vascular endothelial growth factor-A (VEGF-A), and binding of class 3 semaphorins to neuropilin/plexin complexes can be competed for by VEGF-A, resulting in antagonistic effects toward each other. Neuropilins are non-signaling receptors that occur in complex with VEGF receptor 2 (VEGFR2) on angiogenic endothelial cells and enhance binding of VEGF-A to this receptor (Guttman-Raviv et al 2006). Since several classes of plexins are also expressed on endothelial cells during ▶ angiogenesis, it is likely that multicomponent complexes of neuropilins, plexins, and VEGF receptors exist. This is further supported by the finding that an association between PLXNA1 and VEGFR2 (and Off-track) is required for Sema6D signaling. Thus, it is likely that a delicate balance between semaphorins and
Characteristics Plexin-associated molecules: Plexins are receptors for the membrane-bound neuropilins and semaphorins, which exist both as secreted and membrane-bound forms. Identified receptor-ligand pairs are PLXNA1-Sema3A, PLXNA1-Sema6D and PLXNB1-Sema4D, PLXNB3-Sema5A, and PLXNC1-Sema7A, PLXND1-Sema3C, and
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VEGF-A provides a fine-tuning mechanism to regulate angiogenesis. Class B plexins have been found in a complex with c-Met, the receptor for hepatocyte growth factor (HGF), and Ron, the receptor for macrophage-stimulating protein (MSP). These proteins are also known as the scatter factor receptors, and c-Met is critically involved in invasive growth (cell scattering) during development but also during tumor cell ▶ invasion and ▶ metastasis. Except for direct phosphorylation upon binding of scatter factor/HGF, c-Met is also directly activated upon binding of sema4D to a PLXNB1/ c-Met complex, resulting in an invasive cell phenotype. Taken together, most plexins exist in multicomponent receptor complexes, and the outcome of activation of these complexes is determined by a concerted action of different growth factors. Plexin Function Initially, neuropilins and semaphorins were discovered as determinants of patterning of the central nervous system. Most work has been done on the class 3 semaphorins which act via the plexin A family as chemorepulsive cues, i.e., growth factors and ▶ chemokines that repel cells, during axonal growth (Carmeliet and Tessier-Lavigne 2005). There are distinct similarities between development of nerves and vessels. Both display directed growth and branching morphogenesis in an orderly fashion. In this respect it is perhaps not surprising that factors that are involved in axon guidance also play a role in angiogenesis (Oh and Gu 2013). A first indication for this came from studies, in which expression of plexin D1 was demonstrated in developing vasculature during mouse embryogenesis (van der Zwaag et al. 2002). A later study showed that plxnd1 was essential for angiogenesis: plxnd1 knockout mice die within 1 day after birth due to cardiovascular defects. Maldevelopment of the vasculature was also observed in plxnd1 loss of function mutants of zebrafish. Evidence is emerging now that plexins play distinct roles not only during developmental angiogenesis but also during tumor angiogenesis.
Plexins
At the moment an involvement in tumor biology of the B and D plexins is most clear. Therefore, these two family members will be dealt with in more detail. Plexins in Tumor Biology Plexin D1
The occurrence of PLXND1 on developing vasculature has resulted in research toward a potential role of this family member during angiogenesis in tumor development: angiogenesis is critically involved in growth and metastasis of solid tumors, and plexin D1 is prominently expressed in angiogenic tumor blood vessels. Plexin D1 is also expressed on tumor cells in a wide variety of human tumors (Roodink et al. 2005). Semaphorins 3C and 3E are ligands for PLXND1. Whereas Sema3C binding to PLXND1 requires the involvement of neuropilins, this interaction is not needed for Sema3E binding. Sema3C seems to be involved in chemoresistance of tumor cells whereas sema3E has been identified from array analyses as a protein, implicated in tumor progression and metastasis. Whereas plexin D1 may trigger a signaling pathway toward apoptosis, this pathway is inhibited by sema3E binding, suggesting that a sema3E/pexin D1 axis is essential for survival of cancer cells (Luchino et al. 2013). These findings point at plexin D1 and semaphorins as attractive therapeutic, but also diagnostic targets. Especially the fact that plexin D1 is expressed by activated tumor vessels and cancer cells will allow simultaneous targeting of multiple tumor compartments, e.g., for targeted drug delivery. The ▶ signal transduction pathways activated by plexin D1 have not yet been elucidated, but studies on other plexins suggest that active cytoskeletal rearrangements are induced upon plexin D1 activation via the small RhoGTPases. Whereas activation of the Rac RhoGTPase leads to lamellipodia formation and extension of cellular processes, the RhoA GTPase results in the retraction of cell processes via depolymerization of F-actin. Plexin D1 contains a consensus Rac-binding site as well as two conserved arginines that in PLXNA1 are crucial for
Plexins
semaphorin-mediated cellular collapse. It is not known which conditions favor Rac or RhoA activation (Kruger et al. 2005). It is clear though that sema3E-plexin D1 signaling is involved in cellular migration, cancer cell invasion and metastasis (Casazza et al. 2010). B Plexins
Another plexin that is expressed on both endothelial cells and tumor cells is plexin B1, a receptor for semaphorin 4D. It has been demonstrated that plexinB1 is present in a complex with the scatter factor receptors Met and Ron. These two tyrosine kinase receptors are implicated in the invasive growth program of tumors. C-Met is the prototype receptor for hepatocyte growth factor (HGF) and is, upon activation by this ligand, phosphorylated on two tyrosines that are docking sites for a number of signal transduction molecules such as phosphatidyl inositol 3-kinase (PI3K), SRC, GRB2, GAB1, and signal transducer and activator of transcription 3 (STAT3). C-Met is also associated with integrins, adhesion molecules that modulate cellular interactions with extracellular matrix components. C-Met is a prototype oncogene that upon constitutive activation results in malignant tumors. Constitutive activation may be the result of activating mutations, but overexpression of c-Met in itself may cause receptor oligomerization and phosphorylation. Sema4D binding to plexin B1 in the complex also results in tyrosine phosphorylation of c-Met. Furthermore, endogenous overexpression of plexin B1 in epithelial cancers results in constitutive activation of c-Met. Thus, the B plexins may play an important role in c-Met activation and tumorigenesis. Intracellular signaling from a PLXNB1/ Sema4D complex has been elucidated to a large extent. Upon ligand binding, PlexinB1 interacts at its carboxyterminal domain with leukemiaassociated rho guanine exchange factor (LARG), an exchange factor for the small GTPase Rho. Also binding of PDZ-Rho-GEF has been demonstrated. Plexins have also been shown to activate r-RAS, resulting in downregulation of integrinmediated adhesion to the extracellular matrix. The resulting loss of cell adhesion is implicated
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in invasive growth (Trusolino and Comoglio 2002). In summary, plexins constitute a family of receptor proteins which occur in multicomponent complexes on the cell membrane. Evidence for an important role in tumor biology is emerging in the last years. Multiple ligands for the plexins have been identified, and intracellular signaling pathways initiated by plexin activation result in cytoskeletal rearrangements which may lead to extension but also collapse of cellular processes, events that are indispensable for cellular migration.
Cross-References ▶ Apoptosis ▶ Cancer ▶ Chemotherapy ▶ MET ▶ Metastasis ▶ Migration ▶ Rho Family Proteins ▶ Semaphorin ▶ Signal Transducers and Activators of Transcription in Oncogenesis ▶ Signal Transduction ▶ Targeted Drug Delivery
References Carmeliet P, Tessier-Lavigne M (2005) Common mechanisms of nerve and blood vessel wiring. Nature 436:193–200 Casazza A, Finisguerra V, Capparuccia L, Camperi A, Swiercz JM, Rizzolio S, Rolny C, Christensen C, Bertotti A, Sarotto I, Risio M, Trusolino L, Weitz J, Schneider M, Mazzone M, Comoglio PM, Tamagnone L (2010) Sema3E-Plexin D1 signaling drives human cancer cell invasiveness and metastatic spreading in mice. J Clin Invest 120(8):2684–98 Guttmann-Raviv N, Kessler O, Shraga-Heled N et al (2006) The neuropilins and their role in tumorigenesis and tumor progression. Cancer Lett 231:1–11 Kruger R, Aurandt J, Guam KL (2005) Semaphorins command cells to move. Nat Rev Mol Cell Biol 6:789–800 Luchino J, Hocine M, Amoureux MC, Gibert B, Bernet A, Royet A, Treilleux I, Lécine P, Borg JP, Mehlen P, Chauvet S, Mann F (2013) Semaphorin 3E suppresses tumor cell death triggered by the plexin D1 dependence
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3628 receptor in metastatic breast cancers. Cancer Cell 24 (5):673–85 Oh WJ, Gu C (2013) The role and mechanism-of-action of Sema3E and Plexin-D1 in vascular and neural -development. Semin Cell Dev Biol 24:156–162 Pasterkamp J, Kolodkin A (2003) Semaphorin junction: making tracks toward neural connectivity. Curr Opin Neurobiol 13:79–89 Roodink I, Raats J, van der Zwaag B et al (2005) Plexin D1 expression is induced on tumor vasculature and tumor cells: a novel target for diagnosis and therapy? Cancer Res 65:8317–23 Trusolino L, Comoglio PM (2002) Scatter-factor and semaphorin receptors: cell signalling for invasive growth. Nat Rev Cancer 2:289–300 Tseng CH, Murray KD, Jou MF, Hsu SM, Cheng HJ, Huang PH (2011) Sema3E/plexin-D1 mediated epithelial-to-mesenchymal transition in ovarian endometrioid cancer. PLoS One 6(4):e19396 van der Zwaag B, Hellemons AJ, Leenders WP, et al (2002) PLEXIN-D1, a novel plexin family member, is expressed in vascular endothelium and the central nervous system during mouse embryognesis. Dev Dyn. 225:336–43
Plexopathy ▶ Peripheral Neuropathy
PLG61 ▶ Epidermal Growth Factor Receptor
PLP ▶ Parathyroid Hormone-Related Protein
PLXN ▶ Plexins
PMP ▶ Pseudomyxoma Peritonei
Plexopathy
Podoplanin Andreas Wicki1 and Gerhard Christofori2 1 Department of Medical Oncology, University Hospital, Basel, Switzerland 2 Department of Biomedicine, University of Basel, Basel, Switzerland
Synonyms Aggrus; E11 antigen; gp36; gp38; OTS-8; PA2.26; RANDAM-2; RTI40; T1a2
Definition Human podoplanin is a type-1 transmembrane glycoprotein consisting of 162 amino acids, nine of which form the intracellular domain. The extracellular domain is extensively O-glycosylated. Depending on glycosylation, the molecular mass is between 36 and 45 kDa. Podoplanin is physiologically expressed in kidney podocytes, skeletal muscle, placenta, lung, heart, myofibroblasts of the breast and salivary glands, osteoblasts and mesothelial cells, and on the apical surface of alveolar type I cells. Occasionally, focal expression of podoplanin can be found in circumscribed areas of the basal layer of the human epidermis. Podoplanin is also expressed in lymphatic endothelium, but not in blood vessels. Pathological expression of podoplanin is observed in many human cancers, in particular squamous cell carcinomas.
Characteristics In tumors, podoplanin is expressed by the cancer cells themselves, where it is involved in tumor progression. In addition, it is expressed in lymphatic endothelial cells and serves as an immunohistochemical marker for ▶ lymphatic vessels.
Podoplanin
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Podoplanin Expression in Human Tumors The expression of podoplanin is upregulated in testicular carcinoma in situ and in many invasive human cancers, in particular squamous cell carcinomas of the skin, larynx, lung, cervix, mouth, and esophagus, as well as invasive tumors of germinal cells and the central nervous system. Podoplanin expression has also been reported in mesothelioma and several human sarcomas, but not in adenocarcinomas such as colorectal or prostate cancer. Podoplanin can either be selectively expressed on the outer edge of the tumor mass or diffusely throughout the cancerous tissue (Fig. 1). The expression of podoplanin in a single cell layer at the tumor surface is most often observed in squamous cell carcinoma, whereas sarcomas tend to express podoplanin more diffusely. Clinical studies indicate that the expression of podoplanin in human cancer may positively correlate with tumor progression and in some instances a poor prognosis (e.g., malignant astrocytoma of the brain). The physiological role of podoplanin remains in great parts unknown. Podoplanin-deficient mice die at birth owing to respiratory failure and exhibit a phenotype of alveolar hypoplasia, dilated malfunctioning lymphatic vessels, and lymphoedema. In addition, podoplanin has an
extracellular platelet aggregation-stimulating domain and is therefore able to promote hemostasis. Regulation The podoplanin gene promoter is characterized by the absence of a consensus TATA and CAAT box, the presence of multiple Sp1 binding sites, and a high GC-content. This promoter structure is mostly found in ubiquitously expressed or growth-related genes. In human sarcoma cell lines, the basal transcription of podoplanin is regulated by the transcription factors Sp1 and Sp3 and presumably by other not yet identified factors. In lymphatic endothelium, podoplanin is an early responder to Prox-1, a master regulator of lymphatic vessel formation. In human carcinoma cells, upregulated expression of podoplanin is observed upon treatment of cells with EGF, FGF-2, TNF-a, or bradykinin. The expression of podoplanin is increased in mouse skin during tissue regeneration after wounding or by treatment with the carcinogen phorbol-12-myristate-13-acetate. Podoplanin expression is also induced by 12-Otetradecanoylphorbol-13-acetate in mouse osteoblastic cells, and it is constitutively expressed in oncogenic Ras-transformed cells.
P Tumor
Lymphatic vessels
Podoplanin, Fig. 1 Histological section of a human squamous cell carcinoma. The cells of the tumor bulk express E-cadherin (brown staining) and form an invading conus which protrudes into the surrounding tissue. Podoplanin (red staining) is expressed by cells of the
Invasive front
invasive front and by the lymphatic endothelium (as indicated by the arrows). Size bar = 100 mm (Microphotograph courtesy of D. Kerjaschki, MUW, Vienna)
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Podoplanin and Tumor Invasion Podoplanin induces tumor invasion in vitro and in vivo. Transfection of podoplanin into human cancer cells usually results in increased spreading of the cells on ▶ fibronectin, a component of the extracellular matrix. Podoplanin also enhances cell migration and invasion through a collagen IV containing basal membrane-like substrate. The enhanced migration is accompanied by a strong polarization of the cells. Depending on the cell of origin, podoplanin promotes collective or single cell invasion. During collective cell invasion, epithelial cancer cells remain attached to each other, since they continue to express ▶ E-cadherin, a cell adhesion molecule required for the formation of epithelial adherens junctions. In a transgenic mouse model of ▶ insulinoma, podoplanin was shown to shift the invasion pattern from single to collective cell invasion. Podoplanin also mediates single cell invasion upon loss of E-cadherin (e.g., in MDCK cells, an epithelial canine kidney cell line). Single cell invasion and loss of E-cadherin are often associated with ▶ epithelial-mesenchymal transition (EMT), a phenomenon that also occurs during embryogenesis, for example, during neurulation or gastrulation. During both collective and single cell invasion, podoplanin promotes phosphorylation of ▶ ERM-proteins, in particular ezrin. Upon phosphorylation, ezrin associates with the cell membrane and reorganizes the actin cytoskeleton. The expression of podoplanin also affects cell morphology. Stress fibers, which are often found in quiescent cells, are lost and filopodia are formed. Both cell migration and membrane motility are increased, and the formation of multiple microspikes of the cell membrane is induced. Podoplanin modulates the activity of the family of Rho GTPases, in particular RhoA, Cdc42, and Rac. The modulation of RhoA signaling can directly translate into an increased cell movement. However, depending on the cell type, migration and invasion are induced by either up- or downregulation of distinct Rho GTPases. Podoplanin and Metastasis The extracellular portion of podoplanin contains a platelet aggregation domain. Indeed, the
Podoplanin
expression of podoplanin on circulating tumor cells increases the formation of thrombotic cancer cell emboli, thereby increasing the efficiency of metastasis formation. In a mouse model, acetylsalicylic acid inhibits platelet aggregation and reduces the incidence of metastasis after intravenous injection of podoplanin-expressing Chinese hamster ovary (CHO) cells. Thus, podoplanin may be involved in the transport of tumor cell-platelet clusters in the bloodstream. Podoplanin as a Marker for Tumor-Associated Lymphatic Vessels Podoplanin is expressed in lymphatic endothelial cells, but not in blood endothelium. Therefore, podoplanin is frequently used as a selective marker for lymphatic vessels. Together with Lyve-1, another lymphatic endothelium specific marker, it is employed to visualize and quantify lymphatic vessels. Tumor-associated lymphatic endothelial cells are thought to be involved in intralymphatic transport of cancer cells, and the lymphatic microvessel density is important for prognosis. For example, the density of lymphatic vessels is an independent prognostic factor for the prediction of melanoma metastasis and patient survival. Conclusions Podoplanin promotes tumor invasion and metastasis formation in both carcinoma and sarcoma. However, in carcinoma in situ and in regenerating epithelium, its role has remained elusive. Alone or together with Lyve-1, podoplanin is an important marker for the assessment of lymphatic microvessel density in tumors.
Cross-References ▶ E-Cadherin ▶ Epithelial-to-Mesenchymal Transition ▶ ERM Proteins ▶ Fibronectin ▶ Insulinoma ▶ Lymphangiogenesis ▶ Lymphatic Vessels
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References
Podosomes and Invadopodia Hantusch B, Kalt R, Krieger S et al (2007) Sp1/Sp3 and DNA-methylation contribute to basal transcriptional activation of human podoplanin in MG63 versus Saos-2 osteoblastic cells. BMC Mol Biol 8:20 Martin-Villar E, Megias D, Castel S et al (2006) Podoplanin binds ERM proteins to activate RhoA and promote epithelial-mesenchymal transition. J Cell Sci 119:4541–4553 Schacht V, Dadras SS, Johnson LA et al (2005) Up-regulation of the lymphatic marker podoplanin, a mucin-type transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors. Am J Pathol 166:913–921 Wicki A, Christofori G (2007) The potential role of podoplanin in tumour invasion. Br J Cancer 96:1–5 Wicki A, Lehembre F, Wick N et al (2006) Tumor invasion in the absence of epithelial-mesenchymal transition: podoplanin-mediated remodeling of the actin cytoskeleton. Cancer Cell 9:261–272
See Also (2012) Carcinoma. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 657. doi:10.1007/978-3-642-16483-5_848 (2012) Collective Cell Invasion. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 896. doi:10.1007/978-3-642-164835_1262 (2012) Lyve-1. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2128. doi:10.1007/978-3-642-16483-5_3473 (2012) Microspikes. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2308. doi:10.1007/978-3-642-16483-5_3733 (2012) Outer Edge of the Tumor Mass. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2670. doi:10.1007/978-3-642-164835_4292 (2012) Rho. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3302. doi:10.1007/978-3-642-16483-5_5099 (2012) Sarcoma. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3335. doi:10.1007/978-3-642-16483-5_5161 (2012) Single Cell Invasion. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3412. doi:10.1007/978-3-642-164835_5311 (2012) Thrombotic Cancer Cell Emboli. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3682. doi:10.1007/978-3-642-164835_5797 (2012) Tumor Progression. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3800. doi:10.1007/978-3-642-16483-5_6046
▶ Invadosome
POEMS Syndrome Synonyms Crow-Fukase syndrome, Takatsuki disease or PEP syndrome
Definition Is a rare medical syndrome named for its main clinically recognizable features: Polyneuropathy (peripheral nerve damage), Organomegaly (abnormal enlargement of organs), Endocrinopathy (damage to hormone-producing glands)/Edema, M-protein (a monoclonal immunoglobulin produced by plasma cells) and Skin abnormalities, such as hyperpigmentation and hypertrichosis.
Cross-References ▶ Plasmacytoma
Pol I Transcription ▶ RNA Polymerase I Transcription
Poly(ADP-Ribosyl)ation Alexander Bürkle Department of Biology, University of Konstanz, Konstanz, Germany
Definition Poly(ADP-ribosyl)ation is a dynamic posttranslational modification of glutamate, aspartate, or
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Poly(ADP-Ribosyl)ation
protein
O O OH OH O O NH2
O P O N
O
O
O P O NH2
O
O
O
OH
O P O OH
O
OH N
n x
protein
N
OOH OH O
NH2 O
N
O P O
NAD+
N
N
O
O
N
N
N
N
O O P O
O OH
NH2
O P O
OH
O
OH HO
N
O
O O
N
N
O
O O
O
O
NH2
n x
n
OH
HO O
N
NH2 N
N N
O
O P O
O P O
O
O O P O
N
branch
NH2 N
O P O
N
O
O
O
O
OH O
N N
OH
O
poly(ADP-Ribose)
Poly(ADP-Ribosyl)ation, Fig. 1 Schematic representation of the structure of poly(ADP-ribose) (Reprinted from Mangerich and Bürkle 2012)
lysine residues of proteins, with NAD+ serving as a precursor (Fig. 1), and represents an immediate eukaryotic cellular response to DNA damage as induced, e.g., by ionizing radiation, alkylating agents, or oxidants (Bürkle and Virág 2013; Hottiger 2015). Poly(ADP-ribosyl)ation is catalyzed mostly by the 113-kD enzyme poly (ADP-ribose) polymerase-1 (PARP-1; proposed nomenclature: ARTD1). A number of additional polypeptides are also able to catalyze poly (ADP-ribosyl)ation, and sequence homology searches have revealed several different gene loci in the human genome that comprise the so-called PARP signature sequence. The other members of the PARP protein family include PARP-2 (ARTD2), PARP-3 (ARTD3), vPARP (ARTD4), tankyrase-1 (ARTD5), and tankyrase2 (ARTD6), and collectively they may account for up to 25% of the total cellular poly(ADP-ribose) production. The major enzyme catalyzing the catabolism of poly(ADP-ribose), by hydrolyzing the ribose-ribose linkages in the polymer, is poly
(ADP-ribose) glycohydrolase (PARG). The single PARG locus has been shown to encode several polypeptides arising from differential splicing and displaying differential subcellular localization. Additional proteins involved in poly(ADP-ribose) catabolism are ADPribosylhydrolase 3 (ARH3) and terminal ADPribose protein glycohydrolase (TARG1/ C6orf130).
Characteristics Catalytic Function of PARP-1 and Life Cycle of Poly(ADP-Ribose) PARP-1 has been detected in most eukaryotes and displays a characteristic and highly conserved domain structure, which can be further broken down into subdomains. While the protein is constitutively and abundantly expressed in all proliferating and some non-proliferating cells, its catalytic activity is stimulated dramatically by
Poly(ADP-Ribosyl)ation Poly(ADP-Ribosyl)ation, Fig. 2 Life cycle of poly (ADP-ribose). PARP-1, poly(ADP-ribose) polymerase-1; PARG, poly (APD-ribose) glycohydrolase
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Alkylating agents ionizing radiation reactive oxygen species UV-B apoptosis
DNA strand breaks
Binding and activation of PARP-1 ATP NAD+ Mono-ADPribosylation of proteins Cyclic ADP-ribose
Poly(ADP-ribose) PARG Nicotinamide
O -acetylADP-ribose
ADP-ribose
Free ADP-ribose
DNA single-strand or double-strand breaks. Its DNA-binding domain, which is located at the amino-terminus, binds to single- or double-strand breaks in DNA via two zinc fingers. Binding of PARP-1 to broken DNA induces an immediate and dramatic activation of the catalytic center residing in the carboxy-terminal NAD+ -binding domain of the enzyme. PARP-1 is catalytically active as a dimer. In intact cells the enzyme itself is the major target protein (“acceptor”) for covalent modification with poly(ADP-ribose). This “automodification” reaction is thought to occur mostly on a specific domain located between the DNA-binding and the NAD+-binding domain, respectively. However, in living cells a large number of additional acceptor proteins have been identified (Daniels et al. 2015). The half-life of poly(ADP-ribose) is very short under conditions of DNA breakage, due to rapid degradation by PARG and other catabolic enzymes. Hence, the existence of poly(ADP-ribose) in intact cells is highly dynamic and transient. The life cycle of poly(ADP-ribose) is depicted schematically in Fig. 2. A number of molecular functions for PARP-1 and/or poly(ADP-ribose) have been proposed, and the following list is by no means exhaustive. It must be emphasized that many of the claims are based on work on subcellular systems, and the relevance for the in vivo situation is not known.
Molecular Functions Related with Regulation of DNA Strand Breaks and of DNA Repair
• Direct control of the activity of DNA-processing enzymes (DNA polymerases, ligases, topoisomerases, etc.) by their covalent modification with poly(ADP-ribose). • Poly(ADP-ribosyl)ation of histones as a mechanism to induce localized chromatin relaxation, thus allowing access of DNA repair enzymes to the damaged site. • Poly(ADP-ribose) as a “histone shuttle”; highaffinity non-covalent binding of (unmodified) histones to automodified PARP-1 should lead to localized chromatin relaxation, thus allowing access of DNA repair enzymes to the damaged site. • PARP-1 as a component of a multi-protein complex carrying out DNA base-excision repair; such a protein complex would also comprise XRCC1, DNA polymerase-b, and DNA ligase III. PARP-1 would detect DNA strand breaks at the original lesion site and could recruit the other partners. PARP-1 automodification might serve to regulate formation or function of such a complex. • Physical and functional interaction of PARP-1 and poly(ADP-ribose) with the double-strand break-associated proteins DNA-dependent protein kinase (DNA-PK) and “ataxia telangiectasia mutated” (ATM).
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• Contribution of PARP-1 to the back-up pathway of non-homologous end joining (bNHEJ) of DNA double-strand breaks. • Signaling of DNA damage through non-covalent binding of poly(ADP-ribose) with p53, p21WAF, etc. Molecular Functions Related with the Maintenance of Genomic Stability
• Protection of “open” DNA strand breaks by PARP-1 binding in order to prevent nonspecific DNA degradation and/or undesirable recombination. PARP-1 automodification might then serve to weaken the interaction with DNA, thus allowing repair activities to proceed. It should be noted that a primary role of PARP-1 and/or poly(ADP-ribose) in DNA repair (see above) would of course indirectly contribute to the maintenance of genomic stability under conditions of genotoxic stress. • Regulatory function of PARP-1 at the centrosome, thus preserving cellular euploidy. • Stabilizing function of poly(ADP-ribose) at the mitotic spindle. • Involvement of several PARP family members in telomere function. • Physical interaction and functional cooperation of PARP-1 with the Werner syndrome protein (WRN). Molecular Functions Related with DNA Replication
• PARP-1 as a component of a multi-protein DNA replication complex comprising enzymes for leading and lagging-strand DNA synthesis, some of which are potential targets for poly(ADP-ribosyl)ation (DNA polymerase-a, RPA, topoisomerase I, PCNA, DNA ligase I). • PARP-1 as a mediator of replication fork reversal under conditions of replicative stress (Ray Chaudhuri et al. 2012). Molecular Functions Related with Gene Expression
• Poly(ADP-ribosyl)ation as a regulator of chromatin compaction and DNA methylation status. • PARP-1 as a transcriptional cofactor for B-MYB and for AP2 (independent of the catalytic function of PARP-1)
Poly(ADP-Ribosyl)ation
• PARP-1 as a cofactor for NFkB-mediated transactivation. • PARP-1 as interaction partner of transcription factors such as with retinoid X receptors (RXR), p53, Oct-1, PC1, E47, TEF-1, and DF1–4. Molecular Functions Related with Energy Metabolism and Mitochondrial Changes
• Massive poly(ADP-ribose) synthesis leading to NAD+ depletion and/or release of apoptosis-inducing factor (AIF) from mitochondria and consequently cell death. Cellular Functions Depending on the intensity of DNA damage inflicted to the cells and the cellular proliferation/differentiation status, PARP-1 has two contrasting functions: Cytoprotection and Maintenance of Genomic Stability
To study the cellular function of poly (ADP-ribosyl)ation, various strategies have been employed to abrogate it. These include competitive low molecular weight ▶ PARP inhibitors, PARP-1 siRNA, or Parp1 gene disruption in the mouse germ line. The results have consistently revealed that poly(ADP-ribosyl)ation significantly contributes to the recovery of proliferating normal and malignant cells from low-level DNA damage as induced, e.g., by alkylating agents or ionizing radiation, both in vivo and in cell culture. This effect has been linked mechanistically with an involvement of PARP-1 in DNA base-excision repair. Furthermore, there is clear evidence that poly(ADP-ribosyl)ation counteracts the induction of genomic instability by DNA damage, as assessed by several biological markers such as chromosomal aberrations, sister-chromatid exchange, gene amplification, or mutagenesis. In addition, fibroblasts from Parp1-deficient mice display morphological abnormalities and reduced growth rate. These protective functions of PARP1, as well as its role in maintaining telomere length in mice, are in line with correlative data showing an association of mammalian and human longevity with high cellular poly(ADP-ribosyl) ation capacity.
Poly(ADP-Ribosyl)ation
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Cell Death Induction
the execution phase of apoptosis, representing one of the most frequently used biochemical markers of apoptosis. This cleavage is thought to abrogate the responsiveness of PARP-1 to DNA strand breaks and thus to limit NAD+ consumption. PARP-2 also undergoes caspase-mediated cleavage during apoptosis.
