T-Cell Receptor Gene Rearrangement T-Cell Response

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T-Cell Receptor Gene Rearrangement through contact inhibition or other means. Devia▶ V(D)J Recombination

T-Cell Response Periasamy Selvaraj Department of Pathology, Emory University School of Medicine, Atlanta, GA, USA

Definition Expansion of antitumor T cells in response to growth of cancer or to a ▶ cancer vaccine or a cancer immunotherapy.

Characteristics In living organisms, the growth and division of cells are tightly controlled and highly regulated process that maintains the integrity of cellular architecture and survival. Cell growth can occur in our body to replace a dead cell, to heal a wound, to maintain normal homeostasis, or to generate immune cells during an immune response. The number of divisions that cells undergo during these processes is highly controlled, and the cells stop dividing when they receive signals either # Springer-Verlag Berlin Heidelberg 2017 M. Schwab (ed.), Encyclopedia of Cancer, DOI 10.1007/978-3-662-46875-3

tions from normal regulatory mechanisms may result in uncontrolled cell growth and the development of various types of cancers in our body. Many genes in the human body play pivotal roles in regulating the normal metabolic and growth patterns of cells. Mutations in these genes lead to the production of altered proteins that may result in the uncontrolled cell growth that is observed in cancer. Role of Immunosurveillance in Antitumor Immunity: Gene mutations occur constantly through the life span of a living organism, resulting in the occasional production of abnormal cells. Most mutant cells with defective metabolism die of a process called apoptosis. The mutant cells that are able to survive with altered proteins will be recognized by the immune system and destroyed by a process termed immunosurveillance. There is strong evidence for the existence of such immunosurveillance against cancer. Cancers develop in high frequencies in people with immunodeficiency diseases associated with immune system defects such as AIDS, or in animals that have an incomplete immune system, suggesting that cancer-specific immunity is a major mechanism for elimination of cancers from the body. This immunity is provided by the cytotoxic activity of tumor-specific T cells that recognize tumors and destroy them. However, there are many ways that cancer cells circumvent this immunosurveillance. Some cancers secrete

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immunosuppressive factors such as TGF-b, which attenuates T-cell immune response. Downregulation of MHC class I molecules that stimulate the immune system and upregulation of molecules that induce T-cell apoptosis are other mechanisms cancers use to inactivate antitumor T cell immune responses. Molecular Requirements of T Cell Responses: T cells are divided into two major subsets. CD4 antigen-expressing T cells are termed “helper T cells,” whereas CD8 antigen-expressing T cells are termed “cytotoxic T cells.” It has been shown that CD4+ T cells provide help by secreting cytokines for the generation of robust CD8+ T cells that can kill tumor cells. Since T cells play a crucial role in the development of antitumor immunity, most cancer immunotherapy is designed to directly or indirectly activate tumorspecific CD4+ and CD8+ T lymphocytes and induce immunological memory against tumors. Studies have shown that both subsets of T cells play an important role in the antitumor immune response. Advances in understanding the molecular and cellular requirements for antigen-specific immune responses have led to a number of promising immunotherapeutic strategies for inducing antitumor T-cell responses for the treatment of cancer. Many of these strategies include vaccination with ▶ dendritic cells (DCs) engineered to express tumor antigens, cytokine-transduced tumor cells, peptide vaccines, DNA vaccines, heat shock proteins, hybrid tumor cells, and tumor cells transduced with costimulatory molecules. The rationale for the vaccination strategies mentioned above is that antigen-specific T cells can be stimulated effectively by providing stimulatory signals arising from either tumors themselves (direct priming) or through antigenpresenting cells (APCs) of the host (indirect priming). Normally, T cells are educated in the thymus to react against cells expressing MHC molecules that display peptides derived from foreign or altered proteins. Since tumors express altered proteins, the peptides derived from these altered proteins are associated with MHC molecules and presented to T cells that are specific to the altered protein. For an optimal immune response,

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antigen-specific T cells require at least two specific signals. One of the signals is provided by engagement of the T-cell receptor (TCR) with peptide-bearing MHC molecules on the APC. The second signal (costimulatory signal) can be delivered by the interaction of various adhesion molecules on the surface of T cells and the APC, one of which is the interaction of CD28 expressed on T cells and B7-1 (CD80) expressed on APCs such as DCs. The absence of a second signal results in Tcell clonal anergy, thus preventing the development of tumor-specific T cells (Fig. 1). Tumor cells, which lack costimulatory molecules such as B7-1, are poorly immunogenic since they fail to deliver the costimulatory signal necessary for the generation of an antitumor T-cell immune response. Therefore, one approach to improve the immunogenicity of tumor cells has been to introduce costimulatory adhesion molecules such as B7-1 onto the tumor surface by gene transfection. This B7-1 expression results in the induction of T-cell mediated antitumor immunity and subsequent tumor rejection in animals. These studies also demonstrated that costimulation is required only for the initial stimulation and expansion of tumor-specific CD8+ cytotoxic T cells (CTLs) and not required for the killing of tumor cells by CTLs. This basic understanding of the role of T-cell immunity in eliminating tumors has given rise to many immunotherapeutic approaches focused on expanding tumor-specific T cells in a tumor-bearing host using vaccination and other immunotherapy approaches. Pathways of Tumor Antigen-Specific T-cell Stimulation In Vivo: Tumors transduced with the B7-1 molecule have been suggested to prime T cells directly, whereas DNA vaccines, peptide vaccines, and cytokine-transduced tumors may stimulate T cells indirectly through host APC. However, studies suggest that tumors transduced with B7-1 molecule use both priming pathways to induce an antitumor immune response. The indirect pathway could occur through host APCs, mainly by DCs taking up the tumor antigens and processing and presenting them to CD8+ T cells through cross-priming. B7-1 gene-transfected tumor cell vaccines, once thought to work only by activating CD8+ T cells directly, have now

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T-Cell Response, Fig. 1 T cells require two signals to expand and become effective cytotoxic T cells. In the absence of a second signal, T cells undergo energy or apoptosis. Thus, tumors by not expressing the second signaling molecule can prevent generation of an antitumor T-cell response

been shown to activate T cells indirectly through cross-priming by professional APCs. The enhancement of cross-presentation by tumors expressing B7-1 has also been attributed to the enhanced recognition of these tumors by ▶ natural killer cells. NK cells have been shown to express CD28 and cross-linking of CD28 on NK cells by B7-1 results in the release of factors such as TNF-a and IFN-g, which subsequently stimulate DCs. It has been shown that activated human NK cells can also directly interact with CD4+ T cells and costimulate TCR-induced proliferation, suggesting a possible cross talk between CD4+ T cells and NK cells during antigen-specific immune response. Many studies have shown that antigen-specific CD8+ CTLs can be generated without CD4+ T-cell help. DCs have been shown to play a major role in this CD4+T cell-independent expansion of CD8+ CTLs. Antigen-specific CD8+ CTLs developed in the absence of CD4+ T-cell help are capable of providing protective antitumor immunity in mice. However, studies show that CD4+ T cells play a major role in maintaining the CD8 T-cell memory developed during antigen exposure. The CD8+ memory T cells developed in the absence of CD4+ T-cell help are defective in responding to

antigens in a secondary challenge with antigen, suggesting that for induction of an optimal antitumor immunity, both CD4+ T cells and CD8+ T cells are required. These observations demonstrate that interaction of immune cells such as DCs, NK cells, and T cells that are activated by direct and indirect pathways cooperates to produce an optimal antitumor T-cell immune response during vaccination. Thus, the introduction of immunostimulatory molecules directly into the tumor by gene transfer offers an attractive approach to improve the immunogenicity of tumor cells. Accordingly, the expression of costimulatory molecules such as B7-1 or cytokine molecules by gene transfer results in the induction of tumor immunity capable of inducing wild-type tumor rejection in animals. Apart from these vaccine strategies, it has been shown that adoptive transfer of ex vivo expanded and activated tumor-infiltrating T cells reduced tumor burden in animals and humans. Studies have also shown that CD4+ CD25+ regulatory T cells (Tregs) (T regulatory cells) play an important role in suppressing antitumor immunity in a host. The depletion of Tregs has been shown to increase potency of many antitumor immunotherapeutic methods.

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Methods to Detect Antitumor T Cell Responses: Studying T cell immune responses during vaccination or other types of immunotherapies is valuable in understanding the nature of the immune response. Moreover, identifying a correlation between characteristics of antitumor T-cell response and clinical efficacy of a treatment modality will be useful in predicting clinical outcome of a therapy at an early stage. For example, T-cell populations carrying longer telomeres will be the dominant population in mice that are treated with the most potent vaccine since it has been shown that the presence of this T-cell population is a predictive indicator of robust antitumor immunity. Similarly, better therapeutic efficacy was observed when a T-cell population expressing TCR containing specific Vb was expanded during a vaccine administration. Tumor antigen-specific CD8+ T cells can be quantified by CTL assays and intracellular IFN-g staining of cells obtained from the blood in humans or the spleen in the case of mice. Limiting dilution assays or CD8 and IFN-g staining can be used to determine the frequency of activated CD8+ T cells. Increase in IFN-g has been shown to occur during antigen-specific CD8+ T-cell activation. If the frequency of IFN-g staining cells is too low to be detected by the intracellular cytokine staining method, then the ELISPOT assay to determine IFN-g secreting cells can be carried out. Alternatively, expansion of tumor antigenspecific T cells can be monitored using MHC tetramers. Although measuring antigen-specific T cells using MHC tetramers is a very sensitive assay, it indicates the mere physical presence of an antigen-specific T cell but does not indicate whether the expanded antitumor T cells are functionally active or not. Therefore, MHC tetramer assays are normally combined with functional assays such as CTL assay or intracellular IFN-g staining to determine the antitumor efficacy of an immunotherapeutic method. Lessons from Human Immunotherapy: Based on the knowledge obtained from in vitro experiments and animal models of antitumor T-cell responses and vaccines, many clinical trials have been conducted. Both cell-based and vaccinebased therapies have been employed. In cellbased therapies, patients were administered

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ex vivo expanded tumor-infiltrating T cells or DCs loaded with tumor antigens. In vaccinebased therapies, cancer cells or cellular fragments or tumor-associated antigens were modified with adjuvants such as cytokines or other immunostimulatory molecules and administered to induce expansion of tumor antigen-specific T cells. Some of the trials showed moderate success, whereas some of them were not as effective as observed in animal models. Interestingly, in many patients, although vaccination produced strong antitumor T-cell responses, the regression of the tumor did not occur, suggesting there is a disconnect between the expansion of antitumor-specific T cells and efficacy of a cancer vaccine. This could be due to the lack of homing of antitumor T cells to tumor site, or tumors may secrete immunosuppressive factors that lead to the inactivation of cytotoxic T cells. Also, comparison of the results of T-cell response, as measured by MHC tetramer assays and functional assays, showed that not all the tumor antigen-specific T cells expanded during a vaccination are functionally active, suggesting aberrations in the development of T cells during vaccination. These results emphasize the need for further studies on the nature and mechanisms of T-cell responses during various therapeutic approaches. Developments have demonstrated that one of the immunosuppressive mechanisms during cancer and chronic viral infections is the expression of immune checkpoint inhibitor molecules such as PD1 and CTLA-4 on the activated T cells. Antibodies blocking these molecules have approve approved for therapeutic use in cancer. A careful manipulation of the induction of antitumor T-cell immune responses, immunosuppressive mechanisms and homing of T cells to tumors will lead to the development of more effective therapies to treat various types of cancer and chronic viral infections.

References Ha SJ, Mueller SN, Wherry EJ, Barber DL, Aubert RD, Sharpe AH, Freeman GJ, Ahmed R (2008). Enhancing therapeutic vaccination by blocking PD-1-mediated inhibitory signals during chronic infection. The J Exp Med 205:543–55

Tachykinins Keilholz U, Martus P, Scheibenbogen C (2006) Immune monitoring of T-cell responses in cancer vaccine development. Clin Cancer Res 12:2346s–2352s Peggs KS, Allison JP (2005) Co-stimulatory pathways in lymphocyte regulation: the immunoglobulin superfamily. Br J Haematol 130:809–824 Rosenberg SA (2001) Progress in human tumour immunology and immunotherapy. Nature 411:380–384 Smyth MJ, Dunn GP, Schreiber RD (2006) Cancer immunosurveillance and immunoediting: the roles of immunity in suppressing tumor development and shaping tumor immunogenicity. Adv Immunol 90:1–50 Sharma P, Allison JP (2015) The future of immune checkpoint therapy. Science 348:56–61 Zitvogel L, Tesniere A, Kroemer G (2006) Cancer despite immunosurveillance: immunoselection and immunosubversion. Nat Rev Immunol 6:715–727

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Tachykinins Florin Tuluc Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA The Children’s Hospital of Philadelphia, Philadelphia, PA, USA

Synonyms Neurokinins

Definition

T-Cells Recognizing Autoantigens ▶ Autoimmunity and Cancer

t(8;21)

Tachykinins comprise a family of peptides found in all species from amphibians to mammals. Their name is derived from their ability to trigger rapid contraction of gut smooth muscle. The structure of tachykinins is characterized by a conserved COOH-terminal sequence (Phe-X-Gly-Leu-MetNH2, where X is a hydrophobic amino acid residue).

▶ Chromosomal Translocation t(8;21)

Characteristics

t(8;21)(q22;q22) ▶ Chromosomal Translocation t(8;21)

T1a2 ▶ Podoplanin

TACE ▶ ADAM17

Tachykinin Genes The tachykinin family includes several distinct peptides with broad tissue distribution and functions; the undecapeptide substance P (SP) is the most well-known member of this family. The name tachykinin has been coined in connection with the ability of these peptides to produce rapid contraction of intestinal muscle in experimental models on isolated organs, in contrast with the slower acting bradykinin. Tachykinins are encoded by preprotachykinin genes (TAC1, TAC3, and TAC4) which are responsible for the production of long precursor polypeptides; the active tachykinins are generated by proteolytic cleavage of the precursors. All TAC genes can generate multiple transcript variants through alternative splicing, and thus multiple peptide products can be produced by a single

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Tachykinins Sequence

Tachykinin

Tachykinins encoded by TAC1 gene RPKPQQFFGLM-NH2 HKTDSFVGLM-NH2 DAGHGQISHKRHKTDSFVGLM-NH2 DADSSIEKQVALLKALYGHGQISHKRHKTDSFVGLM-NH2

Substance P Neurokinin A Neuropeptide Y Neuropeptide K

Tachykinins encoded by TAC3 gene DMHDFFVGLM-NH2

Neurokinin B

Tachykinins encoded by TAC4 gene

ASQFFGLM-NH2 TGKASQFFGLM-NH2 DGGEEQTLSTEAETWVIVALEEGAGPSIQLQLQEVKTGKASQFFGLM-NH2 DGGEEQTLSTEAETWEGAGPISIQLQLQEVKTGKASQFFGLM-NH2 KKAYQLEHTFQGLL-NH2 VGAYQLEHTFQGLL-NH2

Hemokinin 1 (4-11) Hemokinin 1 Endokinin A Endokinin B Endokinin C Endokinin D

Tachykinins, Fig. 1 The sequence of amino acid residues in human tachykinins. The conserved amino acid residues at the NH2-terminus are highlighted in yellow; note that the last amino acid residue (L) of endokinin B and endokinin D

is different from the rest of the family (M). Common sequences among groups of peptides derived from TAC1 and TAC4 genes are highlighted in green, cyan, and gray

gene. The posttranslational processing of the polypeptide precursors adds an additional layer of complexity to the generation of bioactive tachykinins which serves as a regulatory mechanism of biosynthesis. The TAC1 gene was described first; it has seven exons in its structure and can be alternatively spliced into four possible transcripts a, b, g, or d which undergo proteolytic cleavage to generate SP, NKA, neuropeptide gamma (NKg), and neuropeptide K (NPK). NKA is also known as neurokinin a, substance K, or neuromedin L. The name TAC2 was initially assigned to the gene encoding the precursor of NKA, but later it was discovered that TAC2 and TAC1 are identical; the designation TAC2 is not in use any longer. TAC3 gene encodes the precursor of NKB and TAC4 gene encodes six peptides: four peptides are termed endokinins (A, B, C, and D), hemokinin 1 (HK1), and its shorter derivative, HK1 (4–11) (Fig. 1). The sequence of amino acid residues in tachykinins is well conserved within mammalians, with the exception of the HK-1 peptide which is different among species. The COOHterminal sequence (Phe-X-Gly-Leu-Met-NH2, where X is a hydrophobic amino acid residue) is

highly conserved among tachykinins (Fig. 1), and this sequence is required for the interaction with neurokinin ▶ receptors on the membrane of target cells. NPK and NPg are NH2-terminally extended forms of NKA, while HK1, EKA, and EKB are extended versions of HK1 (4–11). NPg lacks residues 3–17 of NPK. Endokinins C and D (EKC, EKD) have a COOH-terminal sequence only partially conserved, they do not bind with high affinity to any of the tachykinin receptors, and for these reasons they are not regarded a true tachykinins but rather as tachykinin gene-related peptides. The biological roles of tachykinins are broad, and they have been studied in major systems and organs (nervous, immune, gastrointestinal, respiratory, urogenital, skin, hematopoietic bone marrow) and pathological processes (▶ inflammation, pain, viral infections, ▶ cancer). The first biologic activity of SP was reported by Euler and Gaddum in an extract of intestine and brain horse that could be prepared as a powder that was called “preparation P” and later “substance P.” The other tachykinins derived from TAC1 and TAC3 were later discovered in tissue extracts based on their biologic activity on smooth muscle. The HKs and EKs were discovered by analysis of the TAC4 gene rather than by bioassays of tissue extracts.

Tachykinins

Tachykinin Receptors The existence of multiple tachykinin receptors was initially suggested by the different rank order of tachykinin potencies in bioassays performed on various tissues. Cloning of three genes (TACR1, TACR2, TACR3) encoding distinct neurokinin receptors (NK1R, NK2R, NK3R) clarified and categorically established the existence of these three receptor subtypes. SP binds with higher affinity to NK1R than to NK2R and NK3R, thus yielding lower EC50 values in tissues that express NK1R as compared to tissues in which the other two receptors are expressed. NKA binds with higher affinity to NK2R while NKB prefers NK3R. All three TACR genes have five exons and four introns, and they can generate multiple mRNA transcripts through alternative splicing to produce receptor isoforms. The first splice variant identified in human tissues at mRNA and protein level is an isoform of NK1R which lacks 96 amino acid residues at the C-terminus. This “tailless” NK1R isoform is generated when the intron between exons 4 and 5 is not removed, and, as a result, a premature stop codon is encountered before the start of exon 5. At least two more NK1R isoforms were reported but they are less well characterized. All NKRs are classic seven transmembrane domain receptors that include in their structure an extracellular amino terminus, seven transmembrane domains, three intracellular loops, three extracellular loops, and an intracellular carboxyl terminus. They are coupled to heterotrimeric G proteins from the Gq/11 family which lead to the activation of phospholipase Cb, transient increase in intracellular inositol 1,4,5trisphosphate (IP3) and diacylglycerol followed by sustained increase in cytosolic calcium concentration, and activation of protein kinase C (PKC). Subsequently, a cascade of intracellular events occurs and initiates cellular responses that depend on the activated effectors. NK1R and NK2R were reported to induce adenylyl cyclase activation and production of cAMP via the Gs protein. Furthermore, NK1R was found to inhibit of adenylyl cyclase via the pertussis toxinsensitive Gi protein in rat submandibular cells. NK2R couples to Gs and Gq/11 while signaling

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downstream of NK3R seem to be restricted to proteins from the Gq/11 family. Phosphorylation of a plethora of intracellular proteins is triggered after binding of agonists to NK1R; among these proteins are b-arrestins, extracellular signal-regulated kinases (ERK), G proteincoupled receptor kinases (GRK), PKC, Akt, and myosin light-chain kinase (MLCK). The outcomes of the signaling events initiated by tachykinins depend on the target cell type and include alterations of cell shape and motility, excitability, antiapoptotic effect, release of secretory vesicles, etc. The signaling events are terminated through receptor desensitization and also by mechanisms that remove tachykinins from the extracellular microenvironment (reuptake, degradation). The combined actions of G protein-coupled receptor kinases (GRKs) and b-arrestins are responsible for the desensitization of NK1R: upon NK1R activation, at least three distinct GRK proteins (GRK2, GRK3, and GRK5) become capable to phosphorylate amino acid residues located in the intracellular C-terminal domain of NK1R. This event favors physical interaction of the receptor with b-arrestin followed by the formation of endosomes that internalize NK1R together with the bound agonist. Endothelinconverting enzyme-1 is a membrane metalloendopeptidase able to degrade SP inside the endosomes and contributes to the termination of the signaling events initiated by the internalized complex that includes SP, NK1R, b-arrestin, and other signaling molecules. The truncated NK1R is resistant to desensitization because it lacks GRK phosphorylation sites and does not interact with b-arrestins. NK1R becomes resensitized to SP responses following dissociation from b-arrestin and redistribution on plasma membrane (a process known as receptor recycling); thus NK1R becomes again susceptible to binding to extracellular agonists. After stimulation, a substantial proportion of desensitized NK1Rs remain at the plasma membrane, where they resensitize by a mechanism dependent on ▶ phosphatase 2A by dephosphorylation. NK1R is by far the most studied neurokinin receptors, while the functions of NK2R and NK3R are known in less detail.

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At least one cell-surface metalloendopeptidase (neprilysin) is capable of degrading SP in the extracellular fluid and also participates in the termination of NK1R signaling. SP is implicated in a variety of responses, including pain transmission, exocrine and endocrine secretion, vasodilatation, cell proliferation, and modulation of the immune and inflammatory responses. NK1R participates in a variety of complex brain functions, such as neuronal sensory transmission associated with emesis, pain, depression, anxiety, and central responses to stress. NKA has a wide distribution in both the central and peripheral nervous systems and is implicated in pain and inflammatory responses. NKA also participates in triggering bronchoconstriction in individuals with asthma, and it is also regarded as an important mediator in neurologic and psychiatric conditions such as epilepsy, schizophrenia, and depression. NKB is produced in the placenta and the plasma levels of NKB are elevated in preeclampsia. However, whether high NKB levels are causative or merely associated with preeclampsia is not clear, since numerous other vasoactive peptides are overproduced during preeclampsia. Because tachykinins are involved in a variety of physiologic processes and pathologic conditions, a great effort has been directed by pharmaceutical companies to develop effective and safe drugs that can be used to block or activate selectively NKR subtypes, especially NK1R antagonists. Multiple antagonists have been used in clinical trials on patients with a variety of pathologic conditions, including pain, irritable bowel syndrome, asthma, depression, anxiety, panic disorder, posttraumatic stress disorder, prurigo, overactive bladder syndrome, alcoholism, alopecia, HIV infection, and cancer. Implications of Tachykinins in Malignancies Evidence from multiple studies suggests that tachykinins are involved in cancer biology in multiple ways. Some tumors express NKRs and tachykinins from infiltrating nerves, from intra- or peritumoral blood vessels, and from immune cells or produced by the tumor cells themselves can influence proliferation, ▶ apoptosis, and metastasis of cancer cells in a paracrine, neurocrine, or

Tachykinins

autocrine manner. SP induces mitogenesis in normal and tumor cells, has antiapoptotic effect, and controls the ▶ migration of tumor cells. Tachykinins can induce normal cells around the tumor to release cytokines or growth factors that can affect tumor cell growth and survival. There is also evidence that in the case of certain types of tumors (e.g., ▶ lung cancer), NKR agonists may exert anticancer effects. Despite the large number of experimental studies that bring evidence that SP seems to favor the proliferation of human cells and NK1R antagonists have an opposite effect, there are no effective chemotherapeutic anticancer drugs in this pharmacological class that passed the stage of clinical trials. The main therapeutic application of NK1R antagonists remains the prevention and treatment of ▶ chemotherapy-induced nausea and vomiting (CINV) and postoperative nausea and vomiting (PONV). Breast Cancer

▶ Breast cancer is a common human malignancy and it is one of the top leading causes of deaths in women worldwide. The high mortality of this disease is due mainly to the fact that this malignancy is difficult to detect in early stages, before ▶ metastases spread to distant organs. Multiple cell lines derived from breast cancers and malignant breast biopsies have increased levels of TAC1, TACR1, and TACR2 transcription products and produce high levels of SP. NK1R and NK2R antagonists inhibit growth of human breast carcinoma cells in culture and after xenograft implantation in mice, both the presence and absence of exogenous SP and NKA. Multiple mechanisms that mediate the effects of tachykinins on breast cancer cells have been described; one of them involves RE-1 silencer of transcription (REST), which inhibits the transcription of TAC1 in breast cancer cell lines and in primary breast cancer cells. Furthermore, overexpression of the truncated form of NK1R induces transformation of breast cancer cells, suggesting that blocking the truncated version of this receptor could be a therapeutic strategy. ErbB is a family of four structurally related receptor tyrosine kinases which includes ErbB1

Tachykinins

(endothelial growth factor receptor/EGFR, Her1), ErbB2 (Her2, Neu), ErbB3 (Her3), and ErbB4 (Her4). Overexpression and excessive signaling through EGFR or Her2 are critical factors in the development and aggressive behavior in many human malignancies. HER2-positive primary breast tumors were found to express SP and NK1R, and their presence correlated with poor prognosis factors. SP can activate HER2 in breast cancer cell lines and primary cultures derived from breast cancer samples. NK1R antagonists or downregulation of NK1R using ▶ RNA interference techniques decreased the expression of EGFR and HER2. Thus, it seems that SP modulates EGFR and HER2 activation in breast cancer cells, acting to enhance malignancy and therapeutic resistance. Thus, an important oncogenic cooperation between NK1R and HER2 with potential therapeutic applications has been postulated. It has been suggested that SP facilitates ▶ blood-brain barrier breaching by breast cancer cells through changes in microvascular endothelial cell ▶ tight junctions, thus promoting the development of brain metastases. Therapies based on inhibiting the effects of SP may help prevent the breaching of blood-brain barrier by breast cancer cells, thus decreasing the risk of brain metastasis. The efficacy of such a therapy in humans is yet to be demonstrated. Lung Cancer

TAC1 and its products are present in some lung malignant tumors; by the other side, downregulation of NK1R and NK1R antagonists favors the development of bleomycin-associated lung adenocarcinoma. Blockade of the NK1R in mice treated with bleomycin interferes with the activity of several proteins that are implicated in the control of apoptosis (e.g., the death receptor Nur77, Bcl2, and Bak). Consistent with these observations, treatment of mice with aerosolized SP prevents, at least to some extent, cigarette smoke-induced lung damage and tumor development. Brain Cancer

Tumors derived from nervous tissue often express tachykinins and the NK1R. For instance, NK1R is

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expressed in U373MG where it triggers signaling events that induce growth and survival. Notably, NK1R blockade reduces basal Akt phosphorylation which suggests the existence of a constitutively active receptor or autocrine stimulation due to the release of peptides with NK1R agonist activity. HK1 causes NK1R-mediated expression of the matrix metalloproteases by glioma cells, which promotes migration. Neuroblastoma cell lines (e.g., CHP212, SY5Y) express full-length and truncated NK1R as well TAC1, and NK1R silencing blocks mitogenesis. Stimulation of the P2X7 nucleotide receptor triggers proliferation of neuroblastoma cell lines in part due to SP release, also suggesting the existence of an autocrine pathway. It has been suggested that NK1R antagonists may be used as a therapeutic approach for brain cell tumors. Colon Cancer

SP and NK1R are implicated in proliferation and tumorigenesis in colon epithelium. SP activates multiple signaling pathways linked to proliferation of human NCM460 colonic epithelial cells. The NK1R is also expressed in SW-403 ▶ colorectal cancer cells, and the NK1R antagonists block proliferation in these cells in the presence or absence of exogenous SP, suggesting the existence of an autocrine mechanism. Furthermore, it has been shown that the truncated form of NK1R is preferentially upregulated in colonic epithelial cells of patients with ulcerative colitis who develop colonic carcinoma, suggesting that the truncated NK1R has tumorigenic potential, possibly favored by its diminished propensity to desensitization and endocytosis. TAC1 is a frequent target of methylation in primary colon cancers. It has been suggested that TAC1 expression is reduced in colon carcinogenesis through hypermethylation, and this may be a part of carcinogenesis mechanisms in this type of tumor. Pancreatic Cancer

Among gastrointestinal malignancies, ▶ pancreatic cancer has the lowest survival rates. Several pancreatic cancer cell lines also express the NK1R, and SP stimulates growth of these cells

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by an NK1R-dependent mechanism. NK1R is upregulated in pancreatic tumors, especially in advanced tumors with a poor prognosis. Melanoma

SP and the NK1R are expressed by ▶ melanomas and melanoma cell lines and NK1R blockade inhibits cell growth. Although SP and the NK1R can directly promote melanoma cell growth, NK1R signaling seems to inhibit melanoma development in vivo indirectly. SP infusion protects mice against melanoma cell growth, and this protection is lost in mice depleted of T and natural killer cells. The adoptive transfer of these cells from SP-treated mice also suppressed tumor growth, suggesting that SP can prime the immune system to defend against melanoma tumorigenesis. Hematopoietic Malignancies

Substance P is expressed in human blast cells in individuals with ▶ acute lymphoblastic leukemia (ALL). The expression of NK1R was reported on several cell lines derived from ALL cells and knocking down their expression resulted in decreased cell viability. Blocking NK1R with different antagonists resulted in inhibition of ALL cell growth and increased apoptosis. Despite the substantial efforts done to develop potent NKRs antagonists, none of these antagonists had shown effective antitumor activity in clinical trials. NK1R antagonists are used effectively for the prevention and treatment of CINV. Aprepitant for the Treatment CINV Approximately 70–80% of patients with cancer experience CINV if no prophylactic therapy is administered. Emesis is usually a symptom feared by patients; it can lead to dehydration, electrolyte imbalance, esophageal tears, and deterioration of nutritional status, and overall it decreases dramatically the quality of life; severe emesis can be a cause of low adherence to therapy. Furthermore, inadequate emesis control triggers anticipatory nausea and vomiting in 10–40% of the patients, which is often refractory to standard antiemetic medications. The incidence and severity of CINV depend mainly on the

Tachykinins

emetogenic potential of the drugs used for cancer treatment, dosage, schedule, and route of administration of the chemotherapic drugs used but also on patient-related factors such as age, sex, and history of alcohol use. Most antiemetics that are in use nowadays rely on a mechanism of action based on blocking receptors for neurotransmitters in the brain and in the peripheral nervous system; they include antagonists at serotonin 5-HT3 receptors, dopamine receptors, and NK1R receptors. In addition, corticosteroids are also used for their antiemetic effect. Because emesis involves the participation of multiple mechanisms, it is not surprising that single agent therapies do not achieve complete protection against the various phases of emesis; for this reason combinations of antiemetic agents are usually administered. The combination of a corticosteroid (usually dexamethasone) and antagonists at serotonin 5-HT3 receptor (e.g., ondansetron) is one of the most common CINV prevention regimens; however, this combination often fails to prevent CINV with late onset. In patients with tumors treated with highly emetogenic chemotherapy, a standard regimen that includes aprepitant in addition to dexamethasone and ondansetron results in significantly higher rate of complete response (no emesis and no rescue therapy) than with the control regimen of ondansetron and dexamethasone. The efficacy of aprepitant is maintained during multiple cycles of chemotherapy. Thus, for these reasons the use of an NK1R antagonist for CINV prophylaxis in acute and delayed phases for highly emetogenic chemotherapy is recommended by current guidelines for CINV management. ® Aprepitant (Emend ) and fosaprepitant, a prodrug of aprepitant designed for intravenous administration, are NK1R antagonists approved by FDA in the USA. In October 2014 FDA approved netupitant, another NK1R antagonist, in combination with the 5-HT3 antagonist palonosetron to treat nausea and vomiting in patients undergoing cancer chemotherapy. Aprepitant is a selective, high-affinity antagonist that blocks the binding of substance P to NK1R. Aprepitant crosses the blood-brain barrier and it effectively blocks NK1R both in CNS and in

Tachykinins

periphery. Aprepitant is metabolized mainly by ▶ cytochrome P450 (CYP) 3A in the liver and it is excreted as metabolites in the urine and bile and the rate of metabolism is affected by coadministration of inhibitors or inducers of this isoenzyme. Plasma concentrations of aprepitant are higher when drugs such as ketoconazole, itraconazole, nefazodone, troleandomycin, clarithromycin, ritonavir, and nelfinavir, which are strong inhibitors of CYP3A4, are administered together with aprepitant. Conversely, strong CYP3A4 inducers (e.g., phenytoin, rifampicin, carbamazepine) may decrease plasma levels of aprepitant and reduce the antiemetic effect. Aprepitant itself is a moderate CYP3A4 inhibitor, and the standard 3-day antiemetic regimen (125 mg on day 1 followed by 80 mg/day on days 2 and 3) may increase plasma concentrations of coadministered drugs that are metabolized by CYP3A4 (e.g., dexamethasone, methylprednisolone, diltiazem). Furthermore, aprepitant has inducer effect on other metabolic enzymes (CYP2C9, CYP2D6), and its coadministration may result in lower plasma levels of warfarin, tolbutamide, paroxetine, and phenytoin and oral contraceptives. Many other NK1R antagonists (e.g., casopitant, maropitant, nepadutant, saredutant, talnetant) were tested in various experimental models and in clinical trials.

Cross-References ▶ Acute Lymphoblastic Leukemia ▶ Apoptosis ▶ Blood-Brain Barrier ▶ Breast Cancer ▶ Cancer ▶ Chemotherapy ▶ Colorectal Cancer ▶ Cytochrome P450 ▶ Endosomal Compartments ▶ G Proteins ▶ Inflammation ▶ Lung Cancer ▶ Metastasis ▶ Migration ▶ Pancreatic Cancer

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▶ Phosphatase ▶ Receptors ▶ RNA Interference ▶ Tight Junction

References Curran MP, Robinson DM (2009) Aprepitant: a review of its use in the prevention of nausea and vomiting. Drugs 69(13):1853–1878 Steinhoff MS, von Mentzer B, Geppetti P, Pothoulakis C, Bunnett NW (2014) Tachykinins and their receptors: contributions to physiological control and the mechanisms of disease. Physiol Rev 94:265–301 Tuluc F, Lai JP, Kilpatrick LE, Evans DL, Douglas SD (2009) Neurokinin 1 receptor isoforms and the control of innate immunity. Trends Immunol 30(6):271–276

See Also (2012) Adenylyl cyclase. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 57. doi:10.1007/978-3-642-16483-5_92 (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) Autocrine. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 311. doi:10.1007/978-3-642-16483-5_468 (2012) Bradykinin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 468. doi:10.1007/978-3-642-16483-5_701 (2012) CAMP. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 603. doi:10.1007/978-3-642-16483-5_788 (2012) Central nervous system. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 744. doi:10.1007/978-3-642-16483-5_1024 (2012) Endothelin converting enzyme. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1254. doi:10.1007/978-3-642-164835_1903 (2012) Epilepsy. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1290– 1291. doi:10.1007/978-3-642-16483-5_1951 (2012) ERK. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, pp 1307–1308. doi:10.1007/978-3-642-16483-5_1987 (2012) Heterotrimeric G-proteins. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1689. doi:10.1007/978-3-642-164835_2701 (2012) Immune system. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 1815. doi:10.1007/978-3-642-16483-5_2980 (2012) Neurotransmitters. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer, Berlin/Heidelberg, p 2505. doi:10.1007/978-3-642-16483-5_4049

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Takatsuki Disease or PEP Syndrome ▶ POEMS Syndrome

Tamoxifen V. Craig Jordan Breast Medical Oncology, MD Anderson Cancer Center, Houston, TX, USA

Definition Tamoxifen is a nonsteroidal antiestrogen used for the treatment and prevention of ▶ estrogen receptor (ER)-positive breast cancer. Tamoxifen is the most studied anticancer agent.

Takatsuki Disease or PEP Syndrome

Characteristics Background Tamoxifen (ICI 46,474) was first described as an effective postcoital contraceptive in rats, but the drug induces ovulation in subfertile women. The compound was subsequently reinvented throughout the 1970s as an agent to be targeted to OER-positive breast cancers for use as a longterm adjuvant therapy with potential use for the chemoprevention of breast cancer (▶ estrogenic hormones, ▶ hormones, and ▶ cancer). Adjuvant Therapy Studies throughout the 1980s and 1990s demonstrated that long-term adjuvant tamoxifen therapy (5 years) produced dramatic increases in diseasefree survival and overall survival. These data were observed in patients who were classified as stage I and stage II breast cancers, and the compound is effective in both pre- and postmenopausal women. Tamoxifen is cheap and effective with availability in generic form in countries throughout the world. Tamoxifen is credited for increasing survivorship and saving the lives of 500,000 women. The appropriate use of tamoxifen as the gold standard for the endocrine treatment of breast cancer throughout the 1990s is credited in contributing to the decrease death rate from breast cancer observed in the United States and other countries around the world. However, concerns about side effects (see below) and the development of drug resistance have led to the development of the aromatase inhibitors as a substitute for tamoxifen in the adjuvant treatment of breast cancer in postmenopausal women. Current studies demonstrate that aromatase inhibitors have an improved side effect profile compared to tamoxifen and improved disease-free and overall survival. Chemoprevention Laboratory studies first showed that tamoxifen could prevent the development of carcinogeninduced rat mammary tumors. Randomized clinical trials around the world have demonstrated that tamoxifen can reduce the incidence of breast cancer by 50% in high-risk pre- and

Tamoxifen

postmenopausal women. High risk refers to the mathematical model (Gail model) that is available on the Internet through the National Cancer Institute in the United States that will determine the 5 year and lifetime risk of a woman developing breast cancer. Some of the risk factors used in the Gail model are age, start of menses, termination of menses, age when the woman had children, if the woman had children, breast biopsies, ductal hyperplasia, and first-degree relatives developing breast cancer. Concerns about the side effects of tamoxifen (see below) have focused on the use of tamoxifen in the premenopausal population where the risk/benefit ratio is high. In other words, tamoxifen effectively reduces the incidence of breast cancer but the side effect profile is low. Most importantly, after women take a 5 year course of tamoxifen during their premenopausal years, the beneficial effects of tamoxifen in preventing breast cancer may last for up to another 10 years, but the menopausal side effects of hot flashes, night sweats etc. will disappear. Studies of a related compound, raloxifene that is used for the prevention of osteoporosis, show that this selective estrogen receptor modulator (SERM) will prevent breast cancer in women taking raloxifene to prevent osteoporosis. A clinical trial in the United States called the Study of Tamoxifen and Raloxifene (STAR) demonstrated in postmenopausal women that tamoxifen and raloxifene were equally effective in preventing an increase in invasive breast cancer but raloxifene had a superior side effect profile. Mechanism of Action Tamoxifen is lipophilic and well absorbed from the gastrointestinal tract. Patients accumulate tamoxifen over the first 4 weeks of treatment when they reach steady state. The drug has a long biological half-life so that even if treatment is stopped, the drug can be detected in the blood for up to 6 weeks. Tamoxifen is a prodrug that is metabolically activated by the CYP2D6 enzyme system to the compounds 4-hydroxytamoxifen and endoxifen both of which have a high affinity with the OER. It is important to note that

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CYP2D6 enzyme system can be blocked by certain selective serotonin reuptake inhibitors (SSRIs), and this may impair the antitumor actions of tamoxifen. Patients often take SSRIs to reduce the incidence of hot flashes in patients taking tamoxifen. Tamoxifen binds to the ligand-binding domain of the OER in the breast tumor and causes a conformational change that is distinct from the natural estradiol OER complex. As a result, tamoxifen is unable to cause gene activation and completely mimic estrogen action. However, tamoxifen is not a complete antiestrogen. The tamoxifen estrogen receptor complex retains estrogen-like actions which can switch on and switch off target sites around a patient’s body. For example, tamoxifen is an antiestrogen in the breast but has estrogen-like properties in bones and in the uterus. Tamoxifen is classified as a selective estrogen receptor modulator. Side Effects Tamoxifen exhibits specific estrogen-like effects and also antiestrogenic effects. Tamoxifen is sufficiently estrogenic to stimulate the uterine endometrium and enhance the growth of OER-positive ▶ endometrial cancers. Tamoxifen causes a fivefold increase in endometrial cancer compared with women not taking tamoxifen. In other words, if a thousand 60 year postmenopausal women were followed for endometrial cancer, one woman per year would develop endometrial cancer. In contrast, if those same women were taking tamoxifen, five women would develop endometrial cancer (low grade, early stage) per year. Additionally, the estrogen-like effects of tamoxifen are reflected in an increase in thromboembolic disorders in postmenopausal women. It is important to note that endometrial cancer and blood clots are not elevated in premenopausal women. There is also a significant rise in the diagnosis of cataracts and cataract operations. The STAR trial demonstrated that raloxifene does not increase the risk of endometrial cancer. There were fewer hysterectomies, fewer cataracts, fewer cataract operations, and a lower overall incidence of blood clots (STAR trial Vogel et al. 2010).

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Tankyrases

References Early Breast Cancer Trialists Collaborative Group (2005) Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet 365:1687–1717 Jordan VC (2003) Tamoxifen: a most unlikely pioneering medicine. Nat Rev Drug Discov 2:205–213 Jordan VC (2007) Chemoprevention of breast cancer with selective oestrogen-receptor modulators. Nat Rev Cancer 7:46–53 Jordan VC, Brodie AMH (2007) Development and evolution of therapies targeted to the estrogen receptor for the treatment and prevention of breast cancer. Steroids 72:7–25 National Surgical Adjuvant Breast and Bowel Project Study of Tamoxifen and Raloxifene (STAR) P-2 Trial: Preventing breast cancer. Cancer Prev Res 3:696–706. STAR trial Vogel et al (2010) Update of the National Surgical Adjuvant Breast and Bowel Project Study of Tamoxifen and Raloxifene (STAR) P-2 Trial: Preventing breast cancer. Cancer Prev Res 3:696–706

reproductive tissues (i.e., testis and ovary), whereas tankyrase-2 exhibits rather ubiquitous expression.

Characteristics

Definition

Structure Tankyrase-1 has four characteristic domains: HPS, ANK, SAM, and PARP. The N-terminal HPS domain is a homopolymeric run of histidine, proline, and serine residues, the functional significance of which is unknown. The ANK domain is composed of a long stretch of 24 ANK repeats, providing a platform for protein-protein interactions. Distinct from those of ankyrins, tankyrase1’s ANK domain is further divided into five, well-conserved subdomains, ARC (ANK repeat cluster) I-V. Each ARC works as an independent, ligand-binding site. The SAM (sterile alpha motif) domain is another module for protein-protein interaction and contributes to self-multimerization of the protein. The C-terminal PARP domain catalyzes ▶ poly(ADP-ribosyl)ation of acceptor proteins by using NAD as a substrate. This posttranslational modification gives drastic negative charges to the acceptor proteins and often disrupts interactions between the acceptor proteins and their target DNA. Tankyrase-2 is a closely related homologue of tankyrase-1. Amino acid identities between ANK, SAM, and PARP domains of tankyrase-1 and tankyrase-2 are 83%, 74%, and 94%, respectively. The most striking feature of tankyrase-2 is the absence of an N-terminal HPS domain. Tankyrase-1 and tankyrase-2 can associate in intact cells via their SAM domains to form a multimer.

Tankyrases are members of the poly(ADP-ribose) polymerase (Parp) family that regulate telomere length in ▶ telomerase-positive human cells. There are two related homologues, tankyrase-1 and tankyrase-2. Tankyrase-1 is a protein of 1,327 amino acids and 142 kDa. The gene maps to 8p22–p23. Tankyrase-2 is a protein of 1,166 amino acids and 127 kDa. The gene maps to 10q23. Tankyrase-1 is relatively abundant in

Intracellular Distribution Tankyrase-1 is found at various intracellular loci, including telomeres, mitotic ▶ centrosomes, Golgi apparatus, and nuclear pore complexes. Telomeric localization of tankyrase-1 is mediated by its interaction with a telomeric repeat-binding factor 1 (TRF1), which directly binds the doublestrand telomere DNA (TTAGGG)n. During mitosis, tankyrase-1 concentrates around the

Tankyrases Hiroyuki Seimiya Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Koto-ku, Tokyo, Japan

Synonyms TNK; TRF1-interacting, ADP-ribose polymerases

ankyrin-related

Tankyrases

pericentriolar matrices. This accumulation depends on tankyrase-1’s interaction with nuclear/mitotic apparatus protein (NuMA), which plays an essential role in organizing microtubules at the spindle poles. In the Golgi apparatus, tankyrase-1 is peripherally associated with the Golgi membranes. In adipocytes and myocytes, it is colocalized with GLUT4 (glucose transporter 4) storage vesicles in the juxtanuclear region of the cells, where it specifically binds to insulinresponsive aminopeptidase (IRAP). The intracellular localization of tankyrase-2 has been characterized less often than tankyrase1. So far, it has been reported that tankyrase-2 localizes predominantly to a perinuclear region, similar to the properties of tankyrase-1. Upon subcellular fractionation, both tankyrase-1 and tankyrase-2 are predominantly recovered in the low-density microsomal fraction, which contains vesicular ▶ endosomal compartments. Binding Partners To date, various tankyrase-1- and tankyrase-2binding proteins have been reported with a consensus RXX(P/A)DG motif as a canonical tankyrase-binding site. Such proteins include TRF1, NuMA, IRAP, Grb14 signaling adaptor protein, tankyrase-binding protein of 182 kDa (TAB182), Mcl-1 apoptotic regulator, and Epstein-Barr virus nuclear antigen-1 (EBNA-1). TRF1, NuMA, IRAP, TAB182, EBNA-1, and tankyrase-1 and tankyrase-2 themselves have been shown to be poly(ADP-ribosyl)ated by tankyrases. PARP inhibitory compounds, such as 3-aminobenzamide, PJ-34, and 4-amino-1,8naphthalimide, block this poly(ADP-ribosyl) ation. Functions Depending on the binding partner and subcellular localization, tankyrase-1 is involved in several distinct biological events, including telomere elongation, cell division control, and insulinstimulated glucose uptake. Also, the coexistence of multiple ARCs and an oligomer-forming SAM domain is implicated in the master scaffolding function of tankyrase-1 (and tankyrase-2), which could work as an intracellular “molecular lattice.”

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Telomere Elongation

Classical DNA replication machinery cannot replicate the very ends of linear DNA; end replication problem. Accordingly, native capping structures at the ends of chromosomes, telomeres, gradually erode after each round of the cell cycle in somatic cells. Most immortalized cells, including germ cells and 80–90% of cancer cells, maintain their telomere length by activating telomerase. Telomere elongation by telomerase requires its enzyme activity and accessibility to the substrate, 30 -overhang of telomere DNA (also called as the telomeric G-tail). Telomere access to telomerase is repressed in cis by the telomeric protein TRF1. Thus, TRF1 directly binds an array of doublestrand telomere DNA (TTAGGG)n and recruits additional telomere-binding proteins, such as TIN2 (TRF1-interacting nuclear factor 2), TPP1 (originally designated PTOP (POT1- and TIN2organizing protein), PIP1 (POT1-interacting protein 1) or TINT1 (TIN2-interacting protein 1), and POT1 (protection of telomeres 1), to the chromosome ends. The resulting TRF1-TIN2-TPP1POT1 complex diminishes accessibility of telomerase to the telomeres. According to this mechanism, longer telomeres provide more binding sites for TRF1 and therefore become a less reactive substrate for telomerase. Conversely, shorter telomeres provide fewer binding sites for TRF1 and become a more reactive substrate for telomerase. This balance between open and closed states of the telomeres stabilizes the length of telomeric TTAGGG tracts at each chromosome end of telomerase-positive cells. Tankyrase-1 enhances telomere access to telomerase and contributes to telomere elongation: Tankyrase-1 binds to the N-terminal acidic domain of TRF1 via its ANK domain. As described above, the ANK subdomain, ARC, plays a role in association with TRF1. While each of five ARCs can independently recognize TRF1, ARC V, the one closest to the C-terminal, is the most important for telomeric function of tankyrase-1. Interaction between ARC V and TRF1 enables tankyrase-1 to poly(ADP-ribosyl)ate TRF1. This posttranslational modification eliminates telomere binding of TRF1, resulting in dissociation of the TRF1TIN2-TPP1-POT1 complex from telomeres.

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Telomere-unbound TRF1 is degraded by ubiquitindependent proteolysis; ▶ ubiquitination. These alterations induce a telomere open state and facilitate telomere elongation by telomerase. It is notable that tankyrase-1 enhances telomere access of telomerase but does not increase the enzyme activity of telomerase. Indeed, tankyrase-1 overexpression induces telomere elongation in a telomerasedependent manner. Tankyrase-1 can form a ternary complex with TRF1 and TIN2. In this complex, poly (ADP-ribosyl)ation of TRF1 is prevented by TIN2. So far, however, when and how tankyrase-1 is activated or inactivated is not fully understood. Like tankyrase-1, tankyrase-2 also can recognize and poly(ADP-ribosyl)ate TRF1. However, the extent of functional redundancy and specificity between tankyrase-1 and tankyrase-2 are largely unknown. Cell Division Control

Since ▶ siRNA-mediated knockdown of tankyrase-1 causes mitotic arrest of multiple cell types, tankyrase-1 is thought to be required for proper cell division. In normal cell division, each pair of sister chromatids is equally divided into two daughter cells. In some tankyrase-1 knockdown cells, however, sister chromatids can separate at the centromeres and arms but not at the telomeres. Accordingly, cell division is interrupted and abnormal chromosome distribution and spindle morphology occur. In other tankyrase-1 knockdown cells, mitotic arrest occurs with intact sister chromatid cohesion (arrest at the metaphase). Tankyrase-1 is also required for the assembly of bipolar spindles. It recognizes a centrosomal protein NuMA via its ANK domain and poly(ADP-ribosyl)ates NuMA in intact cells. Consistently, these two proteins are colocalized at the mitotic centrosomes. Meanwhile, tankyrase-1 is phosphorylated during mitosis by glycogen synthase kinase 3 (GSK3). GSK3 inhibitors, such as lithium chloride and indirubin, inhibit this phosphorylation. Currently, the functional significance of NuMA’s poly(ADP-ribosyl) ation and tankyrase-1’s phosphorylation during mitosis remains unknown.

Tankyrases

Insulin-Stimulated Glucose Uptake

The GLUT4 vesicle is an endocytic compartment within adipocytes and myocytes. This storage vesicle contains the glucose transporter protein, GLUT4, and it regulates glucose uptake upon stimulation with insulin; major fractions of the GLUT4 vesicles usually reside in the trans-Golgi reticulum. Insulin stimulation induces exocytic translocation of the vesicles toward the cell surface, where GLUT4 facilitates glucose uptake. In adipocytes, insulinstimulated translocation of GLUT4 is mediated by IRAP, another GLUT4 vesicle-resident protein. Tankyrase-1 is implicated in regulation of GLUT4 translocation. As mentioned, tankyrase1 directly binds IRAP via its ANK domain and can poly(ADP-ribosyl)ate IRAP. Tankyrase-1 knockdown by siRNAs attenuates the insulinstimulated translocation of GLUT4 vesicles and subsequent glucose uptake. This inhibitory effect of siRNA is reproducible with PJ-34, a PARP inhibitor that is effective against tankyrase-1 (and other PARPs). Tankyrase-1 knockdown does not attenuate the upstream insulin signaling, such as phosphorylation of the ▶ insulin receptor, IRS-1 (insulin receptor substrate-1), Akt (AKT signal transduction pathway in oncogenesis), GSK3, and p42/p44 ERKs (extracellular signalregulated kinases, ▶ MAP kinase). Clinical Aspects Telomere synthesis by telomerase is the Achilles’ heel of unlimited proliferation of most cancer cells. Continuous treatment of cancer cells with telomerase inhibitory drugs (▶ small molecule drugs) shortens telomeres and eventually induces cellular senescence, ▶ apoptosis, or both. Thus, telomerase inhibitors have the potential to benefit cancer patients; ▶ molecular therapy. One concern is that telomere shortening per se compromises the effect of telomerase inhibitors since shorter telomeres have fewer TRF1 and therefore allow easier access to residual telomerase activity. This phenomenon results from incomplete shutdown of telomerase activity. In addition to enzyme activity, accessibility to telomeres could be a rational target for telomerase inhibition. In fact, tankyrase-1 modulates

Targeted Drug Delivery

the impact of telomerase inhibitors on human cancer cells. First, tankyrase-1 overexpression, which removes TRF1 from telomeres, confers resistance to telomerase inhibitors. PARP inhibitors, such as 3-aminobenzamide and PJ-34, reverse this drug resistance. Second, even in cells that do not overexpress exogenous tankyrase-1 (but do express endogenous tankyrase-1), these PARP inhibitors enhance telomere shortening by means of telomerase inhibitors, such as MST-312. Accordingly, the cells undergo earlier crisis. Telomerase inhibitor resistance caused by telomere shortening per se is also reversed by 3-aminobenzamide. These observations suggest that tankyrase-1 could be a target for cancer therapy; Chemotherapy of Cancer, Progress and Perspectives. Pathologically, tankyrase-1 gene expression is elevated in some tumors but not in others.