In stark contrast to the cytoprotective function mentioned above, PARP-1 overactivation may lead to cell suicide due to severe and irreversible depletion of NAD+ and consequently of ATP pools (Fig. 2). By comparing Parp1-deficient and wild-type mice and derived cells and by using competitive PARP inhibitors, such a cytotoxicity mechanism has been identified to be operative in several non-proliferating cell types. These include (1) pancreatic islet cells exposed to relevant DNA-damaging compounds (reactive oxygen species, nitric oxide metabolites, streptozotocin), (2) neurons after regional ischemia-reperfusion damage of the brain (known to induce widespread release of reactive oxygen species and nitric oxide in the affected area, thus leading to the loss of many neurons and to brain infarct), and (3) dopaminergic neurons exposed to 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP; a drug known to induce the release of reactive oxygen species only in the dopaminergic neurons of the substantia nigra, thus leading to selective neuronal death and Parkinson syndrome). In each case it was shown that the cells from Parp1-deficient mice were highly protected from cell death and the animals displayed increased resistance to clinical disease. Likewise, administration of PARP inhibitors in vivo led to prevention of cell death in post-ischemic heart and skeletal muscle and renal tubular cells. Data reveal that, apart from NAD+ depletion, cell death by active PARP-1 can also be mediated by poly(ADP-ribose) itself. It was shown that the polymer can leave the nucleus and then trigger the release of apoptosis-inducing factor (AIF) from the mitochondria. AIF, in turn, translocates to the nucleus and induces high molecular weight DNA fragmentation and caspase-independent cell death. It should be noted that classical ▶ apoptosis is associated with dramatic changes concerning the poly(ADP-ribosyl)ation system. After an initial burst of cellular poly(ADP-ribose) formation early in apoptosis, there is a well-documented proteolytic cleavage of PARP-1 into two fragments by activated caspase-3/caspase-7 during
Clinical Relevance Genetic Cancer Risk Assessment
Genetic polymorphisms and any resulting functional polymorphisms in cellular proteins involved in recognition and processing of DNA damage can be expected to contribute to the genetic risk profile of an individual with regard to the development of organ-specific cancers or cancer in general. The active human PARP1 gene locus on chromosome 1q42 displays several polymorphisms, such as a polymorphic dinucleotide repeat located in the promoter region and base substitutions in the coding region. Interestingly, the V762A polymorphism in PARP-1 leads to reduced enzymatic activity of PARP-1 and is associated with increased risk of lung, prostate, and esophageal cancer. These observations are perfectly in line with the positive role PARP-1 activity plays in DNA repair and maintenance of genomic stability. According to this, a diminished enzyme activity based on a less active genetic variant can be expected to be limiting for the efficiency of the clearance of DNA damage and thus increase the risk of damage accumulation and genomic instability, which will act as a driving force for carcinogenesis. Therapy
For several decades cytotoxic tumor therapy, as delivered by the application of cytostatic drugs or ionizing radiation, has been the most frequently administered form of cancer therapy. The sensitizing effect of PARP inhibitors in proliferating cells undergoing sublethal ▶ DNA damage has long been considered as a potentially useful mechanism to render conventional cytotoxic treatment regimens more effective at eradicating tumor cells in the body. Available data from cell culture
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Polyamines
experiments suggest that the most suitable cytotoxic agents to be combined with PARP inhibitors would be methylating agents, bleomycin, and ionizing radiation, but not antimetabolites. Therefore, large numbers of novel PARP-inhibitory compounds have been synthesized and characterized, some of which act in submicromolar (rather than millimolar) concentrations. Animal work revealed that combination treatment of tumors with the alkylating agent temozolomide and PARP inhibitors increases the efficacy of temozolomide. Interestingly, it was reported that tumor cells with nonfunctional BRCA1 or BRCA2 proteins can be sensitized to cell death by mere PARP inhibition in the absence of any DNA-damaging treatment (reviewed in: Mangerich and Bürkle 2011). Apparently DNA single-strand breaks arising spontaneously at rather low frequency will accumulate in under these conditions and are converted into double-strand breaks during DNA replication. As BRCA proteins are crucial for the signaling and proper repair of double-strand breaks by homologous recombination, repair is blocked and cells die as a result of the accumulating double-strand breaks. The possible clinical use of this kind of “synthetic lethality” is currently explored in a number of clinical phase 1 or 2 trials and could form the basis for a highly selective future cancer chemotherapy protocol, largely devoid of the severe side effects of classical cytostatic agents, in patients with BRCA1- or BRCA2-deficient tumors. The underlying principle is that the PARP inhibitor would kill the tumor cells, yet spare nonmalignant cells as those would retain at least one functional copy of these genes.
Cross-References ▶ Apoptosis ▶ BRCA1/BRCA2 Germline Breast Cancer Risk ▶ DNA Damage ▶ PARP Inhibitors
References Bürkle A, Virág L (2013) Poly(ADP-ribose): PARadigms and PARadoxes. Mol Aspects Med 34:1046–65 Daniels CM, Ong SE, Leung AK (2015) The promise of proteomics for the study of ADP-ribosylation. Mol Cell 58:911–24 Hottiger MO (2015) Nuclear ADP-ribosylation and its role in chromatin plasticity, cell differentiation, and epigenetics. Annu Rev Biochem 84:227–63 Mangerich A, Bürkle A (2011) How to kill tumor cells with inhibitors of poly(ADP-ribosyl)ation. Int J Cancer 128:251–65 Mangerich A, Bürkle A (2012) Pleiotropic cellular functions of PARP1 in longevity and aging: genome maintenance meets inflammation. Oxid Med Cell Longev 2012. Article ID 321653, 19. doi:10.1155/2012/ 321653 Ray Chaudhuri A, Hashimoto Y, Herrador R, Neelsen KJ, Fachinetti D, Bermejo R, Cocito A, Costanzo V, Lopes M (2012) Topoisomerase I poisoning results in PARP-mediated replication fork reversal. Nat Struct Mol Biol 19:417–23
See Also (2012) AP2. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 230. doi:10.1007/978-3-642-16483-5_342 (2012) Poly(ADP-Ribose) Polymerase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/ Heidelberg, p 2935. doi:10.1007/978-3-642-164835_4655 (2012) Telomere. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3637. doi:10.1007/978-3-642-16483-5_5716
Polyamines Gaetano Marverti Department of Biomedical Sciences, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy
Definition
Mutations
and
The polyamines putrescine (1,4-diaminobutane), spermidine (1,8-diamino-4-azaoctane), and spermine (1,12-diamino-4,9-diazadodecane, 3) are ubiquitous polycationic compounds that are found in significant amounts in nearly every
Polyamines
prokaryotic and eukaryotic cell type. Spermidine and spermine primarily exist in aqueous solution at pH 7.4 as fully protonated polycations, thus able to interact with negatively charged molecules, such as DNA, RNA, proteins, or phospholipids. Polyamines are widely distributed in nature and are known to be required in micromolar to millimolar concentrations to support a wide variety of cellular functions, mostly linked with cell growth, survival, and proliferation. Polyamines are important players in plant growth, stress, longevity, and disease resistance, but they are also involved in diseases, for example, Alzheimer or infectious diseases. The main research area for the involvement of polyamines in diseases is cancer, as high levels of polyamines are observed in cancer cells.
Characteristics Polyamine Metabolism and Transport The regulation of polyamine levels is achieved by a combination of synthesis, catabolism, and transport. The three main sources for polyamines in organisms are food intake, cellular synthesis, and microbial synthesis in the gut. Polyamines are synthesized from the amino acids arginine, ornithine, and methionine. The first step in the pathway is the production of ornithine from arginine by the mitochondrial enzyme arginase. Ornithine is then decarboxylated by ornithine decarboxylase (ODC) to produce putrescine (Fig. 1). ODC expression is tightly regulated from transcription to posttranslational modifications. ODC antizyme (AZ) directly inhibits ODC activity and is also responsible for facilitating ODC degradation by targeting it to the 26S proteasome. In parallel to putrescine production, L-methionine is converted into S-adenosylL-methionine (AdoMet), which is then decarboxylated by AdoMet decarboxylase (AdoMetDC) to produce decarboxylated AdoMet (DcAdoMet). DcAdoMet is then used as an aminopropyl group donor either to putrescine by spermidine synthase to produce spermidine or to spermidine to produce spermine by spermine synthase.
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The polyamines spermine and spermidine are synthesized from arginine. Arginase converts arginine to ornithine, and ornithine decarboxylase (ODC) catalyzes decarboxylation of ornithine to form putrescine, a polyamine precursor containing two amine groups. ODC, a rate-limiting enzyme with a short half-life, is inhibited by antizyme (AZ), and antizyme is inhibited by an antizyme inhibitor (AZI). AZ also inhibits polyamine uptake. ODC is also inhibited by the enzyme-activated, irreversible inhibitor DFMO, DL-a-difluoromethylornithine. S-adenosylmethionine decarboxylase (AdoMetDC) is involved in the decarboxylation of Sadenosylmethionine. Spermidine synthetase and spermine synthase are constitutively expressed aminopropyltransferases that catalyze the transfer of the aminopropyl group from decarboxylated Sadenosylmethionine to putrescine and spermidine to form spermidine and spermine, respectively, and MTA, 50 -methylthioadenosine. Polyamine degradation is achieved by spermine/spermidine N1acetyltransferase (SSAT) and N1-acetylpolyamine oxidase (APAO). In addition, spermine oxidase (SMO) specifically oxidizes spermine. Polyamines are transported across the membrane by the polyamine transporter (Soda 2011). The higher polyamines spermidine and spermine can be converted back to putrescine (Fig. 1). The rate-limiting enzyme of polyamine catabolism is the cytosolic spermidine/spermine N1-acetyltransferase (SSAT). SSAT acetylates both spermine and spermidine. Acetylated spermine and spermidine then move into the peroxisome where they are oxidized by N1-acetylpolyamine oxidase (APAO). Byproducts of this oxidation include hydrogen peroxide (H2O2) and acetamidopropanal. SSAT is absolutely necessary for the formation of putrescine from spermidine. Spermine can also be back-converted into spermidine by spermine oxidase (SMO) in the cytoplasm. In contrast with APAO, the preferred substrate of SMO is spermine itself and not its acetylated derivative, acetylspermine. Eukaryotic polyamine uptake is known to be an energy-dependent and carrier-mediated process and is critical in maintaining cellular
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Polyamines
Polyamines, Fig. 1 Polyamine metabolism. The polyamines spermine and spermidine are synthesized from arginine. Arginase converts arginine to ornithine, and ornithine decarboxylase (ODC) catalyzes decarboxylation of ornithine to form putrescine, a polyamine precursor containing two amine groups. ODC, a rate-limiting enzyme with a short half-life, is inhibited by antizyme (AZ), and antizyme is inhibited by an antizyme inhibitor (AZI). AZ also inhibits polyamine uptake. ODC is also inhibited by the enzyme-activated, irreversible inhibitor DFMO, DL-α-difluoromethylornithine. S-adenosylmethionine decarboxylase (AdoMetDC) is involved in
the decarboxylation of Sadenosylmethionine. Spermidine synthetase and spermine synthase are constitutively expressed aminopropyltransferases that catalyze the transfer of the aminopropyl group from decarboxylated Sadenosylmethionine to putrescine and spermidine to form spermidine and spermine, respectively, and MTA, 5’-methylthioadenosine. Polyamine degradation is achieved by spermine/spermidine N1-acetyltransferase (SSAT) and N1-acetylpolyamine oxidase (APAO). In addition, spermine oxidase (SMO) specifically oxidizes spermine. Polyamines are transported across the membrane by the polyamine transporter
polyamine homeostasis. Although much is recognized about polyamine transport system in bacteria, in yeasts, and in plants, no polyamine transporter has been identified in mammals. Alternatively, it is thought that polyamine uptake in
mammals could be performed by endocytosis. The polyamine exporter should exist as separate system from the uptake transporter in human cancer cells. Finally, a diamine transporter has been identified in colon epithelial cells, which could be
Polyamines
responsible for putrescine as well as acetylated polyamine excretion (Soda 2011). Antizyme (AZ), the protein responsible for ODC inhibition and degradation, also enhanced polyamine and acetylpolyamine excretion. Antizymes are small proteins regulated by polyamines that inhibit polyamine biosynthesis and uptake in mammalian cells. In addition, antizyme functions are also regulated by antizyme inhibitors, homologue proteins of ornithine decarboxylase lacking enzymatic activity. There are two antizyme inhibitors (AZIN), known as AZIN1 and AZIN2, that bind to AZs and negate their effects on polyamine metabolism. Antizyme and its endogenous antizyme inhibitor have emerged as prominent regulators of cell growth, transformation, centrosome duplication, and tumorigenesis (Soda 2011). Polyamine Levels and Cancer Cell Growth Polyamines are essential for cell proliferation, and their levels are elevated in many human tumors. ODC and other polyamine metabolic proteins are essential for normal cell and tissue functions, including growth, development and tissue repair. Polyamine biosynthesis is up-regulated in actively growing cells, including cancer cells, therefore polyamine concentration as well as gene expression and activity of enzymes involved in polyamine biosynthesis, especially ODC, are higher in cancer tissues than in normal surrounding tissues. ODC is considered a proto-oncogene product, and a marker of carcinogenesis and tumor progression. Enhanced levels of ODC activity compared with normal tissues have been detected in several solid tumors, such as tissue specimens from patients suffering from prostate cancer, familial adenomatous polyposis, nonmelanoma skin cancer. Moreover, ODC induction and increased levels of polyamines have been associated with breast cancer, etc. Studies on skin carcinogenesis established that ODC is both necessary and sufficient for the onset of tumours in mice (Soda 2011; Rial et al. 2009). Both blood and urine polyamine concentrations are often increased in different cancer patients. Moreover, these levels decrease after tumor eradication and increase after relapse, indicating that polyamines synthesized by cancer tissues are
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transferred to the blood circulation and kidney, where they are excreted into the urine. Polyamines are also produced in other parts of the body and can be transported to various organs and tissues. Polyamines absorbed by the intestinal lumen are distributed to almost all organs and tissues in the body. Since at least two sources of intestinal polyamines are postulated: foods and intestinal microbiota, decrease in blood polyamine levels can be successfully achieved by eliminating intestinal microbiota in addition to restricting food polyamines. To this purpose, the combination of a low-polyamine diet with DFMO, an inhibitor of gut microflora metabolism (metronidazole) and an inhibitor of APAO, had been proposed. Improvements have been observed in a patient with simply a low-polyamine diet alone (Soda 2011; Rial et al. 2009). Polyamine concentrations in the blood vary considerably among healthy individuals. In addition, high levels of polyamines have also been found in diseases other than malignancy, such as psoriasis, diabetes, cystic fibrosis, and muscular dystrophy, after metabolic, mechanical, chemical, and ischemia injury, indicating a limited usefulness of polyamine measurements in body fluids as diagnostic tool for cancer patients. Patients with increased polyamine levels either in the blood or urine have more advanced disease and worse prognosis compared to those with low levels, regardless of the type of malignancy. Because polyamines are essential for cell growth, the increased capability of polyamine synthesis could reflect enhanced tumor proliferation. Therefore, inhibition of polyamine synthesis and availability by cancer cells could retard cancer cell growth. Inhibition of polyamine synthesis by DL-a-difluoromethylornithine (DFMO), an inhibitor of ODC that catalyzes the first rate-limiting step in polyamine biosynthesis, with or without methylglyoxal-bisguanylhydrazone (MGBG), an inhibitor of S-adenosylmethionine (SAM) that is required for polyamine synthesis, successfully suppressed tumor growth and prolonged survival of tumorbearing animals and also successfully suppressed the progression of neoplastic disease in humans. Increased polyamine synthesis appears to be accompanied by cancer invasiveness as ODC
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overexpression enhances the invasive characteristics of cancer cells. In contrast, inhibition of polyamine synthesis by the ODC inhibitor DFMO attenuates the invasive characteristics of cancer cells. Polyamines are necessary for blood vessel development (angiogenesis) occurring in response to damage to normal tissues or tumor growth; thus, increased polyamine synthesis is also accompanied by angiogenesis that is stimulated by cellular production of several factors, including vascular endothelial growth factor. Inhibition of polyamine synthesis by DFMO has been shown to exert its antitumor activity by inhibiting the proliferation of endothelial cells in tumor models. Spermine and spermidine help cancer cells escape immune system detection also by inhibiting the production of tumoricidal cytokines, such as tumor necrosis factor (TNF), and chemokines in vitro, while they do not inhibit production of transforming growth factor beta, which has immunosuppressive properties. ODC overexpression also led to increased phosphorylation of mitogen-activated protein kinase (MAPK), suggesting an interaction between polyamine pathway and MAPK signaling pathway. Along with ODC degradation, antizyme also facilitates degradation of a set of cell cycle regulatory proteins, including cyclin D1, Smad1, and Aurora A kinase, as well as Mps1, a protein that regulates centrosome duplication. Antizyme has been reported to function as a tumor suppressor and to negatively regulate tumor cell proliferation and transformation. Antizyme inhibitor (AZIN) binds to antizyme and suppresses its known functions, leading to increased polyamine synthesis, increased cell proliferation, and increased transformation and tumorigenesis. Gene array studies show antizyme inhibitor to be amplified in cancers of the ovary, breast, and prostate (Soda 2011; Rial et al. 2009). It has been shown that AZIN1 fulfils an essential regulatory function in polyamine homeostasis and cell proliferation since the cellular content of AZIN1 increased transiently after induction of cell proliferation by diluting cells in fresh medium and inhibition of polyamine biosynthesis
Polyamines
induced an even larger increase in the cellular AZIN1 content. This increase was not a consequence of changes in cell cycle progression but appeared to correlate with the cellular depletion of polyamines. Moreover, induced overexpression of AZIN1 resulted in an increased cell proliferation with a concomitant increase in ODC activity and putrescine content. During mitosis, AZIN1 was localized in a pattern that resembled that of the two centrosomes, confirming earlier observations (Silva et al. 2015). Other metabolic enzymes like spermidine synthase and spermine synthase have yet to be convincingly coordinated with tumourigenesis in humans. Increased SMO expression has been demonstrated in inflammatory-associated cancers. For example, the infection of gastric epithelial cells with Helicobacter pylori (H. pylori) upregulates SMO expression and results in H2O2 production, increased DNA damage, and apoptosis. These effects were attenuated by eradication of H. pylori as well as upon treatment with the SMO inhibitor MDL 72,527 or by knockdown of SMO via RNAi. These studies established SMO as a downstream target of H. pylori infection and, consequently, a contributor to the development of gastric cancer (Silva et al. 2015). Furthermore, SMO expression was shown to be elevated in the tissue samples of patients with prostatic intraepithelial neoplasia (PIN) and prostate cancer when compared to normal prostate tissue samples. SMO has also been shown to be increased in ulcerative colitis, another inflammatory condition leading to a high incidence of colon cancer (Nowotarski et al. 2013). The combinations of polyamine synthesis inhibitors appear to be a promising approach to enhance the anticancer activity of chemotherapic drugs against cancer cells. In this regard, the levels of polyamines affect the efficacy of mononuclear platinum anticancer agents for the treatment of many forms of cancers with a potential to improve response to evade resistance development and toxic side effects. BBR3464 is a promising trinuclear platinum anticancer agent, which is a polyamine mimic. The cytotoxic effect of
Polyamines
BBR3464 on LU-HNSCC-4 cells was increased after cells were pretreated with the analog DENSPM or DFMO, and the interaction was found to be synergistic. In contrast, the interaction between cisplatin and DFMO or DENSPM was near-additive to antagonistic. The intracellular levels of the polyamines putrescine and spermidine were decreased after treatment with DFMO, and treatment DENSPM resulted in an increase in putrescine level and concomitant decrease in spermidine and spermine levels. The uptake of BBR3464 was significantly increased after pretreatment of the cells with DFMO and varied dependent on the concentration of DENSPM. The uptake of cisplatin was unchanged (Kjellström et al. 2012). Polyamines seem also to be involved in vitamin K1 antitumor effects on colon cancer cells. Vitamin K1 treatment caused a significant antiproliferative effect and induced apoptosis in human colon cancer cell lines, with the involvement of the MAPK pathway and a significant decrease in the polyamine biosynthesis. Therapeutically, combinations of vitamin K1 with polyamine inhibitors and/or analogues may represent a suitable option for chemoprevention and/or treatment in future strategies for colorectal cancer management (Orlando et al. 2015). Polyamines also play a significant role in the connection between the composition and quality of intestinal flora and the neoplastic transformation of gastric mucosa. Although several evidences are available on the use of mono- or mixed cultures of live microorganisms administered in adequate amounts (probiotics) against the carcinogen Helicobacter pylori, little is still known about the potential cross-interactions among probiotics and cell proliferation. These small amines are required in both preneoplastic and neoplastic tissue to sustain the cell growth, and the evidences suggest that probiotics may act as antineoplastic agents in the stomach by affecting also the polyamine content and functions (Russo et al. 2014). New antitumor strategies could take advantage of the direct relationship between glucose deprivation and polyamine metabolism impairment,
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leading to cell death and its apparent dependence on n-myc. Combined therapies targeting glucose metabolism and polyamine synthesis could be effective in the treatment of n-myc-expressing tumors. Glycolysis inhibition was able to trigger signaling events leading to the reduction of the oncogene N-Myc protein levels, known to potentiate polyamine metabolism. A subsequent decrease of both ornithine decarboxylase expression and polyamine levels, accompanied by cell cycle blockade preceding cell death in neuroblastoma, the most frequent extracranial solid tumor in children, harboring the amplification of n-myc oncogene in 25% of the cases, is associated with treatment failure and poor prognosis (Ruiz-Pérez et al. 2015). Mechanisms of Polyamine Upregulation in Cancer ODC and polyamine content are increased in many cancers arising from epithelial tissues, such as the skin and colon. For example, breast and colon cancer cells have four to six and three to four times the polyamine content of the respective normal cells. Polyamines exert their effects in eukaryotic cells in part by regulating specific gene expression. In murine and human colonic mucosal tissue, ODC is negatively regulated by the adenomatous polyposis coli (APC) tumor suppressor gene. APC is mutated or deleted in the germline of people with familial adenomatous polyposis (FAP), a genetic syndrome associated with a high risk of colon cancer, and in somatic colon epithelial cells in most sporadic, or nongenetic, forms of colon cancer. Loss of APC function causes an increase in ODC activity and polyamine biosynthesis and tumor formation in a murine model of human FAP. Treatment of these mice with the ODC inhibitor DFMO suppresses intestinal tumor formation. In colon tumor cells, loss of APC function leads to increased expression of the MYC oncogene, aberrant expression of which is associated with the development of Burkitt lymphoma and several epithelial cancers, in addition to colorectal cancer. ODC is a direct transcriptional target of MYC. MYC plays a central role in providing for the
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bioenergetic and biomass needs of proliferating cells, and polyamines are essential cell constituents supporting many of these functions. Studies with transgenic cancer models also support the finding that the effect of MYC on tumor initiation and progression can be attenuated through the repression of polyamine production. High-risk neuroblastomas (an often lethal embryonal tumor in which MYC activation is paramount) deregulate numerous polyamine enzymes to promote the expansion of intracellular polyamine pools. Selective inhibition of key enzymes in this pathway, e.g., using DFMO, reduces tumorigenesis and synergizes with chemotherapy to regress tumors in preclinical models. In addition to APC and MYC, induction of ODC expression is involved in RAS-dependent fibroblast transformation. ODC has been shown to cooperate with RAS in skin carcinogenesis in mice. The expression of SSAT is also negatively downregulated by the KRAS oncogene, which is commonly mutated in human colon cancer and gastrointestinal cancers. SSAT was also identified as a gene that promotes resistance to ionizing radiation (IR), and is overexpressed in brain tumors, correlating with poor outcomes. The biologic significance of elevated SSAT expression in glioblastoma multiforme (GBM), the most common and severe form of brain cancer, lies in its contribution to cell radio-resistance and that SSAT may potentially be a therapeutic target to sensitize GBM to cancer therapies. Knockdown of SSAT using shRNA and siRNA approaches in multiple cell and neurosphere lines resulted in sensitization of GBM cells to radiation in colony formation assays and tumors, and decreased tumorigenesis in vivo. Mechanistically, it was found that SSAT promotes acetylation of histone H3, suggesting a new role of SSAT in chromatin remodeling and regulation of gene expression. In particular, SSAT depletion led to a dramatic reduction in BRCA1 expression, explaining decreased homologous recombination capacity (Brett-Morris et al. 2014). ODC is also regulated by androgens in the prostate gland, and the gene encoding ODC is markedly induced in human prostate cancer.
Polyamines
ODC activity is now known to be induced in normal tissues by a range of environmental and genetic cancer risk factors. For example, ultraviolet light induces Odc gene expression in rodent models of squamous cell skin cancer, and inhibitors of ODC suppress this process. In addition, asbestos, the causative agent of the deadly lung cancer mesothelioma, is a potent inducer of ODC. DFMO, the irreversible suicide inhibitor of ODC, suppressed the expression of several genes involved in two types of cell-cell interactions, tight junctions and gap junctions. Tight junction proteins have been thought to act as tumor suppressors, whereas gap junction proteins have been implicated in cell communication involved in carcinogenesis. Cancer cells are devoid of checkpoints regulating their growth, which generally means a loss of regulation over cell cycle. Natural polyamines are needed for the normal cell cycle progression. Many results indicate that S-phase is the most sensitive phase to polyamine depletion, since polyamine biosynthesis varies bicyclically during the active cell cycle, with one peak in conjunction with the G1/S transition and a second at the end of S-phase and G2-phase. These changes are linked to the interaction of polyamines with oncogenes, cyclins, and CDKs (cyclin-dependent kinases), which drive the cell cycle. The most common type of cell death induced by anticancer drugs is apoptosis, and polyamines have been found to both induce and inhibit apoptosis in mammalian cells. However, the bifunctional regulation of this process remains to be fully understood. It has been proposed that the anti-age activity of exogenous spermidine is related to its ability to promote autophagy in many model organisms. Autophagy is a type of cell death characterized by the enzymatic degradation of macromolecules and organelles in double- or multimembrane autophagic vesicles. This process has important physiological functions, including the degradation of misfolded proteins and organelle turnover. The switch between autophagy and apoptosis has been proposed to constitute an important
Polyamines
regulator of cell death in response to chemotherapeutic drugs. Exogenous polyamines have a role in the cell death decision between roscovitineinduced apoptosis or autophagy in MCF-7 and MDA-MB-231 breast cancer cells (Arisan et al. 2015). Development of Polyamine Analogs for Cancer Chemotherapy and Their Mechanism of Action The requirement for polyamines in mammalian cell growth and the dysregulated polyamine metabolism in tumor cells suggested polyamine metabolism as a logical target for cancer therapy. Initially, several inhibitors of the biosynthetic enzymes ODC and SAMDC have been developed. Among these, DFMO, the enzymeactivated, irreversible inhibitor of ODC, was used in Phase II cancer clinical trials. However, it showed limited therapeutic efficacy since decreases in all three polyamines and the inhibition of the exogenous polyamines are needed to prevent cancer cell growth, not just loss of putrescine and spermidine as occurs with DFMO. In addition, side effects such as thrombocytopenia, anemia, and reversible ototoxicity made it an undesirable chemotherapeutic agent. Because of the limited success of polyamine biosynthetic inhibitors as chemotherapeutic agents, polyamine analogs have been developed to target the several polyamine feedback mechanisms which autoregulate their synthesis. Unlike DFMO, polyamine analogs utilize the polyamine transport system and compete with the uptake of the natural polyamines. In addition, polyamine analogs indirectly downregulate ODC activity through induction of ODC antizyme (AZ). Some analogs induce the activity of the rate-limiting catabolic enzymes, SSAT and SMO (PAOh1), producing cytotoxic H2O2, and therefore are more effective tumor growth inhibitors. Different approaches about polyamine-DNA interactions suggest that DNA is the prime target of polyamines, which play a critical role in DNA conformational transitions. Owing to the
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structural similarity, polyamine analogs interfere with the normal polyamine-DNA interaction affecting the polyamine role in DNA conformational transitions, condensation/decondensation, and stabilization, as demonstrated in analogtreated cancer cells more sensitive to nuclease digestion than untreated cells. Consequently, analogs interfere with cell cycle regulation, mitochondrial functions such as depletion of mitochondrial DNA, tubulin polymerization, polyamine metabolic enzymes, and apoptosis, with the expression of specific genes associated with the regulation of tumor cell proliferation, differentiation, and apoptosis. In this regard, for example, bis(ethyl)polyamine analogs have been shown to decrease c-myc oncoprotein and downregulated NK-kB signaling pathway. Many analogs of polyamines, with changes in the length of the methylene bridging region or terminal amino group substitution, were developed and tested for their cytotoxic effects against cancer cells. Among these, both symmetrically and unsymmetrical, terminally alkylated polyamine analogs and conformationally restricted symmetrically alkylated polyamine analogs and oligoamines have been developed and tested. The symmetrically substituted polyamine analogs have been the most extensively studied. They enter the cell using the polyamine transport system and specifically slow the synthesis of polyamines through downregulation of the biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), but they are sufficiently different not to be able to mimic the function of natural polyamines in cells. The most interesting feature of the cellular response to the polyamine analogs is the induction of SSAT or, in some cases, the “superinduction” of several thousandfold, in cultured tumor cells, that leads to the induction of apoptosis. SSAT induction by polyamine analogs appears regulated through NF-E2-related transcription factor (Nrf-2), constitutively bound to a 9-bp consensus sequence that functions as a polyamine response element (PME), in the SSAT promoter.
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Bis(ethyl) derivatives of spermine were superior to spermidine and putrescine derivatives in inhibiting cell growth and depleting polyamine levels. The most successful of the symmetrically substituted polyamine analogs to date are the N, N’-bis(ethyl)polyamines: bis(ethyl)norspermine (BENSpm; BE-3-3-3), bis(ethyl)spermine (BESpm; BE-3-4-3), and bis(ethyl) homospermine (BEHSpm). Phase I and II clinical studies involved only BENSpm revealing minimal toxicity, but no significant clinical effects in patients with breast or lung malignancies. The cytotoxic effects of polyamine analogs against tumor cells are attributed, in part, to the depletion of intracellular polyamine levels and occur at concentrations less than 10 mM. Unlike DFMO, which requires mM concentration to inhibit tumor cell growth targeting specifically ODC, polyamine analogs have multiple target sites competing with natural polyamines for uptake via transport channels and downregulate the polyamine transporter. In addition, they are also able to replace structurally similar natural polyamines and inhibit their function, thereby driving cells to the apoptotic pathway. Analoginduced apoptosis is partly due to oxidative stress resulting from H2O2 production as a by-product of SSAT and PAO activities induced by analogs. However, polyamine analog-induced apoptosis may occur through multiple structure-related and cell type-specific mechanisms, both caspase dependent and independent, evidenced in breast, melanoma, and lung cancer cell lines. Thus, polyamine depletion as well as overproduction can lead to apoptosis. The inhibition of ODC activity can lead to G1 arrest in a variety of cell lines including tumor cells. The most active analogs can disrupt polyamine metabolism and lead to the cell cycle arrest in cancer cells. Furthermore, polyamine analogs are not efficient substrates for the catabolic enzyme SSAT and, therefore, accumulated in treated cells; however, they can induce SSAT, resulting in the degradation and depletion of natural polyamines. For these reasons, polyamine analogs have been tested on many tumors also reaching clinical
Polyamines
trials, such as on lung and brain cancer, melanoma, and breast and prostate cancer, in which the majority of the studies were conducted with bis(ethyl)spermine derivatives which caused superinduction of SSAT and production of hydrogen peroxide which correlated with cell growth inhibition. However, this correlation was not always observed. Antitumor activity of BENSpm was also demonstrated using several xenograft models. The compound also has greater antitumor activity compared with the closely related compound BESpm and entered Phase I and II of clinical trials, showing a maximum tolerated dose of 185 mg/m2/day. Studies using polyamine/metal complexes as cancer chemotherapeutics demonstrate that complexes in which the metal centers were separated by diaminoalkanes, but not by spermidine, represent antitumor agents that are more potent than cisplatin against murine leukemia cells, murine solid tumor cells, and four human tumor cells in vitro and had good activity against 59 resistant cell lines (Nowotarski et al. 2013). Polyamines and DNA Promoter Methylation The structural and functional similarities between SMO and LSD1 (lysine-specific demethylase1) that demethylates mono- and dimethyl lysine 4 of histone H3, and is a FAD-dependent amine oxidase that contains an active site with 60% homology to SMO, and the demethylation of lysine residues by LSD1 that occurs through this oxidase activity, suggested another potential target for polyamine analogues. Based on previous data indicating that guanidine compounds are capable of inhibiting the polyamine oxidases, of the 13 compounds initially tested, 9 were able to noncompetitively inhibit recombinant LSD1 activity by over 50% at 1 mM concentrations. Polyaminohydroxamic acid (PAHA) derivatives that incorporated structural features of the polyamines spermidine and spermine and the hydroxamic acid moiety that is common amongst potent HDAC inhibitors, maintain a high affinity for DNA and can enter the cell via the polyamine transport system, inhibited HDAC activity by at
Polyamines
least 60%. In a subsequent study, 15 additional PAHA analogues and polyaminobenzamides (PABAs) were studied. PABAs incorporated the benzamide moietyof MS-275 as well as some of the structural features of spermidine and spermine. Two PABA compounds and 7 PAHA compounds inhibited HDAC activity by over 50%. In addition to the (bis)guanidine and biguanide inhibitors of LSD1, specific oligoamine analogues have also been demonstrated to be effective inhibitors of LSD1. Most importantly, both classes of inhibitors have demonstrated significant in vivo inhibition of tumour growth in established tumours, and both have demonstrated a synergistic antitumour response when combined with the DNA methyltransferase inhibitor 5-azacitidine. Interestingly, a study has demonstrated a synergistic effect on gene expression when CGC11144 and DFMO were used in combination in HCT116 cells. To further support a role for polyamines in epigenetic changes is the demonstration that the inhibition of ODC by AZ1 causes the accumulation of decarboxylated dcSAM which subsequently acts as a competitive inhibitor for methylation reactions resulting in a genome-wide hypomethylation of CpG islands. Additionally, AZ1 causes histone H3 lysine 9 dimethylation in UMI human oral cancer cells (Nowotarski et al. 2013). Polyamine Pathway in Cancer Chemoprevention Increased polyamine synthesis and inflammation have long been associated with intraepithelial neoplasia, which are risk factors for cancer development in humans. Targeting polyamine metabolism (by use of polyamine synthesis inhibitors or polyamine catabolism activators) and inflammation (by use of nonsteroidal anti-inflammatory drugs) has been studied for many cancers, including colon, prostate, and skin. Several non-steroidal anti-infiammatory drugs (NSAIDs), the use of which is associated with decreased risk of epithelial cancers, activate the transcription of SSAT, the first enzyme in
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polyamine catabolic pathway. Experimental studies indicate that combinations of DFMO, a selective inhibitor of polyamine synthesis, and NSAIDs are potent inhibitors of colon and intestinal cancer development in murine models. Clinical studies have shown that DFMO is well tolerated and can prevent the development of precancerous lesions in the skin. A clinical trial of DFMO showed that the 1 year treatment duration reduced prostate volume and serum prostate-specific antigen doubling time in men with a family history of prostate cancer. A second, clinical trial of DFMO in combination with sulindac, a NSAID in patients with prior colon polyps found that the 3-year treatment was associated with a 70% reduction of all, and over a 90% reduction of advanced and/or multiple metachronous colon adenomas. SMO may also be a rational chemopreventive target owing to its involvement in inflammationassociated carcinogenesis. H. pylori infection of gastric epithelial cells results in a rapid induction of SMO, increased ROS, and significant DNA damage. Additionally, patients with active H. pylori infections demonstrate increased SMO expression. SMO induction was also obtained by inflammatory cytokines in non-tumorigenic lung epithelial cells, and an in vivo colon cancer model, in response to enterotoxigenic Bacteroidesfragilis (ETBF) infection. Concurrently with increased SMO expression there was an increase in the levels of ROS. Treatment of animals with the SMO inhibitor MDL 72,527 significantly reduced the ETBF-induced burden accounting for the major role played by SMO (Nowotarski et al. 2013).
Cross-References ▶ ABC Drug-Transporters ▶ Adenocarcinoma ▶ Adenoma ▶ Alkylating Agents ▶ Amine Oxidases ▶ Angiogenesis ▶ Apoptosis ▶ Apoptosis Induction for Cancer Therapy
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▶ Arginine ▶ Autophagy ▶ Bcl2 ▶ Blood-Brain Barrier ▶ Brain Tumors ▶ Breast Cancer ▶ Cancer ▶ Cancer Epigenetics ▶ Carcinogenesis ▶ Cervical Cancers ▶ Chemoprevention ▶ Chemotherapy ▶ Cisplatin ▶ Clinical Trial ▶ Clusterin ▶ Colorectal Cancer ▶ Combinatorial Cancer Therapy ▶ Comet Assay ▶ Cyclin D ▶ Cyclin-Dependent Kinases ▶ Cyclins ▶ Cytokine ▶ DNA Damage ▶ DNA Oxidation Damage ▶ Drug Resistance ▶ Epigallocatechin ▶ Epigenetic ▶ Epigenetic Modifications ▶ HDAC Inhibitors ▶ Lung Cancer ▶ Mitochondrial DNA ▶ MYC Oncogene ▶ Oncogene ▶ Oral Cancer ▶ Ovarian Cancer ▶ Ovarian Cancer Chemoresistance ▶ Ovarian Cancer Chemotherapy ▶ Ovarian Serous Carcinoma ▶ Oxidative Stress ▶ Pancreatic Cancer ▶ Programmed Cell Death 4 ▶ Prostate Cancer ▶ Prostate Cancer Chemotherapy ▶ Prostate Cancer Clinical Oncology ▶ Prostate-Specific Antigen ▶ Proteasome ▶ Protein Kinase C Family ▶ Reactive Oxygen Species
Polyaromatic Compounds
▶ SiRNA ▶ Skin Cancer ▶ Stress ▶ Stress Response ▶ Topoisomerases ▶ Tumor Suppression ▶ Vascular Endothelial Growth Factor
References Arisan ED, Akkoç Y, Akyüz KG, Kerman EM, Obakan P, Çoker-Gürkan A, Palavan ÜN (2015) Polyamines modulate the roscovitine-induced cell death switch decision autophagy vs. apoptosis in MCF-7 and MDA-MB-231 breast cancer cells. Mol Med Rep 11 (6):4532–4540 Brett-Morris A, Wright BM, Seo Y, Pasupuleti V, Zhang J, Lu J, Spina R, Bar EE, Gujrati M, Schur R, Lu ZR, Welford SM (2014) The polyamine catabolic enzyme SAT1 modulates tumorigenesis and radiation response in GBM. Cancer Res 74(23):6925–6934 Kjellström J, Oredsson SM and Wennerberg J (2012) Increased toxicity of a trinuclear Pt-compound in a human squamous carcinoma cell line by polyamine depletion. Cancer Cell Int 12:20 Nowotarski SL, Woster PM, Casero RA Jr (2013) Polyamines and cancer: implications for chemoprevention and chemotherapy. Expert Rev Mol Med 15, e3 Orlando A, Linsalata M, Tutino V, D’Attoma B, Notarnicola M, and Russo F (2015) Vitamin K1 exerts antiproliferative effects and induces apoptosis in three differently graded human colon cancer cell lines. BioMed Res Int 2015. Article ID 296721 Rial NS, Meyskens FL, Gerner EW (2009) Polyamines as mediators of APC-dependent intestinal carcinogenesis and cancer chemoprevention. Essays Biochem 46:111–124 Ruiz-Pérez MV, Medina MÁ, Urdiales JL, Keinänen TA, Sánchez-Jiménez F (2015) Polyamine metabolism is sensitive to glycolysis inhibition in human neuroblastoma cells. J Biol Chem 290(10):6106–6119 Russo F, Linsalata M, Orlando A (2014) Probiotics against neoplastic transformation of gastric mucosa: effects on cell proliferation and polyamine metabolism. World J Gastroenterol 20(37):13258–13272 Silva TM, Cirenajwis H, Wallace HM, Oredsson S (2015) Persson L (2015) A role for antizyme inhibitor in cell proliferation. Amino Acids 47:1341–1352 Soda K (2011) The mechanisms by which polyamines accelerate tumor spread. J Exp Clin Cancer Res 30:95
Polyaromatic Compounds ▶ Polycyclic Aromatic Hydrocarbons
Polycomb Group
Polycomb Group Talha Anwar1 and Celina G. Kleer2 1 Medical Scientist Training Program and Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA 2 Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA
Definition The polycomb group (PcG) is a family of proteins required for proper eukaryotic gene expression that determines cell fate (▶ epigenetics, ▶ epigenetic gene silencing). They exert their role mainly by repressing gene transcription through ▶ epigenetic modifications on specific histone tails that are inherited through cell divisions, thus functioning as the cellular memory. They are involved in the maintenance of cell type and differentiation and play a role in tumor development.
Characteristics Cell differentiation depends upon keeping certain genes “on” and other genes “off” through generations. The differential gene expression patterns that define a cell type are maintained during DNA replication and at mitosis so that the daughter cells maintain the differentiated cell type of the parental cell. This is possible because cells have a transcriptional memory. Perturbance of the transcriptional memory can lead to severe developmental defects and to malignancy. The molecular basis of the cellular memory is controlled mainly by two groups of proteins: the polycomb group (PcG) and the trithorax group (TrxG). The PcG and TrxG are chromatin-associated proteins which function by repressing and activating transcription, respectively. While PcG proteins act mainly by repressing gene expression, TrxG proteins, operationally defined as antagonistic of PcG proteins, activate gene expression. Some proteins possess functions of both PcG
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and TrxG proteins. PcG and TrxG proteins have been well conserved throughout evolution. There are at least 20 PcG and TrxG proteins in Drosophila, and many have mammalian counterparts. Biochemically, they function in large protein complexes. In the following discussion, we will concentrate on the PcG proteins. Studies in Drosophila melanogaster have been instrumental in our understanding of proteins involved in transcriptional maintenance. The formation of Drosophila melanogaster relies on the expression pattern of a cluster of genes (homeotic genes) along the anterior-posterior axis during development. The PcG proteins maintain the correct spatial and temporal expression pattern of the homeotic genes through transcriptional repression. PcG mutants in Drosophila express homeotic genes outside the body segments; they are abnormally expressed, causing marked transformations in body plan. PcG Protein Complexes PcG proteins function by forming two large multimeric complexes, termed polycomb repressive complexes (PRCs). The PRC2, composed EED, SUZ12, EZH1 or EZH2, is involved in the initiation of gene repression. The PRC1 contains multiple proteins including BMI1, HPC proteins (CBX2, CBX4, CBX7, CBX8), and RING proteins (Table 1). One of the hallmarks of transcriptionally active genes is that their nucleosomes contain acetylated histone H3. When genes are repressed, the histones are generally deacetylated and H3 is trimethylated on specific lysine residues (especially K9 and K27). The functional conservation between Drosophila and human PcG proteins in methylating histone H3 on lysine 27 has resulted in the development of a model for PcG-mediated gene silencing. In this model, at the initiation of gene repression, PRC2-associated histone deacetylases remove the acetylation of H3 and allow EZH2 to trimethylate lysine 27 and to a lesser extent lysine 9 of histone H3 (H3K27me3 and H3K9me3, respectively). The PRC1 can recognize the trimethylation mark through the chromo domain of the polycomb protein, leading to the recruitment of the PRC1 maintenance complex. This interaction is proposed to target the
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Polycomb Group
Polycomb Group, Table 1 PcG proteins and their altered expression in human malignancies Drosophila Initiation complex-PRC2 Extra sex combs, Esc Enhancer of zeste, E (z)
Human
Suppressor of zeste, Su(z)12 Maintenance complex-PRC1 Polycomb, Pc
SUZ12
Polyhomeiotic, Ph
Sex combs extra, Sce Posterior sex combs, Psc
Human cancer type
EED EZH1 EZH2
CBX2/Pc1/M33 CBX4/Pc2 CBX6 CBX8/Pc3 Rae28/EDR1
EDR2 EDR3 RING1A/RNF1 RING1B/RNF2 Bmi1/RNF51
Lymphoma, melanoma, carcinomas of the breast, prostate, bladder, colon, liver Carcinomas of the breast, colon, liver
Acute lymphoblastic leukemia
Lymphoma, leukemia, medulloblastoma, neuroblastoma, non-small cell lung cancer
RNF110/Mel18 RNF134 NSPC1 RNF3 RNF159 YY1 Pleiohomeotic, PHO
PRC1 to specific repression sites. It should be noted that this model has not been yet confirmed in mammalian cells. The precise mechanisms by which the PcG proteins inhibit transcription are still unclear. One suggested mechanism by which the PcG proteins can block the transcription initiation machinery is through PRC1 ubiquitin E3 ligase activity of histone H2A lysine
119 (H2AK119), a modification associated with gene repression. Another suggested mechanism of PcG-mediated repression is through PRC2 methyltransferase activity on lysine 26 of the linker histone H1 (H1K26). Methylated H1K26 can influence the chromatin structure. Furthermore, EZH2, a member of the PRC2, has been found to recruit DNA methyltransferases (DNMTs) to specific target genes. DNA hypermethylation of gene promoter regions results in transcriptional repression. By repressing specific genes, PcG proteins are involved in regulating a wide variety of fundamental cellular processes such as stem cell maintenance, cell fate, cell division, and neoplastic cell transformation.
PcG Proteins and Stem Cell Maintenance An intriguing function of the PcG proteins is their emerging role in the maintenance of embryonic and adult stem cells. Stem cells have the ability to self-replicate and give rise to specialized cells, processes that require a transcriptional memory system that maintains the cell type identity from one generation of cells to another. This function is underscored by studies showing that targeted disruption of EZH2 or EED results in early embryonic lethality. Consistent with a critical role in the maintenance of stem cells, PcG proteins are able to silence genes with roles in embryonic development and genes responsible for cellular differentiation. Among these targets are homeodomain-containing transcription factors of the Dlx, Irx, Lhx, and Pax gene families with functions in the early development of the neural and hematopoietic systems and members of the Fox, Sox, Gata, and Tbx transcription factor families which not only have roles in development but also in disease. For example, Gata3 has been found to play a role in breast cancer, with strong association with estrogen receptor expression. Furthermore, PcG target genes in signal transduction pathways required for embryonic differentiation as well as for maintenance and proliferation of stem cells in tissues, such as transforming growth factor and fibroblast growth factor families, Hox, Notch (▶ NOTCH/
Polycomb Group
JAGGED signaling), Hedgehog, and ▶ Wnt signaling pathways. PcG Proteins in Human Malignancies and Their Clinical Relevance Lack of differentiation, or anaplasia, is a hallmark of cancer which results from normal cells “forgetting” their cellular identity. Thus, it is not surprising that dysregulation of the transcriptional maintenance system can lead to malignancy. Current data suggest that altered expression of the PcG proteins may influence tumor development mainly by transcriptional repression of tumor suppressor genes and by maintaining the cells in a stem cell state. This is particularly intriguing in light of the stem cell model of carcinogenesis which proposes that human tumors contain and arise from transformed stem cells and early progenitor cells that have sustained specific genetic alterations. At least three mammalian PcG proteins, Bmi1, Rae28, and Mel18, are already implicated in the self-renewal of hematopoietic and neuronal stem cells. Although this theory is under extensive investigation and many questions need to be addressed, PcG proteins play a central role in the process of neoplastic transformation. The first connection between PcG proteins and cancer development came from studies on Bmi1, which was found to cooperate with the protooncogene c-myc (▶ Myc oncongene) to promote the generation of B- and T-cell lymphomas and later shown to inhibit c-myc-induced apoptosis through repression of the Cdkn2a locus. This tumor suppressor locus is frequently inhibited in human tumors. Further studies showed that other PcG proteins are also strongly associated with neoplastic transformation and progression. Although in human malignancies PcG proteins have primarily been found to be dysregulated in cells of hematologic origin, for example, through activating mutations of EZH2, they also play an important role in the development of carcinoma of different organs, melanoma, and neural tumors. EZH2 is overexpressed in multiple types of lymphoma and in solid tumors including carcinomas of the breast, prostate, colon, bladder, liver, and melanoma. In adenocarcinomas of the prostate
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gland, elevated levels of EZH2 protein are able to distinguish patients with localized prostate cancer that are more likely to experience tumor recurrence after prostatectomy. In breast tissues, overexpression of EZH2 occurs before histological evidence of malignancy, and its overexpression in breast cancer is associated with larger tumors, the presence of lymph node and distant metastasis, and a worse clinical outcome. Furthermore, in breast cancers, increased levels of EZH2 are associated with absent estrogen (▶ estrogen receptor) and progesterone receptors. Another PcG protein, SUZ12, is upregulated in both breast and colon cancers. The specific mechanisms leading to the overexpression of PcG genes and the events resulting in the localization of PcG proteins to different target sites await further investigations. It is clear that the PcG proteins are overexpressed in several human malignancies and that in carcinomas of the breast and prostate, their upregulation is associated with an aggressive and metastasizing clinical course. For these reasons, the use of PcG proteins as potential tissue ▶ biomarkers of cancer diagnosis, prognosis, and prediction to treatment response holds promise. Because PcG proteins appear to be expressed in certain cancers such as breast cancers before histological atypia, they may mark epithelium at higher risk for development of carcinoma. Importantly, given their role in malignant transformation of hematopoietic and solid human tumors, components of the PcG family may provide attractive opportunities for therapeutic intervention (▶ Epigenetic Therapy).
References Ding L, Kleer CG (2006) Enhancer of zeste 2 as a marker of preneoplastic progression in the breast. Cancer Res 66:9352–9355 Gil J, Bernard D, Peters G (2005) Role of Polycomb group proteins in stem cell self-renewal and cancer. DNA Cell Biol 24:117–125 Shilatifard A (2006) Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression. Annu Rev Biochem 75:243–269 Sparmann A, van Lohuizen M (2006) Polycomb silencers control cell fate development and cancer. Nature 6:846–856
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Polycyclic Aromatic Hydrocarbons Brinda Mahadevan1 and Andreas Luch2 1 Abbott Nutrition, Regulatory Affairs, Abbott Laboratories, Columbus, OH, USA 2 German Federal Institute for Risk Assessment (BfR), Berlin, Germany
Synonyms Polyaromatic compounds
Definition Polycyclic aromatic hydrocarbons (PAHs) belong to the more general class of polycyclic aromatic compounds that contain two or more aromatic rings fused together in a linear or angular configuration. PAHs consist of carbon and hydrogen only. Hence, polycyclic aromatic compounds containing heteroatoms (e.g., N, S, and O) in their molecular structure would not belong to the group of PAHs in a narrow sense of the chemical nomenclature.
Characteristics Anthropogenic Origin and Carcinogenicity PAHs are formed during incomplete combustion processes of organic matter. Due to the abundant use of fossil energy sources, PAHs are readily detectable as ubiquitous contaminants in the environment. Humans are exposed to complex mixtures of PAHs in the atmosphere from combustion sources such as cigarette smoking, vehicle emissions, and fossil fuels; and in foodstuffs from pyrolysis products such as overbarbequed meat or charbroiled food. In addition, industrial and work place exposures occur through coal gasification, aluminum production, steel founding, and coal tar production. The great interest in this group of chemicals originated on the observation in animal tumor models that some member compounds possessed strong carcinogenic activity in skin, lung, breast, and other organs. Early observations
Polycyclic Aromatic Hydrocarbons
of individual cancer cases occurring from occupational exposures to mixtures highly contaminated with PAHs lend support to the notion that these compounds can also act as carcinogens in man. Epidemiological data identified the occupational exposure to complex PAH-containing mixtures such as coal tar or coal tar pitches (consisting of up to 50% of PAHs with 1–2% benzo[a]pyrene, B[a]P; see Fig. 1) as being carcinogenic to humans (IARC classification Group 1). Epidemiological meta-analyses confirmed that heavy exposures to mixtures of PAHs entails a substantial risk to develop cancer in lung and skin, and possibly also in the bladder (less consistent evidence), larynx, kidney, and pancreas (uncertain evidence). No matter what kind of PAH source, humans are always exposed to mixtures of aromatic hydrocarbons with different degrees of biological activity. For instance, more than a hundred different PAHs can be detected in the air. The composition of these PAH profiles vary depending on the environmental compartment. Since there is no international agreement on which panel of individual PAHs should be analyzed and reported in order to characterize distinct emission sources, PAH lists released from different organizations may contain different compounds. Sixteen different PAHs such as pyrene, B[a]P, benz[a] anthracene, benzo[b]fluoranthene, benzo[k] fluoranthene, indeno[1,2,3-cd]pyrene, dibenz[a, h]anthracene, and others are prioritized by the US Environmental Protection Agency. Lower molecular weight PAHs such as naphthalene, phenanthrene, anthracene, or fluoranthene are also listed due to their occurrence in environmental samples. From all hydrocarbons detectable in the human environment, the most intensively studied example, B[a]P, has been traditionally used as an indicator for carcinogenic PAHs. Molecular Mode of Action PAHs can be readily absorbed from lung, skin, and gut. Rapid absorption of B[a]P and other PAHs from the gastrointestinal tract or from skin has been widely demonstrated in animal models, and sufficient evidence is also available for humans. On the other hand, absorption through
Polycyclic Aromatic Hydrocarbons
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Polycyclic Aromatic Hydrocarbons, Fig. 1
the lungs may be affected by the size of particles at which most of the airborne PAH fraction is adsorbed. It has been shown that a considerable fraction of B[a]P adsorbed on soot, diesel exhaust, or carbon black particulate matter can be retained for up to weeks in the respiratory tract. The slow passage through bronchial mucosa can therefore result in very high local concentrations of these compounds directly within one of their main target tissues, the lungs. To react with cellular macromolecules, however, lipophilic and chemically inert compounds such as PAHs require enzymatic biotransformation into electrophilically reactive descendants (metabolites). In a landmark paper from 1964, Brookes and Lawley reported on the tissue binding levels of six PAHs, i.e., naphthalene, dibenz[a,c] anthracene, dibenz[a,h]anthracene, B[a]P, 3methylcholanthrene, and 7,12-dimethylbenz[a] anthracene in mouse skin in vivo. The authors concluded that there is a significant positive correlation between the binding to DNA and the carcinogenic potency of these compounds. On
the contrary, this correlation was not found for the binding to proteins or RNA. It became clear that vicinal dihydrodiol epoxides of PAHs that contain the epoxy moiety in a sterically crowded bay or fjord region are the actual DNA-binding metabolites that mediate the biological effects associated with their parent structures (Fig. 1). By direct application of these reactive descendants, it has been demonstrated that their mutagenic potency correlates to the level of PAH–DNA adduct formation. Moreover, the tumor-inducing potencies of different PAHs in the lungs of strain A/J mice, another experimental tumor model, were found to correlate to the timeintegrated DNA adduct levels (TIDAL) calculated as area under the curves of total dihydrodiol epoxide–DNA adduct levels during a time course of 30 days after injection. This parameter represents the total effective molecular dose delivered to target lung DNA, and it linearly correlated to the PAH doses administered. The intimate relationships between DNA binding level and mutagenicity and the TIDAL and carcinogenicity
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observed in mice in vivo support the notion that the DNA binding level can serve as an important biomarker for the tumor threat that may result from exposures of humans to carcinogenic PAHs. Hence PAH-DNA damage is considered to be causative and directly related to tumor formation by representing the first important step during the multistage process of PAH-induced carcinogenesis. The PAH–DNA adduct level at a given time point is an integrated product of compound’s toxicokinetic and toxicodynamic behavior, including metabolic activation and detoxification prior to covalent binding, as well as the effectiveness of the repair of those DNA lesions that have been formed. PAHs would not be carcinogenic if they were not metabolized by cytochrome P450dependent monooxygenases (CYP enzymes) and microsomal epoxide hydrolase (mEH) through subsequent steps of epoxidation and hydrolysis (Fig. 1). Deletion of the genes encoding the enzymes involved in this activation route (e.g., CYP1B1, mEH), or of the gene encoding the regulator protein involved in the induction of these enzymes (arylhydrocarbon receptor protein), renders mice resistant to the biological effects of potent PAHs such as B[a]P, 7,12dimethylbenz[a]anthracene, dibenzo[a,l]pyrene, and others. Metabolic activation of PAHs has been shown to be highly stereoselective. As demonstrated for a wide range of carcinogenic PAHs, the initial epoxidation hydrolysis sequence produces a dihydrodiol with (R,R) configuration in high enantiomeric excess. Subsequent epoxidation at the vicinal double bond then predominantly generates the (R,S) dihydrodiol (S,R) epoxide with the epoxide moiety trans to the benzylic hydroxy group and spanning into a bay or fjord region. For B[a]P, all four possible stereoisomeric bay region 7,8-dihydrodiol 9,10-epoxides (B[a]PDE) are depicted in Fig. 1, with (+)-anti-B[a]PDE as the major species formed during bioactivation. Depending on the activation system, very small amounts of the other isomers may also be generated. However, B[a]P-induced DNA damage in vitro or in vivo predominantly results from
Polycyclic Aromatic Hydrocarbons
covalent interaction of (+)-anti-B[a]PDE, most of which is trapped by 20 -deoxyguanosine residues via trans opening of the epoxide moiety [(+)-trans-anti-B[a]PDE-N2–DNA adduct; Fig. 1]. If not repaired, this adduct causes nucleotide misincorporation at the opposite DNA strand during the next round of DNA replication, thus leading to base substitutions or frame-shift mutations. In the case of B[a]P, data also suggest that poor enzymatic repair of its predominant (+)-trans-anti-B[a] PDE-N2–DNA adduct is actually preceded by an insufficiently activated DNA damage checkpoint. At nontoxic doses of the dihydrodiol epoxide, a significant number of synchronized cells in vitro were found to enter the synthesis (S) phase of the cell cycle. The failure to induce a proper DNA damage arrest along with insufficient enzymatic repair increases the likelihood of transforming mutations because DNA replication continues on a damaged template via engagement of error-prone (Y-family) polymerases during translesional synthesis. Analysis of the cell cycle and the expression profiles of human epithelial cells or tumor cells of epithelial origin in culture revealed that the DNA damage arrest was insufficient even at a DNA damage level of about 180,000 anti-B[a]PDE–DNA adducts per cellular genome. Bay or fjord region dihydrodiol epoxides of other carcinogenic PAHs were also identified as the actual metabolites that mainly mediate cellular binding to DNA and thus the genotoxic effects of their parent structures. Covalent PAH–DNA adducts are fixed as mutations if left to errorprone excision repair, misrepair, or replication errors during early S phase. Most importantly, if these fixed mutations occur in proto-oncogenes and/or tumor suppressor genes, they can contribute to the aggravation of neoplastic growth through the processes of tumor promotion and progression. Members of the oncogene family RAS have been found to be commonly mutated in human cancers and animal models for chemical carcinogenesis. Codon 12 (within exon 1) of KRAS is the most frequently affected codon in human cancers including human lung adenocarcinomas. Using human bronchial epithelial cells, it
Polycystic Kidney Disease
was demonstrated that the first dG residue in codon 12 is a preferential binding site for the DNA-damaging dihydrodiol epoxide metabolite of B[a]P (at N2). As compared with other sites, the “hotspot” character of codon 12 in target cells of chemical lung tumorigenesis results from a synergism between the preferential binding of the carcinogenic metabolite and a poor repair of those lesions that had been formed. On the other hand, binding studies of B[a]P at dG residues located in the DNA binding domain of the tumor suppressor TP53 gene (exons 5, 7, and 8) have revealed that the codons and the positions within the codons affected matched between human bronchial epithelial cells exposed to anti-B[a] PDE and the TP53 mutational hotspot pattern registered in the database of human lung cancer. This observation provided strong evidence for an etiological role of B[a]P in the causation of the disease. Given the chemical complexity of most environmental matrices, it seems difficult, if not, impossible to uncover any causative relationship between certain forms of human cancer and the exposure to particular carcinogenic compounds. In addition to epidemiological hints, collective evidence from molecular toxicology and molecular epidemiology may nevertheless be able to point to the role of individual compounds (or single classes of compounds) and to extract their contribution from the overall biological response on environmental mixtures. One of the most well worked-out example is the crucial role of carcinogenic PAHs (i.e., B[a]P) in the etiology of human lung cancer based on their presence in cigarette smoke. Their important role in tumor initiation is supported by several lines of evidence such as: (i) increased levels of PAH activating enzymes (CYP1A1 and CYP1B1) in lung cancer patients compared with controls, (ii) a correlation between pulmonary CYP1A1 levels and bulky anti-B[a]PDE–DNA adduct levels in human lung tissue from cancer patients, (iii) increased levels of anti-B[a]PDE-DNA adducts in lung tissue of smokers compared with nonsmokers, and (iv) the coincidence of mutational hotspots (G to T transversions) in KRAS (codon 12) or TP53
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(codons 157, 248, 273) and anti-B[a]PDEN2–DNA adduct hotspots as the preceding lesions found at the same sites.