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Tarceva ▶ Erlotinib

Targeted Drug Delivery Duxin Sun Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI, USA

Synonyms Drug targeting; Site-specific drug delivery

Definition Cross-References ▶ Drug Resistance ▶ PARP Inhibitors

References Chang P, Coughlin M, Mitchison TJ (2005) Tankyrase-1 polymerization of poly(ADP-ribose) is required for spindle structure and function. Nat Cell Biol 7:1133–1139 Dynek JN, Smith S (2004) Resolution of sister telomere association is required for progression through mitosis. Science 304:97–100 Seimiya H (2006) The telomeric PARP, tankyrases, as targets for cancer therapy. Br J Cancer 94:341–345 Seimiya H, Muramatsu Y, Ohishi T et al (2005) Tankyrase 1 as a target for telomere-directed molecular cancer therapeutics. Cancer Cell 7:25–37 Smith S, Giriat I, Schmitt A et al (1998) Tankyrase, a poly (ADP-ribose) polymerase at human telomeres. Science 282:1484–1487

Tanshinone C ▶ Cryptotanshinone

Targeted drug delivery is to site specifically deliver or activate the therapeutic compounds in tumor. Thus, the targeted drug delivery is expected to enhance drug efficacy by increasing local active drug concentration in tumor and to decrease side effect by minimizing drug exposure in normal tissues.

Characteristics Anticancer drugs possess a greater potential of toxicity and much narrower therapeutic index than any other categories of medication. ▶ Chemotherapy is often dose and toxicity limited. A delicate dose regimen is usually required to balance drug efficacy, drug toxicity, and drug resistance; a high dose might cause toxicity while a low dose might induce drug resistance. In addition, anticancer drugs are usually designed to act on the fast proliferating ▶ cancer cells. However, rapid proliferation is also the feature of some normal cells such as the bone marrow, hair follicles, and intestinal epithelium. Although tremendous effort has been explored to

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improve the protocol of chemotherapy, the success is very limited to enhance drug efficacy and to reduce drug toxicity. Several targeted drug delivery technologies have been studied to improve chemotherapy by enhancing drug efficacy and reducing drug toxicity, which have achieved certain degree of success. Intratumoral Drug Administration The simplest form of targeted drug delivery is local intratumoral drug administration. The therapeutic compounds can be directly injected into tumor tissues to transiently increase local drug concentration with minimal or no exposure to normal tissues. However, this method may not be applicable for many cancers. It is also difficult to maintain effective drug concentration in tumors for a prolonged time, and thus repeated dosing may be required. Liposomal Drug Delivery Chemotherapeutic compounds can be encapsulated in liposomes. Liposomal drug formulation achieves passive drug targeting in tumor by enhanced permeability and retention (EPR) effect. Tumor tissues have abnormal vasculature. Since these vasculatures are hyperpermeable with no lymphatic drainage, the liposome will be delivered to the tumor tissues by blood circulation and trapped in the tumors. The drug in the liposome will be gradually released to achieve anticancer efficacy. Therefore, liposomal drug formulation will passively enhance drug accumulation in tumor and decrease exposure to susceptible healthy tissues. The successful liposomal formulation of doxorubicin has been used clinically for cancer treatment. Several generations of liposome formulations have been tested. First generation of liposome passively enhances drug accumulation in tumors and decreases the exposure in normal tissues, but it is rapidly cleared in the blood (within minutes). Second generation of liposomes (PEGylated long-circulating liposomes and ▶ immunoliposome) increases the half-life in blood circulation and tumor targeting.

Targeted Drug Delivery

Tumor-Activated Prodrug (TAP) Therapy The first strategy in TAP therapy is site-specific prodrug activation. Prodrug is an inactive form of a therapeutic compound by chemical modifications. The inactive prodrug can be activated by an overexpressed enzyme in tumor to achieve sitespecific activation, while the prodrug remains inactive or less activated in normal tissues to reduce toxicity. The site-specific activation of the prodrug in tumor increases the anticancer efficacy. The clinically used anticancer drug Xeloda (capecitabine) provides a best example for this strategy. Xeloda is the first oral chemotherapy drug for the treatment of metastatic colorectal cancer. Xeloda is a 5-fluorouracil (5-FU) prodrug, which is orally administered. Three enzymes (carboxylesterase, cytidine deaminase, and thymidine phosphorylase) activate Xeloda to produce active 5-FU. Xeloda is hydrolyzed by carboxylesterase into 50 -deoxy-5-fluorocytidine (50 -DFCR). Subsequently, 50 -DFCR is converted by deaminase to 50 -deoxy-5-fluorouridine (50 -DFUR). Finally, 50 -DFUR is converted into 5-FU by thymidine phosphorylase. Due to the high level of thymidine phosphorylase in tumor, more activation of Xeloda is observed in tumors than in normal tissues to exhibit better anticancer efficacy than 5-FU. The second strategy of TAP is to directly link the prodrug to a targeting moiety (antibody or other ligands) for targeted drug delivery. The antibody-prodrug conjugate binds to the antigen on the tumor cells. The drug–antibody–antigen complex is rapidly internalized into the cancer cells to achieve high drug concentration in targeted cells. For example, anti-CD33 antibody can be conjugated with calicheamicin to target acute myeloid leukemia cells due to the high level of CD33 on the cell surface. Antibody ▶ trastuzumab-taxane conjugate shows 55–200fold more potent than ▶ Taxol. Daunorubicin and methotrexate have been linked to antiMM46 antibody or anti-EL4 monoclonal antibody for treatment of human melanoma. Most of those TAP therapies show better efficacy in vitro and targeted drug delivery in animal models. There are still many challenges in TAP therapy such as rapid clearance in blood, low

Targeted Drug Delivery

potency, inefficient internalization of conjugates, poor penetration, and premature release of the drugs. Antibody-Directed Enzyme Prodrug Therapy Antibody-directed enzyme prodrug therapy (ADEPT) is a two-step process. In the first step, a drug-activating enzyme is targeted to tumors by a tumor-targeting antibody. In the second step, a nontoxic prodrug is administered systemically and converted to the active drug with high local concentration in tumors by the localized antibodyenzyme conjugate. Meanwhile, the prodrug remains inactive (without drug-activating enzyme) in normal tissues and thus decreases its nonspecific toxicity. ADEPT provides many advantages: (i) Amplification effect: each localized antibody–enzyme molecule converts a large number of nontoxic prodrugs to potent active drugs and increases the local active drug concentration in tumor. (ii) Bystander effect: the locally activated drug molecules with high lipophilicity diffuse into the cancer cells regardless of the heterogeneous antigen expression. The bystander effect addresses the issues of poor tumor penetration of the antibody–enzyme conjugate. (iii) The antibody–enzyme conjugate does not need to be internalized into each cancer cell for prodrug activation. The enzymes in ADEPT could be a bacterial enzyme without mammalian homologs to minimize nonspecific prodrug activation in normal tissues, such as carboxypeptidase G2, cytosine deaminase, beta-lactamase, and penicillin G amidase. Other bacterial enzymes with mammalian homologs or mammalian enzymes with low expression in normal tissues can also be used, such as beta-glucuronidase, alkaline phosphatase, and alpha-galactosidase. Gene-Directed Enzyme Prodrug Therapy GDEPT is also a two-step process. First, a gene that encodes a drug activation enzyme is delivered to tumor cells. The delivered gene will express the drug activation enzyme in the tumor only. In the second step, a nontoxic prodrug is delivered and is converted into active drug in tumor cells by the expressed enzyme. Many enzymes and drugs can be used in this system, for instance, cytosine

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deaminase for 5-fluorocytosine prodrug activation, thymidine kinase for ganciclovir and arabinonucleoside prodrug activation, carboxypeptidase G2 for benzoic acid mustard prodrug activation, carboxypeptidase A for methotrexatealanine prodrug activation, galactosidase for daunorunbicin-galactose prodrug activation, glucuronidase for epirubicin-glucuronide prodrug activation, alkaline phosphatase for doxorubicin phosphate prodrug activation, and cytochrome P-450 for cyclophosphamide and ifosfamide prodrug activation. Folate-Targeted Drug Delivery Many cancer cells overexpress folate receptor on the cell surface. For example, 80% of metastatic ▶ breast cancer and 90% of ▶ ovarian cancer are folate receptor alpha positive. Folate is absorbed by its carrier and can also be taken up by cells through folate receptor-mediated endocytosis. Folate-drug conjugate can bind to folate receptor to achieve targeted drug delivery. Many compounds can be delivered by folate conjugates such as small molecules of chemotherapeutic agents, protein complexes, radioimaging agents, genes, and antisense oligonucleotides. Transferrin-Targeted Drug Delivery Tumor cells have been reported to have high level of transferrin receptor. Transferrin-drug conjugates can bind to transferrin receptor and be internalized inside the cancer cell. This strategy has advantages for tumor tissue distribution and prolonged half-life. For instance, transferrindoxorubicin has the potential to circumvent cardiotoxicity. Transferrin-diphtheria toxin selectively killed brain tumor cells with high level of transferrin receptor, although these conjugates also show neurological side effects due to low level of transferrin receptor in normal brain endothelial cells. Albumin-Drug Conjugate for Targeted Delivery Distinctive characteristics of the tumor tissue with lack of lymphatic drainage lead to accumulation of plasma albumin. Thus, albumin-drug conjugate can achieve targeted drug delivery. For instance,

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methotrexate (MTX) to albumin (1:1 molar ratio) was accumulated at higher level (14% accumulation) in xenograft tumors compared to MTX alone (0.4% accumulation) and thus showed better efficacy.

Targeted Drug Design

Targeted Drug Design ▶ Drug Design

Cross-References

Targeted Radioimmunotherapy

▶ Breast Cancer ▶ Cancer ▶ Chemotherapy ▶ Immunoliposomes ▶ Ovarian Cancer ▶ Taxol ▶ Trastuzumab

▶ Ionizing Radiation Therapy

Targeted Toxins ▶ Immunotoxins

References Brannon-Peppas L, Blanchette JO (2004) Nanoparticle and targeted systems for cancer therapy. Adv Drug Deliv Rev 56:1649–1659 Hood JD, Bednarski M, Frausto R et al (2002) Tumor regression by targeted gene delivery to the neovasculature. Science 296:2404–2407 Rooseboom M, Commandeur JNM, Vermeulen NPE (2004) Enzyme-catalyzed activation of anticancer prodrugs. Pharmacol Rev 56:53–102 Schrama D, Reisfeld RA, Becker JC (2006) Antibody targeted drugs as cancer therapeutics. Nat Rev Drug Discov 5:147–159 Springer CJ, Niculescu-Duvaz I (2000) Prodrug-activating systems in suicide gene therapy. J Clin Invest 105:1161–1167

See Also (2012) CD33. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 698. doi:10.1007/978-3-642-16483-5_927 (2012) Doxorubicin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1159. doi:10.1007/978-3-642-16483-5_1722 (2012) Endothelial Cells. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1251. doi:10.1007/978-3-642-16483-5_1896 (2012) Folate Receptor Alpha. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1440. doi:10.1007/978-3-642-16483-5_6960 (2012) Liposomes. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2063. doi:10.1007/978-3-642-16483-5_3388 (2012) Methotrexate. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2274. doi:10.1007/978-3-642-16483-5_3680

Targeted Viruses ▶ Oncolytic Virus

Targeting Cancer Stem Cells Nino Rainusso Department of Pediatrics, Section of HematologyOncology, Baylor College of Medicine, Texas Children’s Cancer and Hematology Centers, Houston, TX, USA

Synonyms Cancer stem cell therapies; Targeting tumorinitiating cells

Definition The cancer stem cell (CSC) model posits that malignant tumors are generated by unique cancer cells that possess the properties of self-renewal and differentiation. Increasing evidence has shown that this tumor cell subpopulation is

Targeting Cancer Stem Cells

resistant to radiation and chemotherapies. Therefore, targeting cancer stem cells may eliminate tumor regrowth, overcome tumor resistance, and ultimately improve patient outcomes. Because of the potential issue of cellular plasticity where more differentiated cells might acquire CSC-like properties, most investigators have proposed simultaneously targeted both populations, combining CSC-targeted therapies with the standard of care.

Characteristics The CSC paradigm is one of the models that explain the striking intratumoral heterogeneity in several cancers, where observed cell diversity is most likely a consequence of both the hierarchical tumor organization with CSCs at the top of the pyramid as well as mutations and branched evolution. CSCs initiate tumorigenesis based on their properties to self-renew and to differentiate in multiple hematologic and solid tumor cell types. CSCs also share other characteristics with normal – embryonic and adult – stem cells such as quiescence, enhancement of DNA repair capacity, apoptosis resistance, increased membrane transporter activity, and ability to migrate. Thus, CSC features allow them to generate new tumors, drive the metastatic process, and resist conventional cancer therapies. Cancer Stem Cells Are Resistant to Radiation Therapy Cancer stem cells have been found to be resistant to conventional radiotherapy in malignant gliomas and breast cancer. The mechanisms of CSC radioresistance include intrinsic properties such as favored activation of DNA-damage checkpoints or increased capacity to modulate the intracellular levels of reactive oxygen species (ROS). Tumor-specific factors, such as the total number of CSCs and their subsequent increase between irradiation fractions, along with greater CSC expansion under hypoxic conditions, comprise additional mechanisms that permit CSC to strive.

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Cancer Stem Cells Are Resistant to Chemotherapy Adult and embryonic stem cells have a greater capacity to repair DNA damage than their offspring. Similarly, CSCs utilize different molecular pathways to overcome the cytotoxic effect caused by chemotherapy. For instance, hyperactivation of the ATR/Chk1 or the PI3K/AKT pathways confers chemoresistance in some solid tumors. Cancer stem cells also express high levels of drug pumps such as ABC transporters that potentially efflux diverse anticancer drugs. This capacity has been exploited to identify this tumor cell subpopulation using both the aldehyde dehydrogenase 1 and the side population assays. The hallmark of anticancer treatment is the elimination of rapidly dividing cells using different drug combinations. Therefore, cancer cells that remain quiescent may escape the cytotoxic effect of conventional cancer treatment. Using label-retaining techniques, CSCs have shown to be more quiescent and consequently chemoresistant in leukemia and certain solid tumors. Cancer Stem Cells Have Deregulated Embryonic Signaling Pathways Cancer stem cells have been characterized in leukemia and solid tumors mainly by their ability to grow as clonogenic spheres under serumfree conditions, their expression of certain cell surface markers, and their capacity to generate tumors using limiting dilution transplantation in immunodeficient mice. The efforts to isolate and identify CSCs have brought the opportunity to discover critical molecular pathways that differentiate this tumor cell compartment from the bulk of tumor cells. Similarly to embryonic and adult stem cells, CSCs utilize different signaling pathways such as Notch, Hedgehog, and Wnt to undergo self-renewal and differentiation. Moreover, the biological processes of metastasis and stem cell migration have related set of genes and signaling pathways. Thus, targeting these deregulated embryonic molecular pathways offers the possibility to significantly alter tumor formation and to block cancer progression.

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Targeting Cancer Stem Cells A number of experimental agents that selectively target the Notch, Hedgehog, and Wnt signaling pathways have been evaluated in cancer cell lines and preclinical animal models. This therapeutic approach includes using small molecule inhibitors, monoclonal antibodies, or immunotherapy. Proof of concept and phase I/II trials are currently under development either in combination with chemotherapy or as a single agents. Clinical data from these trials has shown mixed efficacy on tumor response although the majority of results are not yet available. The majority of agents under development are oral and have well-tolerated side effects (e.g., diarrhea, nausea, vomiting, fatigue). The development of anti-CSC therapy has brought new challenges that are related to the inherent CSC properties or the design and evaluation of clinical trials. Novel CSC-targeted therapies will require combining different agents that eliminate CSCs at different time points. Interaction or cross talk between diverse signaling pathways may allow CSCs to escape from single targeted therapies. Moreover, aberrant molecular pathways could be dissimilar between CSCs in primary and metastatic tumors, and tumor cells may acquire only transitory stem-like cell properties after undergoing an epithelial-tomesenchymal transition. The influence of the tumor microenvironment on CSCs accentuates the biological complexity in defining specific anti-CSC targets but opens up the possibility to modify the immune system for therapeutic intervention. In addition, anti-CSC therapy should not harm normal stem cells and have minimal toxicity. This issue is especially important in children and adolescents that have growing organs and may be more susceptible to stem cell-directed therapies. For example, the inhibition of the Wnt and Hedgehog signaling pathways may cause permanent bone defects and affect the normal skeletal development. There is increasing clinical interest in finding therapies to eliminate CSCs with the hope to cut the “root of evil” in every single tumor. Conventionally, the response to radiation therapy and

Targeting Cancer Stem Cells

chemotherapy in cancer patients has been assessed by changes in tumor size. However, this approach may not be useful to evaluate CSC-targeted therapies considering that these cells may be scarce and their elimination may not be rapidly reflected by a change in overall tumor volume. Novel approaches are necessary to evaluate the impact of CSC-directed therapies, which may include the development of better preclinical models, identification of CSC biomarkers that could be linked with tumor response, and the development of specific imaging techniques to assess CSC activity. In summary, CSCs are radiation and chemoresistant due to their intrinsic properties and tumor-related factors. Similar to normal embryonic and adult stem cells, CSCs use the Notch, Hedgehog, and Wnt signaling pathways to regulate self-renewal and differentiation. Initial attempts to target several of these pathways and to decrease tumor progression are ongoing. Preliminary results have shown that adequate cancer control would require multiple agents to eliminate both CSCs and the bulk of tumor cells. In addition, novel methods to evaluate the clinical efficacy of anti-CSC therapies are required.

Cross-References ▶ Adult Stem Cells ▶ Aldehyde Dehydrogenases ▶ Cancer Stem Cells Targeted Drug Development ▶ Embryonic Stem Cells ▶ Epithelial-to-Mesenchymal Transition ▶ Hedgehog Signaling ▶ Notch/Jagged Signaling ▶ Side Population Cells ▶ Wnt Signaling

References Krause M, Yaromina A, Eicheler W et al (2011) Cancer stem cells: targets and potential biomarkers for radiotherapy. Clin Cancer Res 17:7224–7229 Maugeri-Sacca M, Vigneri P, De Maria R (2011) Cancer stem cells and chemosensitivity. Clin Cancer Res 17:4942–4947

TAT Protein of HIV Rosen JM, Jordan CT (2009) The increasing complexity of the cancer stem cell paradigm. Science 324: 1670–1674 Takebe N, Harris PJ, Warren RQ et al (2011) Targeting cancer stem cells by inhibiting Wnt, Notch and Hedgehog pathways. Nat Rev Clin Oncol 8:97–106

Targeting Tumor-Initiating Cells

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encountered in persons infected with HIV. How does this small (about 101 amino acids) Tat protein do this? Tat has a capacity to bind a striking number of different proteins, nucleic acids, and even polysaccharides. Studies on Tat indicate that it has a highly flexible structure, allowing binding to different partners that has linked Tat to numerous events in AIDS and intrigued many researchers, making Tat one of the most extensively studied HIV proteins.

▶ Targeting Cancer Stem Cells

Tasigna™ ▶ Nilotinib

TAT Protein of HIV Adriana Albini1 and Douglas Noonan2 1 IRCCS Multimedica, Milano, Italy 2 University of Insubria, Varese, Italy

Definition Tat is a small viral protein that is encoded by the spliced two-exon tat gene in the HIV genome, responsible for transactivation of the HIV genome.

Characteristics The HIV Tat protein gets its name from its principal activity; Tat stands for transactivator, which means that it binds to DNA and activates the transcription of DNA into RNA. The Tat protein has an important role in controlling the transcription of the lentivirus HIV genome from its built-in “promoter,” known as the long terminal repeat (which refers to its structure) or LTR, to make the RNA that forms new HIV virus particles. In addition to this major role, Tat has also been implicated in a wide variety of pathologies

Transcriptional Regulation: Control of HIV Replication The HIV LTR acts as a gene promoter. The gene promoter is a portion of DNA that mediates the binding of RNA polymerase (usually through a series of other proteins that bind to the promoter DNA as well) at the beginning of the gene to be transcribed into RNA. This interaction of proteins with DNA controls the transcription of each gene so that it occurs at a certain moment. In the absence of Tat, very little RNA is transcribed from the HIV LTR promoter. When Tat is present, the rate of transcription shoots up several hundredfold, making the transcription of the HIV genome efficient. After HIV infects a cell, it is reverse transcribed into DNA, which is then integrated into the genome of the cell (similar to retroviruses). The viral DNA is then packaged along with the rest of the cellular DNA by winding onto the histone proteins. In this state, the HIV LTR promoter is rather inactive. In fact, in order to be transcribed, the promoter region of any gene must be unwounded from these histones. The HIV DNA is bound to the histones in a very specific manner, with one histone group just prior to, and another just after, a site which binds DNA-binding proteins of the cell known as SP1 and NFkB. The activity of these proteins may be enough to allow the binding of an RNA polymerase, known as RNA polymerase II or RNAP II, to the HIV LTR. The binding of RNAP II to the LTR alone is not enough for efficient transcription. In the absence of Tat, it does not appear to be able to advance forward to synthesize RNA beyond the first 44 nucleic acid base pairs. The major role for Tat is to unleash this machinery and send it to work.

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When Tat is present, it binds to a segment of this short initially polymerized RNA, which forms a peculiar loop known as the TAR (transactivation responsive) element. The TAR-bound Tat then brings in a series of other proteins that allow the transcription process to proceed. Tat has been demonstrated to play a key role in the unwinding of HIV DNA from histones, which is one key for allowing RNA transcription. To do this, Tat binds to a group of proteins known collectively as Tat-associated histone acetyltransferase (TAH). The TAH complex can be formed by different cellular proteins such as p300 or its close relative CBP, along with P/CAF and/or TAF250. These TAH proteins have an enzymatic activity that transfers an acetyl group to histones and are known as histone acetyltransferases. The recruitment of the TAH complex to the HIV LTR region by TAR-bound Tat leads to acetylation of the histones that bind to the LTR, causing changes in their conformation that facilitate RNA transcription. In fact, in cells where p300 and P/CAF are limiting, addition of Tat increases transcription only about sevenfold, while addition of both Tat and p300-P/CAF allows increases transcription about 80-fold. The TAH complex also appears to acetylate the Tat protein itself. This appears to lower the binding of Tat for the TAR, but increases the binding of Tat to another complex of proteins known as the Tat-associated kinase (TAK) or positive-acting transcription elongation factor complex (P-TEFb). This complex of proteins consists of cyclin-dependent kinase 9 (cdk9) and one of the cyclin T isoforms (T1, T2a, T2b). This protein complex can directly bind Tat. More importantly, the kinase activity of the TAK complex phosphorylates (adds a phosphate group to) the RNA polymerase RNAP II. This phosphorylation appears to alter the activity of the RNAP II, improving its ability to transcribe the HIV genome. Together, the TAH and TAK complexes brought in by Tat unleash the RNAP II to finish the job it started, giving the 100-fold improvement of transcription, and therefore HIV replication, observed when Tat is added to HIV-infected cells. Tat also transactivates several cellular genes in addition to the HIV LTR. The activation of these

TAT Protein of HIV

genes is also thought to contribute to the pathogenesis of HIV. The genes include the cytokines IL-6, TNF, and IL-1, which are known to be increased in AIDS patients and may have detrimental effects on the overall function of the immune system. AIDS-Associated Pathologies: A Direct Contribution by Tat One of the most striking properties of Tat is its ability to exit from cells, where it is released into the extracellular environment. Several studies have shown that the HIV-1 Tat protein can exit from cells, including HIV-infected cells. As the Tat gene does not encode a signal peptide, the release of HIV Tat has been suggested to occur via an alternative secretion pathway, like that demonstrated for some cytokines. It may also come from cells dying due to HIV. The Tat that is found extracellularly appears to be intact and active, and substantial levels of Tat protein have been observed and found in the serum of many HIV patients. Antibodies can be made against Tat in AIDS patients, indicating that it is released, and interestingly an inverse correlation between anti-Tat antibodies and patient survival has been reported in some studies. These data suggest that extracellular Tat may favor HIV replication and have spawned tests on the possibility of using Tat as part of an AIDS vaccine. A wide range of activities have been attributed to the Tat protein released extracellularly. Several studies have demonstrated that the HIV Tat protein or peptides based on Tat are capable of entering cells cultured in vitro. The Tat that enters cells is capable of transactivating the HIV LTR. Tat and Tat peptides have even been used to deliver other proteins into cells, and a peptide based on Tat may find use as a signal to move drugs into cells. In addition to getting into other cells, the Tat that is released also appears to bind to several cell surface proteins, including specific receptors. These activities of Tat have been linked to many of the pathological alterations found in HIV infection. For example, some groups of AIDS patients frequently have ▶ Kaposi sarcoma, an otherwise rare, benign vascular tumor. Unlike most Kaposi, the Kaposi sarcoma associated with AIDS is very malignant and could be life-threatening for many of these patients. Tat was first linked to Kaposi

TAT Protein of HIV

sarcoma when Kaposi-like lesions were found on mice genetically engineered to express Tat. Soon after, Tat was shown to be a growth factor for Kaposi cells, but the reason for this was not known. Later studies showed that Tat could bind to KDR (VEGFR2), a receptor for the growth factor ▶ VEGF, on the surfaces of endothelial and Kaposi sarcoma cells. VEGF (vascular endothelial growth factor) is important in the formation of new blood vessels, as is its receptor KDR. The ability of Tat to bind and activate KDR means that Tat could stimulate the formation of vessels found in Kaposi tumors, as well as the growth of cells of the tumor itself. We now know that Kaposi sarcoma is due to an infection with a herpesvirus, HHV8, that occurs when the immune system is unable to control this virus. All Kaposi cells, whether from aggressive AIDS or benign sporadic of iatrogenic (posttransplant), have KDR; however, the Tat stimulation of KDR and perhaps other receptors appears to make AIDS Kaposi potentially lethal. Many Tat proteins have an RGD sequence, three amino acids found in many proteins of the extracellular matrix. Through this RGD sequence, Tat can bind to cell surface integrins, proteins that are normally involved in binding to extracellular matrix molecules. Studies have shown that several integrins bind the Tat protein. Tat-integrin binding has been shown to trigger events typical of integrinextracellular matrix ligand interactions, including activation of p125 focal adhesion kinase. The binding of Tat to these receptors has also been linked to Kaposi sarcoma and other activities of HIV Tat. The immune suppression seen with AIDS appears to affect cells that are not infected with HIV aside from those harboring the virus. Several studies have shown that there is immune suppression of non-HIV-infected cells from AIDS patients and that the number of immune suppressed cells seems to exceed that of the potentially HIV-infected cells. Proteins released from HIV-infected cells are clearly potential candidates for mediating this immune suppression. Tat has been linked to induction of T-cell anergy (lack of activity), T-cell ▶ apoptosis (programmed death), but also to a T-cell hyperactivation that appears to prime cells for infection by HIV. These events are

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probably all closely linked to the same phenomenon. The potential receptor system(s) involved in these activities of Tat include CD26. CD26 is a dipeptidyl peptidase that is known to cleave and alter the activities of chemokines, molecules whose receptors are very important cell surface receptors for HIVand that can regulate HIV infection. Tat has been shown to significantly increase the expression of two key chemokine-HIV receptors, CXCR4 and CCR5, by monocytes and T lymphocytes, potentially increasing HIV infection. The Tat protein has been reported to act as a growth factor and protect transfected cell lines from apoptosis. Tat has been consistently found to upregulate the expression of CD95-Fas, a protein that signals cells to die. The increase in apoptosis is typical for partially activated T cells, as its entry into anergy resulting from an incomplete stimulation of T cells. Tat may be capable of partial, but incomplete, T-cell activation. HIV does not readily infect resting T cells; T-cell activation is a key requisite for HIV infection of these cells. A partial T-cell activation may be sufficient for HIV infection yet detrimental to the host immune response, a potential role that Tat may fulfill. Extracellular HIV Tat has been shown to have wide ranging effects on lymphatic cells such as monocytes, macrophages, dendritic cells, and even natural killer cells. Tat has been reported by several groups to be a strong chemoattractant for monocytes. This activity could contribute directly to the recruitment of potentially “infectible” cells toward an HIV-infected cell producing and releasing Tat protein, an activity which may have a direct affect on the establishment and spread of HIV infection in the host. Tat has been shown to bind to and activate several chemokine receptors (including CCR3 and CKCR4) and mimic the chemoattractant properties of chemokines. Chronic inflammation is frequently associated with cancer, and inflammatory cells can promote tumor growth and tumor angiogenesis; thus, Tat may influence the AIDS-associated tumor microenvironment through direct and indirect mechanisms. Tat can even inhibit HIV infection in high doses by binding chemokine receptors, although the physiological relevance of this observation is not yet clear.