Cross-References ▶ Carcinogen Metabolism ▶ Toxicological Carcinogenesis
References ATSDR (1995) Toxicological profile for polycyclic aromatic hydrocarbons (PAHs). Agency for toxic substances and disease registry. US Department of Health and Human Services, Atlanta Grimmer G (1983) Environmental carcinogens: polycyclic aromatic hydrocarbons. CRC Press, Boca Raton Harvey RG (1991) Polycyclic aromatic hydrocarbons: chemistry and carcinogenicity. Cambridge University Press, Cambridge, UK Luch A (2005) The carcinogenic effects of polycyclic aromatic hydrocarbons. Imperial College Press, London
Polycystic Kidney Disease Yiqiang Cai Section of Nephrology, Yale University School of Medicine, New Haven, CT, USA
Synonyms Inherited human polycystic kidney disease
Definition Polycystic kidney disease is a common genetic disease characterized by accumulation of multiple fluid-filled cysts in each kidney and other organs. The renal cysts originate from the renal tubular epithelial cells lined by a single layer of cells that have higher rates of cellular proliferation and
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apoptosis and are less differentiated than the normal tubular cells. Progression of cysts in the kidneys could ultimately cause end-stage renal disease.
Characteristics PKD (Polycystic Kidney Disease) consists of two forms: ADPKD (Autosomal Dominant Polycystic Kidney Disease) and ARPKD (Autosomal Recessive Polycystic Kidney Disease). ADPKD is the most common genetic disease affecting 1 in 500 to 1 in 1,000 in adults of all ethnic groups worldwide. ARPKD is far less common, affecting 1 in 6,000 to 1 in 40,000 at a far younger age, including newborns, infants, and children. While kidney cysts arise from all the segments of the nephron and collecting ducts in ADPKD, they arise from collecting ducts in ARPKD. Extrarenal systems are affected in PKD. Clinical Features
Polycystic Kidney Disease
ARPKD
ARPKD is an important childhood nephropathy. The clinical phenotype is characterized by the combination of renal cystic disease and congenital hepatic fibrosis. Affected children often present in utero with enlarged, echogenic kidneys, as well as oligohydramnios secondary to poor urine output. Approximately up to 30–50% of affected neonates die shortly after birth as a result of severe pulmonary hypoplasia and secondary respiratory insufficiency. Those who survive the perinatal period have a 56–67% probability of survival to age 15 without ESRD, and prolonged survival to age 55 has been reported. Those survivors express widely variable disease phenotypes with systemic hypertension, renal insufficiency, and portal hypertension due to portal tract fibrosis as the most common clinical features. Long-term survivors often develop sequelae of portal hypertension including esophageal varices, hepatosplenomegaly, and hypersplenism. Other clinical manifestations include growth retardation, urinary tract infection, and hyponatremia.
ADPKD
It is estimated that there are over 12.5 million affected ADPKD individuals worldwide. The development of bilateral, multiple, focal renal cysts characteristics of ADPKD lead to the clinical manifestations which include abdominal mass, chronic flank or back pain, gross hematuria, urinary tract infection, and urolithiasis. Affected individuals typically present in the third and fourth decade, and ESRD (end-stage renal disease) usually develops in the sixth to eighth decades of life. Extrarenal cysts in ADPKD can arise from liver (70%), pancreas (5%), ovaries, and choroid plexus (rare). Cardiovascular abnormalities include mitral valve prolapse (25%), mitral regurgitation, aortic insufficiency, and tricuspid regurgitation. Hypertension and left ventricular hypertrophy are common. Other extrarenal manifestations include cerebral and aortic aneurysms, cerebral dolichoectasis, and colonic diverticuli. A striking feature of ADPKD is the variability of the phenotype: the severity of the disease, the age of onset of ESRD, and the spectrum of extrarenal manifestation vary widely between affected individuals.
Molecular Basis ADPKD is caused by mutations on a single gene of either PKD1 or PKD2, while typical cases of ARPKD are resulted from mutations on PKHD1 (polycystic kidney and hepatic disease 1) gene. Mutations have been identified throughout the gene of any among those three genes in affected PKD individuals without evidence for clustering. Both ADPKD and ARPKD might be recessive at molecular level: with two germline mutations in ARPKD or one germline mutation plus one somatic mutation caused by “second hit” in ADPKD generating PKD phenotypes. ADPKD
(a) PKD1, located on chromosome 16p13.3 and responsible for 85% of affected individuals of ADPKD, is comprised of 46 exons distributed over 52 kb of genomic DNA. The first 34 exons are duplicated several times elsewhere on chromosome 16, which has made routine mutation analysis technically difficult. PKD1 produces a 14 kb mRNA transcript. Polycystin-1 (PC-1), gene product of PKD1,
Polycystic Kidney Disease
is an integral membrane protein of 4,302 amino acid and has molecular weight of about 500,000 D. PC-1 contains an 11 membrane spanning domain with a 3,000 amino acid extracellular amino (NH2)-terminus and a 220 amino acid cytosolic carboxyl (COOH)-terminus. The large extracellular NH2-terminus contains domains many of which are novel and of unknown function; however, unique array of distinct protein motifs, including leucine-rich repeats flanked by cysteine-rich domains, a C-type lectin domain, a WSC domain, and 16 immunoglobin-like domains named PKD repeats, suggests PC-1 may function as a receptor since many of such motifs are involved in protein-protein or proteincarbohydrate interactions. Closer to the first transmembrane domain, there is a region of homology to the sea urchin egg jelly receptor and a consensus G-protein-coupled receptor proteolytic site (GPS site). The extracellular NH2-terminal fragment (NTF) is cleaved at GPS site where the resulting COOH-terminal fragment (CTF) contains all the membrane spans and cytosolic tail. In addition, between the first and second transmembrane spans, there is a domain homologous to lipoxygenase (PLAT domain). The intracellular COOHterminus contains a coiled-coil domain which mediates protein-protein interaction including the interaction with polycystin-2 (PC-2). Several potential phosphorylation sites are suggested within the COOHterminus. PC-1 expressed widely in vivo including kidney, brain, heart, and muscle. Subcellular expression of PC-1 is somewhat controversial as it has been detected in apical and basolateral membrane, tight junctions, adherens junctions, desmosomes, focal adhesions, and primary cilia, the key player for cystogenesis (further discussion see section “cilia and PKD” below). Functions of PC-1 might be exerted through signal transduction including Wnt, JAK-STAT, AP-1, NFAT and B-Raf/ERK-dependent pathway, ion-channel regulation, cell proliferation regulation,
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G-protein coupled signaling, cellular adhesion properties, and transcription; however, precise function of PC-1 demand further investigation. (b) PKD2, the second gene responsible for 15% of affected individuals of ADPKD, is located on chromosome 4 q21-23. PKD2 gene contains 15 exons covering 46 kb of genomic DNA and encodes a 5.3 kb mRNA transcript that is translated into an integral membrane protein, polycystin-2 (PC-2). PKD2 is approximately 25% homologous to a region of the PKD1 gene. Unlike PKD1, PKD2 gene is not duplicated on the chromosome. PC-2 is composed of 968 amino acids and has molecular weight of about 110,000 D. PC-2 contains six transmembrane spans and both intracellular NH2- and COOH-termini. The transmembrane segments of PC-2 are about 50% identical to the last six transmembrane segments of the 11 transmembrane segments of PC-1. PC-2 is a nonselect cation channel sharing structural features with the transient receptor potential (TRP) channel family. The intracellular COOH-terminus of PC-2 contains a motif known as EF hand that can bind calcium and an ER (endoplasmic reticulum) retention domain, deletion of which cause disruption of PC-2 localization in ER. PC-2 is expressed widely in all tissues tested so far, including kidney, heart, brain, lung, pancreas, liver, testis, ovary, vascular smooth muscle, and intestine. At the subcellular level, located primarily in the ER, PC-2 is also found in basolateral plasma membranes, lamellopodia, and mitotic spindles, in addition to the primary cilia (further discussion see section “cilia and PKD” below). PC-2 channel conducts divalent cations including calcium. ARPKD
PKHD1 (polycystic kidney and hepatic disease 1), the gene responsible for ARPKD, is located on chromosome 6 p21.1-p12. PKHD1 is very large and consists of at least 86 exons extending over 469 kb of genomic DNA on chromosome 6. The gene undergoes a complex pattern of alternative
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splicing to generate mRNA transcripts ranging in size from 8.5 to 13 kb. PKHD1 gene is expressed in kidney with higher level and in liver and pancreas with lower level. Distribution of PKHD1 mRNA transcript includes renal tubules, the bile ducts, blood vessels, testis, and dorsal root ganglia. Polyductin (or Fibrocystin), encoded by PKHD1 gene, is composed of 4,074 amino acids and predicted to contain a large extracellular domain, a single transmembrane span, and a short intracellular COOH-terminus. Polyductin has molecular weight of about 500,000 D and is expressed in cilia, cilia-associated structure basal bodies, and possibly in the plasma membrane. Functions of polyductin remain elusive. Cilia and PKD
PKD is a human cilial disease. Cilia are microtubule-based organelles that project like antennae from the surface of almost every vertebrate cell. Ultrastructurally, cilia consist of membrane that is continuous with the cell plasma membrane but is a separate domain with unique complement of proteins and a central axoneme that is composed of microtubules. Cilia originate from the basal body, an intracellular organelle containing a pair of centrioles and surrounding matrix. There are two types of cilia: motile cilia and nonmotile primary cilia in the body. The axonemes of typical motile cilia contain nine peripheral bundles of microtubules and two central microtubules (9+2 pattern), whereas the axonemes of primary cilia contain nine peripheral bundles of microtubules (9+0 pattern). While motile cilia move fluid past cells, nonmotile primary cilia transduce a multitude of sensory stimuli, including chemical concentrations of growth factors, hormones, odorants, and developmental morphogens, as well as osmolarity, light intensity, and fluid flow. Primary cilia have been identified in all segments of the nephron from Bowman’s capsule to collecting ducts. Abnormal structure and function of the renal primary cilium and its associated structures, i.e., centrosome and basal body, play key role in the pathogenesis of PKD. PC-1, PC-2, and Polyducin
Polycystic Kidney Disease
are localized in primary cilia of renal epithelia. While structural disruption may be important in cystogenesis of ARPKD, dysfunction of the polycystins (PC-1 and PC-2)-functional axis, rather than structural defect in cilia, play a key role in cystogenesis in ADPKD. The pathway in such axis is defined as polycystin-dependent inhibition and cilia-dependent activation manner. Furthermore, within this axis, polycystin-1 serves as a central determinant of cyst formation as a ratelimiting component. Abnormal trafficking and maturation of polcysytin-1, which is also regulated by polycystin-2, underlies to cyst formation in PKD. Animal Models Animal models for PKD have arisen through spontaneous mutations (such as cpk, bpk, jck, kat, pcy, and Han:SPRD-cy), chemical-induced mutagenesis (jckp), and gene-targeting approach (conventional or conditional knockout models for Pkd1, Pkd2, and Pkhd1). The pathology in these models resembles that seen in human PKD with regards to the localization, progression and morphology of cysts, as well as involvement of the liver and pancreas. Homozygous of genetargeting mouse models of Pkd1, Pkd2, and Pkhd1 are embryonic lethal; however, an unique and most relevant mouse model that recapitulates ADPKD is the Pkd2ws25 mouse line, which develop kidney and liver cysts postnatally. The Pkd2ws25 allele was generated fortuitously by a targeting event that duplicated the first axon of Pkd2. The duplicated exon is able to undergo intragenic recombination in somatic cells at a relatively high frequency to yield either a wildtype or a mutant Pkd2 locus. The spontaneous LOH (loss of heterozygosity) in the mouse carrying both the Pkd2 null allele and a Pkd2ws25 allele would result in focal cysts formation, which offering the experimental evidence supporting the “two-hit” hypothesis of cysts formation in ADPKD. Proteins encoded by genes responsible for the PKD animal models, including cystin (cpk), polaris (Tg737orpk), NPHP3 (pcy), Nek-8 (jck), Pc-1 (Pkd1), Pc-2 (Pkd2), and polyductin
Polycystic Kidney Disease
(Pkhd1 and pck), are localized in primary cilia. In addition, the proteins encoded by the NPHP gene family (NPHP1-4) and BBS gene family (BBS18), responsible for the nephronophthisis I–IV and Bardet-Biedl syndrome respectively, are localized in cilia or cilia-associated structure. Nonmammalian PKD models have provided significant insights into cyst development. Most notable among these are the C. elegans, the chlamydomonas, and zebrafish. PKD related genes in C. elegans including lov-1 (PKD1), pkd-2 (PKD2), osm-5 (Tg737orpk), Nphp1, and Nphp4 are identified. Mutations in lov-1 and pkd-2 cause mating behavior defects associated with cilia-mediated sensory reception, whereas mutations in osm-5 result in defects in cilia formation as seen in Tg737orpk mice. Chlamydomonas expresses many proteins that are mutated in murine and human PKD, including PC-2, polaris (IFT88), Kif3A (FLA10), and BBS proteins. Those proteins localize in flagella or cilia, and their function is required for both assembly machinery such as intraflagella transport (IFT) and normal signaling activity of this organelle. In zebrafish, knock-down of zebrafish homologues such as inv and Nek8 produce a distinctive renal cystic phenotype. Therapy and Perspective Therapeutic studies on animal models: Therapeutic tests of V2R (vasopressin-2 receptor) and mTOR (mammalian targets of rapamycin) inhibitors on animal models of PKD result in promising outcomes. Cyst fluid contains many hormonal activities, including ADH (antidiuretic hormone) and epidermal growth factor (EGF) as well as lipophilic substance, capable of stimulating the accumulation of cAMP (cyclic adenosine monophosphate). In the epithelial cells of distal tubules and collecting ducts, ADH activates the V2R, a Gas-coupled receptor linked to adenylate cyclase. PC-32160, a V2R antagonist, could prevent renal accumulation of cAMP and influences disease course in animal models of PKD including PCK rat (ARPKD), pcy mouse (adolescent nephronophthisis), and Pkd2ws25/ (Pkd2 genetargeting mouse). OPC-41061, a structural
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analogue of OPC-31260, also inhibits development of polycystic kidney disease in the PCK rats. Like OPC-31260, OPC-41061 significantly decreased renal cAMP levels, kidney weights, cyst and fibrosis volumes, and mitotic and apoptotic indices. It also inhibits activation of Ras and ERK. None of those antagonists prevent development of fibropolycystic liver disease, consisting with the absence of V2R in liver. mTOR, a key player in proliferation of activated T-lymphocytes, is a member of the phosphoinositide kinase-related kinases and phosphorylates target protein such as S6 kinase and 4E binding protein on serine/threonine residue. Inhibition of mTOR by treatment of rapamycin was effective in reducing cyst and kidney volume in two models of recessive PKD, the Tg737orpk and the bpk mice. In addition, rapamycin was also effective in pcy mouse model even when started at a later time point. Reagents which have been successfully tested in different model systems are the EGFR tyrosine kinase inhibitor (Han:SPRD rat), caspase inhibitor (Han:SPRD rat), taxol (cpk mouse), methylprednisolone (Han:SPRD rat), bicarbonate (Han: SPRD rat), and lovastin (Han:SPRD rat). Clinical trials: Clinical trials on mTOR inhibitors showed that no significant influences on cysts progression by sirolimus and rapamycin, respectively. Rapamycin was not approved by US Food and Drug Administration (FDA). Clinical trials on the vasopressin V2 receptor, Tolvaptan, have shown that it delays the increase in total kidney volume, slows the decline in renal function, and reduces kidney pain. Tolvaptan has significant adverse effects including aquaretic effects and elevation of aminotransferase enzyme concentrations with the potential for acute liver failure. Tolvaptan (also: Samsca, JINARC) has been approved for the treatment of ADPKD in Europe, Japan, and Canada. However, the US FDA did not approve Tolvaptan and requested additional information from the pharmaceutical company Ostuka. Development of renal cysts is the key phenotype of PKD. Maintenance of an intact, normal functional signaling pathway, mediated by cilia
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Polycythemia
and its resident proteins, polycystins and polyductin, play key role in preventing cysts formation. Enhanced understanding of the molecular mechanism regulating the polycystins and polyductin signaling would facilitate the intervention of effective targets which could restore the normal functional axis of polycystins and polyductin.
Cross-References ▶ Ca Homeostasis ▶ Cystic Fibrosis ▶ Hyperplasia ▶ Vasopressin
Polycythemia Fulvio Della Ragione1 and Silverio Perrotta2 1 Department of Biochemistry and Biophysics, Second University of Naples, Naples, Italy 2 Department of Pediatrics, Second University of Naples, Naples, Italy
Synonyms
2+
Erythrocytosis
Definition References Cai Y, Fedeles SV, Dong K, Anyatonwu G, Onoe T, Mitobe M, Gao J-D, Okuhara D, Tian X, Gallagher A-R, Tang Z, Xie X, Lalioti MD, Lee A-H, Ehrlich BE, Somlo S (2014) Altered trafficking and stability of polycystins underlie polycystic kidney disease. J Clin Invest 124(12):5129–5144 Chebib FT, Sussman CR, Wang X, Harris PC, Torres VE (2015) Vasopressin and disruption of calcium signalling in polycystic kidney disease. Nat Rev Nephrol 11(8):451–464 Fedeles SV, Tian X, Gallagher AR, Mitobe M, Nishio S, Lee SH, Cai Y, Geng L, Crews CM, Somlo S (2011) A genetic interaction network of five genes for human polycystic kidney and liver diseases defines polycystin1 as the central determinant of cyst formation. Nat Genet 43(7):639–647 Guay-Woodford LM (2003) Murine models of polycystic kidney disease: molecular and therapeutic insights. Am J Physiol Renal Physiol 285:F1034–F1049 Igarashi P, Somlo S (2002) Genetics and pathogenesis of polycystic kidney disease. J Am Soc Nephrol 13:2384–2398 Ma M, Tian X, Igarashi P, Pazour GJ, Somlo S (2013) Loss of cilia suppresses cyst growth in genetic models of autosomal dominant polycystic kidney disease. Nat Genet 45(9):1004–1012 Torres VE, Harris PC (2006) Mechanisms of disease: autosomal dominant and recessive polycystic kidney diseases. Nat Clin Pract Nephrol 2:40–55 Walz G (2006) Therapeutic approaches in autosomal dominant polycystic kidney disease (ADPKD): is there light at the end of the tunnel? Nephrol Dial Transplant 21:1752–1757 Yoder BK, Mulroy S, Eustace H, Boucher C, Sandford R (2006) Molecular pathogenesis of autosomal dominant polycystic kidney disease. Expert Rev Mol Med 8:1–22
A condition in which the hemoglobin amount and the erythrocytes count are increased over the normal values.
Characteristics The Greek term polycythemia (literally, many cells in the blood) corresponds, in the clinical setting, to the word erythrocytosis. This condition exists when hemoglobin amount and the erythrocytes count are increased as the consequence of a buildup of the total volume of red cells. It is possible to distinguish primary and secondary forms as well as congenital or acquired polycythemias. Primary polycythemias show an enhanced capability of the erythroid progenitors to respond to normal levels of circulating cytokines as the consequence of genetic mutations (both germline inherited and somatically acquired) that cause an increased proliferation or a decreased apoptosis of erythrocytes precursors. From a functional point of view, in these forms, there is a dissociation between the total oxygen-carrying ability (i.e., the total hemoglobin content) and the oxygen tissutal requirement or the status of oxygensensing pathways. It is possible to identify forms of primary polycythemias both congenital and acquired.
Polycythemia
The primary congenital and familial polycythemia is an autosomal dominant disorder characterized by low serum erythropoietin (Epo) levels, in vitro hypersensitivity of erythroid progenitors to Epo, normal leukocyte and platelet counts, vitamin B12 level, and bone marrow cytogenetics. A number of cases of primary congenital and familial polycythemias are due to mutations of the erythropoietin receptor gene. Particularly, the mutated receptors lack C-terminal domains and show ineffectiveness of the mechanism(s) by which the receptor activation is shut down by inhibitory loop(s). This results in an excess of Epo stimulatory activity on erythroid precursors. The commonest acquired form of primary erythrocytosis is the polycythemia vera (PV) also known as polycythemia rubra vera. In a frequent number of cases of PV, an excess of white blood cells and platelets, a low Epo level, and splenomegaly are also evident. Thus, PV is classified as a myeloproliferative disease. This erythrocytosis rarely occurs in people younger than 20 years since it usually develops very slowly. The frequency is about 1–2 individuals affected over 100,000. PV has been ascribed to a mutation of the gene encoding the kinase JAK2 (i.e., the change of the nucleotide G at the 1849 position into T that corresponds to the substitution of valine to phenylalanine at the residue 617 of the protein). About 90% of patients with PV have this mutation, but the precise percentage of them that shows the mutation in homozygosity or heterozygosity has not been definitely determined. The JAK2 mutation results in a nonsynonymous amino acid substitution located in the JH2 pseudo-kinase auto-inhibitory domain. The change renders the enzyme constitutively active and leads to cytokine hypersensitivity and Epo independence of erythroid precursor growth and erythrocytosis in a mouse model. Moreover, the constitutive JAK2 activation is accompanied by the enhancement of growth activating pathways (ERK) and antiapoptotic mechanisms (Akt). Although the JAK2V617F mutation seems to explain some features of PV, it is becoming increasingly evident that the genetic change may
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not be the initial clonogenic event in PV. Indeed, the analysis of single colonies of erythroid precursors from PV patients suggests the existence of molecular changes distinct (but yet unknown) from JAK2V617F mutation. In its early stages, PV usually doesn’t cause any signs, while as the disease progresses, several symptoms might occur. These include headaches, weakness, dizziness (vertigo), and/or a ringing noise in the ear (tinnitus). In some cases, individuals with polycythemia vera experience itching (pruritus), especially after a hot bath. Affected individuals often have an abnormally enlarged spleen (splenomegaly) and/or liver (hepatomegaly). In some cases, patients may have associated conditions including high blood pressure (hypertension), the formation of blood clots (thrombosis), rupturing of and loss of blood (hemorrhaging) from certain blood vessels, and/or Budd–Chiari syndrome, a rare disorder characterized by obstruction (occlusion) of veins of the liver (hepatic veins). Secondary polycythemias are characterized by a physiological responsiveness of bone marrow erythroid progenitors to cytokines and by increased serum levels of factors driving erythropoiesis, including Epo, insulin growth factor 1, cobalt, etc. Therefore, secondary polycythemias must be regarded both (i) as a normal response of bone marrow to oxygen requirements of tissues and (ii) as a mutation (germline or somatic) affecting the oxygen-sensing pathway and its target (see above). In other words, in secondary polycythemias, normal erythroid progenitors are stimulated by alteration in external factors. Like primary, also secondary polycythemias might be distinguished in congenital and acquired forms. Several congenital forms have been identified including high affinity hemoglobins due to globin gene mutations or bisphosphoglycerate (BPG) deficiency, congenital methemoglobinemia, and cyanotic congenital heart or lung diseases. Particularly, cyanotic congenital heart disease represents an important cause of polycythemia in young children worldwide. In all these hereditary
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conditions, there exists a pathological low tissue oxygen tension that represents the initial event to activate mechanisms stimulating an increased erythropoietic activity. So far, there are more than 100 mutations of hemoglobin leading to an increase in oxygen affinity and a reduction in oxygen delivery resulting in compensatory polycythemia. These mutations affect the cooperative binding between the globin subunits (a1/b2 interface) or interfere with the binding of BPG. These mutations are dominantly inherited and cause a benign polycythemia. P50 (partial pressure of oxygen where hemoglobin is 50% oxygenated) offers the best initial screening to detect high oxygen affinity accurately and can be estimated mathematically from venous blood gas measurements. BPG deficiency is a rare congenital red cell disorder and results from a deficiency of biphosphoglyceromutase (BPGM). BPG is present in very high concentrations in red blood cells. It binds hemoglobin and allosterically changes its configuration thereby modulating its ability to bind oxygen. A decreased BPG level shifts the oxygen dissociation curve of hemoglobin to the left (increases hemoglobin affinity for oxygen), resulting in decreased delivery of oxygen to the peripheral tissues and compensatory polycythemia. BPGM mutation should be considered in the setting of a low P50 and after globin gene mutations have been definitely excluded. An important form of secondary congenital erythrocytosis, due to peculiar mutations of the von Hippel–Lindau (VHL) gene, has also been identified. The normal response to acute hypoxia (low oxygen pressure) includes hyperventilation, pulmonary vasoconstriction, systemic peripheral vasodilation, and tachycardia. The mechanisms by which these complex systemic responses are activated and modulated are still limited explained and, in some cases, remain elusive. Conversely, it is now clear that intracellular responses to hypoxia are coordinated by the hypoxia-inducible factor (HIF) family of transcription factors, which regulate (both directly or indirectly) the expression of numerous genes in any given cell type.
Polycythemia
About these downstream target HIF genes, it appears important to cite Epo, SLC2A1 (also known as Glut-1, encoding facilitated glucose transporter member 1 of solute carrier family 2), transferrin, transferrin receptor, vascular endothelial growth factor (VEGF), and a number of genes coding for enzymes of the anaerobic glucose metabolism. The VHL tumor suppressor protein is an essential component of the degradation pathway through which some members of HIF family are primarily regulated by oxygen pressure. Indeed, the HIF-alpha is synthesized continuously but is rapidly destroyed in the presence of O2. Oxygen-dependent prolyl hydroxylases hydroxylate specific residues in HIF-1, increasing its affinity for VHL. The binding of VHL to hydroxylated HIF-alpha then targets the protein for destruction by the ubiquitin–proteasome pathway. Under hypoxic conditions, HIF-alpha hydroxylation is inhibited and thus there is rapid accumulation of the transcription factor and the upregulation of hypoxia-responsive genes, including Epo gene. In the classical VHL-associated cancer syndrome, affected individuals are heterozygous for a germline VHL mutation that predisposes to specific tumors. Known clinical manifestations are confined to cancers and discrete benign lesions that arise following somatic inactivation of the second allele. However, alterations of hypoxiadependent pathways are limited to cancer cells and only rarely result in polycythemias. Chuvash polycythemia (CP) is a form of erythrocytosis endemic in Chuvashia (a republic of the Russian Federation) where approximately several hundred cases are recognized among a population of about 1.5 million people. An additional cluster has been subsequently identified in Ischia island (Italy) and a number of cases in other region of the world. CP patients are homozygous for 598 C > T mutation in VHL gene that reduces but does not abolish HIF-alpha activity. The VHL mutation diminishes HIF-alpha degradation by pathologically upregulating HIF-alpha target genes, including Epo. CP is characterized by congenital erythrocytosis, but has yet to be extensively phenotyped. The CP is a non-benign
Polycythemia
hematological disease associated with lower peripheral blood pressures, higher estimated pulmonary artery pressures, varicose veins, vertebral hemangiomas, arterial and venous thrombosis, major bleeding episodes, cerebral vascular events, and premature mortality due in part to cardiovascular and thrombotic events. Moreover, erythroid progenitors of CP patients are hypersensitive to Epo by a molecular mechanism totally obscure. This last property is, however, reminiscent of primary hereditary polycythemias, thus rendering, at least in part, uncertain the classification of CP as a primary or a secondary erythrocytosis. Because CP is characterized by a germline mutation in the VHL gene, it has been hypothesized that homozygotes for this mutation might develop certain vascular tumors similar to those associated with the classic VHL syndrome. However, in no case, spinocerebellar hemangioblastomas, renal carcinomas, and pheochromocytomas typical of classical VHL tumor predisposition syndrome have been found, and no increased risk of cancer has been demonstrated. Through experiments conducted on CP patients, it has been demonstrated the potential role of the VHL pathway in cardiopulmonary physiology. In particular, patients with CP have an elevated basal ventilation and pulmonary vascular tone, with extremely high ventilatory, pulmonary vasoconstrictive, and heart rate responses to acute hypoxia. The abnormalities they displayed mimicked those caused by acclimatization to hypoxia at high altitude. It has been estimated that the VHLC598T mutation arose in a single ancestor between 12,000 and 51,000 years ago. It is possible that the wide dissemination from the original founder may be associated with some survival advantages for heterozygotes carrying this mutation. Such an advantage might be related to a subtle improvement of iron metabolism, erythropoiesis, embryonic development, energy metabolism, or some other yet unknown effect. An intriguing possibility is raised by the demonstration of a protective role for HIF-alpha in protecting against preeclampsia, the leading cause of maternal and
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fetal mortality worldwide. Another positive role of a mildly augmented hypoxic response is an improvement in the bactericidal action of neutrophils, as observed in HIF1-alpha knock-in mice. Finally, other VHL mutations have been detected in either homozygotes or compound heterozygotes affected by secondary congenital polycythemias. It is tentative to classify these cases and Chuvash erythrocytosis as VHL-dependent polycythemias. Although several genetic alterations responsible for secondary polycythemias have been identified, more than 50% of these erythrocytoses with normal or increased serum levels of Epo (and other cytokines) do not have a definite molecular basis. These cases include both diseases inherited with a recessive or a dominant fashion. Secondary polycythemias due to acquired conditions include a large array of causes, all resulting in low peripheral pressure of oxygen. Among these, high-altitude dwelling, chronic obstructive pulmonary disease, sleep apnea, and cyanotic heart disease result in secondary polycythemia as a physiological adaptation. In addition, kidney transplantation has polycythemia as a complication. The cause of this acquired secondary erythrocytosis might be correlated to specific pharmacological treatments required during the posttransplantation period.
References Ang SO, Chen H, Hirota K et al (2002) Disruption of oxygen homeostasis underlies congenital Chuvash polycythemia. Nat Genet 32:614–621 Gordeuk VR, Stockton DW, Prchal JT (2005) Congenital polycythemias/erythrocytoses. Haematologica 90:109–116 James C, Ugo V, Le Couedic JP et al (2005) A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 434:1144–1148 Khan J, Wei JS, Ringnér M et al (2001) Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks. Nat Med 7:673–679 Tefferi A (2006) Classification, diagnosis and management of myeloproliferative disorders in the JAK2V617F era. Hematology Am Soc Hematol Educ Program 2006:240–245
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Polymeric Nanoparticles ▶ Nanospheres
Polymeric Nanoparticles
rich in polyphenols include tea, soybean, berries, chocolate, wine, apple and orange juices, black beans, tomato, sweet peppers, broccoli, and onion.
Characteristics
Polymorphonuclear Leukocyte Elastase ▶ Neutrophil Elastase
Polyneuropathy ▶ Peripheral Neuropathy
Polyomavirus Enhancer Binding Protein 2aB ▶ Runx1
Polyphenols Elvira de Mejia Department of Food Science and Human Nutrition, University of Illinois, UrbanaChampaign, IL, USA
Definition Polyphenols are plant substances possessing more than one aromatic ring bearing one or more hydroxyl groups, including their functional derivatives, and may occur as unconjugated aglycones or as conjugated with sugars, organic acids, amino acids, or lipids. Examples of polyphenols are epigallocatechin gallate, ▶ genistein, ▶ resveratrol, quercetin, and rutin. Common dietary sources
Polyphenols consist of a family of diverse compounds, which comprises chalcones (butein), dihydrochalcones (tephropurpurin), flavanones (naringenin), flavones (apigenin), dihydroflavonols (engeletin), flavonols (quercetin), flavanols (catechins), ▶ isoflavones (genistein), proanthocyanidins (propelargonidins), and anthocyanidins (delphinidin). Their main physiological function is as antioxidants. Therefore, a long-term consumption of a diet rich in plant foods containing polyphenols may offer some protection against chronic diseases, including ▶ cancer. Thus, fruits, vegetables, tea, red wine, and cocoa consumption have been suggested to have the capacity to reduce cancer development. ▶ Flavonoids may also exert other effects unrelated to their antioxidant capacity, for example, ▶ anti-inflammatory effects and inhibition of tumorigenesis. Cancer has been associated with ▶ oxidative stress and mechanisms involving inflammation, aberrant signaling pathways, and ▶ Gap junction intercellular communication. It is possible that the presence in the diet of compounds with the capacity to scavenge free radicals like polyphenols may play a role in oncogenesis. While the free radical scavenging and antioxidant properties of phenolics are well established, emerging literature reports suggest that their chemopreventive effects may also be attributed to their ability to modulate components of cell signaling pathways. Different polyphenols have different degrees of absorption in humans; however, it is believed that isoflavones are the best absorbed even though this parameter is influenced by the matrix of the diet and enhanced by a high fat diet. Studies in humans and animals have indicated that some polyphenols can be absorbed in the small intestine (5–10%), and most of them enter the circulation as methyl, sulfate, and glucuronide conjugates; of these, only a very small amount (5–10%) enter the plasma as unchanged plant polyphenols. The
Polyphenols
90–95% ingested total polyphenols are fermented in the colon, and a variable portion of these (5–50%) are absorbed mainly as conjugates of microbial metabolites. It is clear that the major part of polyphenols consumed never reach the plasma and systemic circulation so the tissues most exposed are those of the oro-gastrointestinal tract. Animal studies have shown that diets rich in polyphenols that reach the colon may protect rodents from carcinogenesis. Inside the human body, flavonoids themselves are of little or no direct antioxidant value; however, inducing phase II enzymes can help in the elimination of mutagens and carcinogens and may be of value in cancer prevention. Polyphenols could also induce mechanisms that help kill cancer cells and inhibit tumor invasion. Cellular and Molecular Studies The biological mechanisms related to the chemopreventive activities of polyphenols are believed to occur by the regulation of signaling pathways such as ▶ nuclear factor-kappa B, activator protein-1, or mitogen-activated ▶ protein kinases. By modulating cell signaling pathways, polyphenols activate cell death signals and induce ▶ apoptosis in precancerous or malignant cells resulting in the inhibition of cancer development or ▶ progression. However, regulation of cell signaling pathways by dietary polyphenols can also lead to cell proliferation/survival or inflammatory responses due to increased expression of several genes. Dietary polyphenols can exert their effects on these pathways separately or sequentially, and in addition, the occurrence of cross talk between these pathways can also take place. Polyphenols can also behave as detoxifying enzyme inducers, modulating gene expression including induction of phase II enzymes, such as glutathione S-transferases and quinone reductase, which usually leads to protection of cells/tissues against exogenous and/or endogenous carcinogenic intermediates. Phase II gene inducers also activate ▶ MAPK kinases that are involved in the transcription activation of antioxidant response element-mediated reporter gene. Genistein, an isoflavonoid with phytoestrogenic properties, in animal models has
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shown to have a very complex effect on carcinogen-induced mammary cancer, and great care is required in extrapolation of this information to human ▶ breast cancer. Some unconjugated isoflavones from fermented soybean and ▶ tamoxifen promoted an additive reduction in the number of mammary tumors in rats. Isoflavonoids have biphasic effects on the proliferation of breast cancer in culture; genistein at low concentrations can stimulate the growth of estrogen receptor-positive breast cancer cells, but it does not stimulate the growth of ▶ estrogen receptor-negative breast cancer cells. Phytoestrogen-responsive genes characterized from these cells can be used to clarify the role of isoflavones in cancer prevention. Of course many other mechanisms of action have been suggested for isoflavones and in particular for genistein. An important aspect of cancer risk is the involvement of the inflammatory response; thus, soy isoflavones may have potentially protective benefits at sites of inflammation due to their antioxidant action and could contribute to anticancer ability because ▶ reactive oxygen species could initiate signal transduction through the mitogenactivated protein kinases. Clinical Studies Human studies are still contradictory and no final conclusions can be drawn on the effect of polyphenols and cancer. Flavonoids may be capable of exerting antioxidant effects in humans with the possibility of direct radical scavenging, downregulation of radical production, elimination of radical precursors such as hydrogen peroxide, metal chelation, inhibition of xanthine oxidase, and of course elevation of endogenous antioxidants. Dietary polyphenols as health-promoting dietary antioxidants have a broader mechanism of action than simple radical scavenging and radical suppression. Studies with pre- and postmenopausal women administered beverages and supplements with isoflavones gave no conclusive results on breast cancer risk factors, and more studies are needed to clarify the effect of isoflavones and breast cancer in women. In cultures where the intake of soy is high and consequently dietary isoflavones, breast-
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fed infants are exposed to high levels without adverse effects, and it has been observed that early exposure may even protect against cancer. Safety and efficacy of isoflavones in humans is a topic that needs further investigation. While experimental models have suggested that flavonoids attenuated cancer risk, epidemiological studies have failed to demonstrate a clear effect for tea, although there is moderate evidence for a slightly positive or no effect of black tea consumption on colorectal cancer. Studies on cancer have been limited by sample sizes and insufficient control of confounder factors. In addition, caution must be exerted when polyphenols bypass the gastrointestinal tract (intravenous injections) or mega doses of these compounds (purified and presented in the form of tablets or capsules) are taken due to possible adverse effects such as nephrotoxicity. The available evidence for tea polyphenols tentatively supports their advancement into phase III clinical intervention trials aimed at the prevention of progression of prostate intraepithelial neoplasia, leukoplakia, or premalignant cervical disease. In the case of curcumin and soya isoflavones, more studies in premalignacies seem appropriate to optimize the nature and design of suitable phase III trials. There is insufficient evidence from human research as yet to claim benefits of polyphenols in relation to cancer prevention. Epidemiological data that suggest tea consumption contributes to cancer prevention do exist; however, these failed to differentiate between green, black, and oolong tea. Studies of colorectal cancer suggested either a slightly positive effect or null effect. In conclusion, further mechanistic insights are needed as well as an accurate knowledge of the concentrations of the chemopreventive agents and their metabolites occurring in humans. Only small amounts of flavonoids may be necessary to see medical benefits. In terms of safety, consistent with the expectation that dietary constituents are harmless and well tolerated, unexpected cases of severe toxicity associated with the consumption of polyphenols have been rare. Severe adverse effects have only been reported for a very limited number
Polyphenols
of cases when consuming daily gram amounts of green tea polyphenols in the form of green tea extracts or when administering high doses of quercetin intravenously in cancer patients.
Cross-References ▶ Anti-Inflammatory Drugs ▶ Apoptosis ▶ Breast Cancer ▶ Cancer ▶ Epigallocatechin ▶ Estrogen Receptor ▶ Flavonoids ▶ Gap Junctions ▶ Genistein ▶ Isoflavones ▶ MAP Kinase ▶ Nuclear Factor-kB ▶ Oxidative Stress ▶ Progression ▶ Protein Kinases ▶ Phytoestrogens ▶ Reactive Oxygen Species ▶ Resveratrol ▶ Tamoxifen
References Fresco P, Borges F, Diniz C et al (2006) New insights on the anticancer properties of dietary polyphenols. Med Res Rev 26:747–766 Gardner EJ, Ruxton CH, Leeds AR (2007) Black tea – helpful or harmful? A review of the evidence. Eur J Clin Nutr 61:3–18 Thomasset SC, Berry DP, Garcea G et al (2007) Dietary polyphenolic phytochemicals – promising cancer chemopreventive agents in humans? A review of their clinical properties. Int J Cancer 120(3):451–458 World Cancer Research Fund, American Institute for Cancer Research (2007) Food, nutrition, physical activity, and the prevention of cancer: a global perspective. AICR, Washington, DC
See Also (2012) Antioxidant Capacity. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 216. doi:10.1007/978-3-642-164835_329
Polyunsaturated Fatty Acids (2012) Carcinogen. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 644. doi:10.1007/978-3-642-16483-5_839 (2012) Leukoplakia. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2028. doi:10.1007/978-3-642-16483-5_3332 (2012) MAPK. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2167. doi:10.1007/978-3-642-16483-5_3532 (2012) Mitogen-Activated Protein Kinase. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2336. doi:10.1007/978-3-642-164835_3770 (2012) Scavenge Free Radicals. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3340. doi:10.1007/978-3-642-16483-5_5166 (2012) Xanthine Oxidase. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3963. doi:10.1007/978-3-642-16483-5_6270
Polyserase (TMPRSS9) ▶ Serine Proteases (Type II) Spanning the Plasma Membrane
Polyunsaturated Fats ▶ Polyunsaturated Fatty Acids
Polyunsaturated Fatty Acids Catalina A. Rosselló1, Manuel Torres1, Xavier Busquets2 and Pablo V. Escribá2 1 University of the Balearic Islands, Palma de Mallorca, Spain 2 Department of Biology, University of the Balearic Islands, Palma de Mallorca, Spain
Synonyms Polyunsaturated fats; PUFAs
Catalina A. Rosselló and Manuel Torres have equally contributed to this work.
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Definition Fatty acids are unbranched hydrocarbons with a carboxyl (COOH) group at one end. Polyunsaturated fatty acids (PUFAs) are fatty acids that contain more than one double bond in their backbone, and they are abundant in cell membranes where they are mainly incorporated into membrane phospholipids. Both natural and synthetic PUFA derivatives exhibit activity against cancer growth. PUFAs can be classified into different groups according to their chemical structure: – Methylene-interrupted polyenes: These fatty acids have two or more cis double bonds that are separated by a single methylene bridge (-CH2- unit). – Conjugated fatty acids. – Other PUFAs. The carbon next to the carboxyl group is known as the a carbon and the next carbon is the b carbon, and so forth. The last position is considered as a “o”. The nomenclature for PUFAs is quite straightforward, and for instance, 18:4 o-3 or 18:4 n-3 (stearidonic acid) indicates an 18-carbon chain with 4 double bonds, where the first double bond lies between the third and fourth carbons from the CH3 end. The physiological properties of unsaturated fatty acids largely depend on the position of the first unsaturated bond relative to the end position. The essential fatty acids, a-linolenic acid (ALA, 18:3 o-3) and linoleic acid (LA, 18:2 o-6), must be acquired from the diet, and they are the starting point for the synthesis of longer and more unsaturated PUFAs: arachidonic acid (ARA, 20:4 o-6), eicosapentaenoic acid (EPA, 20:5 o-3), and docosahexaenoic acid (DHA, 22:6 o-3: Fig. 1). LA is abundant in nearly all commonly available vegetable oils, including corn, sunflower, safflower, and olive oil. Plants can also synthesize ALA and sources of this fatty acid include soybeans; walnuts; dark green leafy vegetables like kale, spinach, broccoli, and Brussels sprouts; and seeds or their oils, such as flaxseed, mustard seed, and rapeseed (canola). The majority of these oils
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Polyunsaturated Fatty Acids
Polyunsaturated Fatty Acids, Fig. 1 Pathways for the desaturation and chain elongation of o-3 and o-6 fatty acids
are also rich in LA. The conversion of ALA to DHA in humans is very inefficient since the same enzymes are involved in the o-3 and o-6 pathways. Indeed, the first enzyme in the bioconversion sequence, D-6 desaturase, is the rate-limiting step in this pathway. Therefore, an EPA- and DHA-enriched diet is recommended to attain the required amounts of these fatty acids.
Characteristics PUFA Intake and Cancer Risk The balance of o-6 to o-3 intake has a strong impact on health. In Western societies, this ratio is about 10–20:1, while in other cultures this ratio is as low as 1–2:1, as has also been seen historically. Indeed, total fat intake and the o-6 to o-3 ratio in Western diets have increased significantly
since the Industrial Revolution. Epidemiological studies, including correlation and migration studies, suggest a protective effect of o-3 PUFAs against cancer, while high o-6 PUFA intake promotes cancer. Several studies have shown that certain eicosanoids can accelerate cancer growth and that enzymes that convert fatty acids to eicosanoids are upregulated in malignant tissues. This is especially true for the o-6 ARA and cyclooxygenase-2 (COX-2). However, not only is the amount of ARA higher in cancer tissue, but the concentration of the o-3 fatty acids, DHA and EPA, is also usually lower compared to the corresponding normal tissue. Accelerated cancer growth could therefore be the combined effect of both increased o-6 levels and decreased o-3 levels (Fig. 2). Omega-3 fatty acid intake is associated with a lower incidence of breast cancer, prostate cancer,
Polyunsaturated Fatty Acids
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Polyunsaturated Fatty Acids, Fig. 2 The chemical structure of o-3 PUFAs and their natural hydroxylated polyunsaturated derivatives that display anti-tumorigenic properties. DHA docosahexaenoic acid, EPA eicosapentaenoic acid, ARA arachidonic acid, PG prostaglandin,
4-HDHA 4-hydroxy-DHA, 17-HDHA 17-hydroxy-DHA, LT leukotriene, 15-HEPE 15-hydroxy-EPA, 13-HODE 13-hydroxy-octadecadienoic acid, 7,17-diHDHA 7,17dihydroxy-DHA or protectin D5, 10,17-diHDHA 10,17dihydroxy-DHA or protectin DX, RV resolvins
colon cancer, and kidney cancer. Observational studies that support the theory of cancer prevention by o-3 fatty acids have been performed on the Inuit population of Alaska, where the high o-3 intake is associated with a diet rich in seal meat and fish, and on the Japanese population that consumes large amounts of fish. DHA levels in the Inuit population are severalfold higher than in Caucasians, and the occurrence of childhood cancer is significantly lower than in a North American population. In addition, the incidence of certain cancers in Japan (e.g., breast cancer) has increased with the consumption of more “westernized” food and the corresponding change in lifestyle. In addition, evidence supporting a protective role of o-3 PUFAs against cancer has come from case-control studies performed in Japan and Scotland. In the Japanese study, there was an inverse trend between the risk of distal colon cancer and o-3 PUFA consumption. In the Scottish study, significant dose-dependent reductions in colon cancer risk were associated with increased intake of total
o-3 PUFAs, as well as separate consumption of EPA or DHA. Cellular and Molecular Mechanisms Mediating v-3-PUFA Anticancer Activity Omega-3 PUFAs, like DHA and EPA, have proven anticancer properties in vitro and in vivo, although the mechanisms underlying these benefits are not clear. By contrast, o-6 PUFAs like ARA or LA are considered to possess tumor-promoting effects. In this context, the pro-tumorigenic effect of o-6 PUFAs is principally attributed to o-6 eicosanoids enzymatically generated by cancer cells, such as prostaglandins and leukotrienes. Interestingly, both o-6 eicosanoids and the enzymatic activity responsible for their generation are increased in many types of cancer. Omega-3 PUFAs are generally regarded as safe compounds, i.e., well tolerated and causing few side effects. Hence, at concentrations that inhibit cancer cell growth, PUFAs exert few or no cytotoxic effects on normal cells. The anticancer effects of omega-3 have been shown in a variety
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of tumors, and, for example, DHA supplementation reduces tumor size in animal models of breast cancer (xenograft or chemically induced). Similarly, o-3 PUFA dietary supplementation reduces human prostate tumor size in mouse xenograft models, inhibiting tumor growth and decreasing serum ▶ prostate-specific antigen (PSA) levels. In this context, mice with prostate tumors in which the PUFA o-6:o-3 ratio is lower than 5 have a longer lifespan. Moreover, o-3 supplementation induces tumor suppression and inhibits metastasis in mice with colon cancer, and monotherapy with DHA inhibits tumor growth in mice with pancreatic cancer and in rats with neuroblastoma xenografts. Omega-3 PUFAs Regulate Membrane Lipid Composition and Structure
Omega-3 PUFAs, like DHA, modify the properties of cell membranes, including their fluidity, permeability, phase behavior, fusion, and membrane-associated protein activity. Phospholipids with DHA regulate plasma membrane organization, which in turn influences protein activity and cell function. Moreover, DHA can also modify specific membrane microdomains like lipid rafts, highly ordered cholesterol- and sphingomyelin-rich structures that mediate a variety of tumor-promoting activities in cancer cells. In cancer cells, the incorporation of PUFAs disrupts lipid rafts and favors the formation of liquiddisordered PUFA-rich/cholesterol-poor non-raft domains. Both DHA and EPA decrease the sphingomyelin content in lipid rafts in certain cancer cells, which might reflect the activation of sphingomyelinase activity and the ensuing production of ceramide, a well-known mediator of cellular stress and apoptosis. The anticancer effects of PUFAs have been associated with the regulation of the lipid raft structure and the subsequent alterations to the expression and/or activity of proteins involved in cancer cell growth (e.g., CXCR4 and EGFR). Omega-3 PUFAs and Bioactive Hydroxylated Polyunsaturated Metabolites
PUFAs are precursors of biologically active metabolites, such as eicosanoids, resolvins, and
Polyunsaturated Fatty Acids
protectins. Most of these metabolites are naturally generated hydroxylated derivatives, and many show anticancer properties (Fig. 2). In this regard, the incorporation of o-3-PUFAs (such as DHA and EPA) into glycerophospholipids is more efficient than that of o-6 PUFAs, and consequently, a decrease in the ARA levels in membranes reduces ARA-derived eicosanoid production. Therefore, the antiproliferative and pro-apoptotic effects of o-3 PUFAs are in part due to the protection afforded to the cell from ARA-derived eicosanoid pro-inflammatory and pro-tumorigenic activities. Omega-3 PUFAs affect the formation of certain hydroxylated lipid mediators that are generated enzymatically by COX and lipoxygenase (LOX). While DHA is believed not to be a substrate for COX enzymes, COX-2 can act on EPA and transform it into the o-3 prostaglandin, termed prostaglandin E3 (PGE3). PGE3 inhibits cancer cell proliferation and invasion, and it induces cancer cell apoptosis. In addition, PGE3 also hinders angiogenesis by inhibiting the expression of angiopoietin-2 (Ang2) and matrix metalloprotease-9 (MMP-9). Another EPA-derived lipid mediator generated via COX is D12-prostaglandin J3 (D12-PGJ3), which inhibits leukemia progression in animal models. Unfortunately, EPA is a poor substrate for COX enzymes, compared with ARA, so that generation of these EPA-derivatives is not favored enzymatically. LOX transforms PUFAs into leukotrienes, hydroxylated forms of PUFAs. Among the LOX isoforms, 5-LOX is classically implicated in cell proliferation and tumorigenesis. However, 5LOX also transforms DHA into the antiangiogenic 4-hydroxy-docosahexaenoic acid (4-HDHA), and it can produce leukotriene-B5 (LTB5), a hydroxyl derivative of EPA with antiproliferative properties. DHA and EPA may also be substrates for 15-LOX, which yields 17-hydroxy-docosahexaenoic acid (17-HDHA) and 15-hydroxy-eicosapentaenoic acid (15-HEPE), respectively. Both hydroxyl derivatives have anti-inflammatory and anticancer activities, and 17-HDHA in particular inhibits prostate cancer cell proliferation at doses much lower than
Polyunsaturated Fatty Acids
those of DHA itself. Other 15-LOX derivatives of DHA are 17-hydroperoxy-DHA (17-HpDHA), 10,17-dihydroxy-DHA (10,17-diHDHA or protectin DX) and 7,17-dihydroxy-DHA (7,17diHDHA or protectin D5), which also inhibit the proliferation of prostate cancer cells. Interestingly, the LA metabolite 13-hydroxy-octadecadienoic acid (13-HODE) produced via 15-LOX also exhibits similar anticancer activity (Fig. 2). Resolvins (RVs) are hydroxylated metabolites derived from EPA and DHA that possess potent anti-inflammatory properties at concentrations in the nanomolar to picomolar range. Compounds derived from DHA are classified as RVs of the D-series or protectins, while those formed from EPA are designated as RVs of the E-series. DHA can be hydroxylated on carbon 17 by 15-LOX or acetylated by COX-2, leading to stereoselective formation of 17S- or 17R-HDHA, respectively. These derivatives can be further hydroxylated to give rise to the trihydroxy derivatives, the D1, D2, D3, and D4 17-(S/R)-resolvins (D-series RVs), and the dihydroxy compound 17-(S/R)-protectin or neuroprotectin (NPD1). EPA can be stereoselectively hydroxylated to 18-(S/R)-hydroxyeicosapentaenoic acid (18-HEPA), which is further processed to form the E1, E2 and E3 18-(S/R)-resolvins (E-series RVs). Both 17-HDHA and 18-HEPA serve as markers for resolvins and protectins, and remarkably, their presence in blood is directly related to the intake of o-3 PUFAs in animal models. The anticancer properties of RVD1 and RVD2 have been studied in lung cancer cells, where they failed to regulate cancer cell proliferation, but they did inhibit the transforming growth factor beta-1 (TGFb-1)mediated epithelial-mesenchymal transition. Since this transition is considered a key event for tumor cells to initiate metastasis, RVD1 and RVD2 have been attributed anti-metastatic properties (see Fig. 2). By contrast, the anticancer activity of other resolvins remains uncertain, such as E-series RVs. Similarly, 2-hydroxylated PUFA derivatives (e.g., 2-hydroxy-DHA, 2-hydroxy-linoleic acid, etc.) have shown efficacy against various types of cancer (patent WO2010106211 A1), and some of them are
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under clinical development (see below), although their mechanisms of action remain unresolved. Hydroxylated PUFAs are autocrine and/or paracrine mediators that regulate inflammation and cellular homeostasis. Many of them are short-lived molecules, produced and acting locally in response to cellular stimuli, rapidly degraded or catabolized to maintain homeostasis. Consequently, these PUFA derivatives are thought to exert their biological function by cellular mechanisms that extend beyond the regulation of lipid membrane composition and structure. On the one hand, the oxidative metabolism of o-3 PUFAs is different in cancer and normal cells, with a strong production of cytotoxic oxidative o-3 PUFA lipid mediators in cancer cells. In the case of DHA, DHA-derived lipid mediators are neurotoxic to neuroblastoma cells due to the accumulation of the highly cytotoxic 17-HpDHA, an intermediate product in the synthesis of resolvins and protectins. Indeed, the production of protectins and resolvins seems to be blocked in neuroblastoma cells, and thus, the accumulation of 17-HpDHA leads to oxidative stress, growth arrest, and apoptosis. By contrast, 17-HpDHA is fully converted into resolvins and protectins in normal neurons. Hence, cancer cells appear to be subjected to increased oxidative stress as a consequence of membrane DHA enrichment. Accordingly, various antioxidant molecules can inhibit o-3 PUFA-induced apoptosis, suggesting the involvement of lipid peroxidation-derived reactive oxygen species (ROS) in the cytotoxic action exerted by o-3 PUFAs in cancer cells. On the other hand, many of these lipid mediators bind to specific receptors that trigger or modulate cellular signaling cascades. Binding of Omega-3 PUFAs and of Their Bioactive Lipid Mediators to Fatty Acid Receptors
Fatty acids are ligands for cell-surface G proteincoupled receptors (GPCRs) and peroxisome proliferator-activated receptors (PPARs). Several studies report that free fatty acids (FFA) can bind to and activate several GPCRs, including GPR40, GPR41, GPR43, GPR84, and GPR120. GPR40 (also known as the FFA Receptor 1) and GPR120
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(FFA Receptor 4) specifically bind o-3 PUFAs as agonist ligands. Moreover, GPR40 is overexpressed and activated in breast cancer cells, in which its activation promotes proliferation, whereas GPR120 activation by EPA or DHA inhibits the lysophosphatidic acid- and EGF-induced proliferation and migration of human prostate cancer cells. Finally, it is worth noting that the inhibitory effect of the DHA-hydroxyl derivative RVD1 on the TGFb-1-mediated epithelial-mesenchymal transition of lung cancer cells is exerted through binding to another GPCR, GPR32, also known as the lipoxin A4 receptor. PPARs (PPARa, PPARb/d, and PPARg) are a superfamily of ligand-activated transcription factors and nuclear hormone receptors. Growing evidence has demonstrated that PPARg serves as a tumor suppressor in cancer. Omega-3 PUFAs can induce apoptosis in human prostate cancer cells by activating PPARg and upregulating the PPARg target gene, syndecan-1 (SDC-1). Interestingly, loss of cell-surface SDC-1 has been reported in many carcinomas, such as skin cancer and colorectal adenocarcinomas favoring the acquisition of a metastatic phenotype by tumor cells, whereas DHA mediates SDC-1 upregulation in human breast cancer cells and mammary glands, and in the liver in animal models. Furthermore, several PUFA lipid mediators exert their anticancer properties via mechanisms involving PPARg activation, such as 4-HDHA, 17-HDHA, 10,17- and 7,17-dihydroxy-DHA, and 13-HODE. Indeed, it has been suggested that o-3 PUFAs induce cell apoptosis in prostate cancer through a mechanism involving 15-LOX, provoking the SDC-1dependent suppression of phosphorylation in the PDPK1/AKT and MEK/Erk (MAPK) pathways that unavoidably leads to Bad activation and cancer cell apoptosis. These data suggest that DHA-hydroxylated derivatives are preferential ligands for PPAR-g rather than FFA. Omega-3 PUFA-Based Therapeutic Strategies and Clinical Trials in Cancer The epidemiological evidence available, combined with the known effects of o-3 PUFAs on cancer in animal and cell models, has motivated
Polyunsaturated Fatty Acids
the development of clinical intervention trials using fish oil or o-3 PUFAs to give nutritional supplementation or prevent and treat cancer. Most trials aim to upregulate o-3 PUFAs, downregulate o-6 PUFAs, and/or decrease the o-6/o-3 ratio in cell membranes. Nutritional supplements containing o-3 PUFAs are beneficial to reduce cancer-associated weight loss (cachexia). In addition, administration of o-3 PUFAs either before or after major abdominal surgery for cancer reduces inflammatory cytokines and improves liver and pancreatic function. The anti-inflammatory effects of fish oil o-3 fatty acids may help tolerate chemotherapy or radiotherapy in cancer patients (ClinicalTrials. gov identifier NCT01871350). Moreover, DHA also has interesting synergistic effects with other compounds, such as curcumin. Therefore, PUFAs could be considered as powerful nontoxic adjuvants in canonical anticancer treatments, especially for patients suffering from late-stage metastatic breast cancer. Omega-3 supplements for subjects with a high risk of developing colon cancer reduce ARA in the rectal mucosa and cell proliferation to those levels found in low-risk subjects. Moreover, o-3 PUFAs may slow tumor progression in prostate cancer patients (ClinicalTrials.gov identifiers: NCT00798876, NCT00253643, NCT01869764, NCT02176902, NCT00402285). Dietary interventions with o-3 PUFAs have also been assessed for the prevention and/or treatment of other types of cancer, including breast and skin cancer or lymphoma (ClinicalTrials.gov identifiers: NCT01869764, NCT01478477, NCT02150525, NCT01032343, NCT00003077, NCT00455416). Other therapeutic approaches involve the direct administration of hydroxylated bioactive derivatives from DHA and/or EPA such as natural or synthetic resolvins. A number of hydroxylated PUFAs are currently under development for the treatment of cancer, and like other hydroxylated compounds, their mechanism of action follows the principles of ▶ Membrane-Lipid Therapy (Fig. 3). For instance, a-hydroxy-linoleic acid is under clinical investigation for the treatment of advanced solid tumors (ClinicalTrials.gov
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References Azrad M, Turgeon C, Demark-Wahnefried W (2013) Current evidence linking polyunsaturated fatty acids with cancer risk and progression. Front Oncol 3:224 Gleissman H, Johnsen JI, Kogner P (2010) Omega-3 fatty acids in cancer, the protectors of good and the killers of evil? Exp Cell Res 316:1365–1373 Ibarguren M, López DJ, Escribá PV (2014) The effect of natural and synthetic fatty acids on membrane structure, microdomain organization, cellular functions and human health. Biochim Biophys Acta 1838:1518–1528 Lee HJ, Park MK, Lee EJ, Lee CH (2013) Resolvin D1 inhibits TGF-b1-induced epithelial mesenchymal transition of A549 lung cancer cells via lipoxin A4 receptor/formyl peptide receptor 2 and GPR32. Int J Biochem Cell Biol 45:2801–2807 Serini S, Fasano E, Piccioni E, Cittadini ARM, Calviello G (2011) Dietary n-3 polyunsaturated fatty acids and the paradox of their health benefits and potential harmful effects. Chem Res Toxicol 24:2093–2105
Population Candidate Gene Association Study ▶ Case Control Association Study Polyunsaturated Fatty Acids, Fig. 3 Threedimensional structure of PUFAs and a-hydroxylated PUFAs used for nutraceutical or pharmaceutical treatment of diverse human conditions, including cancer. In column “a” are the native forms and in column “b” the a-hydroxylated form of (1) linoleic acid, (2) a-linolenic acid, (3) g-linolenic acid, (4) arachidonic acid, (5) eicosapentaenoic acid, and (6) docosahexaenoic acid
Population-Based Cancer Research ▶ Cancer Epidemiology
Positron Emission Tomography identifier NCT02201823). This compound has been described as an inhibitor of the mammalian target of rapamycin, mTORC1/C2. Interestingly, the mTOR signaling pathway is upregulated in many types of cancer, and it seems to be involved in ARA-induced proliferation and angiogenesis of breast tumors.