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The Tat protein appears to inhibit dendritic cell phagocytosis and natural killer cell function, apparently by blockage of certain calcium channels. Finally, Tat has been linked to AIDSassociated neurocognitive disorders. Tat induced alterations in the brain involve a variety of pathways, including neuron excitotoxicity, oxidative stress, calcium dysregulation, damage to the blood-brain barrier, and signaling perturbations involving many receptor and non-receptor tyrosine kinases as well as some adaptor proteins. Tat also appears to alter the developing dopamine system in children with HIV.

Tax Binding Protein-181

Tax Binding Protein-181 ▶ Mitotic Arrest-Deficient Protein 1

Tax Helper Protein ▶ GLI Proteins

Taxol Conclusion Tat is known to have a major role in HIV replication through a complex series of interactions with nuclear proteins. In addition, Tat outside the cell appears to be able to stimulate through, or interfere with, several cell surface receptors, sending signals that may be a root cause of many pathologies found in HIVinfected patients.

References Albini A, Soldi R, Giunciuglio D et al (1996) The angiogenesis induced by HIV-1 Tat is mediated by the flk-1/ KDR receptor on vascular endothelial cells. Nat Med 2:1371–1375 Fields JA, Dumaop W, Crews L et al (2015) Mechanisms of HIV-1 Tat Neurotoxicity via CDK5 Translocation and Hyper-Activation: Role in HIV-Associated Neurocognitive Disorders. Curr HIV Res 13:43–54 Fitting S, Booze RM, and Mactutus CF (2015) HIV-1 Proteins, Tat and gp120, Target the Developing Dopamine System. Curr HIV Res 13:21–42 Gallo RC (1999) Tat as one key to HIV-induced immune pathogenesis and Tat (correction of Pat) toxoid as an important component of a vaccine. Proc Natl Acad Sci U S A 96:8324–8326 Jeang KT, Xiao H, Rich EA (1999) Multifaceted activities of the HIV-1 transactivator of transcription, Tat. J Biol Chem 274:28837–28840 Noonan DM, Albini A (2000) From the outside in: extracellular activities of HIV Tat. In: Jeang KT (ed) Advances in pharmacology: HIV: molecular mechanisms and clinical applications. Academic, San Diego, pp 229–250 Rubartelli A, Poggi A, Sitia R et al (1998) HIV-I Tat: a polypeptide for all seasons. Immunol Today 19: 543–545

Spyros D. Georgatos and Panayiotis A. Theodoropoulos Department of Basic Sciences, The University of Crete, School of Medicine, Heraklion, Crete, Greece

Definition Taxol (paclitaxel) was first isolated in 1971 from a crude extract of Taxus brevifolia, a scarce, slowgrowing yew plant found in the forests of the Pacific Northwest. It is a diterpenoid containing the characteristic taxane ring (Fig. 1). Total chemical synthesis of this compound was achieved in 1994, opening the way for the production of various analogues. Currently, taxol is commercially prepared by hemisynthesis, in which a synthetic side chain is attached to natural products isolated from the needles of Taxus plants. Enzymatic conversion of various taxanes to 10-deacetylbaccatin III (a precursor for taxol hemisynthesis) has been reported.

Characteristics Mode of Action Due to its hydrophobic character, taxol readily crosses the plasma membrane. Once in the cytoplasm, the drug binds with high affinity to the b-subunit of tubulin, modifying and stabilizing microtubules. When modified by taxol, these

Taxol

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Taxol, Fig. 1 Chemical structure of taxol

O O H3C 5′ O

H3C

NH

12

11

OH

O CH3

10 CH3

9

7 8

CH3

O

H

13 2′ OH

O

O H

O

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Taxol (taxolA)

cytoskeletal structures exhibit decreased dynamic instability and increased rigidity. As a result, the function of the microtubule-based machines (e.g., ▶ mitotic spindle) is compromised and the cells cannot divide properly. Taxol also binds to ▶ BCL-2, a protein involved in the process of programmed cell death (▶ apoptosis). The cellular effects of taxol vary depending on dose and treatment scheme. In the range of nanomolar concentrations, it induces sustained mitotic arrest (Fig. 2), inhibits protein prenylation, and triggers apoptosis. At micromolar doses, it promotes synthesis and release of cytokines, such as ▶ tumor necrosis factor (TNF) and interleukins (IL1 and IL8), increases tyrosine phosphorylation by ▶ MAP kinases, induces early response genes, and stimulates production of ▶ nitric oxide. How taxol exerts its cytotoxic action remains elusive. Structure-activity studies differentiate microtubule stabilization from other effects, supporting a direct effect on the genetic and signal transduction machinery. However, other studies suggest that the drug acts by inducing cytoskeletal damage. Observations show that taxol activates Raf-1 kinase and induces phosphorylation of Bcl-2. Phosphorylation of the latter may, in turn, lead to dissociation of Bcl-2/Bax complexes, unleashing Bax into the cell and thus triggering apoptosis. Although these hypotheses are intuitively

T Taxol, Fig. 2 Confocal microscopy image, showing human cervical carcinoma cells in mitosis. Cells were treated with 10 nM taxol for 20 h. Immunostaining was done with anti-tubulin antibodies and counter-staining was done with propidium iodide. Chromosomes are shown in red, the mitotic spindle in green

attractive, neither of them could fully account for the cell killing action of taxol: programmed cell death is also induced by other microtubulestabilizing drugs which are free of genomic side

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Taxol

Taxol, Fig. 3 Nuclear lamina and pore complex lesions after treatment of human endometrial carcinoma cells with 10 nM taxol. Left: indirect immunofluorescence using antilamin B antibodies. Right: indirect immunofluorescence

using anti-nucleoporin antibodies. The interruption of the nuclear lamina and the formation of large pore clusters are evident in these images

effects (e.g., ▶ Taxotere), while apoptosis can still occur independently of Raf-1 phosphorylation, or after Bcl-2 is dephosphorylated by cellular phosphatases. Taxol and other microtubule-acting agents affect dramatically the architecture of the cell nucleus. It is widely known that cells treated with nanomolar amounts of taxanes or Vinca alkaloids develop lobulated nuclei or multiple micronuclei and missort key cellular constituents. Also, taxol affects the nuclear envelope. Even at nanomolar concentrations, the drug induces focal unraveling of the nuclear lamina and extensive clustering or ectopic localization of nuclear pore complexes (Fig. 3). Cells that possess a defective nuclear envelope and which are treated with taxol remain alive for at least 24 h after the end of the treatment, but are unable to import karyophilic proteins, such as the transcription factor NFkB, into the nucleus. It has been proposed that inhibition of NFkB import may render the cells prone to programmed cell death.

neck, esophageal, bladder, and lung carcinomas. In addition, several schedules of combination therapy have been developed as alternatives for patients with advanced cancer. Pilot studies show that taxol can enhance radiation sensitivity of tumor cells, potentiate tumor response, and increase the therapeutic ratio of radiotherapy.

Clinical Pharmacology

Structurally and Functionally Related Compounds

Absorption and Excretion

The drug is administered as a 3-h or 24-h infusion. It undergoes ▶ cytochrome P450-mediated metabolism to 6-OH derivatives and other products. Less than 10% of a dose is excreted intact from the urine. Toxicity

The principal toxic effect of taxol is neutropenia. Several other toxic effects, such as myalgias, mucositis, hypersensitivity reactions, stockingglove sensory neuropathy, and disturbances of the cardiac rhythm, have also been encountered.

Antitumor Activity

Taxol as a single chemotherapeutic agent has been proven effective against a variety of tumors, including ovarian, breast, head and

Structurally Related Compounds

• ▶ Docetaxel (Taxotere), produced by semisynthesis (1986) from 10-deacetyl baccatin

Taxotere

III, a taxoid precursor. It is the second member of the taxane class to reach clinical use. • Several chemically synthesized taxoids bearing substitutions or modifications. Nontaxane, Microtubule-Stabilizing Agents

• Estramustine, a conjugate of estradiol and nornitrogen mustard • ▶ Epothilones A and B, two macrolides isolated from myxobacterium, Sorangium cellulosum • A family of marine-derived compounds extracted from sponges (discodermolide and laulimalide) or corals (sarcodictyins A–F and eleutherobin)

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antitumor activity against a range of human cancers, including ▶ breast, ▶ lung, ▶ prostate, and ▶ ovarian cancers. It is a semisynthetic molecule belonging to the taxane family and is closely related chemically and pharmacologically to the naturally occurring drug ▶ Taxol (▶ paclitaxel). Taxotere binds to the b-tubulin component of microtubules, a key cytoskeletal protein in cells, and stabilizes the structure of the microtubule polymer, prevents its disassembly, and suppresses microtubule dynamics. These actions interfere with a number of cellular functions in which the microtubules are involved.

Characteristics References Haldar S, Basu A, Croce CM (1997) Bcl-2 is the guardian of microtubule integrity. Cancer Res 57:229–233 Nicolaou KC, Yang Z, Liu JJ et al (1994) Total synthesis of taxol. Nature 367:630–634 Rodi DJ, Janes RW, Sanganee HJ et al (1999) Screening of a library of phage-displayed peptides identifies human bcl-2 as a taxol-binding protein. J Mol Biol 285:197–203 Rowinsky EK, Donehower RS (1995) Paclitaxel (Taxol). N Engl J Med 332:1004–1014 Theodoropoulos PA, Polioudaki H, Kostaki O et al (1999) Taxol affects nuclear lamina and pore complex organization and inhibits import of karyophilic proteins into the cell nucleus. Cancer Res 59:4625–4633

Taxotere Edward L. Schwartz Department of Medicine (Oncology), Albert Einstein College of Medicine, Bronx, NY, USA

Synonyms Docetaxel

Definition Taxotere is a widely used ▶ cancer chemotherapeutic drug with well-documented clinical

Chemistry The antitumor activity of the taxanes was first observed in preclinical models using a crude extract of the bark of the Pacific yew tree Taxus brevifolia, and Taxol was subsequently (1971) identified as the active constituent. Both Taxotere and Taxol are diterpenoid compounds consisting of a 15-member taxane ring system linked to an unusual 4-member oxetan ring. Limited supplies of the bark from the T. brevifolia tree prompted the search for alternative derivatives and sources of starting material. An inactive compound, 10-deacetylbaccatin III, found in the needles (which are more abundant and also are a renewable source) of several yew species was used as starting material for the synthesis of Taxotere, which was first reported in 1986. Taxotere and Taxol differ in the nature of the substitutions at the C-10 position of the taxane ring and on the ester side chain attached to C-13, and these differences are the basis for the increased water solubility and greater potency of Taxotere, compared to Taxol (Fig. 1). Mechanisms of Action Microtubules are a component of the cell’s ▶ cytoskeleton and form a well-organized network of hollow tubes in which one end is anchored at the ▶ centrosome (the microtubuleorganizing center) and the opposite, free end extends out into the cytoplasm. Microtubules are

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Taxotere, Fig. 1 Chemical structure of Taxotere (docetaxel)

composed of noncovalent heterodimers of a- and b-tubulins, which are arranged head to tail to form helical polymers. There is an exchangeable GTP-/GDP-binding site on b-tubulin which mediates the rapid assembly and disassembly of the microtubules. The free ends of the microtubules undergo frequent periods of slow growth and rapid shortening, a process called dynamic instability, and microtubules also exhibit treadmilling, in which the loss of tubulin at one end is matched by the addition of tubulin at the opposite end. This dynamic behavior is required for the proper functioning of the microtubules. During mitosis, microtubules capture and are responsible for the alignment of the chromosomes and for their subsequent separation to the two daughter cells during anaphase. Properly functioning microtubules are required for the migration of most cell types, and they also play an important role in cell signaling and the intracellular transport of proteins, vesicles, and mitochondria. Much of the early work on the binding and effect of the taxanes on microtubules was done using Taxol. Taxol preferentially binds to the microtubule polymer, rather than to unassembled tubulin, at a site that is distinct from the GTP/GDP site and which also differs from the sites at which colchicine, vinblastine, and podophyllotoxin bind. Studies suggest that in their interactions with purified microtubules, Taxotere and Taxol are qualitatively indistinguishable. Although Taxotere and Taxol have the same apparent binding site on tubulin, Taxotere binds with a 1.9-fold greater effective affinity.

The taxanes promote microtubule assembly, even in the absence of added GTP, by altering the tubulin dissociation constants at both ends of the microtubules. The taxanes reduce the critical concentration of tubulin required for microtubule assembly, with the required concentration 2.1fold lower in the presence of Taxotere than with Taxol. In addition to promoting both the nucleation and elongation phases of polymerization, they also produce microtubules that are extremely stable and are resistant to depolymerization. The effects on microtubule assembly are maximal at Taxol or Taxotere concentrations that are equimolar to that of tubulin, and evidence indicates that they bind with a stoichiometry of 1 per ab-tubulin dimer. In cells treated with stoichiometric concentrations of taxanes, there is an increase in the microtubule-polymer mass along with the formation of abnormal polymers and a characteristic intracellular “bundling” observed. While these concentrations of the drugs are obtained clinically, it is only for brief periods of time after drug administration. At substoichiometric concentrations, the taxanes effects on microtubule dynamics are more prominent than are their actions on microtubule mass, and the suppression of mitotic spindle dynamics plays a central role in their cytotoxic actions. By interfering with the mitotic spindle, the taxanes block cancer cell proliferation by causing cell cycle arrest at the metaphase/anaphase boundary. The resultant blocking or slowing of mitosis leads to cell death via the induction of the intrinsic mitochondrial pathway of apoptosis. Taxotere is transported out of the cells by the ABC transporter class of membrane transport proteins, which include P-glycoprotein, MDR1, and MDR2. Overexpression of these pumps and the accompanying multidrug resistance (MDR) phenotype can lead to profound cellular resistance to the taxanes. Microtubule-related determinants may also confer resistance to the taxanes, and these include the levels of expression of microtubule-associated regulatory proteins, the extent and nature of posttranslational modification of tubulin, and the varying levels of expression of different tubulin isotypes. Interestingly, cancer cells made resistant to Taxol do not necessarily

Taxotere

show the same degree of resistance to Taxotere, and this may be due to the observation that Taxotere is retained intracellularly longer than is paclitaxel. In addition to effects on tumor cell proliferation and apoptosis, Taxotere is a potent inhibitor of angiogenesis, and this is likely due to its direct effects on endothelial cells. While it inhibits endothelial cell proliferation at concentrations comparable to those which inhibit cancer cells, it also inhibits endothelial cell motility, invasiveness, and tubule formation in vitro at concentrations substantially (10- to 100-fold) lower than required to cause cell cycle arrest or apoptosis. Based on pharmacokinetic analyses of Taxotere, cancer patients are exposed to these anti-angiogenic concentrations of drug for extended periods of time. Taxotere also has anti-angiogenic activity in several in vivo preclinical models, and these in vivo anti-angiogenic actions were probably not directly due to the antiproliferative or cytotoxic activities of the drug, as other agents that inhibited endothelial cell proliferation in vitro did not affect angiogenesis in vivo. The mechanisms for these actions are not fully understood and may include effects on signaling pathways, including those mediated by cell surface receptors for the angiogenic factor ▶ vascular endothelial growth factor (VEGF). The effects of the taxanes on microtubule dynamicity at the low concentrations that inhibit angiogenesis appear to be qualitatively different from those at concentrations that block cell proliferation. Clinical Use Taxotere is a highly effective drug with a spectrum of anticancer activity that is virtually identical to that of Taxol. Taxotere initially received regulatory approval for use in patients with metastatic or locally advanced ▶ breast cancer who had failed other drug therapies. In addition to increasing survival when used as second-line therapy in patients with advanced disease, subsequent studies showed it was active in the adjuvant treatment (▶ adjuvant therapy) of patients with local breast cancer after definitive local treatment. It has been shown to increase survival in unresectable metastatic ▶ non-small

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cell lung cancer and is one of the most effective agents in the treatment of ovarian cancer and in hormone-refractory ▶ prostate cancer. The wide spectrum of activity that Taxotere was found to have in preclinical models has been confirmed in the clinic, and antitumor activity has been noted in ▶ bladder, ▶ endometrial, ▶ esophageal, ▶ gastric, head and neck, and small cell lung carcinomas and in lymphoma and melanoma. Taxotere is often used in combination with other chemotherapeutic drugs, including ▶ cisplatin, cyclophosphamide, doxorubicin, and prednisone. In addition to being evaluated in a large number of drug combinations, a range of doses and schedules of Taxotere have been studied in clinical trials over the years. The drug is usually administered once a week or once every 3 weeks as short (1 h) intravenous infusions at doses ranging from 60 to 100 mg/m2. ▶ Neutropenia is the primary toxicity observed at these doses, with a significantly reduced neutrophil count typically first observed on day 8 after treatment. Complete resolution occurs on days 15–21, and the severity of the neutropenia is related to the dosage of the drug and the extent to which the patient’s bone marrow function has been compromised by the concomitant or prior use of other myelosuppressive (▶ myelosuppression) drugs. The neutropenic effect of Taxotere generally is more severe, but shorter in duration, than that of Taxol. A number of other toxic effects have been associated with Taxotere use. Hypersensitivity reactions (dyspnea, bronchospasm, and hypotension) can occur acutely with drug administration, and a unique fluid retention syndrome (peripheral edema, pleural and peritoneal fluid) can occur chronically with multiple courses of therapy. In both instances, the frequency and severity of these toxicities are substantially reduced by premedication with a corticosteroid and H1 and H2 histamine antagonists. Skin toxicities occur frequently, although these are also attenuated by premedication, with the most common manifestation being an erythematous pruritic maculopapular rash on the arms, hands, and feet. Taxotere causes mild to moderate

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neurosensory and neuromuscular effects (numbness, paresthesia), and although common (up to 40% of patients), these occur less frequently and are less severe than in patients receiving Taxol. The relative water insolubility of Taxotere requires it to be formulated for clinical use in the nonionic surfactant polysorbate 80 (Tween 80). Both the hypersensitivity and fluid retention side effects of the drug have been attributed to the use of this vehicle. A number of different alternatives to the use of Tween 80 have been evaluated in preclinical studies, with the objective of developing a less-toxic, better-tolerated formulation, and the identification of ways to better target Taxotere to malignant tissue. These include polyethylene glycol (PEGylated) liposomal Taxotere, immunotargeted liposomal Taxotere conjugates, Taxotere fibrinogen-coated olive oil droplets, Taxotere-encapsulated nanoparticle aptamer bioconjugates, and submicronic dispersion formulations. Some of these formulations have entered early clinical trials.

Cross-References ▶ Adjuvant Therapy ▶ Bladder Cancer ▶ Breast Cancer ▶ BRMS1 ▶ Cancer ▶ Centrosome ▶ Cisplatin ▶ Cytoskeleton ▶ Docetaxel ▶ Endometrial Cancer ▶ Esophageal Cancer ▶ Gastric Cancer ▶ Lung Cancer ▶ Myelosuppression ▶ Neutropenia ▶ Non-Small-Cell Lung Cancer ▶ Ovarian Cancer ▶ Paclitaxel ▶ Prostate Cancer ▶ Taxol ▶ Vascular Endothelial Growth Factor

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References Bhalla KN (2003) Microtubule-targeted anticancer agents and apoptosis. Oncogene 22:9075–9086 Hotchkiss KA, Ashton AW, Mahmood R et al (2002) Inhibition of endothelial cell function in vitro and angiogenesis in vivo by docetaxel (taxotere): association with impaired repositioning of the microtubule organizing center. Mol Cancer Ther 1:1191–1200 Montero A, Fossella F, Hortobagyi G et al (2005) Docetaxel for treatment of solid tumours: a systematic review of clinical data. Lancet Oncol 6:229–239 Ringel I, Horwitz SB (1990) Studies with RP56976 (Taxotere): a semisynthetic analog of taxol. J Natl Cancer Inst 83:288–291 Verweij J, Clavel M, Chevalier B (1994) Paclitaxel (Taxol) and docetaxel (Taxotere): not simply two of a kind. Ann Oncol 5:495–505

See Also (2012) Endothelial cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p. 1251. doi:10.1007/978-3-642-16483-5_1896 (2012) Integrin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p. 1884. doi:10.1007/978-3-642-16483-5_3084 (2012) Microtubules. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p. 2312. doi:10.1007/978-3-642-16483-5_3740 (2012) Mitotic spindle. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p. 2348. doi:10.1007/978-3-642-16483-5_3782 (2012) Paresthesia. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p. 2788. doi:10.1007/978-3-642-16483-5_4390 (2012) Taxane. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p 3614. doi:10.1007/978-3-642-16483-5_5689 (2012) Tubulin. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p. 3792. doi:10.1007/978-3-642-16483-5_6011

T-body ▶ Chimeric Antigen Receptor (CAR)

TCC ▶ Transitional Cell Carcinoma

TCL1

2,3,7,8-TCDD ▶ Dioxin

TCDD ▶ Dioxin

TCL1 Giandomenico Russo Istituto Dermopatico dell’Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Roma, Italy

Definition The TCL1 gene is involved in the generation and/or manifestation of mature forms of leukemias, mainly in the T-Prolymphocytic leukemia (T-PLL) and in chronic lymphocytic leukemia (B-CLL).

Characteristics Clinical Characteristics of the T-PLL T-PLL is a disease that represents 20% of prolymphocytic leukemias. It occurs at an advanced age of 70–80 years, with a slight male predominance. It is, however, quite frequent in patients with the immunodeficiency syndrome ataxia-telangiectasia (AT) (1–5% of these patients develop it). Clinically, it is accompanied by splenomegaly (75%), hepatomegaly (42%), lymphadenopathy (55%), and a high blood count (>200  109/L), with a very bad survival rate (70%), followed by the N3 position of adenine (9.2%) and the O6 atom of guanine (5%). Figure 2 depicts the chemical structure of the naturally occurring bases in DNA (guanine and adenine) and the TMZ-induced alkylated products (O6-MeG, N7-MeG, and N3-MeA). Temozolomide and MGMT One of the major cytotoxic DNA lesions or types of ▶ DNA damage induced by TMZ treatment is the formation of methyl adducts on the O6 position of guanine (O6-MeG; see Fig. 2). However, the cytotoxic guanine lesion O6-MeG is rapidly repaired by a direct reversal reaction conducted by the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT or more commonly referred to as AGT). This DNA repair protein is encoded by the O6-methylguanine DNA methyltransferase gene, MGMT, located on chromosome 10 at position 10q26. The MGMT gene encodes a single mRNA (NM_002412) that translates to a single protein of 207 amino acids (NP_002403). MGMT functions to remove O6MeG lesions in DNA via a suicide reaction in which the O6-Me group on guanine is transferred to the cys145 residue in MGMT, rendering MGMT inactive. Once alkylated on amino acid residue cys145, the MGMT protein is depleted from the

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Temozolomide, Fig. 2 Chemical structures of the guanine and adenine bases in DNA and the temozolomideinduced modification to these bases. The guanine base (a) is modified by temozolomide on the O6-atom to yield O6-methylguanine (b) and on the N7-atom to

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yield N7-methylguanine (c) at a frequency of 5% and >70%, respectively. The adenine base (d) is modified by temozolomide at the N3-position to yield N3-methyladenine (e) at a frequency of 9.2%

Temozolomide

cell within a few hours. Depletion of the MGMT protein is facilitated by the ubiquitin-dependent proteasome pathway. The alkylated form of MGMT is targeted for poly-ubiquitylation, a posttranslational modification that initiates proteasome-mediated degradation. Interestingly, MGMT activity is observed to be elevated in many tumors (in relation to the surrounding normal tissue) such as those derived from colon, lung, pancreatic, and breast cancer, non-Hodgkin lymphoma, myeloma, and glioma. It is now well established that elevated MGMT expression leads to resistance to clinical alkylating agents due to failure to remove the O6-MeG lesion. To improve TMZ efficacy, several strategies have been developed to limit the repair of the O6MeG lesion. MGMT activity can be successfully attenuated by the use of free guanine base derivatives, with alkyl groups at the O6 position, which act as a pseudosubstrate and lead to MGMT depletion. Two of the most promising MGMT-specific drugs are O6-benzylguanine (BG) and O6-(4-bromothenyl)guanine (Patrin, PaTrin-2, lomeguatrib). Both of these MGMTspecific small-molecule drugs are currently undergoing clinical evaluation to be used in combination with TMZ. In addition, other clinical alkylating agents are found to induce the formation of O6-MeG adducts to DNA, including the platinum drugs cisplatin and carboplatin as well as the alkylating agent dacarbazine. Combination treatments with TMZ are therefore being evaluated as a novel approach to deplete MGMT protein levels and thereby increase TMZ efficacy. Interestingly, the dosing or scheduling of TMZ administration can also be altered to maximize MGMT depletion. For example, the standard 5day dosing schedule of TMZ (150–250 mg/m2/ day) leads to complete depletion of MGMT in peripheral blood cells, but MGMT levels will recover within 24 h. The observed TMZ-induced depletion of MGMT suggests that variation in the dosing schedules may improve MGMT depletion and clinical response. Compressed scheduling (five doses every 4 h or once every 8 or 12 h) has shown variation in MGMT depletion as well as elevated myelotoxicity. Alternatively, low (75 mg/m2) extended dosing

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for 6–7 weeks in cycles of 8 weeks resulted in complete MGMT depletion. Clinical benefit to this or other scheduling paradigms is under intense investigation. The TMZ-mediated induction of the O6-MeG DNA lesion and the depletion of MGMT have also been used to improve the effectiveness of ▶ topoisomerase inhibitors. Topoisomerase inhibitors such as ▶ irinotecan (CPT-11) have been shown to have an enhanced efficacy when delivered in combination with TMZ. The increased level of O6-MeG in DNA acts as a topoisomerase I (topo-I) trap and an increase in topo-I/DNA covalent complexes. Pretreatment with interferon-beta was also shown to downregulate MGMT expression and to improve the response of xenografts to TMZ in preclinical testing. Conversely, low or undetectable expression of MGMT is generally considered a strong indicator of potential clinical response to TMZ. Loss of MGMT expression in tumors is generally via elevated methylation and hence inactivation of the MGMT promoter. Although MGMT promoter hypermethylation is associated with hypermethylation of many other promoter regions throughout the genome, ▶ epigenetic regulation (epigenetic gene silencing) is not considered a random process in cancer. The development of methylation-specific PCR provides for a highly sensitive and accurate measurement of the methylation status of the MGMT promoter within the tumor tissue. DNA isolated from the tumor can therefore be analyzed for methylation status. Several studies have demonstrated that MGMT methylation status can be used to predict TMZ response. It is possible that MGMT promoter methylation measurements may be used to prescreen patients for treatment options. However, there are other concerns that define response to TMZ and the cytotoxicity to the O6-MeG DNA lesion. Mismatch DNA Repair and Temozolomide Response The O6-MeG DNA adduct is not inherently cytotoxic by itself. The adduct is stable and genomic DNA containing O6-MeG adducts is efficiently replicated during cell division by human

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replicative DNA polymerases. However, if not repaired by MGMT, either cytosine or thymine may be inserted opposite the O6-MeG DNA adduct during replication. Insertion of thymine would lead to G to A point mutations in subsequent rounds of cell division and DNA replication, consistent with the elevated level of point mutations in oncogenes and tumor suppressor genes found in tumors with a loss of MGMT expression. The cytotoxicity of the TMZ-induced O6-MeG adduct stems from the replication-dependent formation of the O6MeG:T mispair and the recognition of this mispair by the post-replication mismatch DNA repair pathway (MMR). The MMR pathway is a multi-protein DNA repair and ▶ DNA damage signaling pathway that removes errors of DNA replication and functions in meiotic and mitotic recombination as well as in DNA damage signaling and ▶ apoptosis following alkylation damage. Currently, two mechanisms have been proposed for the MMR-dependent cytotoxicity of the TMZ-induced O6-MeG DNA adduct: a “Futile Cycle of Repair” model and a “Direct DNA Damage Signaling” model. In the “Futile Cycle of Repair” model, the MutSa complex (a heterodimeric complex of the two MMR proteins MSH2 and MSH6) recognizes and binds to the O6-MeG:T mispair, recruits the MutLa complex (a heterodimeric complex of the two MMR proteins MLH1 and PMS2) to the mispair, and initiates repair of the newly synthesized DNA strand. Since this repair process involves removal and resynthesis of the T-containing DNA strand, the O6-MeG:T mispair is regenerated during each cycle of repair thereby generating the substrate for another cycle. It is proposed that continued rounds of repair may lead to some aborted repair and the formation of double-strand breaks and cell death. In the “Direct DNA Damage Signaling” model, MutSa binds to the O6-MeG:T mispair and, without repair processing, recruits MutLa and the DNA damage response proteins ATRIP and ATR to initiate the DNA damage checkpoints leading to cell cycle arrest and apoptosis. Critical MMR proteins are therefore required for efficacy of TMZ and hence loss of MMR leads to resistance to TMZ.

Temozolomide

By a mechanism similar to that described above for MGMT epigenetic gene silencing, MMR can be compromised by tumor-specific promoter methylation of critical MMR genes. Whereas improved prognosis has been reported in tumors with loss of MGMT expression due to promoter methylation, poor prognosis is observed when MMR capacity is compromised by methylation of the promoter for the essential MMR genes MLH1, MSH2, and MSH6. Loss of expression or inactivating mutations in MSH6 (an essential component of the MutSa complex) have been observed in TMZ-resistant glioma tumors and in recurrent tumors following TMZ therapy, implicating an essential role for MHS6 in TMZ response. It is likely that all components of the MutSa complex (MSH2 and MSH6) and the MutLa complex (MLH1 and PMS2) may be essential for TMZ efficacy and response to the TMZ-induced O6-MeG DNA adduct. Repair of N7-Methylguanine and N3-Methyladenine DNA Lesions The N7-methylguanine (N7-MeG) and N3-methyladenine (N3-MeA) DNA lesions (Fig. 2) comprise over 80% of the DNA adducts induced by TMZ. Both of these DNA lesions are repaired by the base excision repair (BER) pathway. BER is the predominant DNA damage repair pathway for the processing of small base lesions derived from alkylation damage. BER is normally defined as DNA repair initiated by a lesionspecific DNA glycosylase and completed by either of two sub-pathways: short-patch BER, a mechanism whereby only one nucleotide is replaced, or long-patch BER, a mechanism whereby 2–13 nucleotides are replaced. The majority of repair is currently thought to occur via the short-patch pathway. Repair of N7-MeG and N3-MeA is initiated by the methylpurine DNA glycosylase (Mpg; NM_002434). The paradigm for the short-patch BER pathway initiated by Mpg involves base lesion removal and then AP site hydrolysis by AP endonuclease (Ape1; NM_080649), catalyzing the incision of the damaged strand, leaving a 30 OH and a 50 deoxyribosephosphate moiety (50 dRP) at the margins. DNA polymerase b (Pol b; NM_002690) hydrolyzes

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the 50 dRP moiety and fills the single-nucleotide gap, preparing the strand for ligation by either DNA ligase I (LigI; NM_000234) or a complex of DNA ligase IIIa (LigIIIa; NM_013975) and XRCC1 (NM_006297). Each step throughout BER is coordinated via protein-protein interactions with the XRCC1/DNA ligase IIIa heterodimer and PARP1 (NM_001618), two important BER scaffold protein complexes. Furthermore, PARP1 has been linked more directly to BER as it interacts both physically and functionally with Pol b and LigIIIa, placing it as a member of the short-patch BER pathway. In addition, PARP1 coordinates with long-patch BER proteins to facilitate the repair of longer stretches of DNA. BER removes >80% of the DNA lesions induced by TMZ. The advent of TMZ has therefore increased interest in the development of BER-specific inhibitors since aborted or blocked BER sensitizes most cancer cells to alkylating agents. Blocking almost any step in the BER pathway will improve TMZ efficacy in cell culture assays, suggesting that BER inhibitors (just as observed with inhibitors of MGMT) may prove useful when administered in combination with TMZ. Few small-molecule drugs have been developed that are specific for BER proteins. Lucanthone and CRT0044876 have been reported to inhibit Ape1 and will sensitize cells to TMZ. Methoxyamine is another BER inhibitor; however, this compound does not directly inhibit BER enzymes but instead binds to abasic sites in DNA, making them refractory to further repair, and the methoxyamine-bound abasic site is highly cytotoxic. Methoxyamine is currently undergoing clinical trials in concert with TMZ. The most common BER target for smallmolecule drugs (inhibitors) is PARP1 although PARP1 is also involved in the repair of doublestrand breaks (DSBs) in DNA via a nonhomologous end-joining pathway. PARP1 is the founding member of a large family of ADPribosyltransferase proteins including six PARPs (poly-ADP-ribose polymerases), eleven MARTs (mono-ADP-ribosyltransferases), seven SIRTs, four PARGs (poly-ADP-ribose glycohydrolases),

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and Pi-MARTs. To date, only PARP isoforms 1, 2, and 3 have been found to respond to DNA damage such as that induced by TMZ. Upon binding to a nick (a single-strand break) or a DSB, PARP is activated to ▶ poly-ADP-ribosylate itself and other target proteins and to synthesize poly-ADP-ribose. Inhibiting PARP activation has been shown to significantly improve TMZ-induced tumor cell killing and to improve response to TMZ in preclinical xenograft models. Currently, there are at least six PARP inhibitors undergoing clinical trials as anticancer agents, although the specificity of these for PARP1, PARP2, PARP3, or other PARP-family members remains to be determined. Three of these small-molecule inhibitors undergoing clinical trials are being evaluated for clinical efficacy in combination with TMZ. These clinical trials (phase I and II) are for the treatment of solid tumors, metastatic melanoma, and glioblastoma multiforme. Although TMZ is effective in the treatment of glioblastoma and is being evaluated for ▶ melanoma and other cancers, efficacy can clearly be improved through combination therapy to inhibit one or more DNA repair pathways. Continued development of MGMT and BER inhibitors is expected to have significant impact on response to TMZ in the near future. Further, analysis of these critical DNA repair pathways in tumors can provide valuable biomarkers to anticipate response.

References Almeida KH, Sobol RW (2007) A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification. DNA Repair 6:695–711 Gerson SL (2004) MGMT: its role in cancer aetiology and cancer therapeutics. Nat Rev Cancer 4:296–307 Jiricny J (2006) The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol 7:335–346 Newlands ES, Stevens MF, Wedge SR et al (1997) Temozolomide: a review of its discovery, chemical properties, pre-clinical development and clinical trials. Cancer Treat Rev 23:35–61 Wang JY, Edelmann W (2006) Mismatch repair proteins as sensors of alkylation DNA damage. Cancer Cell 9:417–418

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Temsirolimus Janet E. Dancey Canadian Cancer Trials Group Queen’s University, Kingston, ON, Canada

Synonyms CCI-779; Torisel™

Definition Temsirolimus is a small molecule inhibitor of mammalian target of rapamycin (mTOR) kinase. Aberrant intracellular signaling through mTOR is associated with the cancer cell proliferation. Inhibition of this pathway by temsirolimus results in antiproliferative effects that may result in improved survival in patients with cancer.

Characteristics Temsirolimus is a soluble 42-[2,2-bis (hydroxymethyl)]-propionic ester of the macrocyclic lactone rapamycin (also known as sirolimus). Temsirolimus, sirolimus, and other members of this class of agents inhibit the proliferation of normal and malignant cells by inhibiting the activity of mTOR, an intracellular serine-threonine kinase within the phosphoinositide 3-kinase (PI3K)/Akt signal transduction pathway. Temsirolimus, like sirolimus, reacts with the ubiquitous intracellular FK506-binding protein 12 (FKBP12) to inhibit mTOR function. Mechanism of Action Activation of the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway plays a pivotal role in essential cellular functions such as survival, proliferation, migration, differentiation, and carbohydrate metabolism and is important in the molecular mechanisms of diseases such as diabetes and chronic inflammation, as well as cancer. Normal

Temsirolimus

cells such as lymphocytes, endothelial cells, and fibroblasts as well as cancer cells are dependent of this signaling pathway. The observed antitumor and immunosuppressive properties of temsirolimus and other agents within this class are due to their ability to disrupt mTOR function. Both activating mutations and amplification of oncogenes and loss of tumor suppressor genes occur within the pathway in human neoplasms with remarkable frequency. Activation mutations and amplification of growth factor receptors, PI3K and AKT, and overexpression of PI3K and Akt have been reported in different tumor histologies. Similarly, the loss of tumor suppressor proteins that regulate the PI3K-AktmTOR pathway such as tuberous sclerosis proteins 1 or 2 (TSC1/2), phosphatase and tensin homologue deleted on chromosome 10 (PTEN), and serine/threonine kinase 11 (STK11 also known as LKB1) has been linked to the pathobiology of a number of tumor predisposition syndromes, including tuberous sclerosis syndrome (TSC1/2), ▶ Peutz-Jeghers syndrome (STK11/ LKB1), and ▶ Cowden syndrome (PTEN). In laboratory models, the resultant aberrant activation of the signaling pathway through oncogene stimulation or tumor suppressor gene loss not only leads to a growth advantage during carcinogenesis but also contributes to tumor angiogenesis, metastasis, and resistance to standard cancer therapy. Of interest and relevance to cancer therapeutics development, aberrant pathway activation may also lead to sensitivity to agents that target mTOR. Temsirolimus does not directly interact with mTOR kinase and does not inhibit all mTOR functions. mTOR functions are dependent on its forming complexes with other proteins. Two mTOR-containing complexes have been well characterized: a rapamycin-sensitive complex (also called mTOR complex 1, mTORC1), which includes mTOR accessory protein Raptor along with mammalian ortholog of LST8 (mLST8) (regulatory-associated protein of mTOR), and a rapamycin-insensitive complex (also called mTOR complex 2, mTORC2), which composed of mTOR and Rictor

Temsirolimus

(rapamycin-insensitive companion of mTOR). mTORC1, phosphorylates the well-characterized mTOR effectors S6 kinase 1 (S6K1, also known as p70S6K) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4EBP1). TORC2 controls the actin cytoskeleton as well as Akt. However, mTOR when bound to Rictor can phosphorylate Akt at its hydrophobic motif leading to Akt activation. This positive feedback loop may result in increased phosphorylation of Akt in the presence of temsirolimus inhibition of the Raptor-mTOR complex. Activity in Cancer Models The antiproliferative effects of temsirolimus and other sirolimus derivatives have been evaluated in numerous in vitro and in vivo tumor models. In sensitive cell lines, these agents inhibit tumor and endothelial cell proliferation in picomolar to nanomolar concentrations and may add to the cytotoxicity of other chemotherapeutic agents and radiation. The antiproliferative effects of temsirolimus may be due, at least in part, to its well-characterized inhibitory effects on the activation of S6K1 and 4EBP1. Inhibition of these proteins alters the translation of subsets of mRNAs, particularly those that may be involved in regulating cell cycle progression. In a relatively limited number of tumor models, this class of agents may induce cancer cell death apoptosis or autophagy. The molecular mechanisms leading to apoptosis in cancer cells have not yet been fully deciphered. In addition, mTOR inhibitors can target tumor growth indirectly by inhibiting endothelial cells and pericytes proliferation required for tumor angiogenesis. Activity in Clinical Trials Results from cancer clinical trials suggest that temsirolimus is well tolerated and appears to have antitumor activity. The most common toxicities seen with the drug are mild to moderate skin reactions; stomatitis; reductions in blood counts, particularly platelets; and metabolic abnormalities such hyperlipidemia and hyperglycemia. These adverse effects are reversible with interruption of dosing or, for hyperlipidemia and hyperglycemia,

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with specific treatment. Rarely, pneumonitis has been reported in patients that have received temsirolimus. To date, there has been no evidence of clinically significant immunosuppression with intermittent schedules. Effective target inhibition has been shown through pharmacodynamic assays assessing inhibition of either S6K1 or 4E-BP1 phosphorylation in surrogate tissue from patients treated with temsirolimus. More limited data from baseline and on treatment tumor tissue specimens supports that temsirolimus inhibited mTOR and downstream targets. However, optimal dose/schedule for intratumoral target inhibition has not been well defined due to limitations in numbers of specimens analyzed. Results from four phase I studies evaluating increasing doses of temsirolimus on different schedules and with oral and intravenous formulations have been reported. The weekly intravenous schedule is the one that has been most extensively evaluated in phase II and III studies. Phase II studies of single-agent temsirolimus evaluating different doses of 25, 75, and/or 250 mg weekly IV have been undertaken in broad range of tumor histologies. The most promising antitumor activity has been seen in ▶ mantle cell lymphoma and endometrial carcinoma with objective tumor response rates of 30–40%. Moderate activity has been reported in breast and renal cell carcinoma. Minimal single-agent activity has been seen in small-cell lung carcinoma, melanoma, and glioblastoma multiforme. In general, lower doses appear to be as active as higher doses with better tolerability. Temsirolimus is approved for the treatment of patients with renal cell carcinoma (RCC) worldwide and for relapsed or MCL in many countries although not in the United States. phase III trial of temsirolimus, temsirolimus with interferon versus interferon in poor-prognosis patients with renal cell carcinoma, has been reported. Of the 626 patients, overall survival of patients treated with temsirolimus was significantly prolonged compared to those treated with interferon (median 10.9 months versus 7.3 months, hazard ratio for death 0.73, p = 0.0069). The combination of interferon and temsirolimus did not confer

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greater benefit than interferon alone, possibly due to compromised dose delivery of the agent(s) due to significant. Approval of IV temsirolimus for the treatment of MCL was based on improved PFS seen in a phase III trial conducted in 162 subjects randomly assigned to receive temsirolimus IV 175 mg (3 successive weekly doses) followed by 75 mg weekly, temsirolimus IV 175 mg (3 successive weekly doses) followed by 25 mg weekly, or investigator’s choice of single-agent treatment. Temsirolimus 175/75 mg led to a statistically significant improvement in PFS compared with investigator's choice. The median PFS was 4.8 months in the temsirolimus 175/75-mg group, 3.4 months in the temsirolimus 175/25-mg group, and 1.9 months in the investigator's choice group. The identification of tumor types that respond to mTOR inhibitors remains a major issue for the development of temsirolimus. mTOR is ubiquitously expressed, and therefore, the sensitivity or resistance of specific tissues to temsirolimus cannot be predicted solely on the basis of whether the target protein can be detected in the tumor tissue. Activation status of the PI3K/AKT/mTOR signaling pathway seems to be the most promising strategy to identifying tumor types potentially sensitive to temsirolimus. As a significant number of patients have cancers that are insensitive to temsirolimus, combinations of the mTOR inhibitor with hormonal therapy (hormonal treatment), chemotherapy, or other targeted therapies, based on the rationale that simultaneous inhibition of multiple signaling pathways are under evaluation. Temsirolimus, which inhibits the downstream kinase mTOR, has demonstrated that it may be a useful cancer therapeutic as it may confer clinical benefit to patients with acceptable toxicity. The proof of principle that temsirolimus can improve cancer patient survival has been obtained from a large randomized trial in advanced poor prognostic renal cell carcinoma. The major clinical development challenges will be efficiently identifying the optimal dose, schedule, and combination regimens for patients with susceptible malignancies and monitoring and managing toxicities to optimize the therapeutic index.

Temsirolimus (Torisel )

Cross-References ▶ Cowden Syndrome ▶ Endometrial Cancer ▶ Mantle Cell Lymphoma ▶ Peutz–Jeghers Syndrome ▶ Rapamycin

References Cully M, You H, Levine AJ et al (2006) Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat Rev Cancer 6(3):184–192 Faivre S, Kroemer G, Raymond E (2006) Current development of mTOR inhibitors as anticancer agents. Nat Rev Drug Discov 5(8):671–688 Hudes G, Carducci M, Tomczak P, et al: Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med 356:2271-81, 2007. Hess G, Herbrecht R, Romaguera J, et al: Phase III study to evaluate temsirolimus compared with investigator's choice therapy for the treatment of relapsed or refractory mantle cell lymphoma. J Clin Oncol 27:3822-9, 2009. Samuels Y, Ericson K (2006) Oncogenic PI3K and its role in cancer. Curr Opin Oncol 18(1):77–82 Thomas GV (2006) mTOR and cancer: reason for dancing at the crossroads? Curr Opin Genet Dev 16(1):78–84

See Also (2012) Endothelial cells. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p. 1251. doi:10.1007/978-3-642-16483-5_1896 (2012) Pericyte. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, pp 28142815. doi:10.1007/978-3-642-16483-5_4448 (2012) Small cell lung carcinoma. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p. 3448. doi:10.1007/978-3-642-164835_5368 (2012) Stomatitis. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p. 3535. doi: 0.1007/978-3-642-16483-5_5522 (2012) Tuberous sclerosis syndrome. In: Schwab M (ed) Encyclopedia of cancer, 3rd edn. Springer Berlin Heidelberg, p. 3792. doi:10.1007/978-3-642-164835_6010

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Temsirolimus (Torisel ) ▶ Rapamycin

Tenascin-C

Tenascin-C Gertraud Orend Department of Clinical and Biological Sciences, Institute of Biochemistry and Genetics, Center for Biomedicine, DKBW, University of Basel, Basel, Switzerland

Synonyms Cytotactin; Glial/mesenchymal extracellular matrix protein; GMEM; Hexabrachion; J1 220/200; Myotendinous antigen; Neuronectin

Definition Tenascin-C is the founding member of a family of extracellular matrix glycoproteins comprising tenascin-X, tenascin-R, and tenascin-W in addition to tenascin-C. Its name has been created by Ruth Chiquet-Ehrismann (1986) and represents a combination of the Latin verbs “tenere” (to hold) and “nasci” (to grow, develop, to be born), which provided the roots of the English words “tendon” and “nascent,” and reflects the location and developmental expression of the protein observed at that time.

Characteristics Tenascin-C is part of a tumor-specific stroma of most solid cancers and plays a role in enhancing proliferation, ▶ angiogenesis, and ▶ metastasis during tumorigenesis. Moreover, research data support the possibility that tenascin-C contributes to cancer formation via interference with genomic stability, by blocking the immunosurveillance and by providing a favorable niche for tumor stem cells (▶ cancer stem-like cells, stem cells, and cancer). Its high expression correlates with bad prognosis for disease-free survival in patients with glioma, lung, and colon cancer.

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Molecular Organization The tenascin-C protein, 20 201 amino acids in length giving rise to a 190–300 kDa monomer, is encoded by 60 603 bp, which are organized into at least 28 exons on chromosome 9q33. Tenascin-C is a modular molecule consisting of an N-terminal region containing a chaperone-like sequence forming coiled-coil structures and interchain disulfide bonds that are essential for subunit oligomerization into hexamers. Tenascin-C is comprised of 14.5 epidermal growth factor (EGF)-like repeats, 30–50 amino acids in length, which contain six cysteine residues involved in intrachain disulfide bonds. In tenascin-C, up to 17 ▶ fibronectin type III domains are present that are 90 amino acids in length and that are composed of seven antiparallel b-strands arranged in two sheets. The nature and number of fibronectin type III domains in tenascinC are generated by alternative splicing that is modulated by the proliferative state of a cell, extracellular pH, and TGFb1. At least nine different fibronectin type III domains are differentially included or excluded by RNA splicing. This can generate a considerable diversity among different cancers (Fig. 1) and can cause variable cell responses toward tenascin-C. The C-terminal fibrinogen globular domain resembling the b- and g-chains of fibrinogen, 210 amino acids in length, forms intrachain disulfide bonds (Fig. 1). Induction and Processing Tenascin-C can be induced in a tumor by various pro- and anti-inflammatory cytokines and growth factors that are mostly secreted by stromal cells. In addition, hypoxia (hypoxia and tumor physiology), ▶ reactive oxygen species, and mechanical stress, which are also present in tumor tissue, induce tenascin-C expression. In contrast, glucocorticoids suppress tenascin-C expression. Signaling causing activation of transcription factors such as TCF/LEF, NfkB, c-Jun, Ets, SP1, and Prx-1 is involved in tenascin-C gene transcription. Tenascin-C is cleaved by ▶ matrix metalloproteinases and serine proteases, thus potentially releasing cryptic sites within the fibronectin type III domains of tenascin-C. Cell contact with tenascin-C also induces the expression of matrix metalloproteases, thus presenting a positive

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Astrocytoma Melanoma, breast ductal carcinoma Malignant oral cancer Breast ductal carcinoma in situ Colorectal carcinoma 1 2 EGFR

4 5 A1 A2 A3 A4 B AD2 AD1 C D 6

3

RPTPβ

7 8

Integrin α7β1 Annexin II

Nav1.9/NaN Fibronectin Integrins Perlecan Lecticans Neurocan Heparin Contactin

Integrin αvβ3, Neurocan, RPTPβ, CALEB

Tenascin-C, Fig. 1 Domain structure, binding partners, and expression of tenascin-C in cancer tissue. The N-terminal oligomerization, EGF-like, fibronectin type III and fibrinogen-like domains are schematically depicted as triangle, rhombomeres, boxes, and circles, respectively. The alternatively spliced fibronectin type III domains A1-D are shown in black. An electron micrograph of a tenascin-C hexamer is shown at the left corner. Fibronectin

type III domains specifically detected in certain cancers are highlighted above the model. EGFR, epidermal growth factor receptor; CALEB, chicken acidic leucine-rich EGF-like domain containing brain protein; RPTPb, receptor protein tyrosine phosphatase-b/z; Nav1.9/NaN, sodium channel subunit b2 (Picture was taken from Orend and Chiquet-Ehrismann (2006))

feedback loop between induction of matrix metalloproteases by tenascin-C and cleavage of tenascin-C by these enzymes.

antiadhesive sequences that coexist in the native molecule. These opposing activities arise as a consequence of tenascin-C binding to extracellular matrix components and to cell surface receptors. One mechanism that induces cell rounding involves tenascin-C inhibition of cell adhesion to fibronectin. This occurs through competitive binding of tenascin-C to fibronectin, thus masking the binding site for integrin a5b1 coreceptor syndecan-4 (heparanases). This blocks activation of the small GTPase RhoA and focal adhesion kinase. Activation of oncogenic Wnt (▶ Wnt signaling), endothelin receptor type A, and MAPK signaling induced by tenascin-C and elimination of G0 and G1 cell cycle (cell cycle targets for cancer therapy) transition control could contribute to enhanced tumor cell proliferation by tenascin-C.