Andreas K. Buck1 and Markus Schwaiger2 1 Department of Nuclear Medicine, University of Würzburg, Würzburg, Germany 2 Department of Nuclear Medicine, Technical University of Munich, Munich, Germany
Synonyms Cross-References ▶ Membrane-Lipid Therapy ▶ Prostate-Specific Antigen
Hybrid positron emission tomography/computed tomography; Integrated positron emission tomography/computed tomography
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Definition Positron emission tomography (PET) is a noninvasive nuclear medical imaging modality enabling the visualization and quantification of biological processes. PET provides integral information regarding metabolic activity of the primary tumor and potential lymph node or distant organ metastases. PET can be used for cancer detection, tumor staging and restaging, assessment of response to treatment, and anticancer drug development.
Characteristics Principle of Positron Emission Tomography (PET) PET allows noninvasive assessment of the threedimensional distribution of a positron-labeled compound within the living body. Positrons are antiparticles of electrons and originate from b+ decays of radioactive isotopes such as 11C, 13N, 15 O, 18F, 68Ga, 86Y, or 124I. During b+ decay, a positron and a neutrino are emitted, both sharing a certain amount of kinetic energy. Once the positron is slowed down, a positronium consisting of a positron and an electron is created. The positronium has a very short half-life of 1010 s, and the masses of the positron and the electron are finally transferred into energy. This annihilation results in two gamma quants with an energy of 511 keV each. Decay events are detected by coincidence registration enabling the measurement of activity distribution in a specific transaxial section of the body. Activity distribution can be calculated from respective projections after correction for scatter, attenuation, dead time, and random coincidences. Attenuation correction can be performed using a radioactive transmission source rotating around the patient. Emission and transmission scanning from the scull to the mid thigh usually takes 30–45 min and whole-body scans 60–90 min. The radiation dose of a standard PET examination is low with approximately 7.4 mSv and similar to spiral CT of the thorax.
Positron Emission Tomography
Radiolabeled Biomarkers for PET Imaging Specifically Addressing Metabolic Pathways or Target Molecules Depending on the clinical situation, various radiolabeled pharmaceuticals can be utilized for tumor imaging (Table 1). The most important biomarker for functional diagnosis of tumors is the glucose analog 20 -[18F]-fluoro-20 -deoxy-D-glucose (FDG, 18 F-fluorodeoxyglucose) (Fig. 1). Since conventional imaging modalities such as computed tomography (CT), magnetic resonance imaging, or ultrasound detect malignant lesions because of characteristic morphological alterations, FDG-PET enables the diagnosis of malignant tumors due to an increased glucose metabolism in malignant cells. After intravenous administration, FDG is predominantly taken up by tumor cells. After enzymatic conversion of FDG to FDG-6-monophosphate by hexokinase, the metabolite cannot be further metabolized resulting in an intracellular “trapping” of FDG (metabolic trapping). There are many other radiopharmaceuticals capable of assessing distinct pathophysiological processes (Table 1). As an example, radiolabeled nucleoside analogs such as 30 -deoxy-30 -[18F]fluorothymidine (FLT, F-fluorothymidine) can be used to noninvasively assess the proliferative activity of tumors. With the positron emitter 15O, H2 15O can be synthesized and used for assessment of tumor blood flow. A variety of radiolabeled amino acids such as [11C]methionine (MET, C-methionine), [11C]-leucine (LEU), or [18F]-fluoro-ethyl-tyrosine (FET, F-fluoroethyltyrosine) can be used to evaluate transport rates of amino acids and/or protein biosynthesis. Imidazole derivatives such as [18F]misonidazole (FMISO) can be used to delineate hypoxic tissue areas of the tumor which is particularly useful for radiation treatment planning. Synthesis of phospholipids is increased in many neoplasms leading to increased uptake of [18F]choline and [11C]-choline (CHO, C-choline). [68Ga]-DOTATOC (DOTATOC, [68Ga] DOTATOC) specifically binds to somatostatin receptors and is therefore highly sensitive for detection of neuroendocrine tumors. [18F]galacto-RGD (RGD, F-galacto-RGD) has a high
Positron Emission Tomography
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Positron Emission Tomography, Table 1 Radiopharmaceuticals (tracer) used for PET imaging Radiopharmaceutical (*under investigation) 18 F-fluorodeoxy-glucose (FDG)
11
Native molecule Glucose
C-choline (CHO), F-fluorethylcholine (FEC), 18 F-fluorocholine
Choline
11
Acetate
18
C-acetate
68
Uptake mechanism in cancer Glucose transport/ phosphorylation by hexokinase
Uptake by active transport and phosphorylation by choline kinase; incorporation into phospholipids (cellular membrane) Lipid synthesis (key enzyme, fatty acid synthase, FASE)
Octreotide (somatostatin analog)
Binding to somatostatin receptors (predominantly SSTR-2)
18
Dihydroxyphenylalanaline
Uptake in tumors capable of DOPA decarboxylation
18
F-fluoro-ethyl-tyrosine (FET), 11C-methionine (MET)
Tyrosine methionine
Amino acid transport, protein biosynthesis
18
Peptide containing the sequence RGD (arginine, glycine, aspartate)
Binding to integrin avb3 (vitronectin receptor), expressed on activated endothelial cells
18
Thymidine
DNA synthesis, tumor cell proliferation
Ga-DOTATOC, Ga-DOTATATE
68
F-DOPA
F-galacto-RGD (RGD*)
F-fluorothymidine (FLT*), C-thymidine (THY*)
11
Clinical applications Diagnosis, staging, restaging (e.g., cancer of the lung, breast, colon, rectum, and thyroid; lymphoma melanoma; sarcoma); CUP monitoring of response to therapy (e.g., lymphoma, various cancer types including breast, GI tract, lung*) Staging, restaging (prostate cancer, bladder cancer*); therapy monitoring (prostate cancer*, bladder cancer*)
Staging, restaging, monitoring response to therapy in a variety of cancers Diagnosis, staging, restaging of neuroendocrine tumors, CUP (neuroendocrine) Diagnosis, staging, restaging of neuroendocrine and brain tumors, CUP (neuroendocrine) Diagnosis, staging, restaging of brain tumors; differentiation of scar/ recurrence, monitoring response in various tumor types Assessment of tumor angiogenesis (e.g., melanoma, sarcoma, head and neck cancer, breast cancer), monitoring response to antiangiogenic treatment Assessment of tumor proliferation (monitoring response to cytotoxic treatment in lymphoma, sarcoma, breast cancer) (continued)
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Positron Emission Tomography
Positron Emission Tomography, Table 1 (continued) Radiopharmaceutical (*under investigation) 18 F-fluoroazamycine arabinoside (FAZA*), 18Ffluoromiso-nidazole (FMISO*)
Native molecule Hypoxia markers (no biologic analog)
11
F-fluoro-17-b-estradiol (FES*)
Estradiol
18
Fluoride
F-fluoride
Uptake mechanism in cancer Passive diffusion into hypoxic cells; reactive intermediates are formed by intracellular nitroreductase and trapped within the cell Binding to estrogen receptors Bone mineralization
Clinical applications Assessment of tumor hypoxia (especially for use in tumors of the head and neck, radiation treatment planning) Monitoring of response to antihormone treatment in breast cancer Screening for bone metastases at staging or restaging
CUP cancer of unknown primary
CH2OH
OH
CH2OH 6
OH
6 OH
OH O
OH
O
OH
OH 18F
Glucose
FDG
Positron Emission Tomography, Fig. 1 The glucose analog FDG (20 -[18F]-fluoro-20 -deoxy-D-glucose) is the most widely used radiopharmaceutical for PET imaging. After IV injection, FDG is taken up by glucose transporters Glut-1 (cancer tissue, brain) and Glut-4 (skeletal muscle, heart) and is phosphorylated by hexokinase II. FDG-6-P is
not a substrate for hexokinase phosphate isomerase which is the next enzyme of the glycolytic pathway. Consecutively, FDG accumulates in the cytoplasm (metabolic trapping). The figure shows the structure of the native glucose molecule (left) and the [18F]-labeled analog (right)
affinity to the vitronectin receptor avb3 and can be used as potential surrogate marker of neoangiogenesis. These and many other radiopharmaceuticals specifically address metabolic pathways or bind to specific target structures and therefore enable molecular imaging of cancer. Specific radiotracers are especially helpful for evaluation of new drugs and early response assessment in cancer.
tool for characterization of indeterminate lesions, initial staging, restaging, and assessment of response to therapy in a variety of cancers. Combination of a PET scanner with spiral computed tomography in a single examination (PET/CT, integrated positron emission tomography/computed tomography) allows integrated functional (PET) and morphologic (CT) imaging. Additionally to the results returned by individual modalities, coregistration of CT allows precise localization of PET lesions. The addition of PET to CT leads to an increase of sensitivity as well as specificity for tumor imaging. Moreover, CT data can be used for attenuation correction which leads to a significant reduction of scanning time making
Clinical Applications of PET and PET/CT The introduction of PET to clinical medicine has influenced the management of patients with cancer. In most industrialized countries, PET is now accepted as a both useful and economic diagnostic
Positron Emission Tomography
PET/CT more comfortable for the patient. A standard examination including the head, thorax, abdomen, and pelvis can be performed within 25–30 min. Since its introduction to clinical medicine in 2001, PET/CT represents the fastest growing imaging modality. The Centers for Medicare and Medicaid Services (CMS) approved a variety of clinical indications including staging and restaging of non-small cell ▶ lung cancer, ▶ esophageal cancer, ▶ colorectal cancer, ▶ breast cancer, head and neck cancers (oral squamous cell carcinoma, ▶ oral cancer), malignant lymphoma, and ▶ melanoma. Monitoring response to treatment in breast cancer is also covered. The CMS announced to provide widespread coverage of PET when respective examinations are part of prospective clinical trials. Differentiation of Benign from Malignant Tumors and Detection of the Primary Tumor (Cancer of Unknown Primary) Due to different glucose consumption of benign and malignant lesions, FDG-PET allows assessment of undefined tumors detected by conventional imaging modalities such as CT or MRI. Furthermore, PET sometimes allows detection of malignant lesions even when no or only minimal morphologic alterations are present. Regarding evaluation of indeterminate pulmonary nodules, prospective studies reported sensitivity values for FDG-PET between 89% and 100%, a specificity of 69–100%, and an overall accuracy of 89–96%. FDG is not tumor specific leading also to nonspecific tracer accumulation in benign, predominantly inflammatory lesions. However, surgery may be circumvented in patients with increased perioperative risk if the PET scan is negative. Dynamic data acquisition can further enhance the accuracy of PET imaging. In malignant lesions, a continuous increase of glucose uptake has been described, whereas benign lesions showed an increase of FDG uptake followed by rapid efflux of FDG. Dual time point imaging or delayed PET imaging after 1 and 2 h contributes to better differentiate between benign and malignant tumors. PET can also be used for detection of the malignant primary (cancer of unknown primary,
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CUP). PET is especially useful in detecting primary tumors in the head and neck region. In case of increased cancer biomarkers (▶ clinical cancer biomarkers) or paraneoplastic syndromes, PET can aid in localizing the primary tumor manifestation site. Staging of Cancer, Prognostic Potential of PET For optimal treatment of patients with cancer, precise knowledge of the extent of the disease is crucial (tumor staging). If cancer is detected at a stage in which uncontrolled growth of tumor cells takes place but no tumor manifestations are present in distant organs, surgery is usually performed to obtain ultimate cure. However, if the tumor has already spread to distant organs, cure can usually not be achieved by surgery alone (Fig. 2). In this situation, surgery has to be replaced or supported by systemic chemo- and/or radiotherapy to entirely destroy the primary tumor and metastatic sites or to induce growth arrest in the tumor. In this context, PET has several advantages compared to conventional imaging modalities. Small tumor manifestation sites such as ▶ metastases in the bone, liver, lung, adrenal gland, or in rare locations such as soft tissues, thyroid, or (sub-) cutaneous lesions can be detected. However, micrometastases or single tumor cells can also not be detected with PET. Also, small lung metastases may appear negative at FDG-PET. In principal, staging of all tumors is possible. With the standard radiotracer FDG, PET is highly accurate for staging of non-small cell ▶ lung cancer, thyroid cancer, tumors of the head and neck region (oral squamous cell carcinoma, ▶ oral cancer), colon cancer and ▶ esophageal cancer, malignant lymphoma, sarcoma (▶ osteosarcoma, ▶ Ewing sarcoma), and ▶ melanoma. PET has been demonstrated to cause a change in patient management in 15–40% depending on the type of cancer. Some tumors present without increased glucose consumption such as prostate or neuroendocrine cancer. 11Ccholine PET and 11C-choline PET/CT have been demonstrated to be highly accurate for staging and especially restaging of prostate cancer. 68GaDOTATOC is a new PET tracer for imaging neuroendocrine tumors. A variety of molecular
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Positron Emission Tomography
Positron Emission Tomography, Fig. 2 (a) FDG-PET/ CT in breast cancer (maximum intensity projection); malignoma-associated intense FDG uptake in primary breast cancer, lymph node metastases in the axilla, liver metastases, and bone metastases (arrows). (b) Transaxial PET section showing intense FDG uptake of the primary tumor. (c) Transaxial section of helical CT indicates the
anatomic correlate of primary breast cancer. (d) Fused image. (e) Transaxial PET section, high FDG-uptake in a bone metastasis of the vertebral column. (f) Transaxial section of helical CT indicates a sclerotic lesion corresponding to the PET lesion. (g) Fused image. Intense physiologic uptake of FDG in the brain, the heart, and intestines (*)
probes have been evaluated to address biologic targets or metabolic pathways in vivo (Table 1). In the majority of these compounds, clinical utility remains to be determined. The most important prognostic factor (prognostic biomarker) is the tumor stage at initial presentation. However, risk stratification according to the TNM system is also subject to error, because patients with limited disease undergoing definite therapy may also develop recurrent disease. Other factors such as tumor aggressiveness or metabolic activity of tumors may aid in individual risk assessment. Several studies have correlated the intensity of FDG uptake in the primary tumor to progression-free and overall survival in various cancers. In lung cancer,
intensity of FDG uptake turned out to be an independent prognostic marker. The prognostic potential of PET has also been described for colorectal cancer, breast cancer, and malignant lymphoma. Assessment of Response to Therapy Therapeutic efficiency of chemo- and radiotherapeutic strategies varies significantly between individual patients. Therefore, noninvasive assessment of the performance of a therapeutic protocol in an individual patient is highly desirable. With conventional imaging modalities such as CT or MRI, response (▶ radiological response criteria) to therapy can be detected as early as a reduction in tumor size occurs. On the contrary, PET allows assessment of response to treatment at
Positron Emission Tomography
an earlier time point before tumor shrinking can be detected by conventional imaging. In responding tumors, metabolism of tumor cells is markedly decreased due to the cytotoxic effect of the respective therapeutic regimen. Concomitantly, accumulation of FDG is reduced. This is a sign of an efficient treatment and has a high prognostic value regarding the success of further treatment. In case of a nonresponding tumor, the therapeutic regimen can be altered by changing the combination of cytotoxic drugs or the radiation dose. In ▶ breast cancer, rapid decline of FDG uptake already after one cycle of chemotherapy was demonstrated, whereas in nonresponding tumors, increasing or unchanged FDG uptake was described. A variety of other neoplasms including malignant lymphoma, ▶ gastric cancer and ▶ esophageal cancer, head and neck cancer (oral squamous cell carcinoma, ▶ oral cancer), or non-small cell ▶ lung cancer showed rapid reduction of FDG uptake in responding tumors. Significantly better disease-free and overall survival was described in responders compared to tumors without significant reduction of tumoral FDG uptake (Fig. 3). Clinical studies are needed reporting on the clinical benefit of a PET-guided change of patient management. Restaging of Cancer, Detection of Recurrence After definite surgery or chemo/radiotherapy, examinations and imaging at follow-up are important to early detect disease recurrence originating from residual tumor cells. In daily clinical practice, differentiation between scar tissue and vital tumor tissue is a frequent problem. At anatomically based imaging modalities, both are present as indeterminate tissue formation, and frequently, biopsy is needed for further clarification. Differentiation of scar tissue from vital tumor tissue is a prerequisite of PET imaging. While new onset of cancer tissue is associated with increased metabolism causing increased uptake of, e.g., FDG, scar tissue is frequently associated with reduced metabolism compared to surrounding normal tissue. PET is especially useful in the follow-up of tumor entities such as colorectal (▶ colorectal cancer) and ▶ esophageal cancer, non-small cell ▶ lung cancer, ▶ breast cancer, tumors of the head
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and neck (oral squamous cell carcinoma, ▶ oral cancer), brain tumors (malignant brain tumors), ▶ melanoma, and malignant lymphoma. Restaging with PET is also approved for differentiated thyroid carcinoma with a negative 131I whole-body scan and elevated tumor marker thyroglobulin. Radiation Treatment Planning The use of metabolic information leads to biological target volumes which can have substantial impact on radiation treatment planning (▶ radiation oncology) by increasing or reducing the target volume. The additional identification of tumor manifestation sites which are not visible at conventional staging causes an enlargement of respective target volume. On the other hand, the radiation field can be reduced when nonmalignant lesions such as atelectatic tissue can be reliably characterized as benign (Fig. 4). Consecutively, radiation dose to surrounding normal tissue can be reduced. The use of PET for radiation treatment planning leads to a change of the target volume in up to 60% of patients. This is in part related to pretherapeutic detection of distant metastases, previously unknown metastases in locoregional lymph nodes, or characterization of suspicious lesions as benign. However, PET-based radiotherapy planning is not trivial. Especially the delineation of the primary tumor is subject to a relevant interobserver variability. There is a need for standardized evaluation criteria of PET allowing also the quantification of metabolic changes. The introduction of PET/CT hybrid scanners has lead to a reduction of errors concerning image coregistration. In several prospective studies, it was shown that overall survival of patients receiving PET-guided radiation therapy was significantly longer compared to patients receiving standard treatment. Prospective randomized studies have to be performed demonstrating that the use of PET positively affects patient outcome and overall survival. PET for Anticancer Drug Development PET imaging has unique properties for use in anticancer drug development. Therapeutic efficiency of a novel drug can be evaluated
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Positron Emission Tomography
Positron Emission Tomography, Fig. 3 (a) FDG-PET for response assessment in malignant lymphoma (maximum intensity projection); intense FDG uptake of multiple lesions of high-grade non-Hodgkin B-cell lymphoma (arrows) indicates lymphoma manifestations in the left supraclavicular region and in paraaortic, parailiac, and
inguinal lymph nodes. (b) Corresponding PET image of the same patient 3 weeks after completion of eight cycles of chemotherapy with R-CHOP. No pathologic FDG uptake in residual lymph nodes indicates complete remission of the disease and favorable outcome. (c–f) Corresponding transaxial sections of PET and CT
noninvasively by assessment of specific biologic endpoints such as changes in cellular proliferation (e.g., by the use of [18F]FLT), glucose utilization ([18F]FDG), tissue perfusion ([15O]H2O), metabolism of amino acids ([18F]FET, [11C]MET), or inhibition of ▶ angiogenesis ([18F]galacto-RGD). (Over-)expression of the therapeutic target such as thymidylate synthase, VEGF receptor, ErbB2, or ▶ estrogen receptor status can be quantified with [11C]thymidine, radiolabeled antibodies specifically binding to VEGF or ErbB2, or 18F-fluoro17-b-estradiol, respectively. Assessing biologic endpoints further provides proof of principle of the proposed mechanism of action. PET can also
be utilized for in vivo evaluation of gene expression, e.g., by the use of the substrate [124I]fluoro5-iodo-1-b-D-arabinofuranosyluracil (FIAU) for detection of Herpes simplex virus thymidine kinase type 1 or Na[124I] for detection of sodium iodide symporter expression. Generic endpoints can also be studied by PET. Drugs or biochemical probes can be labeled with positron emitters such as small molecules, proteins, or antibodies. Drugs which have been evaluated so far include 18F-fluorouracil, 18 F-tamoxifen, or 13N-cisplatin. Pharmacokinetics of a drug can be investigated in tumors and normal tissues, in animal models or as part of
Positron Emission Tomography
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Positron Emission Tomography, Fig. 4 FDG-PET/CT for radiation treatment planning of non-small cell lung cancer. (a) Transaxial section of spiral CT shows a central tumor in the left lung which can not be discriminated from adjacent atelectatic tissue. (b) Corresponding section of
FDG-PET indicates high metabolic activity of the malignant primary but anatomic landmarks are missing preventing precise tumor localization. (c) Fused PET/CT image allows exact delineation of the tumor which can be distinguished from adjacent atelectatic tissue
clinical phase I (or phase II) studies. In the future, PET will be increasingly used to assess the efficiency of novel anticancer drugs.
Lardinois D, Weder W, Hany TF et al (2003) Staging of non-small-cell lung cancer with integrated positronemission tomography and computed tomography. N Engl J Med 348:2500–2507 von Schulthess GK, Steinert HC, Hany TF (2006) Integrated PET/CT: current applications and future directions. Radiology 238:405–422 Weber WA (2006) Positron emission tomography as an imaging biomarker. J Clin Oncol 24:3282–3292
Cross-References ▶ Angiogenesis ▶ Breast Cancer ▶ Clinical Cancer Biomarkers ▶ Colorectal Cancer ▶ Esophageal Cancer ▶ Estrogen Receptor ▶ Ewing Sarcoma ▶ Gastric Cancer ▶ Lung Cancer ▶ Malignant Lymphoma: Hallmarks Concepts ▶ Metastasis ▶ Oral Cancer ▶ Osteosarcoma ▶ Radiation Oncology ▶ Radiological Response Criteria ▶ Staging of Tumors
See Also
and
References Grosu AL, Molls M, Zimmermann FB et al (2006) Highprecision radiation therapy with integrated biological imaging and tumor monitoring: evolution of the Munich concept and future research options. Strahlenther Onkol 182:361–368 Juweid ME, Cheson BD (2006) Positron-emission tomography and assessment of cancer therapy. N Engl J Med 354:496–507
(2012) CHO Cells. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 818. doi:10.1007/978-3-642-16483-5_1105 (2012) Computed Tomography. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 964–965. doi:10.1007/978-3-642-164835_1295 (2012) CUP. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 1012–1013. doi:10.1007/978-3-642-16483-5_1410 (2012) FET. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1391. doi:10.1007/978-3-642-16483-5_2150 (2012) FLT. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1423. doi:10.1007/978-3-642-16483-5_2210 (2012) Magnetic Resonance Imaging. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2136. doi:10.1007/978-3-642-164835_3496 (2012) MET, 11C-Methionine. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2254. doi:10.1007/978-3-642-164835_3650 (2012) Metabolic Trapping. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2258. doi:10.1007/978-3-642-164835_3659 (2012) PET/CT, Integrated Positron Emission Tomography/Computed Tomography. In: Schwab M (ed)
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Postirradiation Sarcoma
Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2829. doi:10.1007/978-3-642-164835_4480 (2012) Prognostic Biomarker. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2994. doi:10.1007/978-3-642-16483-5_4759 (2012) RGD, 18F-Galacto-RGD. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3298. doi:10.1007/978-3-642-16483-5_5091
PPAR ▶ Peroxisome Proliferator-Activated Receptor
pRB ▶ Retinoblastoma Protein, Biological and Clinical Functions
Postirradiation Sarcoma ▶ Radiation-Induced Radiotherapy
Sarcomas
After
Preclinical Drug Safety Evaluation ▶ Preclinical Testing
Postnatal Stem Cells ▶ Adult Stem Cells
Preclinical Safety Testing ▶ Preclinical Testing
Postreplication Repair ▶ DNA Damage Tolerance
Preclinical Testing
Postsurgical Systemic Therapy
Peter Greaves Department of Cancer Studies, University of Leicester, Leicester, UK
▶ Adjuvant Chemoendocrine Therapy
Synonyms
pp60c-Src
Preclinical drug safety evaluation; Preclinical safety testing; Toxicity testing
▶ Src
Definition
pp60v-Src ▶ Src
The process of testing potential new therapies in animal and cell-based test systems prior to their study in patients in order to assure their potential safety and efficacy.
Preclinical Testing
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Characteristics
problem under study so that the anticipated result will justify the performance of the experiment.
It was recognized many years ago that potential medicines were often “poisons” so that some form of screening in animal models was required before new drugs could be tried in patients. For all types of medicines, it has been generally agreed that these tests should take the form of both pharmacological and toxicological studies in animals to avoid patients being exposed to drugs that are excessively toxic or without evidence of potential efficacy. Added impetus has come from the Nuremberg Code that was formulated after the trials of the Nazi doctors convicted for the conduct of horrific medical experiments on prisoners. While this code deals primarily with the consent of volunteers and patients, one of its articles places emphasis on the justification for any experiments in humans being based on prior information derived from animal experiments: The experiment should be so designed and based on the results of animal experimentation and a knowledge of the natural history of the disease or other
Hence, the main concept underpinning the preclinical safety testing is the protection of volunteers and patients in the testing of new drugs. The basic paradigm for testing is similar for all drug types and falls into three main phases. Firstly, toxicity testing is often used in the discovery phase to select the least toxic candidate drug from a series of chemicals. Secondly and most importantly, it is used to provide the basic safety data to permit first dosing of a novel agent to volunteers. Finally, further detailed testing using repeated dosing schedules over longer periods and specialist protocols is conducted in parallel with ongoing clinical trials to complete the preclinical safety data. This permits extended dosing of patients and supports eventual marketing of the drug (Fig. 1). Although cytotoxic anticancer drugs represent a special case often needing only modest preclinical testing programs, it needs to be kept in mind that not all drugs used in the therapy of cancer are conventional cytotoxic drugs. Some
Toxicity screen for compound selection
Active compound identified Absorption Distribution Metabolism Excretion
Phase 1
Pharmacokinetics
Phase II
Bioavailability
Range finding studies Single and multiple dose studies in 2 species Mutagenicity & in vitro studies
Chronic studies
Carcinogenicity Phase III
Mutagenicity
Teratology
Fertility studies – perinatal & postnatal
Drug interactions Population kinetics
Marketing for use in patients
Preclinical Testing, Fig. 1 This diagram shows the place of preclinical safety studies within the context of drug discovery and development. So-called screening toxicology might be conducted in rodents prior to entry into formal “single-dose” and “repeat-dose” toxicology studies immediately before testing in humans (red line). In parallel with the conduct of clinical phases I, II, and III, further testing including “chronic” (6 months or more duration)
toxicity and reproductive studies are carried out prior to marketing (green line). Italics show drug kinetics and metabolism studies – performed in both humans and animals – and enable effects in animals and humans to be correlated. Cancer drugs usually do not require the testing marked in gray print, although these are needed for drugs of other types intended for long-term use
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of these such as those used to manage pain, nausea, or vomiting are developed in a manner similar to non-anticancer drugs and consequently often have preclinical testing program analogous to novel drugs intended for long-term use. The use of preclinical safety testing is primarily screening for serious drug toxicity to permit their safe testing in humans. They are no substitute for careful study of new drugs in patients to test their ability to treat disease and for monitoring of adverse effects. Adverse effects occurring in 1 out of 10,000 or 100,000 patients can be devastating but would never be detected in the small number of healthy animals used in safety testing. Discovery Phase Basic toxicity testing has an important place in the selection of a potential new anticancer therapy, despite application of a vast array of novel approaches using molecular biology, combinatorial chemistry, and bioinformatics. The activity of anticancer drugs is typically assessed using a battery of ▶ mouse models for different cancer types. At this stage, informal studies, so-called screening toxicity using small numbers of rodents, often mice, might be conducted. This is often performed when series of potential drugs cause toxicity in particular organs such as the liver and kidney to enable selection of the least toxic for progression to the next phase of formal testing prior to administration to humans. Depending on the nature of the toxicity, these studies usually employ simple endpoints – clinical observation, examination of blood values, and microscopic examination of important organs. Phase I Clinical Study Following identification of a potentially active new drug, the next major step is testing for effects in humans. However, the transition from the laboratory bench to the bedside represents a large step for which preclinical studies are crucial. At this stage, a small number of carefully planned and conducted animal toxicity and safety pharmacology experiments are performed. Typically, two laboratory animal species are used for each new drug. One of these is a rodent usually rat and the other a non-rodent usually dog. These allow the
Preclinical Testing
precise characterization of the effects activity of the drug on body functions and any cellular toxicity. They provide the basis for the design, conduct, and safety monitoring of clinical phase I studies of the new drug in volunteers. While healthy volunteers may be studied for cancer drugs that are not cytotoxic, cytotoxic agents are usually studied in volunteer cancer patients. The paradigm for a toxicity experiment is similar for most types of studies. Animals are dosed drug by the similar route to that intended for use in patients. This is usually done at a clinically relevant dose and usually two higher doses on a body weight or surface area basis. The minimum number of animals of each sex in each dose group is mandated in most government guidelines, notably those of the European Union, the United States, and Japan. These studies are conducted to an agreed international laboratory standard known as ▶ Good Laboratory Practice (GLP). This requires regular auditing of studies as well as inspection of laboratories by government agencies. In all these toxicology studies, the animals are monitored clinically with particular attention being paid to changes in animal behavior that could signify adverse effects on critical functions such as the nervous system. Blood pressure, heart rate, and electrocardiograms are usually monitored in the dog as they are more difficult to perform in rodents that have very fast heart rates. Eyes are also carefully examined using sophisticated optical equipment similar to that employed by an ophthalmologist or optician. Blood is sampled and tested for any biochemical or hematological alterations in a similar manner to human patients, often using identical laboratory analytical apparatus. Levels of drug circulating in the body are also usually monitored in these experiments, often referred to as toxicokinetics. At the end of the dosing period, or if any ill health intervenes, the animals are humanely killed using an anesthetic or barbiturate and subject to a full autopsy examination. Over 30 tissues from all the organs are taken for microscopic examination by an experienced toxicological pathologist. This is to check for any organ damage that could reflect danger to patients if they were to be treated with
Preclinical Testing
the new drug. Organs such as the liver, kidneys, ovaries, and testes are particularly important, as are the lymph nodes, thymus, and spleen for any deleterious effect on the immune system. Additional special studies are performed if there are findings of concern that need elucidation. As many drugs, particularly cancer drugs, are locally irritant to tissues, the gastrointestinal tract is examined closely if the oral route is employed. Likewise, injection sites are also studied microscopically if a parenteral route is used. Severe local irritancy might preclude dosing to humans or would dictate particular caution in use. These studies are designed in a particular way for cytotoxic cancer drugs because these are liable to damage rapidly dividing cells such as those in the bone marrow and gastrointestinal tract. For these drugs, it has been shown that the maximum tolerated dose (MTD) is often similar in mouse, rat, dog, monkey, and man when compared on the basis of mg/m2 of body surface area. Experience has shown that a safe starting dose in humans is one tenth of the MTD based on single-dose toxicity studies. This means that a key study for cytotoxic drugs is a single-dose toxicity study that has sufficient number of animals per dose to establish an MTD. These single-dose studies usually have a follow-up period of clinical examination of the animals for at least 14 days after dosing to exclude delayed toxic effects. Usually two animal species, one rodent and one non-rodent, are used to confirm the MTD. This study is required by government drug regulatory authorities prior to testing cytotoxic cancer drugs in cancer patient volunteers. Repeat-dose toxicity studies are also performed with cytotoxic drugs prior to phase I clinical studies. These are designed to mirror the proposed clinical schedule with particular attention being paid to organ toxicity and reversibility of toxic effects. These are of limited duration usually 2–4 weeks or one or two cycles of treatment in two species. However, increasingly many new anticancer drugs are not cytotoxic because they act through other mechanisms such as modulation of hormones or growth factors. These are tested in a more conventional manner similar to most other
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drugs intended for long-term use. Here, toxicity experiments comprise single-dose studies in rodents and repeated dosing over a period of up to 1 month in two animal species. One species is a rodent, usually laboratory rat, and the other a non-rodent species, usually the beagle dog. Experiments in monkeys are avoided where possible unless the compound being studied has particular pharmacodynamic or metabolic characteristics that demand it. Yet, another variation on the basic preclinical testing scheme is used for biological products. Again the aim is to mirror the clinical treatment schedule. However, as some biological agents do not possess activity in non-primate species, monkey studies are sometimes needed to assure safety in humans. Immunogenic potential is also thoroughly investigated in specialized animal models. At this stage determining the safety profile of a new drug is not limited to animal toxicity experiments. There are also in vitro genotoxicity studies, such as the Ames test which uses bacteria and other experiments using cells to study whether the drug can damage DNA. A standard battery of both in vitro and in vivo genotoxicity experiments is conducted prior to phase II clinical studies for conventional drugs (▶ Micronucleus assay). A particularly important component of preclinical study is the investigation of absorption, distribution, metabolism, and excretion of drug and their metabolites in the blood and tissues of animal species chosen for toxicity testing. This data helps in the interpretation of the relevance of adverse findings in animals for humans because it forms a point of comparison of drug handling between animals and humans. It also helps to validate the relevance of the data obtained in animals (▶ Pharmacokinetics and Pharmacodynamics in Drug Development). At the end of this phase of work, scientists and physicians review the assembled data from all these experiments along with information from studies of drug metabolism prior to design and conduct of first studies in humans. In all cases, a clear rational based on a trade-off between any potential risks with likely long-term benefit would be generated prior to the conduct of any human
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experiment. Moreover, there is a mandatory ethical review of the study protocol by a panel not directly involved in the human studies. The review panel would be provided with a summary of the results of the animal safety information. Government agencies in the United States and in the European Union have approval processes for all such human studies. Phase II and III Clinical Studies In clinical studies in patients, dosing periods are usually lengthened and more subjects are involved. These are accompanied by animal toxicology studies of longer duration and, when appropriate, other specialist studies. The organization of these toxicity studies is similar to the earlier studies, but the period of dosing is extended from a period of up to 1 month to period of dosing of 6 or 9 months or 1 year for most drugs. For anticancer drugs, studies are usually conducted with continuous or intermittent dosing for a period equal to the duration of clinical trials although not longer than 6 months. Further drug kinetics and metabolism studies are conducted at this stage as data from humans is available and enables comparison between species. This also serves to validate the preclinical data. Metabolites may be isolated and their toxicity studied if there is particular concern about the adverse effects of metabolites. Enzyme induction potential may also be tested in animals or in vitro hepatocyte test systems (Pharmacokinetics/ pharmacodynamics). Although for most drugs, special experiments to examine the effects of a new drug on reproductive function and the developing fetus (reproductive toxicity studies) are required to be conducted in two sensitive animal species, conventionally rat and rabbit, however, for most cytotoxic anticancer drugs, these experiments are not usually performed. Although in vitro genotoxicity tests are not required for anticancer drugs prior to phase I and phase II clinical trials, tests such as the Ames test which uses bacteria and other experiments using cells to study whether the drug can damage DNA are usually expected prior to clinical phase III trials and marketing applications. A full battery of in vitro and in vivo genotoxicity tests is
Pregnane X Receptor (PXR)
required before phase II for more conventional drugs (micronucleus assay). The last preclinical experiments that are usually conducted for most drugs are the so-called carcinogenicity studies. Although these are a requirement for most drugs that are going to be used for extended periods in patients, they are not performed for many anticancer drugs. Exceptions exist for drugs such as the selective estrogenmodulating drugs such as tamoxifen that are used for extended periods and for cancer prophylaxis. The studies have typically been mandated in two rodent species, mouse and rat. Again the organization of the studies is similar to those conducted previously except that the dosing period is for 2 years – most of the lifetime of a rodent (▶ Carcinogenesis or ▶ toxicological carcinogenesis). Shorter studies using genetically modified, cancer-prone mice have been accepted by government drug regulatory agencies in place of one of these two large studies (mouse models). At the end of this process, the preclinical information is summarized along with information from clinical trials and manufacturing data and assembled for submission to government drug regulatory authorities to obtain marketing authorization.