Interaction Partners Tenascin-C binds to extracellular matrix molecules such as fibronectin, perlecan, aggrecan, versican, and brevican (Fig. 1), thus potentially forming a tumor-specific extracellular matrix network. Cells can interact with tenascin-C via cell surface receptors including integrins (integrin signaling in cancer) a2b1, a7b1, a9b1, and avb3, syndecan, annexin II, and ▶ epidermal growth factor receptor (EGFR) among others (Fig. 1). Cell Rounding and Tumor Cell Proliferation Tenascin-C has distinct effects on tumor cells and tumor-associated cells such as carcinomaassociated fibroblasts, tumor-associated macrophages, and endothelial cells within the tumor stroma based on as yet poorly understood cell type-specific responses toward tenascin-C splice variants. Tenascin-C contains adhesive and

Metastasis Tenascin-C is expressed around invasive carcinoma cells that have undergone epithelialmesenchymal transition (EMT) (epithelial to

Tenascin-C

mesenchymal transition). Tenascin-C supports tumor cell migration and invasion by mechanisms that are little understood. Tenascin-C provides a substratum that supports migration of several cell types including glioma and laryngeal carcinoma cells. A mechanism by which tenascin-C supports colon carcinoma cell invasion involves secretion of tenascin-C by carcinoma-associated fibroblasts, activation of EGFR, and expression of hepatocyte growth factor and activation of its receptor c-Met. This triggered downstream activation and inhibition of the small GTPases Rac and RhoA, respectively, in the invading carcinoma cells. In addition to an EMT-associated migration, tenascin-C might also promote other forms of migration in cancer cells. Angiogenesis Tenascin-C plays a role during embryonic vascularization and promotes vascular sprouting. It is also expressed during formation of new blood vessels in the adult as, e.g., in granulation tissue of wounds after myocardial infarction, in arthritis, and in neoplastic diseases. In human gliomas, tenascin-C expression correlates with the degree of tumor neovascularization. Tenascin-C may promote angiogenesis by serving as chemoattractant for endothelial cells, by initiating endothelial cell differentiation, survival, and proliferation, events that involve integrin avb3 and vascular endothelial growth factor among not yet identified other molecules. Phenotype of Tenascin-C Knockout Mice and Cancer The tenascin-C sequence is highly conserved among species, which suggests that evolutionary forces prevented loss of this gene because of its importance to life. Tenascin-C knockout (gene knockout) mice are hyperactive among other neurological defects, which would make them an easy prey. Abnormal behavior in these mice might be due to its role as ligand for neuronal receptors. Despite an otherwise apparently normal phenotype, which is likely due to compensatory mechanisms, tenascin-C knockout mice show difficulties in regeneration upon disturbance of tissue homeostasis such as during healing of wounds in the eye

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and in the inflamed kidney. Tenascin-C is expressed in stem cell niches (▶ adult stem cells) such as those of the bone marrow, brain, and skin. Stem cells are required to maintain and restore tissue homeostasis, in particular upon insults to tissues. This may explain the more severe phenotype in injured tenascin-C knockout mice. The first in vivo evidence for a role of tenascin-C in tumor angiogenesis derives from studies with xenografted melanoma cells into mice lacking tenascin-C expression. In these tenascin-C knockout mice, tumor growth and angiogenesis were strongly reduced in comparison to mice exhibiting tenascin-C expression. Clinical Aspects How can we use our knowledge about tenascin-C to combat cancer? Given that expression of tenascin-C correlates with tumorigenesisenhancing events and with a reduced disease-free survival in patients with some cancers, inhibition of tenascin-C expression at the transcriptional level would be the first choice to block tenascinC actions in cancer. Unfortunately, this approach is questionable since many factors and conditions that trigger tenascin-C expression are not specific for tenascin-C alone but affect many other genes. Preventing tenascin-C action in a tumor, e.g., by restoration of syndecan-4 function in gliomas, offers another approach. However, since tenascin-C has many poorly understood effects at the molecular level on the different cell types within cancer tissue, targeting tenascin-C actions may produce undesirable side effects. The most promising approach today is to target tenascin-C with antitenascin-C-directed antibody fragments that are coupled to cytotoxic reagents in a trojan horse-like strategy, which would trigger destruction of the tumor. Tenascin-C-targeting antibodies are in clinical trials and one needs to await the antitumor response rates in cancer patients.

References Chiquet-Ehrismann R, Chiquet M (2003) Tenascins: regulation and putative functions during pathological stress. J Pathol 200:488–499

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4490 Jones PL, Jones FS (2000) Tenascin-C in development and disease: gene regulation and cell function. Matrix Biol 19:581–596 Orend G (2005) Potential oncogenic action of tenascin-C in tumorigenesis. Int J Biochem Cell Biol 37: 1066–1083 Orend G, Chiquet-Ehrismann R (2006) Tenascin-C induced signaling in cancer. Cancer Lett 244:143–163

Tensional Homeostasis Inkyung Kang and Valerie M. Weaver Department of Surgery, University of California, San Francisco, San Francisco, CA, USA

Definition A mechano-regulatory network that integrates physical and biochemical cues from the tissue microenvironment through mechano-responsive elements such as transmembrane integrins to evoke cytoskeletal reorganization and actomyosin contractility, thereby altering signal transduction and gene expression to modulate cell and tissue phenotype.

Characteristics Cells and tissues experience and respond to externally applied mechanical force through mechanoresponsive elements that influence signal transduction and result in the generation of reciprocal intracellular force or contractility. The types of mechanical stress a cell can experience include compressive or tensile stress which is applied perpendicular to the surface of the cell and shear stress which is applied parallel to the surface of the cell. For example, osteoblasts and chondrocytes within bone and cartilage are subjected to compressive force induced by walking, lung alveolar cells experience tensile load resulting from inhalation-induced alveolar sac expansion, and endothelial cells lining the lumens of blood vessels undergo shear force induced by circulating blood flow. Cells integrate external

Tensional Homeostasis

mechanical force on multiple levels. This includes force-dependent changes in the conformation of the plasma membrane lipid bilayers as well as modifications in the orientation and molecular associations of transmembrane proteins. These changes enhance the activity of calcium and potassium ion channels, the extracellular matrix affinity, and cytoskeletal plaque associations of various adhesion molecules including integrins (integrin signaling and cancer). Cells also integrate external force cues to generate reciprocal actomyosin-mediated cell contractility and modulate their mechanical properties through remodeling of the microtubule, intermediate filament, and actin cytoskeletal network. Intracellular mechanical force is transduced to the extracellular microenvironment via functional links between transmembrane receptors that bind to extracellular matrix (ECM) proteins and the intracellular cytoskeletal network and ultimately mediate an equilibrium of extracellular and intracellular forces in the cell. This equilibrium or balance between the extracellular forces and the intracellular forces is called tensional homeostasis. When the extracellular mechanical microenvironment becomes altered, cells and tissues will coordinately respond by adjusting cell-generated mechanical force or contractility, which in turn elicits changes in cell behavior by modifying the activity and function of signaling pathways and gene expression that determine growth, survival, and differentiation. Cells sense and integrate tensional forces by altering the expression and activity of a plethora of putative mechanosensors. Nevertheless, integrins are considered key mechanotransducers by virtue of their external associations with the extracellular matrix and their internal links to various adhesion plaque proteins including vinculin, talin, and ▶ focal adhesion kinase (FAK), which in turn mediate interactions with the cytoskeleton and activate various signaling cascades. Extracellular mechanical force can alter the conformation of an integrin from a low ligand-binding affinity state to high ligandbinding affinity state, the conformation of extracellular matrix proteins such as ▶ fibronectin and collagen I to expose or alter ligand-binding sites, and the conformation of vinculin and talin to

Tensional Homeostasis

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Focal complex

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EGFR

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ERK Rho

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Tensional Homeostasis, Fig. 1 Key molecular pathways that mediate tensional homeostasis in cells and tissues. Changes in the mechanical environment of cell, such as an increase in ECM stiffness or elevated extracellular tension, promote integrin clustering to drive the maturation of nascent focal contacts into focal adhesions. The assembly of focal adhesions is associated with increased Rho GTPase activity and elevated and sustained ERK signaling. The combination of enhanced Rho and ERK activity increases actomyosin-mediated intracellular contractility

by altering the function of Rho kinase (ROCK) and phosphorylated myosin light chain (MLC-P). Elevated cellgenerated force promotes focal adhesion assembly and potentiates growth factor-dependent ERK activation in a feed-forward vicious cycle. Elevated intracellular force also alters ECM deposition and organization by orienting and further stiffening ECM. Oncogenes which promote RAS-dependent ERK activation and Rho GTPase activity additionally contribute to cell-generated forces by regulating ROCK and MLCK and myosin II activity

favor intracellular molecular associations. These mechanically initiated events promote actin assembly and stabilize adhesion plaque protein assembly and clustering of integrins to convert nascent focal complexes into mature focal adhesions. Force-dependent integrin activation and focal adhesion maturation increase the magnitude and duration of adhesion signaling including ERK ▶ MAP kinase and RhoA GTPase (▶ Rho family proteins). Elevated and sustained

activity of ERK and RhoA GTPase drives actomyosin-mediated intracellular contractility by altering the function of Rho kinase (ROCK) and phosphorylated myosin light chain. The elevated intracellular tension in turn promotes focal adhesion maturation, creating a feedback loop of biochemical signaling pathways resulting in an elevated intracellular force generated by actomyosin cytoskeleton and inside-out remodeling of extracellular matrix proteins (see Fig. 1).

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When the balance between the external and intracellular stress is altered, the cell and tissue will adapt to the new mechanical microenvironment challenge, which can result in positive outcomes such as an increase in bone and muscle density due to exercise or in negative outcomes such as atherosclerosis mediated by chronically elevated shear force applied by perturbed blood flow and cardiac hypertrophy due to hypertension. Mechanical compression can also regulate gene expression to influence tissue development as has been documented during embryogenesis. Changes in matrix stiffness determine the lineage commitment of mesenchymal stem cells, such that the cells express neurogenic markers when grown in mechanical environment closer to the stiffness of brain (0.1–1 kPa), myogenic markers at an intermediate stiffness (8–17 kPa), and osteogenic markers at a higher stiffness (25–40 kPa). This lineage commitment is regulated by nonmuscle myosin II and is accompanied by an increase in the size of focal adhesions and in the expression of focal adhesion components including talin and phosphoFAK. These results suggest that a cell dynamically probes its mechanical microenvironment through active engagement of integrin adhesion receptors and generation of actomyosin contractility and that an increase in focal adhesion maturation and intracellular contractility drives downstream signaling events which determine lineage differentiation. Thus, tensional homeostasis is emerging as a critical determinant in cell fate during normal morphogenesis as well as pathophysiological processes. Solid tumors are characteristically stiffer than normal tissue, which allows detecting tumors by palpation. The elevated stiffness is mediated by increased interstitial tissue pressure and changes in the mechanical properties of malignant cells and the surrounding stroma. The tumor stroma is characterized by an increased deposition and reorganization of matrix proteins including collagen, fibronectin and tenascin, and aberrant ECM crosslinking induced by lysyl oxidase, transglutaminase, proteoglycans, and glycation, which contribute to the stiffening of the stroma (▶ extracellular matrix remodeling). In addition, transforming oncogenes such as RAS, ErbB/ HER2 neu, and c-Myc (▶ myc oncogene) can

Tensional Homeostasis

alter the mechano-responsiveness of cells and cooperate with integrin adhesion signaling molecules to enhance cell proliferation, survival, and invasion. Indeed, oncogenes such as Ras and ErbB/HER activate Rho and ERK that induce actomyosin contractility and elevate cellgenerated forces to further promote the assembly and maturation of integrin adhesions and enhance growth factor receptor cross-talk. This raises the intriguing possibility that in addition to promoting cell growth and survival by directly modifying the activity of various signaling molecules, some transforming oncogenes might promote malignancy by altering the cell tensional homeostasis. Consistently, Paszek et al. demonstrated that increasing ECM stiffness from 140 Pa (approximating the compliance of the normal murine breast) to 1,000–5,000 Pa (similar to the stiffness of a malignant murine breast) compromised mammary morphogenesis and induced the malignant phenotype of nonmalignant mammary epithelial cells in culture, as demonstrated by an increase in cell growth and survival, and the loss of mammary tissue integrity (i.e., disruption of cell-cell junctions and loss of tissue polarity). Stiffening of ECM also significantly increased recruitment and activation of FAK and actin-binding proteins such as vinculin to b1 integrin adhesion, which was accompanied by an increase of larger, mature focal adhesions, contractility, and enhanced growth factordependent ERK activation. More intriguingly, malignantly transformed mammary cells with elevated epidermal growth factor receptor (EGFR) signaling that form colonies of disorganized, invasive, and continuously growing cancer cells in response to a compliant normal matrix also exerted higher cell contractility. Strikingly, inhibiting the activity of Rho GTPase or myosin II or ERK was sufficient to reduce the tumor cells contractility and revert the malignant phenotype of these breast cancer cells toward that of a normal breast acini. Likewise, inhibiting Rho- or ERKdependent myosin activity also normalized the phenotype of non-transformed mammary cells interacting with an abnormally stiffened matrix. Together these findings suggest that breast transformation could arise through the combination of

Testicular Cancer

oncogenic mutations that promote cell-generated contractility and a progressive stiffening of the ECM which compromises the tensional homeostasis to elevate cell contractility and to increase focal adhesion assembly, which enhance aberrant cell growth, survival, and invasion. Clinical studies indicate that mammographic density is strongly and reproducibly associated with an increased risk of breast cancer, independent of other risk factors (▶ mammographic breast density and cancer risk). For example, breast cancer risk rises to 30% when greater than 50% of the mammography qualifies as dense. Although the high cancer risk linked with breast density could be attributed to decreased detection sensitivity and increased epithelial mass, data indicate that elevated collagen and proteoglycan content are also risk factors that contribute to the enhanced transformation frequency associated with this condition. Elevated mammographic density frequently precedes ▶ ductal carcinoma in situ (DCIS), and DCIS often occurs predominantly in the mammographically dense areas of the breast. Because higher collagen density and elevated proteoglycan-mediated cross-linking correlate with an increase in ECM stiffness, these findings are consistent with the prediction that mammographic density could promote carcinogenesis by perturbing the cell tensional homeostasis. If true, an increase in matrix stiffening would herald an altered tissue tensional homeostasis and constitute a tractable predictor of future tissue transformation. Accordingly, an improved understanding of the parameters that promote matrix stiffening and alter tissue tensional homeostasis would assist in the development of improved detection, prognosis, and treatment strategies for solid cancers. To summarize, cells and tissues sense and respond to external force through a process called tensional homeostasis that reciprocally alters the external microenvironment through cellgenerated force. Tensional homeostasis is emerging as an important determinant of normal tissue development and adult tissue homeostasis, and studies indicate that an altered tensional homeostasis likely contributes to the pathogenesis of diseases including cancer and atherosclerosis.

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References Boyd NF, Rommens JM, Vogt K et al (2005) Mammographic breast density as an intermediated phenotype for breast cancer. Lancet Oncol 6:798–808 Engler AJ, Sen S, Sweeney HL et al (2006) Matrix elasticity directs stem cell lineage specification. Cell 126: 677–689 Keller R, Davidson LA, Shook DR (2003) How we are shaped: the biomechanics of gastrulation. Differentiation 71(3):171–205 Paszek MJ, Zahir N, Johnson KR et al (2005) Tensional homeostasis and the malignant phenotype. Cancer Cell 8:241–254 Yamaguchi H, Wyckoff J, Condeelis J (2005) Cell migration in tumors. Curr Opin Cell Biol 17:559–564

Teratocarcinoma-Derived Growth Factor-1 ▶ Cripto-1

Teratoma ▶ Ovarian Tumors During Childhood and Adolescence

Testicular Cancer Axel Heidenreich Division of Oncological Urology, Department of Urology, University of Köln, Köln, Germany

T Synonyms Non-seminomatous germ cell tumor; Seminoma; Seminomatous germ cell tumor; Testicular germ cell tumor

Definition About 90% of all testicular tumors are malignant germ cell tumors, and the rest comprise benign

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tumors deriving from Leydig and Sertoli cells and other interstitial components. Testicular germ cell tumors originate from totipotent primordial germ cells, which undergo neoplastic transformation as a result of a number of endogenous, exogenous, hormonal, and genetic, as well as environmental, events. The neoplastic process results in the development of preinvasive ▶ carcinoma in situ (CIS) or TIN representing the common precursor for all testicular germ cell tumors, except spermatocytic seminoma.

Characteristics Testicular cancer represents the most common malignant tumor in young men in the age group of 20–40 years. In 1994, 6,800 new cases of testicular cancer were diagnosed in the USA. There are striking differences in TC incidences around the world, with the highest incidence of 12–14 per 100,000 person-years in Switzerland and Denmark and the lowest incidence of less than one per 100,000 person-years among AfricanAmericans and the Chinese populations. Cryptorchidism is the best-known risk factor and, according to case–control studies, the relative risk for TC is 2.5–8.8. Familial and genetic factors have been suggested to be involved in the development of TC with a six- to tenfold higher risk in first-degree relatives. Diagnosis The majority of patients present with a painless scrotal mass, and the diagnosis is usually established by physical examination of the tumor-bearing and the contralateral testicle, scrotal ultrasonography, and determination of the serum tumor markers alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), and lactate dehydrogenase (LDH). Inguinal exploration and scrotal orchiectomy confirms the diagnosis and is the therapy of choice, revealing accurate information with regard to histopathology and pathological-stage classification. Since benign testicular lesions are recognized with increasing frequency, frozen section analysis should be considered. Contralateral

Testicular Cancer

testicular biopsy to diagnose TIN is recommended in high-risk patients (testis volume 10.000 ng/ml ß-hCG > 10.000 ng/ml LDH > 10x Norm

markers should decline according to their half-life (ß-hCG: 24–36 h, AFP: 5–7 days). Any plateau phase or any delay in decline is predictive for a poor outcome in terms of response to therapy. The prognostic significance of tumor markers at the time of diagnosis becomes evident for advanced disease only, adhering to the International Germ Cell Consensus Classification Group (IGCCCG) classification. The Lugano classification represents the most widely used clinical staging system for testicular cancer (Table 2) and describes the extent of metastatic involvement of the lymph nodes and visceral organs. The IGCCCG has introduced a new staging system for advanced TC defining three prognostic risk groups with regard to therapeutic outcome. Patients are classified to be at good risk (probability of cure 95%), intermediate risk (probability of cure 70%), or poor risk (probability of cure 50%). The IGCCCG classification gives high prognostic evidence and enables an individualized risk-adapted approach in patients with advanced TC. Therapy Once TIN is diagnosed, therapeutic intervention is recommended, since 70% of patients will develop invasive germ cell tumor within the next 7 years. Local radiation therapy with 18 Gy is the therapy of choice in patients with a contralateral invasive

Testicular Cancer, Table 2 Clinical Lugano – classification of TC Stage I

Stage IIA Stage IIB Stage IIC Stage IIIA Stage IIIB

Stage IIIC

Tumor markers normalized or decline according to their half-life after orchiectomy No detectable metastases by imaging studies Primary TC confined to the testicle Retroperitoneal metastases 5 cm Supraclavicular or mediastinal metastases Pulmonary metastases Minimal: 2 cm Extrapulmonary visceral metastases

T germ cell tumor. In patients with unilateral TIN and a contralateral normal testis, inguinal orchiectomy appears to be the preferred management, since local radiation bears the risk of damaging the healthy testicle. Non-seminomatous Germ Cell Tumors (NSGCT) Clinical stage I NSGCT represents a troublesome entity concerning recommendations for optimal management since about 30% of patients will

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exhibit microscopic lymph node disease. Several treatment options such as primary nerve-sparing retroperitoneal lymphadenectomy, primary chemotherapy, and active surveillance have been developed resulting in the same high cure rates of 98%. The European Germ Cell Cancer Consensus Group recommends an individualized, risk-adapted approach based on the results of prospective randomized trials considering the presence or absence of the risk factors vascular invasion (VI) and percentage of embryonal carcinoma (ECA). VI has been identified as the most powerful clinical predictor of lymph node metastasis with 48% of NSGCTs with VI developing metastases, compared to 14–22% of tumors without VI. A combination of VI and ECA might be even more powerful. Nowadays, nerve-sparing RPLND – if performed – is regarded as the standard approach. Up to 10% of patients will suffer from pulmonary relapse within the first 2 years and will be cured by platinum-based chemotherapy. Even in low-volume lymph node disease such as pathological stage IIA, the nerve-sparing RPLND can be performed as bilateral radical surgery without compromising the therapeutic outcome. Primary chemotherapy [two cycles of cisplatin, etoposide, and bleomycin (PEB)] and surveillance (absence of VI) result in relapse rates of only 7% and 14%, respectively. Low-Stage (IIA/B) NSGCT

Low-stage testicular disease comprises clinical stages IIA and IIB associated with a cure rate of 98%. Patients with low-volume disease and abnormal tumor marker levels of AFP, ß-hCG, or LDH are treated with two to three cycles of PEB chemotherapy; patients with negative markers might be offered nerve-sparing RPLND or surveillance; and patients with clinical stage IIB TC will undergo primary chemotherapy depending on the serum tumor marker concentrations with three or four cycles of PEB followed by secondary RPLND in about 30% of cases. Clinical Stages IIC and III

Inductive chemotherapy represents the therapy of choice, with the number of cycles applied

Testicular Cancer

depending on the IGCCCG-based prognostic classification. Patients with “good prognosis” face a long-term survival rate of >90% and are managed by three cycles of PEB. Patients with “intermediate prognosis” face a survival rate of 70–80% and are managed by four cycles of PEB or cisplatin, etoposide, and ifosfamide (PEI). Patients with “poor prognosis” have a survival rate of only about 50%; standard therapy consists of four cycles of PEB or PEI. A major advantage of primary high-dose chemotherapy has not been demonstrated, but this approach is currently being tested in prospective randomized trials. Seminomatous Germ Cell Tumors Clinical Stage I Seminoma

Despite negative CT scans, there is a risk of 12–32% of occult retroperitoneal lymph node metastases depending on the absence or presence negative prognostic markers. The cure rate of clinical stage I seminomatous germ cell cancer is close to 100% and can be achieved by the three different therapeutic options: active surveillance, radiation therapy, and carboplatin monochemotherapy. Adjuvant retroperitoneal radiation therapy to the para-aortic or paracaval region with 20 Gy or adjuvant chemotherapy with one cycle carboplatin AUC 7 is the standard approach for high-risk patients (tumor size >4 cm, rete testis invasion) and results in a relapse-free long-term survival of 97%. Active surveillance represents the most reasonable approach to patients with good prognostic markers associated with a low recurrence rate of about 12%. Treatment of relapses is more intense with systemic chemotherapy of three to four cycles PEB in most cases. Low-Stage (Clinical Stage IIA/B) Seminoma

Radiation therapy with 30 Gy (IIA) and 36 Gy (IIB), including the ipsilateral iliac and inguinal lymph nodes, is one standard therapeutic approach for low-stage seminomas. Relapse-free survival is as high as 92.5% in clinical stage IIA/B; relapse rates are about 5% in stage IIA and about 11% in stage IIB seminomas. Primary

Testicular Cancer

chemotherapy with two cycles of PEB is an alternative to radiation in clinical stage IIB seminoma. Clinical Stages IIC and III

As pointed out for advanced non-seminomatous germ cell tumors, therapy should be initiated according to the IGCCCG classification. For patients with good prognosis, three cycles of PEB chemotherapy are the treatment of choice; in patients with intermediate prognosis, four cycles of PEB chemotherapy are applied. Residual Tumor Resection (RTR) Following Chemotherapy for Advanced Testicular Cancer. RTR represents an integral part of the multimodality treatment of advanced testicular germ cell tumors. The rationale for RTR is to completely resect mature teratoma and vital cancer which will be found in 30–40% and 20% of the patients, respectively. Currently, all residual lesions independent on size should be resected in NSGCT since even small lesions 10% vital cancer cells or those with uncomplete resection might benefit from consolidation chemotherapy with two cycles. Post-chemotherapy or postradiotherapy RPLND in seminomas has only to be performed in lesions with a positive PET scan performed about 6 weeks after chemotherapy or radiation therapy in patients with residual lesions >3 cm. Salvage Chemotherapy, High-Dose Chemotherapy

In seminomas relapsing after first-line radiation therapy, a cure rate of >90% can be achieved by

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cisplatin-based chemotherapy according to the IGCCCG algorithm with regard to advanced seminomas. About 50% of relapsing seminomas following conventional chemotherapy can be salvaged with another combination chemotherapy consisting of platinol, etoposide, and ifosfamide (PEI) (VIP) or vinblastine, ifosfamide, and platinol (VeIP). Currently, a 10% benefit of highdose chemotherapy with regard to survival has been demonstrated; therefore, it seems advisable that all relapsing patients should be treated in a tertiary referral center. In NSGCT relapsing following conventional chemotherapy, salvage rates are as low as 15–40% using standard salvage protocols such as PEI–VIP or PEI–VeIP. In some institutions the addition of paclitaxel to ifosfamide and cisplatin has been favored due to a high response rate >50%. Conventional-dose cisplatin-based salvage chemotherapy can achieve long-term remission in 15–40% of patients. Early consideration of high-dose chemotherapy seems advisable: trials suggest a benefit for the use of high-dose chemotherapy and autologous bone marrow transfer, with 46% and 50% of the patients being alive and disease-free after a median follow-up of 31 months and 30 months, respectively. Options for third-line chemotherapy are combinations such as paclitaxel and gemcitabine, gemcitabine and oxaliplatin or paclitaxel, and gemcitabine and cisplatin, within clinical trials. Genetics With regard to predisposing genetic events, the locus Xq27 predisposes for bilateral TC and bilateral cryptorchidism. Other studies have reported the loci 1p36, 4p14–13, 5q21–21, 14q13–q24.3, and 18q21.1–21.3 to be highly associated with TC. It has been demonstrated that somatic mutations of exons 10, 11, and 17 of KIT occur significantly more often in patients with bilateral TC as compared to patients with unilateral disease. The results indicate that KIT might be involved in the development of familial and a minority of sporadic germ cell tumors and that KIT mutations primarily take place during embryogenesis such that primordial germ cells with KIT mutations are distributed to both testes.

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Currently, all molecular markers such as p53, Ki-67, bcl-2, cathepsin D, and E-cadherin have not been proven to be clinically useful prognosticators; only reverse transcriptase-polymerase chain reaction for AFP, hCG, and germ cell alkaline phosphate (GCAP) mRNA for the detection of circulating tumor cells appears to be an interesting approach, with 60% of clinical stage I testicular cancer patients exhibiting positive signals that turn into negative signals following adjuvant chemotherapy. Future Directions in TC Based on the excellent therapeutic outcome, there appear to be only a few developments possible that will have further impact on the survival of testicular cancer patients. However, there might be many options to improve quality of life either due to reduction of acute toxicity or due to the development of treatment regimes associated with a significantly reduced long-term toxicity. The risk of cardiovascular disease is significantly increased after standard chemotherapy with three to four cycles of PEB and/or salvage treatment (RR = 2.59). The increased risk is not due to an increase in classical cardiac risk factors but directly dependent on first-line therapy. For the future, attempts to minimize treatment should be undertaken especially in patients with good prognosis in whom this type of long-term toxicity might be a greater risk to long-term survival than testicular cancer itself. Elucidation of those mechanisms involved in the development of intrinsic and extrinsic chemorefractoriness in testicular cancer will be a major issue in the future, to apply effective chemotherapeutic protocols and to save even more lives. There are some promising approaches using modern molecular techniques such as gene expression profiling to explore the role of mismatch repair genes, multidrug-resistance genes, and potentially unknown genes. Despite the high cure rates, it will be necessary for testicular cancer to be treated by clinicians and institutions with sufficient experience in diagnosis and management of germ cell tumors. Specific problems such as extended tumor masses, relapsing tumors, or

Testicular Cancer

poor prognosis at initial diagnosis must be referred to tertiary centers having the ability of an interdisciplinary approach.

Cross-References ▶ Carcinoma in Situ

References Albers P, Albrecht W, Algaba F, Bokemeyer C, CohnCedermark G, Fizazi K, Horwich A, Laguna MP, Nicolai N, Oldenburg J (2015) Guidelines on testicular cancer: 2015 update. Eur Urol 68(6):1054–1068 Beyer J, Albers P, Altena R, Aparicio J, Bokemeyer C, Busch J, Cathomas R, Cavallin-Stahl E, Clarke NW, Claßen J, Cohn-Cedermark G, Dahl AA, Daugaard G, De Giorgi U, De Santis M, De Wit M, De Wit R, Dieckmann KP, Fenner M, Fizazi K, Flechon A, Fossa SD, Germá Lluch JR, Gietema JA, Gillessen S, Giwercman A, Hartmann JT, Heidenreich A, Hentrich M, Honecker F, Horwich A, Huddart RA, Kliesch S, Kollmannsberger C, Krege S, Laguna MP, Looijenga LH, Lorch A, Lotz JP, Mayer F, Necchi A, Nicolai N, Nuver J, Oechsle K, Oldenburg J, Oosterhuis JW, Powles T, Rajpert-De Meyts E, Rick O, Rosti G, Salvioni R, Schrader M, Schweyer S, Sedlmayer F, Sohaib A, Souchon R, Tandstad T, Winter C, Wittekind C (2013) Maintaining success, reducing treatment burden, focusing on survivorship: highlights from the third European consensus conference on diagnosis and treatment of germ-cell cancer. Ann Oncol 24 (4):878–888 Cavalli F, Manfardini S, Pizzocaro G (1980) Report on the international workshop on staging and treatment of testicular cancer. Eur J Cancer 6:1367–1372 Daneshmand S, Albers P, Fosså SD, Heidenreich A, Kollmannsberger C, Krege S, Nichols C, Oldenburg J, Wood L (2012) Contemporary management of postchemotherapy testis cancer. Eur Urol 62(5):867–876 Fung C, Fossa SD, Williams A, Travis LB (2015) Longterm morbidity of testicular cancer treatment. Urol Clin North Am 42(3):393–408 Heidenreich A, Srivastava S, Moul JW et al (2000) Molecular genetic parameters in pathogenesis and prognosis of testicular germ cell tumors. Eur Urol 37:121–135 International Germ Cell Consensus Classification Group (1997) A prognostic factor-based staging system for metastatic germ cell cancers. J Clin Oncol 15:594–603 Oldenburg J, Fosså SD, Nuver J, Heidenreich A, Schmoll HJ, Bokemeyer C, Horwich A, Beyer J, Kataja V; ESMO Guidelines Working Group (2013) Testicular seminoma and non-seminoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 24(Suppl 6):vi125–32

Testicular Germ Cell Tumors Schmoll HJ, Souchon R, Krege S et al (2004) European consensus on diagnosis and treatment of germ cell cancer: a report of the European Germ Cell Cancer Consensus Group (EGCCCG). Ann Oncol 15:1377–1399 Skakkebaek NE, Bertlesen JG, Giwercman A et al (1987) Carcinoma in situ of the testis: possible origin from gonocytes and precursor of all types of germ cell tumours except spermatocytic seminoma. Int J Androl 10:19–28

See Also (2012) Germ Cell Tumors. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1541. doi:10.1007/978-3-642-16483-5_6905

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Definition Testicular cancer represents a group of histologically heterogeneous neoplasms typically arising in gonadal tissue and, uncommonly, arising in extragonadal sites such as the retroperitoneum or mediastinum. Tumors of the testis include germ cell tumors (which include seminomas and nonseminomas) or sex cord tumors (includes (▶ Leydig Cell Tumor, Sertoli, and ▶ granulosa cell tumors). With improvement of care, even metastatic disease is now curable in many men with testicular cancer.

Characteristics

Testicular Feminization (TFM) ▶ Androgen Receptor

Testicular Germ Cell Tumor ▶ Testicular Cancer

Testicular Germ Cell Tumors Kamran Zargar-Shoshtari1, Craig Kovitz2, Phillippe E. Spiess3 and Nizar M. Tannir4 1 Department of Urology, Moffitt Cancer Center and Research Institute, Tampa, FL, USA 2 Department of Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA 3 Department of Genitourinary Oncology, Moffitt Cancer Center, Tampa, FL, USA 4 Department of Genitourinary Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA

Synonyms Dysgerminoma; Gonadal neoplasms; Testicular tumors

Incidence Testicular cancers represent only 1% of male tumors and 5% of genitourinary malignancies; however, germ cell tumors are the most common malignancy in men between the ages of 20–40 years. These tumors demonstrate a bimodal distribution in occurrence, and they are most often seen in 15–25 year-old men with a second, smaller peak at about age 60. Overall incidence of seminoma peak is in the 40s and nonseminoma in 30s. It is estimated that in the Western society, 3–10 new cases per year occur per 100,000 males. Over the past few years, the incidence of testicular cancer has significantly increased, with the highest rise reported in the Great Britain, United States, and Northern Europe. Risk Factors Cryptorchidism has been identified as a major risk factor for development of testicular cancer, although only about 10% of cases are associated with this phenomenon. Data suggest that orchidopexy prior to puberty may reduce the risk of testicular cancer. This increased risk is true for the contralateral testicle even if it is descended normally. Additional risk factors include a previous history of testicular tumor as well as the presence of a first degree relative with the disease. Testicular dysgenesis syndrome (cryptorchidism, hypospadias, reduced spermatogenesis, and subfertility) is also linked to the development of

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testicular cancer. Scrotal trauma and toxic exposures have no proven association with the occurrence of germ cell tumors. Histological Classification The main histological categories of germ cell tumors are seminomas (seminomatous germ cell tumor) and nonseminomas (nonseminomatous germ cell tumor). Nonseminomas are further subcategorized as embryonal carcinomas, endodermal sinus tumors (also known as yolk sac tumors), choriocarcinoma, and teratoma. Tumors that contain more than one histological subtype are termed mixed germ cell tumors. For classification and treatment purposes, any tumor not histologically a pure seminoma is classified as a nonseminoma. Non–germ cell tumors are known as sex cord or stromal tumors and include Leydig cell, Sertoli cell, Granulosa cell, as well as some other rare types of stromal tumors. Clinical Presentation and Diagnosis Painless testicular swelling or a palpable testicular mass is the most common form of presentation for testicular malignancy. Associated pain is often related to infarction or bleeding within the tumor. Systemic symptoms at presentation such as abdominal and back pain, anorexia with or without associated weight loss, night sweats, chest pain, and shortness of breath or hemoptysis usually indicate an advanced stage of disease or an extragonadal primary tumor. Testicular ultrasound is the ideal form of radiological examination for assessment of scrotal masses, with sensitivity approaching 100%. Magnetic resonance imaging (MRI) is also an option, although this remains less practical. Additionally, the tumor markers human chorionic gonadotropin (hCG), ▶ alpha-fetoprotein (AFP), and lactate dehydrogenase (LDH) should be obtained at diagnosis. These have value in diagnosis, staging, and evaluation of response to therapy as well as follow up assessment of germ cell tumors. Although not specific for germ cell tumors, AFP is produced by tumors with endodermal sinus or embryonal components as well as by immature teratomas. HCG is a hormonal product of syncytiotrophoblasts and can be expressed

Testicular Germ Cell Tumors

by choriocarcinoma, mixed germ cell tumors and, sometimes, by seminomas. LDH is a cellular protein expressed in numerous tissues and can be produced by nonseminomas. Additional preoperative workup typically includes a chest radiograph and discussion of sperm banking with the patient. When a testicular mass is found on ultrasonography, a radical inguinal orchidectomy is the standard of care. However, in certain circumstances when the patient has extensive systemic disease, systemic chemotherapy should be started prior to an orchidectomy. In this instance, it would be appropriate to proceed with chemotherapy without tissue diagnosis, if the markers are elevated and the clinical picture is compatible with the diagnosis of germ cell tumor. Postoperatively (or preoperatively if this will not delay surgery) an abdominal and pelvic computed tomography (CT) scan should be performed and, if clinically indicated, brain magnetic resonance imaging (MRI) and a bone scan. CT scans can reveal clinically significant lymphadenopathy that will be important in staging and clinical decision-making. The preferred sites of initial spread for right-sided tumors are typically the infrarenal paracaval, interaortocaval, and possibly paraaortic nodes. In contrast, left-sided tumors preferentially spread to the infrarenal paraaortic nodes initially. Direct spread from right side of the retroperitoneum to the left side, but not vice versa, is also possible. Transscrotal biopsies should be avoided as they can disturb lymphatic channels, potentially changing the typically predictable lymphatic spread of these tumors. Organ sparing orchidectomy is possible in certain clinical situations, including synchronous bilateral testicular tumors or in tumor in a solitary testis. Organ sparing surgery can only be considered when there is normal preoperative testosterone levels and when the tumor volume is less than 30% of the total testicular volume and clear surgical margins can be achieved. Staging and Risk Stratification The aim of staging is to classify patients with respect to prognosis and to allow for standardized

Testicular Germ Cell Tumors

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Testicular Germ Cell Tumors, Table 1 International germ cell cancer consensus group classification prognostic risk stratification Good risk

Intermediate risk

Poor risk

Seminoma Any disease primary site No nonpulmonary visceral metastases Normal markers

82% 5-year PFS; 86% 5-year OS Any primary site Nonpulmonary visceral metastases Normal AFP Any hCG Any LDH 67% 5-year PFS; 72% 5-year OS –

Nonseminoma Testis or retroperitoneal primary No nonpulmonary visceral metastases S0 AFP < 1,000 ng/mL S1 hCG < 5,000 mIu/mL LDH < 1.5  (ULN) 86% 5-year PFS; 90% 5-year OS Testis/retroperitoneal primary No nonpulmonary visceral metastases S2 AFP 1,000–10,000 ng/mL hCG 5,000–50,000 mIu/mL LDH 1.5–10  ULN 75% 5-year PFS; 80% 5-year OS Mediastinal primary Nonpulmonary visceral metastases S3 AFP > 10,000 ng/mL HCG > 50,000 mIu/mL LDH > 10  ULN 41% 5-year PFS; 48% 5-year OS

ULN upper limits of normal range, PFS progression free survival, OS overall survival

treatment planning. Primary tumor characteristics, extent and size of lymphadenopathy, presence of visceral metastases as well as post orchidectomy tumor markers are important factors in determining accurate disease stage. The standard risk stratification used for these tumors is that developed by the International Germ Cell Cancer Consensus Group (IGCCCG). Through a retrospective multivariate analysis of nearly 6,000 patients with germ cell tumors, this group identified a number of clinical features which are strongly associated with prognosis: primary disease site, the presence of nonpulmonary visceral metastases, and tumor marker levels after orchidectomy. Based on these characteristics, IGCCCG divided nonseminomatous germ cell tumors into good-, intermediate-, and poor-risk categories and seminomas into good- and intermediate-risk categories (Table 1). This risk stratification system provided the basis for the American Joint Commission on Cancer (AJCC) TNM staging system for germ cell tumors. In general, stage I disease is confined to the testis, stage II disease is limited to the retroperitoneum, and stage III disease involves the nodal disease beyond the retroperitoneum or

nonnodal metastatic disease. Elevated serum tumor markers can help to define higher stages of disease. Management As the biology and management of seminomas and nonseminomas are quite different, we will consider the management of these distinct histologies separately. Seminomas

By IGCCCG risk stratification, all patients with seminomas without nonpulmonary visceral metastases are categorized as having good-risk disease. Amongst these patients are those with stages I and II disease and some with stage III disease. The majority of patients with stage I disease (TxN0M0, based on CT scan staging) will be cured with radical inguinal orchidectomy alone; however, at 5 years, 15–20% of patients with stage I seminoma will relapse, mainly in the infradiaphragmatic lymph nodes. As a result, attempts have been made to identify those features of stage I disease which would indicate a higher

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risk of relapse. It has been reported that there is a subset of patients with stage I seminoma with a primary tumor smaller than 4 cm in size and without rete testis involvement who have a relapse-free survival of 88%, and thus may constitute a group of patients most appropriate for surveillance. Although, the latter finding has not been validated by other studies. At present, surveillance is considered a reasonable option for highly motivated patients with stage I seminoma after orchidectomy. Adjuvant chemotherapy is an alternative choice, with one or two cycles of carboplatin reducing relapse rates to 1–3%. Seminoma is also extremely radiosensitive and adjuvant 20 Gy of radiotherapy to a paraaortic field will reduce the relapse rate to 1–3%. Some have demonstrated that single dose of carboplatin may be less toxic than radiotherapy in stage I seminoma. Patients with stage II disease (infradiaphragmatic nodal disease only) are divided into those with nonbulky disease (those with nodal metastases less than 5 cm) and those with bulky disease (those with nodal metastases greater than 5 cm). For patients with nonbulky disease (stage IIA-B), infradiaphragmatic radiation therapy (30 Gy stage IIA AND 36Gy stage IIB) to include the paraaortic and ipsilateral iliac nodes is the standard therapy. This technique yields a relapse-free survival in stage IIA and IIB of 92% and 90%, respectively. Residual abnormalities are sometimes encountered following radiation therapy, but observation is generally recommended. Alternatively, primary chemotherapy with four cycles of EP (eposide/cisplatin) or three cycles of BEP (▶ Bleomycin/▶ Etoposide/▶ Cisplatin) is an option. There are no phase III trials comparing chemotherapy with radiation in this setting, although chemotherapy offers similar disease-free rates as radiation therapy. Some authorities recommend chemotherapy as the first choice for stage IIB, and radiation therapy as the preferred choice for stage IIA. Retroperitoneal node dissection or surveillance is not recommended in this clinical situation. For patients with advanced seminoma, defined as having stage IIC or stage III disease, chemotherapy is the treatment modality of choice. For

Testicular Germ Cell Tumors

those patients with good-risk disease, chemotherapy with four cycles of EP is generally offered. Bleomycin is generally excluded as the risk of pulmonary toxicity outweighs the small incremental benefit afforded by its use. In the same light, carboplatin has been demonstrated to be inferior to cisplatin and is thus not substituted in this scenario. For those patients with intermediate-risk disease, chemotherapy is usually administered with four cycles of BEP. After completion of chemotherapy, patients with advanced seminoma are restaged with chest, abdominal, and pelvic CT scans as well as serum tumor markers. If no residual mass is found, or the residual mass measures 3 cm in size, a ▶ positron emission tomography (PET) scan is performed 6 weeks after chemotherapy to assess for the presence of viable tumor. In the presence of a positive PET scan, second line chemotherapy or retroperitoneal lymph node dissection can be considered. Patients who are found to have progressive disease after initial therapy are generally treated with salvage chemotherapy. These include VeIP (vinblastine, ifosfamide, cisplatin), TIP (paclitaxel, ifosfamide, cisplatin), or high dose chemotherapy. Nonseminomatous Germ Cell Tumors (NSGCT)

NSGCT are risk stratified according to the IGCCCG classification schema based on the location of the primary tumor, the presence of nonpulmonary visceral metastases, and the level of tumor marker elevation. Treatment options vary by stage and can include observation, chemotherapy, and/or retroperitoneal lymph node dissection (RPLND). Patients with stage IA NSGCT (tumor limited to the testis and epididymis without lymphovascular invasion and normal postorchidectomy tumor markers) are generally managed with either surveillance (in reliable patients) or RPLND. The data from largest surveillance studies suggest a relapse rate of about 30%, with 80% of relapses seen within the first 12 months, 12% seen in the second year, and 6% during the third year, with

Testicular Germ Cell Tumors

1% occurring during the fourth and fifth years. Patients who relapse while on surveillance can be treated with chemotherapy with excellent longterm outcomes. RPLND is often used because it usually leads to accurate staging and can be curative in the majority of patients. The presence of pN1 or pN2 disease at RPLND can then be managed either with surveillance (pN1) or two cycles of EP or BEP chemotherapy (pN2). Patients with N3 disease found at RPLND are typically managed as good-risk advanced stage patients and are treated with EP for four cycles or BEP for three cycles. Patients with stage IB NSGCT can be managed with RPLND, although either active surveillance (for T2 disease only) or chemotherapy with two cycles of BEP is also appropriate. One or two cycles of BEP for stage I disease has reported relapse rates of 1.6–3.1%. Patients with Stage IS disease (persistent marker elevation after orchidectomy) are managed with chemotherapy (either four cycles of EP or three cycles of BEP). Stage IIA NSGCT in the presence of negative postorchidectomy tumor markers are generally approached with either RPLND or primary chemotherapy with EP for four cycles or BEP for three cycles. Those patients with stage IIA disease who have persistent tumor marker elevations are managed with chemotherapy alone. Patients with stage IIB disease and negative markers may undergo RPLND as long as lymph node metastases are within lymphatic drainage sites. PostRPLND management is the same as for those patients who have RPLND for stage I disease. If the patient has multifocal lymph node metastases, adjuvant chemotherapy is the preferred management option. Patients with advanced disease (stage IIC and III) are risk stratified by the IGCCCG into three categories (good, intermediate, and poor). Those with good-risk disease (testis or retroperitoneal primary; no nonpulmonary visceral metastases as well as AFP 2 cm but 4 cm in greatest dimension Tumor >4 cm in greatest dimension Tumor invades adjacent structures (e.g., through cortical bone, into deep extrinsic muscle of tongue, maxillary sinus, skin; superficial erosion of bone/tooth socket by gingival primary is not sufficient to classify as T4) Regional lymph nodes cannot be assessed No regional lymph node metastasis Metastasis in single ipsilateral node, 3 cm in greatest dimension Metastasis in single ipsilateral node, >3 cm but 6 cm Metastasis in multiple ipsilateral nodes, all 6 cm Metastasis in bilateral or contralateral nodes, all 6 cm Metastasis in lymph node >6 cm in greatest dimension Distant metastasis cannot be assessed No distant metastasis Distant metastasis (continued)

Tongue Cancer 0 I II III IV

Tis N0 M0 T1 N0 M0 T2 N0 M0 T3 N0 M0, T1–2 N1 M0, T3 N1 M0 T4 N0–1 M0, anyT N2–3 M0, anyT anyN M1

It has been shown by many studies that tumor thickness, but not the largest dimension, is a significant independent prognostic factor in predicting subclinical nodal metastasis, local recurrence, and survival of oral tongue carcinoma. The thicker the tumor, the higher would be the risk of local recurrence, subclinical nodal metastasis, and treatment failure. It is however still unresolved yet on the best cutoff thickness value for staging purpose. The proposed cutoff thickness values for prognosis or staging by various studies vary between 3 and 9 mm. The other important prognostic factor is histologic feature of perineural spread which is a significant risk factor of local recurrence after surgical treatment. Pretreatment Assessment Preoperative endoscopy and biopsy should be done to confirm the diagnosis and evaluate the extent of local tumor infiltration. Small neck nodes less than 1 cm along the jugular chain may not be palpable with fingers. Ultrasonography of the neck and ultrasound-guided fine-needle aspiration for cytology should be done to screen for presence of small nodal metastasis that may not be palpable. Tumor thickness is an important factor for prognosis evaluation and treatment planning. Preoperative assessment of tumor thickness cannot be done with palpation. Intraoral ultrasonography using 7.5 MHz probe can be used to document the tumor thickness. MRI can also be used to assess the tumor thickness for preoperative evaluation of prognosis. Both T1- and T2-weighted MRI images can show the tumor thickness satisfactorily. MRI images in three-dimensional planes can also help the surgeon in the planning of surgical resection. Treatment Options The treatment of oral tongue carcinoma remains controversial. Brachytherapy alone or surgery

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alone is each commonly used as primary treatment for early carcinomas by radiation oncologists and surgeons. The curative results of brachytherapy and surgery are similar for early stage carcinoma with over 90% of local control. Either surgery or brachytherapy alone is not effective for stage III and IV carcinomas; combined surgery and postoperative chemoradiotherapy are recommended for advanced-stage carcinomas. Other less commonly practiced alternative treatment options include laser surgery or photodynamic therapy for early stage carcinomas. Concurrent intra-arterial regional chemoperfusion of high dose of cisplatin and radiotherapy may be considered as alternative treatment option for advanced T4 stage carcinoma. Controversy of Elective Neck Dissection Versus Observation of N0 Neck Since there is high risk of nodal recurrence of observed N0 neck of early tongue carcinoma, elective neck dissection is commonly practiced in many cancer centers worldwide. In retrospective studies of elective selective neck dissection versus observation of N0 neck, the regional recurrence rates could be reduced from 30 to 50% of observation to 10–15% after for elective selective I, II, III neck dissection. The regional recurrence related mortality rates could be reduced from 20% to 25% for observation to 4–10% for elective selective I, II, III neck dissection group. From the results of retrospective studies, elective neck dissection can reduce both initial regional recurrence rate and regional recurrence related mortality, and the reduction of noderelated mortality contributes to long-term survival benefit. There is however no prospective randomized study comparing the long-term benefit of elective neck dissection compared with observation in the treatment of N0 neck of early stage I and stage II oral tongue carcinoma. There is risk of mortality and morbidity of elective neck dissection. Both elective neck dissection and observation have their proponents in different cancer centers. Instead of performing elective neck dissection for all patients with early tongue carcinoma, patients should be informed of the

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possible choice of both treatment options of either observation or elective neck dissection of N0 neck. Observation is particularly suitable for patients with thin carcinomas of less than 3–4 mm. The risk of nodal recurrence of patients with thin tumors of 3–4 mm is in the range of 10–15%. Patients choosing observation treatment of N0 neck should be advised to have regular follow-up after primary treatment for early detection of nodal recurrence. Early nodal recurrence can be salvaged with modified or radical neck dissection successfully. Of those patients who cannot be closely followed up, elective neck dissection is a more suitable treatment of choice. Outcome of Treatment Local or regional lymph node recurrences account for over 90% of recurrences. Majority of local recurrences cannot be salvaged. Over 90% nodal recurrences of closely observed neck can be successfully salvaged with neck dissection. Of those patients with elective selective neck dissection of cN0 neck, the nodal recurrence rate of pN0 neck is less than 5% and is 30–40% for pN+ neck. Radiotherapy of pN+ neck is therefore advised. The overall 5-year disease-free survival rates are in order of 90–100% for stage I, 60–80% for stage II, 30–50% for stage III, and less than 20% for stage IV.

Topoisomerases

▶ Osteopontin ▶ p21 ▶ Podoplanin ▶ Retinoid Receptor Cross-Talk ▶ Vascular Endothelial Growth Factor

References Tsantoulis PK, Kastrinakis NG, Tourvas AD et al (2007) Advances of the biology of oral cancer. Oral Oncol 43(6):523–534 Yuen APW (2004) Cancer of the tongue: operative techniques in otorhinolaryngology. Head Neck Surg 15:234–238 Yuen APW, Wei WI, Wong YM et al (1997) Elective neck dissection versus observation in the surgical treatment of early oral tongue carcinoma. Head Neck 19:583–588

See Also (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

Topoisomerases Wenjian Ma National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC, USA

Cross-Reference Definition ▶ Adhesion ▶ Akt Signal Transduction Pathway ▶ Angiogenesis ▶ Apoptosis ▶ Bcl2 ▶ Cyclin D ▶ E-Cadherin ▶ Epidermal Growth Factor-like Ligands ▶ Ets Transcription Factors ▶ Furin ▶ Maspin ▶ Matrix Metalloproteinases ▶ Motility ▶ Oral Cancer

DNA topoisomerases are a class of enzymes required for the regulation of DNA folding and supercoiling to maintain chromosome integrity and cellular function. They are essential for relaxing entangled DNA and modulating DNA topology by transiently breaking and rejoining DNA single or double strands. Topoisomerases play a key role during DNA replication, transcription, and chromosome segregation. They are also involved in resolving specific DNA structures and intermediates arising from other cellular processes such as DNA repair and recombination.