References Greaves P, Williams A, Eve M (1997) First dose of potential medicines to humans: how animals help. Nat Rev Drug Discov 3:226–236 Schuster E (1997) Fifty years later: the significance of the Nuremberg Code. N Engl J Med 337:1436–1440
Pregnane X Receptor (PXR) ▶ Steroid X Receptor (SXR)
Preinvasive Breast Cancer ▶ Ductal Carcinoma In Situ
Pre-mRNA Splicing
Preleukemia ▶ Myelodysplastic Syndromes
Pre-mRNA Splicing Dawn S. Chandler Department of Pediatrics, Columbus Children’s Research Institute, Center for Childhood Cancer, The Ohio State University School of Medicine, Columbus, OH, USA
Definition Splicing is a tightly regulated mechanism for the control of gene expression that involves the precise removal of introns from the precursor mRNA molecule and the subsequent ligation of the remaining exons.
Characteristics Premature RNAs that are transcribed within the cell nucleus contain both coding sequences (contained within functional units called exons) and noncoding sequences that are eventually excised. Splicing is the mechanism by which the noncoding portions of the RNA (also known as introns) are removed from pre-mRNAs, via two cleavage-ligation reactions, each involving transesterification at a splice site phosphate (see Fig. 1). Ultimately, the two exons are ligated to generate the spliced mRNA, and the excised intron is released in a lariat configuration that is eventually degraded. The splicing reaction has been well defined and is mediated by the dynamic ordered assembly of numerous spliceosome components directly on the pre-mRNA. To avoid the production of nonfunctional proteins, it is essential that splicing occurs precisely and consistently as introns that are removed incorrectly or not at all can cause prematurely truncated proteins resulting
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from in-frame stop codons located in the intron or in proteins translated out of frame. Splicing is therefore, by necessity, an extremely accurate and specific reaction. The way in which regulation of splicing is achieved is a complex intersection of distinct recognition sequences for binding of the splicing machinery and accessory molecules with the proteins and snRNP complexes that bind to these elements. Since exons are separated by introns and can be literally thousands of base pairs away, the splicing machinery acts to recognize the splice sites and to remodel the RNA such that the splice sites are juxtaposed. In the first step of splicing, the splicing components U2AF and U1 snRNP bind to the 30 and 50 splice sites, respectively. These proteins, along with accessory molecules known as SR proteins that help to stabilize the reaction, form the splicing commitment complex. Differential splicing regulation can be achieved by the relative concentration of these accessory molecules in the nucleus and by the expression of proteins that enhance or inhibit the splicing reaction. Subsequent steps involve the binding of the U2 snRNP to the branch point and the U4/U5/U6 tri-snRNP complex remodeling the RNA and catalyzing the splicing reaction. Several systems have taken advantage of the precise nature of the splicing reaction and have implemented it as a mechanism for regulation of gene expression and function. Alternative RNA splicing is the process by which mRNAs encoding several distinct proteins are produced from one single pre-mRNA sequence by use of differential splice site choices. There is a large degree of diversity in the ways that cells use alternative splicing as a mechanism for gene regulation (see Fig. 2). Exons can encode discrete functional domains, and thus exons that are differentially included may dramatically alter the protein function. The inclusion or exclusion of exons may also change the reading frame of the RNA and thus regulate protein function by affecting whether or not an RNA with an intact open reading frame is produced. Because of the versatility of splicing regulation, alternative splicing is prevalent in several developmental processes including muscle
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Pre-mRNA Splicing, Fig. 1 The catalytic steps of pre-mRNA splicing. The required elements of the pre-mRNA are shown schematically. The exons are depicted as boxes and the intron as a line. The conserved 30 and 50 splice site sequences are depicted along with the relevant phosphate groups (p). The polypyrimidine tract (Py)n and the branch point (A) are also shown. The dashed arrows signify the hydroxyl group attack of the splice site
phosphate. The first transesterification reaction produces two splicing intermediates, the free 50 exon and the intron/ 30 exon in a lariat configuration. The second transesterification reaction results in the ligation of the two exons and the release of the intron lariat that is degraded. The resultant ligated exons make up the mRNA that eventually gets transcribed into protein (Figure adapted from Kramer (1995))
development, neurogenesis, meiosis, and spermatogenesis. However, disruption of the developmental regulation or alteration in appropriate RNA splicing can lead to incorrect expression of alternatively or aberrantly spliced isoforms and can lead to disease.
with defined tissue-specific functions. In the case of the c-Jun amino-terminal kinase 2 (JNK2, ▶ JNK subfamily) that is known to phosphorylate the c-Jun proto-oncogene, there is a well-defined tissue-specific splicing pattern. In neuronal cells, exon 6A is included and 6B excluded, while in nonneuronal cells, 6B is included in the absence of exon 6A. There are several examples of alternative splice site usage (Fig. 2b) for genes that are associated with cancer. The ▶ KRAS tumorsuppressor gene produces oncogenic variants due to alternative 50 splice site selection. These splice variants are overexpressed in prostate cancer and seems to negatively affect the tumorsuppressor activity of its normally spliced
Spliced Forms in Cancer For each of the alternative splicing patterns, there exist multiple examples that exhibit cancerspecific expression. An example of each is discussed below. Mutually exclusive splicing of exons (Fig. 2a) occurs such that only one of a group of adjacent exons is included in an RNA at any one time. This type of splicing regulation is common for control of RNAs that encode proteins
Pre-mRNA Splicing Pre-mRNA Splicing, Fig. 2 Examples of possible alternative splicing patterns. Splicing choices are shown schematically with exons depicted as boxes and introns depicted as lines. A single pre-mRNA may employ multiple of these alternative choices in a combinatorial manner and thus greatly increase the diversity of proteins encoded (Figure adapted from Cartegni et al. (2002))
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counterpart. In another example, the telomerase reverse transcriptase, TERT, utilizes alternative splicing of a 30 splice site to generate TERT alpha. TERT is activated in most human cancers and is overexpressed in high-grade astrocytic tumors. The alpha TERT isoform that results from the use of a downstream 30 splice site lacks 12 amino acids and is deficient for telomerase activity. TERT alpha is furthermore suspected to cause apoptosis and thus provide cancer protection. In these two cases of splicing regulation in cancer, the proteins that result from alternative splicing appear to have antagonistic functions to their full-length counterparts, one
form promotes tumors and the other protects against cancer. Intron retention (Fig. 2c) is yet another mechanism of regulating the splicing and thus function of genes that are involved in cancer. Intron retention occurs when the 50 and/or 30 splice sites are not recognized by the splicing machinery, and the intron is therefore not removed from the transcript. A gene that is thought to regulate invasion and metastasis, CCK2, is altered in various cancers by retention of an intron that encodes a large intracellular loop as part of its transmembrane domain. The splice variant promotes growth via interaction with the ▶ Src tyrosine kinase.
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The misregulation of splicing can occur via two general mechanisms. The first way is via point mutations that affect the cis-regulatory elements important for splicing control. Point mutations that create or destroy splicing signals within the RNA itself can affect splicing and are a prevalent way to destroy RNA splicing in a number of different cancers. In one example, the breast cancer susceptibility gene, BRCA1 (▶ BRCA1/ BRCA2 germline mutations and Breast Cancer Risk), is spliced incorrectly, and the resultant truncated BRCA1 protein leads to breast cancer. In this case, a single nucleotide mutation was identified in exon 18 of BRCA1. This point mutation disrupts an exonic splicing enhancer (ESE) that is known to bind to the SR protein SF2/ASF. This disruption leads to the inability of exon 18 to be recognized by the splicing machinery, and the exon is skipped as in Fig. 2d. The resultant mRNA is in a different reading frame than the normally spliced RNA, and a truncated protein is produced. This example underscores the importance of regulatory element sequence fidelity for efficient splicing and is an important example of a cancer-causing point mutation that leads to a splicing deficiency. The second way to express incorrect splice forms is through the altered expression or function of splicing trans-regulators. Changes in expression levels, phosphorylation state, and subcellular localization of splicing accessory molecules are all known to be ways of regulating specific splice site choices. In the case of the ▶ Mdm2 (or Hdm2 in humans, ▶ MDM genes), the modulator of tumor-suppressor p53 activity, splicing is regulated in normal cells as a response to stress. The resultant splice form is also an example of exon inclusion (normally) versus exon skipping (in stressed cells). As shown in Fig. 3, the internal eight exons are skipped (exons 4–11) making this an extreme example of exon skipping. Although the mechanism of MDM2 splicing regulation is still unknown, it is thought to be a result of transfactor control since this represents a normal cellular process that is reversible once the stress is removed. The MDM2 protein derived from the alternatively spliced form has been shown to bind to the
Pre-mRNA Splicing
full-length MDM2 and interfere with its ability to bind to and regulate p53 (see Fig. 3). In this way, expression of MDM2alt1 facilitates upregulation of p53 activity and promotes the damage response playing an important role in tumor suppression. However, this alternatively spliced form of MDM2 is contradictorily found to be overexpressed in a number of human cancers including gliomas, rhabdomyosarcomas, and breast and ovarian cancers. Although the expression of the MDM2 alternatively spliced form is predicted to suppress tumors by activating the trp53 tumor-suppressor pathway, its prevalent expression in multiple tumor types has led to much speculation about a possible role of MDM2 splicing in tumor formation. One prevailing hypothesis for the role of MDM2alt in tumorigenesis is a cancer progression model in which the expression of the alternative form of MDM2 initially protects the cells by activating trp53, but sustained expression of the MDM2 form and upregulated trp53 puts a selective pressure on the cells that results ultimately in mutations in the p53 gene itself or other genes in the pathway. It is these secondary mutation(s), then, that induce the cancer phenotype. This predicted model mirrors a similar situation in which perpetual myc (▶ Myc oncogene) expression likewise induces trp53 providing cancer protection initially, but secondary mutations in genes of the trp53 pathway lead to cancer, in the end. Although experimental proof for the exact role the MDM2 alternatively spliced form plays in the initiation of cancer is lacking, the expression of alternatively spliced forms of MDM2 is clearly a marker for many different tumor types. In summary, the expression of many alternatively spliced isoforms is associated with cancer. In many cases, the new proteins formed are antiapoptotic or growth-promoting lending clear clues to their roles in tumor formation. In other cases, the role of the alternative forms in tumorigenesis is more elusive. Therapeutic Intervention Since a number of tumor-specific isoforms have been identified that block apoptosis and/or promote cell growth or invasion, modification of the
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Pre-mRNA Splicing, Fig. 3 Alternative splicing of MDM2. The MDM2 pre-mRNA is shown schematically; exons are depicted as boxes and introns as lines. Under normal conditions the MDM2 is spliced to include all exons and encodes the full-length protein as depicted in the left part of the figure. Under stress conditions and in certain cancers, MDM2 alternative splicing causes skipping of exons 4 through 11 and results in the expression of a novel MDM2 protein that lacks the p53 binding domain,
the nuclear localization and export signals (NLS and NES), and the ARF binding domain (as shown in the right part of the figure). The short MDM2 protein negatively regulates its respective full-length counterpart and ultimately activates the p53 pathway. This example of regulated splicing uncovers a novel mechanism by which cellular injury can control distribution and activity of p53 within the cell and possibly lead to cancer
splicing profile in tumors is a possible therapeutic intervention point. There have been several approaches that have been successfully utilized to change splice site choices in a variety of human diseases. Low molecular weight drugs such as neomycin, aclarubicin, and sodium butyrate have been successfully utilized to promote exon inclusion. Likewise, heterologous expression of trans-acting splicing factors can alter splicing patterns of their target genes. These types of therapies must be carefully tested for nonspecific effects as they have the potential to affect the splicing of many genes in addition to the
targeted disease-causing gene. This danger of nonspecificity may be avoided by designing selective agents that only recognize the RNA in question. Antisense oligonucleotides and RNAi technology have been shown to be effective in recognizing specific sequences in the target RNA and successfully modulating splicing. As more becomes known about the splicing of genes and the roles of this process cancer, there is great promise for modulating gene expression by directing splice choices that encode tumorsuppressor proteins and squelch the tumorpromoting alternatively spliced isoforms.
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Cross-References
Preneoplastic Lesions ▶ BARD1 ▶ BRCA1/BRCA2 Germline Breast Cancer Risk ▶ Cancer ▶ JNK Subfamily ▶ KRAS ▶ MDM Genes ▶ MDM2 ▶ MYC Oncogene ▶ Src
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References Black DL (2003) Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem 72:291–336 Cartegni L, Chew SL, Krainer AR (2002) Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet 34:285–298 Chandler D (2007) Splicing of the p53 pathway. In: Venables J (ed) Alternative splicing in cancer. Transworld Research Network, Newcastle Kramer A (1995) The biochemistry of pre-mRNA splicing. In: Lamond A pre-mRNA splicing. R.G. Landes, Austin, pp 35–64 Venables JP (2004) Aberrant and alternative splicing in cancer. Cancer Res 64(21):7647–7654
See Also (2012) Exon. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1360. doi:10.1007/978-3-642-16483-5_2059 (2012) Exonic splicing enhancer. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1361. doi:10.1007/978-3-64216483-5_2060 (2012) Intron. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1904. doi:10.1007/978-3-642-16483-5_3131 (2012) Pre-mRNA. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2972. doi:10.1007/978-3-642-16483-5_4710 (2012) SnRNP. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3460. doi:10.1007/978-3-642-16483-5_5398 (2012) Spliceosome. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3487. doi:10.1007/978-3-642-16483-5_5455 (2012) SR proteins. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3495. doi:10.1007/978-3-642-16483-5_5463
Francesco Feo Department of Biomedical Sciences, Division of Experimental Pathology and Oncology, University of Sassari, Sassari, Italy
Definition The development of primary tumours is often preceded, both in humans and experimental animals (mainly rodents), by the appearance of lesions referred to as preneoplastic, since they consist of genetically and phenotypically altered cells exhibiting a higher risk of malignant evolution than normal cells. These lesions generally lack one of the principal characteristics of neoplastic lesions: the capacity to grow autonomously after cessation of the stimuli that induced the lesion. Nonetheless, the distinction between preneoplastic lesions and benign neoplasias is sometimes difficult, and the terms “preneoplastic” and “premalignant” are often considered synonyms. However, benign tumours, constituted by autonomously growing cells, cannot be strictly classified as preneoplastic but only as premalignant lesions, whereas premalignant lesions can include both preneoplastic lesions and benign tumours.
Characteristics Following the pioneering work of I. Beremblum, on skin carcinogenesis, and E. Farber, on hepatocarcinogenesis, tumorigenesis is considered a multistep process characterized, both in humans and rodents, by the progressive development of preneoplastic, premalignant, and malignant lesions. During this process, genomic instability occurs (▶ Microsatellite Instability), followed by alterations in oncogenes, oncosuppressors, DNA repair genes, with consequent changes in the signal transduction (▶ Signal Transduction) network (Fig. 1). The progressive accumulation of genetic changes generates
Preneoplastic Lesions
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Premalignant Genomic instability
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Accumulation of genetic damage
Preneoplastic Lesions, Fig. 1 Schematic representation of multistage carcinogenesis. Carcinogenesis initiation is associated with the appearance of genomic instability. Clonal expansion of initiated cells leads to the development of preneoplastic lesions carrying various genomic
alterations. The accumulation of these alterations is associated with the acquisition of the capacity of autonomous growth and evolution to neoplastic lesion, which progress to moderately differentiated and poorly differentiated, invasive, carcinomas
autonomously growing premalignant and malignant lesions.
heterogeneous population of foci of altered hepatocytes (FAH) expressing specific patterns of marker genes, such as glutathione S-transferase (GST) 7–7, g-glutamyl transpeptidase, and alterations of carbohydrate metabolism defining different lineages of preneoplastic cells. When the treatment with promoters is suspended before the appearance of neoplastic lesions, early preneoplastic liver lesions partially disappear by a process named “remodeling.” GST 7-7-positive cells that acquire autonomous growth persist and further evolve to premalignant low-grade and high-grade dysplastic nodules and then to welldifferentiated hepatocellular carcinomas (HCCs; ▶ Hepatocellular Carcinoma; ▶ Hepatocellular Carcinoma Molecular Biology), which progress to moderately and poorly differentiated carcinomas. Other hepatocarcinogenesis models are based on the stable transfection of one or two cancer related genes (i.e., c-myc, c-myc plus Tgfa genes) in mouse and, less frequently, rat genome, or in inactivating a gene of intact mice (knockout mice). In these models, generally the liver is dysplastic and progressively becomes adenomatous before the development of HCCs. Alterations of several signal transduction pathways occur in rodent HCC and, at a lower extent, in preneoplastic liver lesions. They include
Some Experimental Models of Preneoplastic Lesions Various preneoplastic lesions have been induced in different organs, including liver, pancreas, lung, colon, skin, thyroid, mammary gland, gall bladder, prostate, etc., in rodents treated with carcinogens and in transgenic mice. Although a close correspondence of experimental and human preneoplastic lesions does not always occur, the study of experimental lesions allowed discovering pathogenetic mechanisms and diagnostic and prognostic markers of different tumor types. Only lesions preceding tumor development in some organs (liver, colon, lung) will be given as examples. In hepatocarcinogenesis rodent models, irreversibly initiated cells by a carcinogenic stimulus can undergo clonal expansion by over-response to the administration for several weeks of promoting agents, such as phenobarbital, 2,3,7,8tetrachlorodibenzo-p-dioxin, chlorinated hydrocarbons, peroxisome proliferators. Only few initiated cells undergo clonal expansion under promoting stimuli, probably because of irreversible change and apoptosis, giving rise to a
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Preneoplastic Lesions, Fig. 2 Schematic representation of signal transduction pathways involving Jak/Stat, Ras/Mapk, Wnt/b-catenin, and Tgf-b1 signaling pathways. Binding to legends or autologous upregulation of epidermal growth factor (EGF) family receptors causes phosphorylation of Jak1 and 2 and activation of Stat3 and formation of active GTP-Ras complex, which activates the Raf/MEK/ERK and Mekk/Jnkk/Jnk pathways. This leads to the phosphorylation of nuclear fetors that, migrating into nucleus, activate numerous target genes. The inactivation of components of the complex Apc/Axin/Gsk3b/b-catenin complex, or the inhibition of Gsk3b by Disheveled (Dsh)
protein by Wnt1/Freezzled activation, suppresses b-catenin phosphorylation and ubiquitination followed by disruption by proteasome. b-Catenin accumulation into the cytoplasm causes its nuclear translocation and activation of various target genes. Activation of Tgf-b receptor (TBR) leads to the formation of Smad2/3/Smad4 heterodimers leading to cell death by apoptosis. Overexpression of Smad7 and/or downregulation of Smad2/3 and Smad4 inhibit the TBR signaling pathway. Enhancing and inhibitory effects are indicated by pointed arrows and blunt arrows, respectively
upregulation of EGF receptor family, resulting in induction of the Ras-dependent activation (▶ Ras) of mitogen-activated protein kinases (MAPK, ▶ Map kinase), JAK-Stat, and Pi3K-AKT signaling, transducing extracellular signals resulting in phosphorylation of transcription factors targeting various genes (Fig. 2). c-Raf/MAP kinase kinaseextracellular signal-related kinases (MEK-ERK) pathway is primarily responsible for responding to cellular proliferation signals, whereas the mitogen-activated protein kinase kinase (MEKK) and c-Jun N-terminal kinases (JNK; ▶ JNK Subfamily) respond to cellular stress signals and treatment with carcinogens. This leads, through upregulation of the c-FOS and c-JUN genes, to the activation of AP-1 and its targets
(i.e., Cyclin D1 gene; ▶ Cyclin D). As a consequence Odc, a c-MYC (▶ Myc Oncogene) target is overexpressed and its gene product, ornithine decarboxylase, activates polyamine synthesis required for nucleotide biosynthesis. Upregulation of c-myc, cyclin D1, and other cyclins and cyclin-dependent kinases, activates cell cycle and cell proliferation. This is favored by downregulation of cell cycle inhibitors, such as p16INK4a and p53, and Tgf-b/Smad signaling. Profound alteration of the Wnt/b-catenin pathway (▶ Wnt Signaling), leading to inactivation of the complex Apc/Axin/Gsk3b/b-catenin, decreases b-catenin disruption via proteasome. Consequent nuclear translocation of b-catenin activates various targets, including c-myc and cyclins.
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Overexpression of the inducible nitric oxide synthase (iNos) contributes to activation of the MAPK cascade and the hypoxia inducible factor-1, leading to an increase in vascular endothelial growth factor (Vegf) expression and angiogenesis. MYBL2 (v-Myb avian myeloblastosis viral oncogene homolog-like 2; B-MYB) is a transcription factor belonging to the MYB protooncogene family that forms an active complex with LIN-9, required for transcription of G2/M genes. MYBL2 upregulation is associated with faster growth and progression of premalignant and malignant liver lesions in chemically treated rats, transgenic mouse model, and humans. MYBL2 activation is crucial for human HCC progression, and the MYBL2-LIN9 complex contributes to survival of DNA-damaged p53/ cells. Finally, results underline the role of ubiquitination of the ERK inhibitor DUSP1 (dual-specificity phosphatase 1), deregulation of FOXM1 (Forkhead box M1B), and proteasome degradation of cell cycle inhibitors, including P21WAF1, P27KIP1, P57KIP2, P130, RASSF1A (Ras-associated factor 1), and FOXO1 (Forkhead box O1) in the progression of rat and human premalignant and malignant liver lesions. Another largely investigated model of multistep tumorigenesis is represented by chemically induced colon-rectal cancer (CRC) of rodents. Aberrant crypt foci (ACF; Colorectal Pre-malignant Lesions) identified in colonic mucosa of rodents treated with chemical carcinogens are considered preneoplastic lesions. These lesions exhibit increased expression of c-fos, decreased c-myc expression and hexosaminidase activity, and loss of transforming growth factor-a (Tgf-a), whereas mutations of Adenomatous polyposis coli (Apc; ▶ APC/b-Catenin Pathway) gene are absent. KRAS mutations occur in azoxymethane-induced ACF in rats. However, various observations strongly suggest that ACF are not preneoplastic, but only hyperplastic lesions. A subset of ACF, called “dysplastic ACF,” consisting of fast growing crypts with altered b-catenin expression associated, in some cases, with b-catenin mutation, are considered true CRC precursors. In colonic mucosa of rats, treated with azoxymethane, mucin depleted foci
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(MDF) have been described. MDF are more dysplastic and more like to express b-catenin than common ACF. b-Catenin accumulating crypts (BCAC) with higher cell proliferative activity than ACF significantly increase with time during rodents colorectal carcinogenesis. The study of the molecular events in early colorectal preneoplastic lesions indicates that various mutations occur and are selected during colorectal carcinogenesis, the main selective factor being represented by those leading to a b-catenin down-regulating function. Lung cancer (▶ Lung Cancer) develops in a gradual and stepwise fashion. Numerous researches have been dedicated to lung tumorigenesis in tobacco smokers. Preneoplastic lesions include focal epithelial cell ▶ hyperplasia, squamous metaplasia, and ▶ dysplasia. These lesions have been induced in rats by various carcinogens, and are followed by the development of adenomas, adenocarcinomas and squamous cell carcinomas. Interestingly, exposure of rats to tobacco smoke induces dose-dependent cell proliferation and squamous metaplasia. These effects are paralleled by activation of Mapk signaling pathways and AP-1 binding to DNA (Fig. 2). This is associated with upregulation of AP-1-dependent cell cycle proteins, such as Cyclin D1 and proliferating cell nuclear antigen (PCNA). Human Preneoplasia Etiologic factors of HCC include cirrhosis induced by hepatitis B virus (HBV) and hepatitis C virus (HCV), alcoholic cirrhosis, exposure to Aflatoxin B1, estrogenic steroids, some naturally occurring carcinogens in food, and some rare genetic syndromes (i.e., hemochromatosis, glycogenosis type I, a1 antitrypsin deficiency). About 4% of HBV infections evolve to persistent hepatitis, 30% of which can further evolve to chronic hepatitis and cirrhosis. HBV-positive cirrhosis is considered a preneoplastic lesion, although the evolution to HCC has been found in only 10% of cases. At difference from HBV infection, HCV-induced hepatitis becomes chronic in the majority of patients. At least 20% of them develop cirrhosis, which in most cases evolves to HCC. In these patients, as well as in
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individuals with alcoholic cirrhosis, the development of FAH and adenomatous nodules, correspondent to those chemically induced in rats and mice or developing in woodchucks viral hepatitis, exhibit several molecular alterations in common with the corresponding lesions of rodent liver. Various preneoplastic lesions have been identified in the gastroenterical tract. About 10% of patients with long-standing grastroesophageal reflux develop the Barrett oesophagus, in which the distal squamous mucosal epithelium is replaced by metaplastic columnar epithelium. This lesion is preneoplastic, and the patients with Barrett oesophagus develop adenocarcinomas, preceded by dysplastic lesions, with a 30–40 times increased rate over the general population. Dysplastic epithelium exhibits cell cycle deregulation, high proliferative rate, upregulation of TP53, and in more advanced stage, amplification of chromosome 4. Further evolution to carcinoma implicates deregulation of Wnt/b-catenin pathway and c-ERB-B2 amplification. Preneoplastic conditions of human gastric cancer have not yet been well characterized to date. Ménétrier disease, resulting from profound ▶ hyperplasia of mucosal epithelium and glandular atrophy, rarely exhibits epithelial metaplasia, a condition that may favor the development of gastric carcinoma. Interestingly, transgenic mice overexpressing Tgf-a at gastric level develop a syndrome similar to human Ménétrier disease. A multistep model from chronic active Helicobacter pylori infection through multifocal mucosal atrophy, intestinal metaplasia, ▶ dysplasia, and carcinoma has been described. During this process, complex interactions between several bacterial, host genetic and environmental factors determine whether H. pylori-infected individual develop cancer. H. pylori infection is characterized by upregulation of various inflammation-associated genes, including chemokines, adhesion molecules, surfactant protein D, and CD74 in infected stomach. Nitric oxide and reactive oxygen species, which may induce DNA damage (▶ DNA Damage Response; ▶ Repair of DNA), are overproduced. The role of these and other factors in the evolution of metaplastic and dysplastic lesions to carcinoma
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is still object of study. H. pylori infection also predisposes to the lymphomatous transformation of the mucosa-associated lymphatic tissue. The molecular profile of preneoplastic lesions is heterogeneous, however, and there are still no molecular markers enabling the distinction between atypical hyperplastic lesions and low-grade noninvasive neoplasia (NiN) or between high-grade NiN and early invasive neoplasia. Indeed, within the spectrum of morphological changes characterizing this multistep evolution, dysplasia (NiN) is the lesion coming closest to the development of invasive adenocarcinoma. Several of the genetic and epigenetic alterations reported in gastric precancerous lesions affect DNA repair system genes, tumor suppressor genes, oncogenes, cell cycle regulators, growth factors, and adhesion molecules. Most knowledge on multistep carcinogenesis in the gastrointestinal tract derives from colorectal tumorigenesis. Inherited syndromes, occurring in less than 10% of patients, include familial adenomatous polyposis (FAP), hamartomatous polyposis, hereditary nonpolyposis colorectal cancer (▶ Lynch syndrome), and common cancer family syndrome, not belonging to aforementioned syndromes, Causative germline mutations of high-penetrance genes affect APC gene for familial adenomatous polyposis, LKB1, SMAD4, and BMPR1 genes for hamartomatous polyposis, various Mismatch repair genes (e.g., hMSH2, hMLH1, hPMS1, hPMS2, hMSH6) (▶ Mismatch Repair in Genetic Instability), for Lynch syndrome I, and AXIN2, TGFbR-2, and POLD genes for Lynch-like syndromes. The homologue of human FAP, the Min mouse strain-1, carries the APCMin mutation and develops intestinal neoplasms. The study of these syndromes allowed identifying early human colorectal preneoplastic lesions and the so-called adenoma-carcinoma sequence, as well as the molecular alterations underlying the sequence of events leading to CRC. The morphological events include early appearance of preneoplastic ACF, followed by premalignant lesions such as adenomas and adenomatous polyps, and, finally, carcinoma development. The appearance of ACF, however, is preceded by numerous molecular events, such as
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germline (in the inherited syndromes) or somatic (acquired first “hit,” in a “multihit” process, in sporadic cases) mutations of APC or mismatch repair genes. The mucosa harboring these mutations is at risk. Inactivation of normal alleles of tumor suppressor genes (i.e., by promoter methylation of APC, MSH2, b-catenin) causes hyperproliferation and appearance of early preneoplastic lesions, which following KRAS mutation evolve to adenomas. The loss of further suppressor genes (i.e., TP53) precedes the appearance of carcinomas whose progression (▶ Progression) is characterized by additional mutations of various oncogenes, chromosomal aberrations, etc. Dysplastic ACF are recognized precursors of CRC, KRAS mutation, in sporadic CRC, and APC mutation, in FAP are followed by the development of dysplastic ACF, adenomas, and CRC. Human ACF have been subclassified as dysplastic, heteroplastic (nondysplastic), and mixed types. Serrated adenoma (SA) is a dysplastic polyp with serrated architecture and can be subclassified as traditional SA or sessile SA. The latter is considered preneoplastic and differs from most lesions in the traditional SA category because of the lack of cytological dysplasia. Studies have uncovered other developmental pathways including a heteroplastic ACF-HP/SAcarcinoma sequence and a heteroplastic ACF–adenoma–carcinoma sequence. The relationship between heteroplastic ACF, HP, and colorectal cancer is less certain than that of dysplastic ACF. Nevertheless, numerous genetic and epigenetic changes have been described in the pathways to CRC via hyperplastic ACF. KRAS mutation leading to hyperplastic nonserrated hyperplastic ACF, followed by chromosome 1p loss may result in CRC development. Serrated hyperplastic ACF arise as a consequence of BRAF mutation and CpG island methylation. This is followed by the development of hyperplastic polyps and SA. The persistence of a CpG island methylator phenotype with methylation of the repair gene hMLH1 leads to the development of CRC with microsatellite instability, in SA, whereas the methylation of MGMT probably associated with KRAS mutation can result in CRC
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development, in absence of microsatellite instability. Four- to five-fold increase in incidence of the gastrointestinal tract cancer occurs in patients with Crohn disease, in which the development of carcinoma is generally preceded by dysplastic lesions of the ileum and/or colon mucosa. The pathogenesis of neoplastic transformation is unknown. Crohn disease seems to occur in predisposed individuals carrying some susceptibility loci on chromosomes 3, 7, 12, or 16. The ileum localization seems to be linked to mutations of NOD2 and CARD15 genes. However, the relationships between these alteration and cancer development are not known. A 20- to 30-fold rise in CRC incidence occurs in patients with ulcerative colitis. Cancer development is preceded by premalignant multifocal dysplastic lesions exhibiting DNA instability. Genomic instability has also been documented in then colon mucosa outside of dysplastic lesions, and it has been hypothesized that some deficit of DNA repair occurs in these patients. Preneoplastic and premalignant lesions have been described for different other human tumours (Table 1). Epidemiological, clinical, and molecular characteristics of a number of these lesions are often incompletely known, but particular attention must be focused on these parameters because of their importance for prevention and early diagnosis of malignancy. Genetic Predisposition to Neoplasia A body of evidence shows the existence of a genetic predisposition to tumours. Strong predisposition by high penetrance mutations of oncogenes, oncosuppressor genes, DNA repair genes occurs for a relatively small number of the so-called hereditary tumours. Genetic predisposition to sporadic tumours depends on the inheritance of several susceptibility or resistance allelic variants, which influence (modify) the behavior of the molecular mechanisms of tumorigenesis and, hence, the phenotypic features of preneoplastic and neoplastic lesions. Several modifier genes have been mapped, but only few genes involved in genetic predisposition have been identified so far. These observations may in some way modify
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Preneoplastic Lesions
Preneoplastic Lesions, Table 1 Preneoplastic and premalignant lesion of various human tissues Tissue Lung
Preneoplastic Morphology Squamous dysplasias, bronchial dysplasia, diffuse idiopathic pulmonary neuroendocrine cell hyperplasia
Molecular markers Deletions of chromosomes, 3p, 9p,17p
Liver
FAH
Pancreas
Fat or papillary mucinous hyperplasia
Oral epithelium
Leukoplakia
HER-2, Ki-67, P21WAF1, CYCLIN D1 upregulation, SMAD 4 underregulation LOH at 3p and 9p
Oesophagus
Barrett epithelium with metaplasia
TP53 mutations, CDX mutations
Stomach
Ménétrier disease with metaplasia
TGF-alpha Table 1. Preneoplastic and premalignant lesion of various human tissues overexpression Overexpression of GASTRIN, COX-2, SURVIVIN, BCL-2 NOD2 and CARD15 mutations TP53 mutations, Nitric oxide overproduction b-Catenin activation
Gastrointestinal tract
H. Pylori atrophic gastritis with intestinal metaplasia Crohn disease Ulcerative colitis
ACF
Urothelial Vulva Uterine cervix Endometrium
Papillary hyperplasia
Premalignanta Morphology Atypical adenomatous hyperplasia;
Dysplastic nodules
LOH at 9q
Atypical hyperplasia
Dysplastic leukoplakia Barrett dysplasia
Molecular markers Activation of RAS signaling (smoking associated). Activation of EGFR signaling (nonsmoking associated). uPA (urokinase type plasminogen activator) downregulation. TP53 mutation Upregulation of MAPK, JAK-STAT, Pi3K-AKT, IKK-NF.kB signaling. Upregulation of ODC, C-JUN, C-FOS, C-MYC, RAS family genes, cell cycle key genes Downregulation of TP53, cell cycle inhibitors HER-2, Ki-67, P21WAF1, CYCLIN D1 upregulation, SMAD 4 underregulation
TP53 overexpression; loss of differentiation-related keratins TP53 mutations, APC LOH, p16INK4a hypermethylation or LOH
Dysplastic lesions
Overexpression of Gastrin, COX-2, survivin, BCL-2
Epithelial dysplasia
TP53 mutation, Nitric oxide overproduction
Adenoma
b-Catenin activation, APC and KRAS mutation, LOH at TP53, SMAD2 and 4 LOH at 9q, TP53 mutations ei5A-1 overexpression P16INK4A overexpression, pRb ubiquitination Loss of PTEN expression
Papilloma VIN I and II Dysplasia (CIN I, CIN II) Atypical hyperplasia
(continued)
Preoperative Chemotherapy
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Preneoplastic Lesions, Table 1 (continued)
Breast
Preneoplastic Morphology Metaplastic surface epithelium and inclusion glands Hyperplastic lesions
Thyroid
C-cell hyperplasia
Skin
Lentiginous melanocytic hyperplasia; Lentiginous junctional nevus
Tissue Ovary
a
Molecular markers
Microsatellite instability Overexpression of BCl-2 and BCL-X
Premalignanta Morphology Atypical hyperplasia Atypical hyperplasia Follicular adenoma (atypical) Dysplastic nevus
Molecular markers TP53 mutation. Loss of BRCA1 and 2 Microsatellite instability Mutations of RAS family genes Overexpression of bFGF, and IL-8
The definition of premalignant lesion is often based on morphologic, clinical, and epidemiologic criteria
the definition of preneoplastic lesion. Indeed, a cell carrying polymorphic variants of cancer modifier genes responsible for increased susceptibility to malignancy, namely, increased cancer risk, could be considered preneoplastic. The identification of modifier genes and the signaling pathways that they influence may lead to the discovery of early diagnostic and prognostic markers as well as therapeutic targets, which may prevent the evolution of preneoplastic cells to full malignancy. Clinical Relevance One of the major challenges of cancer research has been the discovery of tools for efficient prevention of malignancy. In this contest, the identification of very early biochemical, molecular, and morphologic changes, predisposing normal cells to tumorous transformation, plays a pivotal role. In principle, tumor prevention would imply the protection of humans against tumor initiation. When this is not possible, an efficacious preventive strategy may attempt to block the evolution of initiated cells to malignancy. Of course, early identification of precancerous lesions is a prerequisite for efficacious prevention. The recognition of these lesions (i.e., polyps, adenomas, dysplastic nevi, leukoplakias, etc.) may allow their surgical or medical treatments. The knowledge of early molecular alterations, in preneoplastic lesions, is also important to adopt chemopreventive strategies aimed at contrasting the growth and progression of early lesions to cancer and/or to block the
expression of transformation.
genes
involved
in
cell
References Brambilla C, Fievet F, Jeanmart M, de Fraipont F, Lantuejoul S, Frappat V, Ferretti G, Brichon PY, Moro-Sibilot D (2003) Early detection of lung cancer: role of biomarkers. Eur Respir J 39:36s–44s de la Chapelle A (2004) Genetic predisposition to colorectal cancer. Nat Rev Cancer 4:769–780 Feo F, De Miglio MR, Simile MM, Muroni MR, Calvisi DF, Frau M, Pascale RM (2006) Hepatocellular carcinoma as a complex polygenic disease. Interpretive analysis of recent developments on genetic predisposition. BBA Cancer Rev 1765:126–147 Frau M, Biasi F, Feo F, Pascale RM (2010) Prognostic markers and putative therapeutic targets for hepatocellular carcinoma. Mol Aspects Med 31:179–193 Gologan A, Graham DY, Sepulveda AR (2005) Molecular markers in Helicobacter pylori-associated gastric carcinogenesis. Clin Lab Med 25:197–222 Kumar V, Abbas AK, Fausto N (2005) Robbins and Cotran pathologic basis of disease, 7th edn. Elsevier Inc., Philadelphia Suehiron Y, Hinoda Y (2008) Genetic and epigenetic changes in aberrant crypt foci and serrated polyps. Cancer Sci 99:1071–1076
Preoperative Chemotherapy ▶ Induction Chemotherapy ▶ Neoadjuvant Therapy
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Presenilin Yung-Feng Liao Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan
Synonyms g-Secretase; HOP-1; PS; PSEN1; PSEN2; SEL-12
Definition Presenilins (PSs) are known to be the most critical constituents of g-secretase that catalyzes the g-cleavage of b-amyloid precursor protein (APP) to release the C-terminus of amyloid-b (Ab) peptides. The genes encoding PSs were first identified in screens for causative alleles that lead to early onset of Alzheimer disease (AD). The human genome contains two PS alleles: PSEN1 on chromosome 14 encodes presenilin-1 (PS-1), and PSEN2 on chromosome 1 encodes presenilin-2 (PS-2). The nematode worm C. elegans contains two orthologs of presenilins, Sel-12 and Hop-1, that are functionally conserved.
Characteristics Presenilins primarily consist of the catalytic subunit of g-secretase. They have also been implicated in various cellular functions, including protein trafficking, calcium homeostasis, and regulation of b-catenin signaling, in g-secretaseindependent manners. Genetic analysis of families with inherited forms of AD led to the identification of disease-causative mutations in the genes presenilin 1 (PSEN1) on chromosome 14q24.3 and presenilin 2 (PSEN2) on chromosome1q42.2 (Levy-Lahad et al. 1995; Rogaev et al. 1995). There have been more than 150 different AD-causing mutations identified in PSEN1 and PSEN2 alleles (for updated PSEN1/2 mutation
Presenilin
databases, please refer to http://www.alzgene. org/geneoverview.asp?geneid=84 and http:// www.alzgene.org/geneoverview.asp?geneid=55, respectively). Presenilins are an obligate constituent of a larger g-secretase complex that functions as an aspartyl protease (Wolfe et al. 1999). The PS/g-secretase constitutes its active site at the interface of PS heterodimer that is thought to be the active form of PS and consists of a 28-kDa Nterminal fragment (NTF) and a 18-kDa C-terminal fragment (CTF) through a yet unclear proteolytic processing of PS holoprotein. PS heterodimers co-migrate with a high molecular mass of ~250 kDa through density gradients, and detergent-solubilized g-secretase activity can be eluted by size-exclusion chromatography with an estimated molecular mass of 2 megadaltons. g-Secretase activity can be isolated and copurified with PS heterodimers and additional PS-associated membrane proteins, including nicastrin, Aph-1, and Pen-2. The full spectrum of g-secretase activity can be reconstituted by the co-expression of human presenilin, nicastrin, Aph-1, and Pen-2 in yeast, providing the definitive proof for the minimal required constituents of the physiologically active g-secretase (Edbauer et al. 2003). These presenilin cofactors could play distinct functions in the formation of highmolecular-weight enzyme complex, as Aph-1 stabilizes the presenilin holoprotein in the complex and Pen-2 sustains g-secretase activity by enforcing the endoproteolysis of presenilin. Thus, the identification of these presenilin cofactors presents additional targets for the intervention of PS/g-secretase activity and the development of anti-AD therapeutics. The pivotal role of PS/g-secretase in Ab generation and AD pathogenesis is confirmed by the analysis of FAD-associated PS missense mutations that have been shown to intimately correlate with the selective elevation of Ab42 versus Ab40 in human, cultured cells, and transgenic mice. Transgenic mice co-expressing human APP and a PS1 missense mutation also exhibit a substantially accelerated AD-like phenotype with apparent Ab42 plaque formation. Furthermore, patients carrying these PS missense mutations exhibit
Presenilin
relatively more Ab42-containing plaques than those with sporadic forms of AD. The profound phenotype of PS1-null mice, including significantly altered axial skeletal development and somitogenesis, cerebral hemorrhages, and abnormal neurodevelopment with premature depletion of neuronal precursors, results in the prenatal mortality and establishes the definitive requirement of presenilin/g-secretase in embryonic development as well. Primary neurons derived from PS1-deficient embryos exhibit marked reduction in g-secretase activity without significant alteration in the maturation and distribution of APP and the release of soluble a- or b-APP fragments, suggesting an indispensable role of presenilin in g-secretase cleavage of APP (Shen et al. 1997). Function The primary function of presenilins is to form the catalytic subunit of g-secretase that has been a major pharmacological target for the development of AD therapeutics. Presenilin is also indispensable for the g-secretase-mediated processing of Notch whose functions involve in the embryonic development, the maintenance of progenitor cells, the differentiation/maturation of T cells from hematopoietic progenitor cells, and the differentiation of goblet cells of the intestine. Consistent with its essential roles in development, mice deficient in PS1 exhibit similar developmental abnormalities to those with genetic ablation of Notch. Other presenilin functions that do not require g-secretase activity have also been identified in various cellular events, including protein trafficking and turnover, maintenance of calcium homeostasis, and regulation of b-catenin signaling. Presenilin-dependent modulation of intracellular Ca2+ could influence presynaptic neurotransmitter release and long-term potentiation induction as evidenced by the defective transmission in hippocampal neurons with presynaptic depletion of PS1 and could lead to neurodegeneration as well. The complexity of PS functions could be further exemplified by the promiscuous substrate specificity of PS/g-secretase. There have been more than 90 PS/g-secretase substrate proteins being identified. The PS/g-secretase-mediated
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processing is known to govern the physiological properties of its substrate proteins. It is thus understandable that the impairment of PS/g-secretase functions could result in aberrant signaling associated with its particular substrate proteins, for example, Ab-elicited neurodegeneration during the course of AD pathogenesis. This is also believed to be the major reason for the latest setback in the development of PS/g-secretase inhibitor as AD therapy. Because the majority of commonly used g-secretase inhibitors block PS/g-secretase-mediated proteolysis of its substrate proteins without any selectivity, this nonselective PS/g-secretase inhibition could cause an imbalance of its substrate-dependent signaling essential for various cellular functions. This is thought to be the major contributor to the side effects of these g-secretase inhibitors. The current challenge for the clinical use of g-secretase inhibitors is to develop substratespecific g-secretase inhibitors or modulators, for example, ones that can preferentially inhibit Ab production without affecting Notch cleavage for AD therapeutics. PS/g-Secretase-Mediated Notch Signaling and Cancers The membrane receptor Notch is known as a PS/g-secretase substrate, and the PS/g-secretasemediated cleavage of Notch is essential for its downstream signaling. Active Notch signaling is crucial for the maintenance of progenitor cell fate in a dividing and undifferentiated state, and the overactivation of Notch signaling has been shown to cause a number of cancers, including T-cell acute lymphoblastic leukemia, non-small-cell lung cancer, ovarian cancer, breast cancer, and neuroblastoma. Given that activation of Notch signaling does require PS/g-secretase cleavage, PS/g-secretase has also been regarded as a pharmacological target of anticancer drugs through the blockade of overactivated Notch signaling. The efficacy of this approach depends on how efficient the PS/g-secretase inhibitors can prevent the release of the Notch intracellular domain to transcriptionally activate the expression of its downstream target genes involved in tumorigenesis. However, Notch signaling in the skin acts as a
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tumor suppressor, and inhibition of PS/g-secretase could lead to the development of skin cancer by suppressing Notch signaling. It is evident that the substrate specificity/selectivity of PS/g-secretase inhibitor/modulator would dictate how valuable these PS/g-secretase chemical modifiers would be for the development of therapeutic strategies against AD and cancers. The expression of PS1 is found to be downregulated in the tumor reversion models, suggesting that inhibition of PS1 in cancer cells could suppress tumor progression. In a lymphoma cell line U937, the expression of an antisense PS1 cDNA could induce the apoptosis of transfected U937 cells and greatly diminish their tumorpropagating ability in SCID mice. These findings thus implicate that inhibition of PS1 can result in the deficient cleavage of Notch, precluding the nuclear translocation of Notch intracellular domain. It is now believed that downregulation of PS1 could induce tumor reversion. g-Secretase-Independent Functions of Presenilins Accumulated evidence has now suggested that presenilins can exert g-secretase-independent activities, such as the regulation of Wnt/b-catenin signaling pathway, calcium release from the ER, and lysosomal proteolysis. The pivotal role of presenilins in the maintenance of intracellular calcium homeostasis has been explored more extensively among these g-secretase-independent functions. Cortical neurons derived from PS1 knockout mice exhibit increases in calcium currents mediated by L- and P-type voltage-gated calcium channels (VGCCs). In addition, capacitative calcium influx is greatly augmented in cultured cells depleted of PS1 or expressing PS1 FAD mutants. PS1 can also interact with ER-localized sarco/endoplasmic reticulum Ca2+ATPase (SERCA) to maintain the homeostasis of ER calcium store and modulate calcium release through either the inositol-1,4,5-triphosphate receptor (IP3R) or RyR. PSs could form calcium leak channels in the ER, and the dysfunctional PS-mediated calcium leak channels could potentially contribute to aberrant calcium signaling leading to AD pathogenesis. Consistent with its
Prevention
role in the maintenance of cytosolic Ca2+ level, FAD-linked mutations in PS1 have been shown to enhance ER calcium release via IP3R and RyR. Together, presenilins could play pivotal roles in the regulation of calcium homeostasis, synaptic function, and neuronal survival.
References Edbauer D, Winkler E, Regula JT, Pesold B, Steiner H, Haass C (2003) Reconstitution of gamma-secretase activity. Nat Cell Biol 5:486–488 Levy-Lahad E, Wijsman EM, Nemens E, Anderson L, Goddard KA, Weber JL, Bird TD, Schellenberg GD (1995) A familial Alzheimer’s disease locus on chromosome 1. Science 269:970–973 Rogaev EI, Sherrington R, Rogaeva EA, Levesque G, Ikeda M, Liang Y, Chi H, Lin C, Holman K, Tsuda T et al (1995) Familial Alzheimer’s disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzheimer’s disease type 3 gene. Nature 376:775–778 Shen J, Bronson RT, Chen DF, Xia W, Selkoe DJ, Tonegawa S (1997) Skeletal and CNS defects in Presenilin1-deficient mice. Cell 89:629–639 Wolfe MS, Xia W, Ostaszewski BL, Diehl TS, Kimberly WT, Selkoe DJ (1999) Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. Nature 398:513–517
See Also
(2012) γ-Secretase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 3349
Prevention ▶ Cancer Causes and Control
Primary Chemotherapy ▶ Neoadjuvant Therapy
Primary Liver Cancer ▶ Hepatocellular Carcinoma: Etiology, Risk Factors, and Prevention
Primary Myelofibrosis
Primary Myelofibrosis Ayalew Tefferi Division of Hematology, Mayo Clinic College of Medicine, Rochester, MN, USA
Synonyms Agnogenic myeloid metaplasia; Chronic idiopathic myelofibrosis; Idiopathic myelofibrosis; Myelofibrosis with myeloid metaplasia
Definition Primary myelofibrosis (PMF) is a stem cellderived clonal myeloproliferative disorder (MPD) that is characterized clinically by anemia, marked enlargement of the spleen and liver, and severe constitutional symptoms. Peripheral blood findings include the presence of immature myeloid cells including nucleated red blood cells, immature granulocytes, and teardrop-shaped erythrocytes. The bone marrow histology exhibits reticulin and collagen fibrosis, osteosclerosis, and angiogenesis.
Characteristics Background The blood and bone marrow features associated with PMF are discovered either de novo (i.e., PMF) or in the setting of either polycythemia vera (post-PV MF) or essential thrombocythemia (postET MF). PMF is also known by many synonyms. However, the use of the term “PMF” was endorsed by the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT). Historical Perspective The first description of PMF is credited to Heuck (1879). William Dameshek classified PMF as a MPD, along with chronic myeloid leukemia (CML), ET, and PV. In 1960, the Philadelphia chromosome was described in CML, which was
3701
later shown to harbor first t(9;22)(q32;q13) and subsequently the BCR-ABL disease-causing mutation. Accordingly, modern classification systems list PMF, PV, and ET as BCR-ABL -negative classic MPDs. In 1967, the Polycythemia Vera Study Group (PVSG) provided, for the first time, formal criteria for the diagnosis of PMF.Subsequently, a WHO-sponsored committee on classification of hematological malignancies revised the PVSG diagnostic criteria for PMF and reorganized the overall classification system for myeloid neoplasms. Disease Mechanisms In 1978, G6PD-based clonality studies established PMF as a stem cell-derived clonal myeloproliferation. In 2005, a novel gain-offunction (GOF) mutation involving the JAK2 tyrosine kinase (JAK2V617F) was described in ~50% of PMF patients but also in the majority of those with PVas well as ET. In 2006, another GOF mutation involving MPL (MPLW515L/K) was described in ~5% of patients with PMF. JAK2V617F is an exon 14 JAK2 mutation at nucleotide position 1,849 representing a G to T somatic point mutation. The mutation results in the substitution of valine to phenylalanine at codon 617. MPLW515L mutation represents a G to T transition at nucleotide 1,544, resulting in a tryptophan to leucine substitution at codon 515 of the transmembrane region of the MPL receptor. Both of the above mutations induce an MPD phenotype in mice, the former a PV-like disease and the latter a PMF-like disease. In addition to clonal myeloproliferation, PMF is characterized by bone marrow stromal aberration including collagen fibrosis, osteosclerosis, and angiogenesis. In mice, similar histological features have been induced by either systemic overexpression of thrombopoietin (TPOhigh mice) or by megakaryocyte lineage restricted under-expression of the transcription factor GATA-1 (GATA-1low mice).17 In both human PMF and experimental myelofibrosis in mice, the bone marrow stromal changes are believed to be secondary to abnormal release of fibrogenic and angiogenic cytokines including transforming growth factor-b1 (TGF-b), platelet-derived
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growth factor (PDGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), tissue inhibitors of matrix metalloproteinases, and neutrophil-derived elastase and other proteases. Clinical and Laboratory Characteristics The prevalence of PMF is similar in men and women (M:F = 1.6:1) and overall reported incidence figures range from 0.4 to 1.5/100,000. Median age at diagnosis is estimated between 55 and 60 years. Most, but not all, patients with PMF are symptomatic at diagnosis. The typical presentation includes anemia, marked splenomegaly, and constitutional symptoms including fatigue and night sweats. Hepatosplenomegaly in PMF is secondary to extramedullary hematopoiesis (EMH) that might also involve other organs including lymph nodes, pleura, peritoneum, and the paraspinal and epidural spaces. The peripheral blood smear in PMF often shows leukoerythroblastosis (presence of nucleated red blood cells and immature granulocytes) and teardrop-shaped red blood cells. Other laboratory abnormalities at diagnosis include anemia, leukocytosis or leukopenia, thrombocytosis or thrombocytopenia, and increased serum levels of lactate dehydrogenase (LDH). Bone marrow examination reveals both “cellular phase” and “overtly fibrotic” stages of the disease. In both instances, the most characteristic feature is the presence of dense megakaryocyte clusters with atypical megakaryocyte morphology (cloud-like nuclear morphology) that is accompanied by increased granulocyte proliferation and reduced erythropoiesis. Additional histological features of advanced disease include new bone formation and intra-sinusoidal hematopoiesis. Diagnosis Bone marrow examination is essential in the diagnosis of PMF and should be accompanied by mutation screening for BCR-ABL in order to exclude the diagnostic possibility of CML and JAK2V617F in order exclude the possibility of bone marrow fibrosis associated with nonmalignant condition, lymphoid disorder, or metastatic cancer. It should be noted, however,
Primary Myelofibrosis
that JAK2V617F cannot distinguish PMF from other myeloid disorders such as MDS, ET, PV, or other MPD. Therefore, accurate diagnosis requires careful morphological evaluation. Cytogenetic abnormalities occur in approximately half of the patients with PMF at diagnosis and include del(20)(q11;q13), del(13)(q12;q22), trisomy 8, trisomy 9, del(12)(p11;p13), monosomy or long arm deletions involving chromosome 7, and partial trisomy (1q). Although none of these abnormalities are specific to PMF, the presence of either del (13)(q12;q22) or der(6)t(1;6)(q21–23;p21–23) is strongly suggestive of the specific diagnosis. Prognosis and Treatment Causes of death in PMF include development of blast phase PMF, which occurs in ~10% of patients during the first decade of their disease, and infections. Survival in PMF is estimated by the use of one of several prognostic scoring systems (PSSs) that rely on the presence or absence of well-established adverse prognostic features. Among the latter, the Mayo Clinic PSS has been reported to be superior, compared to other PSSs, in delineating both good-risk and intermediaterisk disease categories. The Mayo PSS is based on four adverse prognostic variables: hemoglobin 10 and Gleason score >7, are correlated with increased risk of extra-prostatic spread and are considered to be the key factors in determining the need for staging work-up. PSA levels 10 with high-grade histology or DRE findings suggesting stage T3 disease should undergo staging computed tomography (CT) scan and bone scan (Fig. 5). Magnetic resonance imaging (MRI) is superior to bone scan in evaluating bone metastasis but is so far relatively impractical for routine total body surveys. Instead it is used to determine the etiology of questionable lesions found on bone scan. The use of MRI to stage or determine the location of the local lesion is controversial. Prostate-specific membrane antigen (PMSA) immunoscintigraphy with the indium-labeled immune conjugate CYT-356
3737 Prostate Cancer Clinical Oncology, Table 1 The 1997 UICC/AJC TNM staging system UICC/AJC 2010 T1a
T1b
T1c
T2a T2b T2c T3a T3b T4
N(+) M(+)
Description Tumor incidental histologic finding in 5% or less of tissue resected and Gleason