Topoisomerases

Characteristics To fit into the tiny nucleus, cellular DNA, which has a combined length of several meters, is compacted and bound by proteins to form chromatin, which is twisted and folded into a space of only a few microns. However, excess folding of DNA poses a unique challenge to living organisms and generates topological problems when the DNA helix must be unwound to make its genetic information accessible. This highly supercoiled DNA, in the form of condensed chromatin, must be relaxed during the cell cycle in order for cellular processes such as transcription and replication to occur. In addition, as long as both strands of the double helix are covalently continuous, progressive unwinding of one region of DNA increases overwinding and therefore tension, in adjacent regions. This presents an impediment to further unwinding. These types of topological problems were recognized immediately after the discovery of DNA helical structure, but how cells deal with these problems had been a mystery until the discovery of the first topoisomerase in E. coli by James C. Wang in 1971. Afterward, it was shown that topoisomerases bind to either singlestranded or double-stranded DNA and generate transient DNA strandbreaks, allowing the DNA to be untangled or unwound. Topoisomerase Families Multiple topoisomerases, specializing in different types of DNA manipulation and cellular function, have been found in organisms ranging from virus to human. They can be divided into two major categories based on their DNA cleavage property. Type I enzymes wrap around DNA to make a transient break in only one DNA strand, which is important for the release of underwinding or overwinding forces of the DNA helix. Type II enzymes cleave both strands at the same time to untangle supercoiled DNA. Topoisomerases with an odd number after their names belong to type I (e.g., Top1), and those with even numbers belong to type II (e.g., Top2a). The letters A, B or a, b following Roman numbers are used to distinguish between subfamily members that are different in their sequence/structure, polarity of

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strand cleavage, etc. So far, six topoisomerases have been identified in mammalian cells, namely, TOP1 and TOP1mt (mitochondria form), TOP2a and b, and TOP3a and b. Molecular Mechanism The catalytic reactions of all topoisomerases share some common features. These enzymes contain a nucleophilic tyrosine to promote DNA strand scission and covalently bind to either the 30 - or 50 -end of the broken DNA. Formation of such transient enzyme-DNA adducts is believed to prevent the exposure of the broken DNA ends that may lead to genome instability. Following breakage of the DNA strands, unwinding/relaxation of DNA is through one of the following two mechanisms: Rotation – The nicked DNA strand spins around the phosphodiester bond of the other intact DNA strand. After releasing the overwinding force, the topoisomerase catalyzes religation of the nick to restore the DNA double helix. This reaction does not require ATP or divalent metal binding, and the spin is controlled by friction between the DNA and the enzyme. Strand passage – A single- or double-stranded DNA is nicked and physically opened. A homodimer of the enzyme binds at the break ends to serve as a “gate” allowing the passing of the other intact DNA strands. Finally, it rejoins the broken DNA to finish the unwinding/untangling procedure. This mechanism uses ATP to power strand passage. Depending on their cellular function and the DNA structures to resolve, topoisomerases of the same category may have different enzymatic mechanisms. For example, TOP1 and TOP3 are both type I topoisomerases, but Top1 enzymes unwind/relax DNA through the controlled rotation mechanism, whereas TOP3 uses “strand passage.” These differences are reflected in their cellular function. While TOP1 is required for DNA replication and transcription dealing with the DNA helix, TOP3 plays a role in resolving Holliday junctions during recombination, where two DNA molecules are intertwined. Both Top2

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and Top3 enzymes modulate DNA topology by the strand passage mechanism, but with different targets. Top2 enzymes cleave both strands of a DNA duplex and pass a second intact duplex through the transient break. Top3 enzymes generate a single-strand break in double-stranded DNA, which allows another single-stranded DNA to go through the break. The difference also depends on cell function as Top2 enzymes act on DNA supercoils, intertwined chromosomes, and DNA catenanes. Cellular Functions Topoisomerases recognize specific DNA structures associated with different cellular processes. Their major activity includes modulating the under- or overwound DNA helix, resolving supercoiled or entangled DNA, facilitating decatenation, and participating in chromatin remodeling. They have shown to play important roles in the following cellular processes: Replication – Chromosomal DNA needs to be unwound in order for the replication machinery to gain access. Topoisomerases are needed at a very early stage of replication for replisome assembly. During strand synthesis, they are indispensable since the DNA helix would become either overwound or underwound following the progress of the replication fork, which generates positive supercoils ahead of the replication machinery and negative supercoils behind it. This function requires type 1 topoisomerases. Transcription – Topological problems need to be dealt with when transcribing genetic information from DNA to RNA, which produces DNA supercoiling with the movement of RNA polymerase. If left unchecked, this supercoiling can impede the progress of the transcription protein machinery. Type I topoisomerases are implicated in the removal of supercoils as well as suppressing loop formation. Chromosome segregation – At the G2 phase of the cell cycle, sister chromosomes are catenated or looped around each other. Removal of these catenations is vital for proper chromosome segregation. In addition, organisms with a circular

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genome are also in need of decatenation since the DNA strands are topologically linked or knotted. Topoisomerases II and IV (bacteria) are implicated in these processes. DNA repair and recombination – Topoisomerases also play a role during the repair of DNA damage, especially the repair of DNA doublestrand breaks. Repair of the broken DNA ends often proceeds through a pathway called homologous recombination, which leads to the formation of interlinked intermediates. Resolution of the intermediates was shown to require the function of topoisomerase III, which can introduce single-strand breaks to facilitate the strand exchange between homologous sequences. Other types of recombination events, such as sister chromosome exchange before cell division and the insertion of viral DNA into host chromosomes, also require topoisomerases. Chromosome packaging and condensation – Topoisomerases not only help in relaxing or disentangling DNA, they also participate in chromosome condensation to form the intertwined DNA duplexes and assemble the highly ordered chromatin structure. Topoisomerase II is the major player in this process. It promotes intense supercoiling of the DNA helix to aid in chromosome condensation. This occurs at specific stages of the cell cycle and is regulated by cell cycle control and checkpoint proteins such as p53 and Cdc2. Topoisomerase Inhibition and Cancer Therapy As part of the reaction mechanism, topoisomerases generate transient DNA strand breaks in DNA, which, if not efficiently resealed, can lead to genome instability. Disruption or inhibition of topoisomerase often causes serious problems for DNA metabolism and may even trigger cell death. However, it also generates significant clinical benefits. Some inhibitors targeting bacterial topoisomerases II and IV have been developed as antibiotics to eliminate pathogens and to treat diseases such as tuberculosis and malaria. Most importantly, topoisomerases are especially relevant in cancer.

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Interfering with topoisomerase function is one of the most effective strategies for anticancer therapy. On the one hand, topoisomerases play a central role in various DNA metabolic pathways such as replication that are critical for the fast-growing tumor cells. On the other hand, cancer cells are often defective in one or more of the DNA repair pathways that cannot efficiently deal with topoisomerase-mediated DNA strand breaks. Some chemicals serving as topoisomerase inhibitors prevent enzymatic function by recognizing structural motifs present in the enzymes or by intercalation into specific DNA sequences bound by the topoisomerase. These inhibitors often prevent the nick-resealing step and the dissociation of topoisomerase from DNA, which results in persistent single- or double-strand breaks. In other words, the enzyme is converted into a cellular poison trapped at the ends of DNA strand breaks. While normal cells are capable of repairing such damage to restore cellular function, tumor cells are killed due to gene mutations or deficiency required to repair certain damages, especially DNA double-strand breaks. Most of the inhibitors/drugs selectively target either topoisomerase I or II, and both types of inhibitors have been exploited in cancer therapy. TOP1 inhibitors, such as camptothecin, block DNA replication and interfere with cell proliferation to kill rapidly dividing cancer cells. They have been used to treat ovarian cancers and small-cell lung cancers (SCLC). Topoisomerase II targeting drugs can be classified into two groups, topoisomerase II poisons and topoisomerase II catalytic inhibitors. Topoisomerase II poisons that traps the enzyme onto double-strand breaks are among the most successful anticancer drugs currently in clinical use. So far six top II-related anticancer agents have been approved for use in the United States, more in other countries. For example, the widely used anthracycline drug doxorubicin is highly effective in the treatment of a wide range of cancers including hematological malignancies and many types of carcinoma. Despite their efficacy for many cancers, the use of topoisomerase-inhibiting agents for cancer therapy is limited by common negative effects, such as chronic cardiotoxicity, hematological toxicity, and secondary malignancies.

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References Nitiss JL (2009) DNA topoisomerase II and its growing repertoire of biological functions. Nat Rev Cancer 9(5):327–337 Vos SM, Tretter EM, Schmidt BH, Berger JM (2011) All tangled up: how cells direct, manage and exploit topoisomerase function. Nat Rev Mol Cell Biol 12(12):827–841 Wang JC (2002) Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol 3(6):430–440

Torisel™ ▶ Temsirolimus

Toxicity Testing ▶ Preclinical Testing

Toxicogenomics Scott Auerbach Biomolecular Screening, National Toxicology Program, National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC, USA

Definition Toxicogenomics was first described in 1999 as the application of omic technologies (genomics, transcriptomics, proteomics, and metabolomics) to the study of toxicology. Today the definition should be expanded to include epigenomics. The omic disciplines represented a continuum of biological information starting with the genome, which is then regulated by the epigenome to give rise to the transcriptome, which in turn encodes the proteome whose actions give rise to the metabolome (Fig. 1). Since its inception, The entry “Toxicogenomics” appears under the copyright Springer-Verlag Berlin Heidelberg (outside the USA) both in the print and the online version of this Encyclopedia.

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Toxicogenomics

Toxicogenomics, Fig. 1 Toxicogenomics involves the application of multiple omic disciplines to the study of toxicity and its related diseases. These omics domains represent a continuum of functionally related biological information. The biological entities studied in each domain are distinct; therefore, each omic domain requires distinct measurement techniques to generate quantitative and qualitative information

toxicogenomics has primarily focused on the identification of biomarkers that diagnose or predict disease. In addition, it has been used extensively to shed light on the molecular mechanisms and key events that lead to toxicity.

Characteristics Genomics Genomics is the study of DNA variants (e.g., single nucleotide polymorphisms (SNPs), genomic copy number variations (CNVs)) and their causal role in the manifestation of phenotypes such as disease and chemical toxicity. Approaches, Technology, and Data Analysis

For many years the field of pharmacogenetics/ ecogenetics has focused on single gene genetic association studies to identify the genetic basis for susceptibilities to toxicity. The genes that

served as a focal point of studies were selected based upon biochemistry and molecular biology studies that indicated they play a role in detoxifying/metabolizing xenobiotics. In the broader field of genetics, the first approach developed for querying the entire genome for genotype-phenotype relationships was linkage analysis, which traces transmission of alleles and phenotypes through families to identify regions of the genome that carry an allelic variant which confers a phenotype. Subsequently, with the sequencing of the human genome and the formulation of the human haplotype map, case-control-based genome-wide association (GWA) studies became possible. Finally, in the last 5 years, it has become economically feasible for labs to sequence an entire genome and to identify allelic variants that underlie phenotypes using integrated bioinformatics approaches. The two major technologies that are currently employed for genome-wide genotype to phenotype mapping are SNP genotyping arrays and whole

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exome/genome-wide sequencing. Most SNP arrays work by hybridization methods in which probes representing distinct alleles across the genome are hybridized to label genomic DNA. In the case of exome sequencing, genomic DNA is first enriched through hybridization-based methods for exonic sequence; the enriched DNA is then integrated into sequencing libraries and applied to any number of next-generation sequencing platforms. The output from these machines is a collection of millions of random reads across the genome, which are either de novo assembled into a genome or mapped to a reference genome. Once the mapping is done, variant genomic positions can be identified. Whole genome sequencing is similar with the exception that there is no enrichment for exonic sequence. A number of critical variables go into designing and then subsequently analyzing GWA studies including considerations for statistical power (i.e., population size necessary to detect a statistical association between an allele and a phenotype), genetic stratification between cases and controls, and the potential genic and allelic diversity that underlies the phenotype under study (which relates back to statistical power to detect an association). The data from a GWA study is typically analyzed using a chi-square test to determine if any of alleles are significantly associated with the phenotype under study. Unlike GWA studies, genomic sequencing data has the added complexity of rare mutations. These rare mutations although potentially biologically meaningful create a significant statistical challenge; hence, alternative methods that incorporate prior knowledge of biology (i.e., disease pathways, protein-protein interactions, and evolutionary conservation of the mutated positions) are taken into consideration when determining if a mutation has a plausible causal role in the phenotype under study.

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findings, a number of different resources are often used to provide biological interpretation of associated genes including network-level analysis (i.e., gene coexpression and/or protein-protein interaction networks). In addition extensive literature mining is often performed to piece together plausible biological relationships between the phenotype under study and the associated gene(s). Applications in Toxicology

It is notable that some of the first discoveries in human genetics related to drug toxicity (e.g., isoniazid toxicity and N-acetyltransferase, quinine antimalarial toxicity, and glucose-6-phosphate dehydrogenase). Genome-wide association studies of the side effects of select drugs have identified genetic variation in the HLA class I and class II genes, HLA-A, SLC01B1, CERKL, and CYP2C8, as determinants of susceptibility for drug-induced liver toxicity, skin hypersensitivity reactions, myotoxicity, QT prolongation, and osteonecrosis of the jaw, respectively. It is notable that the genes identified by these studies are disproportionately associated with inflammatory processes and drug metabolism and distribution. In addition to studies in humans, a number of groups are leveraging the power of genetically defined inbred strains of mice to identify genetic determinants of susceptibility to toxicity. One example study was published in 2009 that identified allelic variants in Cd44 as being a determining factor for the susceptibility to acetaminophen-induced liver toxicity. Epigenomics Epigenomics is the study of the complete set of epigenetic modifications (DNA methylation, posttranslational histone modification, and nucleosome positioning) on the genome of a cell. Approaches, Technology, and Data Analysis

Data Interpretation

It is often the case in GWA studies that the genes identified do not have a documented biological link to the disease or phenotype under study. This general observation underscores our limited understanding of gene-phenotype relationships and the complex network biology that underlies phenotype. In order to contextualize the GWA

There are a variety of techniques used to analyze the epigenome. They vary based upon the epigenetic modification that is under study and by the degree to which they resolve the genomic location where changes in an epigenetic mark occur. One of the lowest-resolution assessments involves cleavage of chromatin using nuclease digestion. In this assay regions that are hypersensitive to

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nuclease digestion reflect areas where nucleosomes have been removed and the chromatin structure is open. A slightly higher-resolution analysis involves cross-linking proteins to DNA, shearing the DNA, performing immunoprecipitation with antibodies targeted against any number of epigenetic marks, and then labeling and hybridizing immunoprecipitated DNA to microarrays tiled with probes that survey the landscape of the genome. Alternatively, the immunoprecipitated DNA can be incorporated into sequencing libraries in a technique called ChIP-seq and then sequenced using next-generation sequencing technologies. The resultant data is then mapped to the genome to identify areas of that exhibit differing levels of the epigenetic mark under study. Another approach to mapping one particular type of epigenetic mark (DNA methylation) involves the use of restriction endonucleases that are sensitive to the methylation state of cytosine. With this approach differentially digested DNA is either labeled in hybridized to the genome-wide CpG array, or alternatively fragments are evaluated by next-generation sequencing. Still another technique for evaluating DNA methylation is the use of sodium bisulfite which converts unmethylated cytosine to uracil, but has no effect on methylated cytosine. The converted DNA can then be hybridized to arrays that contain probes that are complementary to either the cytosine or uracil-containing sequence. Alternatively, the bisulfite-treated DNA can be analyzed using next-generation sequencing where the methylation state can be identified for individual nucleotides by mapping sequence reads to a virtual bisulfate-treated or untreated genome sequence. Data Interpretation

Currently there is a limited understanding of how the various epigenetic marks translate into gene regulatory processes. This is particularly true for DNA methylation as the degree of methylation does not correlate with gene expression. Hence, it is often challenging to translate epigenomic findings at a gene level to clear molecular mechanism or biological significance. However, as with other functional genomic data, pathway and gene ontology analysis tools can potentially be

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used (assuming epigenetic marks can be mapped to individual genes) for formulating hypotheses on the role of alterations in the epigenome. Applications in Toxicology

Toxicity-related epigenomics has become an area of significant interest among researchers. One of the primary reasons for this interest in the epigenomics relates to concerns about early life exposure and its impacts on epigenetic patterning, which may lead to altered risk of disease later in life. In addition, studies of identical twins have shown significant epigenetic drift over a lifetime suggesting a strong environmental influence on the epigenome. Studies in rodents have suggested chemicals that alter the epigenome during development may do so through alteration of the methylation associated with parasitic DNA (e.g., retrotransposons) which can lead to expression of these elements and subsequent related genomic instability. In addition, alterations in repeat element DNA methylation have been demonstrated to alter expression of genes proximal to the repeat elements. Still further studies have demonstrated that target organ toxicity at a level that produces cell proliferation causes global DNA hypomethylation, therefore suggesting a dynamic role for epigenomics in tissue repair and adaptation to toxic stress. Transcriptomics Transcriptomics is the genome-wide study of RNA (i.e., mRNA, miRNA, ncRNA) expression levels. Approaches, Technology, and Data Analysis

There are two primary approaches to highdimensional transcriptomic analysis; hybridizationbased microarrays, and direct sequencing of cDNA fragments using next-generation sequencing (RNA-seq or digital gene expression). In a typical microarray experiment, RNA is extracted from a biological sample, reverse transcribed into cDNA, labeled with fluorescent dyes, and then hybridized to a high-density microarray which contains antisense oligonucleotides that correspond to most, if not all, annotated genes for the species under study. Hybridized arrays are then

Toxicogenomics

scanned to determine the degree of hybridization to each spot, and the intensity data that is collected is normalized using any number of algorithms. There are a variety of microarray types; the most common is a 30 in which the probes hybridize with 30 end of a transcript. Arrays have been developed that cover a wider range of the transcriptome. The arrays contain probes that hybridize each exon allowing for the identification of alternative splicing. In the case of RNA-seq, one starts with a similar RNA preparation to that used for microarrays; however, depending upon which RNA species an investigator desires to evaluate, different purification steps are employed. The purified RNA is then sheared to a size that is appropriate for the sequencing technology that will be employed, and then the fragmented RNA will then be incorporated into a cDNA library using primer ligation PCR. The library is then applied to a next-generation sequencer and individual fragments are sequenced and recorded. In a typical RNA-seq experiment, many millions of individual reads per sample are generated that can range in size from 50 to over 500 base pairs in length. The sequences are then mapped back to the genome in essence creating histograms across the genome for every nucleotide, which when normalized and compiled at the gene level corresponds to a detailed picture of transcript expression across the genome. The added value of RNA-seq is most notable when it is performed with whole transcript protocol as it allows for the sequence level identification of alternatively spliced transcripts and fusion transcripts that arise in cancer due to rearrangement of the genome. As with other high-dimensional data types discussed here, there are a number of multivariate approaches that can be used to analyze transcriptomic data. In general these analyses break down into two categories, supervised and unsupervised. The distinction between these approaches relates to the integration of data labels into the analysis (e.g., cancer vs healthy). In unsupervised analysis relationships in the data are driven by the data itself in the absence of sample context, whereas supervised analysis takes into consideration sample type and will

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often prefilter data to identify genes that differentiate phenotypes under study which in turn can be used to generate predictive/diagnostic, machine learning-based models. Another critical concept that needs to be addressed in multivariate data analysis is the issue of multiple testing corrections. This issue arises when there are a large number of statistical tests (e.g., all genes on a microarray; all SNPs on SNP array) in a single comparison (e.g., treated vs control). Just by chance alone 5% of all genes on an array will exhibit significant differential expression if a P 105. One of the primary strengths of NMR-based metabolomics is the robust reproducibility of the method. Further NMR-based metabolomics does not involve fractionation or destruction of the sample, therefore allowing for further study of biospecimens. Its primary liability is its low sensitivity although developments using so-called cryoprobes and low-volume probes have pushed the limits of detection for NMR into the 105 M range. Mass spectral-based metabolomics involves a wide variety of platforms and methodologies. There are two basic approaches to MS-based metabolomics, those that fractionate complex mixtures prior to MS analysis and those in which complex mixtures are directly injected into the mass spectrometer. The inherent challenge with mass spectral-based metabolomics is the simultaneous ionization of multiple analytes. In order to detect an analyte using mass spectrometer, the analyte must be ionized. In metabolomics this is most often achieved by electrospray ionization which applies enough energy to induce ionization without destroying the analyte. In the case of metabolomics, one is trying to apply a charge to many metabolites all it wants; hence, there is competition for applied energy which may favor a subset of metabolites. To limit this competition, fractionation techniques have been coupled with mass spectrometry. The fractionation methods that are used include gas chromatography, highperformance liquid chromatography, and capillary electrophoresis. Gas chromatography provides high chromatographic resolution; however, since this technique can only fractionate volatile chemicals, it is necessary to derivatize many metabolites prior to analysis which can introduce experimental variability. High-performance liquid chromatography has lower resolution than gas chromatography; however, with the development of ultrahigh-pressure liquid chromatography systems that use small particle stationary phases, it is possible to significantly enhance chromatographic resolution of HPLC systems. Capillary electrophoresis theory has greater separation efficiency than high-performance liquid chromatography

Toxicogenomics

and is amenable for use with a greater variety of metabolites than gas chromatography. In order for the analytes to be detected following separation by one of the above techniques, they need to undergo ionization before injection into the mass spectrometer. In GC-MS, electron impact ionization is often used that breaks each analyte into numerous ionized fragments which in turn produces a fragment pattern in the subsequent mass spectral analysis. Fragmentation patterns can then be compared to those of known metabolites therefore allowing for metabolite identification. In LC-MS the most common method of ionization is electrospray ionization (ESI). This form of ionization preserves the molecular ion therefore making the spectral patterns slightly less complex. Direct injection or surface-based metabolomics was developed primarily to avoid the analytical variability introduced by chromatography; however, the lack of fractionation introduces some challenges including salts, charge competition, and the inability to distinguish compounds with the same molecular weight. Matrix-assisted laser desorption/ionization (MALDI) was the first technology used in direct injection metabolomics. When performing MALDI a matrix is applied to the biological sample to allow for desorption and ionization of the metabolites following laserbased energy transfer. Due to matrix-associated challenges with MALDI, such as high background in the low-molecular-weight range and crystal formation that leads to challenges with tissue imaging, a number of other technologies that are matrix independent have been developed. Nanostructure-initiator mass spectrometry (NIMS), secondary ion mass spectrometry (SIMS), and desorption electrospray ionization (DESI) are all matrix-free techniques used for direct injection metabolite studies. Each analytical approach and platform requires distinct approaches to data processing and analysis. In the case of fingerprinting methods, various types of supervised and unsupervised multivariate analysis is performed in the case of NMR following parsing of spectra into frequency bins or in the case of GC/LC-MS following the collection of data features such as chromatographic retention

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time, mass charge (m/z) pairs, and peak intensity values. Analysis of data using multivariate approaches allows for the development of classification models of different biological states which can then be applied to subsequent studies. Often times simple fingerprints are not satisfactory to biologists and specific identification/quantification of analytes is critical to understanding biological processes. In the case of NMR, annotation of spectral peaks is done by comparison to spectral standards that are available through several commercial and publicly available databases. Analyte identification in MS-based metabolite mix is faced with the challenge of deconvoluting complex spectral patterns that arise due to the process of analyte ionization. As with NMR the identification of spectral patterns and assignment to specific molecular species is dependent upon repositories of spectra generated from standards. With respect to quantification, NMR peak integrals are directly proportional to the concentration of the nucleus under study; hence, quantification of metabolites is relatively straightforward following deconvolution of the data. Quantification of metabolites from MS data is slightly more challenging because ionizability varies between metabolites in addition to the challenges associated with ion suppression that arise due to competition for ionization energy. This makes absolute quantification only possible when spectra are calibrated against different concentrations of standards. Hence, it is often the case that quantification occurs in targeted follow-up studies.

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toxicity. This premise was the basis of the COMET consortium which generated spectral profiles from over 100 short-term rodent toxicity studies (Lindon et al. 2005). These profiles were then used to train a number of predictive models. In addition to the efforts of the COMET consortium, metabolomics has been used to identify biomarkers of the hepatic and renal toxicity and to identify diseases that are commonly associated with toxic exposure (e.g., alcoholic liver disease). Metabolomics has also been employed in mechanistic toxicology to characterize a number of processes including microsomal enzyme induction, oxidative stress, myotoxicity, and inflammationassociated hepatotoxicity.

References Afshari CA, Hamadeh HK, Bushel PR (2011) The evolution of bioinformatics in toxicology: advancing toxicogenomics. Toxicol Sci 120(Suppl 1):S225–S237 Daly AK (2012) Using genome-wide association studies to identify genes important in serious adverse drug reactions. Annu Rev Pharmacol Toxicol 52:21–35 Lindon JC, Keun HC, Ebbels TM, Pearce JM, Holmes E, Nicholson JK (2005) The Consortium for Metabonomic Toxicology (COMET): aims, activities and achievements. Pharmacogenomics 6(7):691–699 Merrick BA, Witzmann FA (2009) The role of toxicoproteomics in assessing organ specific toxicity. EXS 99:367–400 Robertson DG, Watkins PB, Reily MD (2011) Metabolomics in toxicology: preclinical and clinical applications. Toxicol Sci 120(Suppl 1):S146–S170 Szyf M (2011) The implications of DNA methylation for toxicology: toward toxicomethylomics, the toxicology of DNA methylation. Toxicol Sci 120(2):235–255

Data Interpretation

Biological interpretation of metabolomics results often involves performing biochemical pathway enrichment analysis. In order to understand where critical perturbations occur in biochemical system, it is often necessary to know the levels at which feedback control function and where rate limiting steps in a biochemical pathway occur.

T Toxicological Carcinogenesis Takuji Tanaka Department of Oncologic Pathology, Kanazawa Medical University, Kanazawa, Japan

Application to Toxicology

One of the initial applications of metabolomics was in the area of toxicity prediction. It was hypothesized that analysis of biofluids would provide metabolite signatures (fingerprints) of

Synonyms Chemical carcinogenesis; carcinogenesis

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Definition In the broadest possible sense, ▶ carcinogenesis is a process of generation of benign and malignant neoplasm. Agents such as viruses, radiation, and chemicals are able to induce ▶ cancer in humans and experimental animals. However, the importance of chemicals as a cause of cancer has long been recognized in basic and clinical studies and is emphasized by the epidemic of tobacco-related lung cancer in the twentieth century. Carcinogenesis may be considered as a form of toxicity in which cells achieve a different steady state from the normal and do not respond normally to homeostatic mechanisms. Carcinogenesis induced by chemicals is called “toxicological (chemical) carcinogenesis.” Basic and clinical research in the field of toxicological carcinogenesis has led to many major advances, ranging from the fields of epidemiology and international human studies to laboratory research on mechanisms involved in the complex processes that are associated with the initiation and development of malignant disease (cancer). Many chemical carcinogens have been identified, and their effects documented in experiments in which animals exposed to the agents at the maximum tolerated dose develop neoplasm. Toxicological carcinogenesis and human cancer epidemiology studies have clearly identified specific chemicals that can act as human carcinogens in both occupational and environmental settings. The main groups of relevance to human disease include ▶ polycyclic aromatic hydrocarbons, aromatic amines, nitrosamines, ▶ alkylating agents, and heterocyclic amines. Cancer resulting from exposure to chemicals in the environment has taken on new importance. Knowledge about the mechanisms and natural history of cancer development from toxicological carcinogenesis as well as epidemiology of human cancer is critical in the control and prevention of human neoplastic disease.

Characteristics Mutagens are agents that can permanently alter the genetic constitution of a cell. The most widely

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used screening test, the Ames test, uses the appearance of mutants in a culture of bacteria of the Salmonella species. Approximately 90% of known carcinogens are mutagenic in this system. Moreover, most, but not all, mutagens are carcinogenic. This close correlation between carcinogenicity and mutagenicity presumably occurs because both reflect ▶ DNA damage. The in vitro mutagenicity assay is a valuable tool in screening for the carcinogenic potential of chemicals. Cultured human cells are also being increasingly used for assays of mutagenicity. Chemical carcinogens may cause development of neoplasm either directly or indirectly. They can be grouped into two main classes according to the mechanism by which they stimulate development of neoplasm: (i) Genotoxic carcinogen causes direct damage to DNA by forming chemical/ ▶ DNA adducts. The abnormal areas of DNA are prone to damage in replication and some adducts are resistant to normal ▶ DNA repair mechanisms. (ii) Non-genotoxic carcinogen is a carcinogen for which there is no evidence of direct interaction with cellular DNA. This type of carcinogen can be divided into two subgroups. Mitogenic carcinogen binds to receptors on or in cells and stimulates cell division without causing direct DNA damage. In experimental skin carcinogenesis such agents have been shown to bind to and activate protein kinase C, causing sustained epidermal hyperplasia. Cytotoxic carcinogen produces tissue damage and leads to hyperplasia with cycles of tissue regeneration and damage. In some cases it is believed that cytokines generated in response to tissue damage act as mitogenic factors. Chemical carcinogens can be further divided into two groups: (i) direct-acting carcinogen (the agent is capable of directly causing neoplasia) and (ii) procarcinogen (the agent requires conversion to an active carcinogen). This conversion takes place through normal metabolic pathways. In procarcinogens the ▶ cytochrome P450 (CYP) monooxygenase system plays an important role in conversion in many instances. ▶ Detoxification reactions also occur, the accumulation of carcinogen being determined by a balance between (i) dose of procarcinogen, rate of

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Toxicological Carcinogenesis, Fig. 1 Toxicological carcinogenesis as a multistep process

detoxification and elimination, and (ii) rate of conversion to the active form. Three stages have been defined in toxicological carcinogenesis (Fig. 1). Studies of toxicological carcinogenesis among experimental animals have shed light on the individual stages in the progression of normal cells to cancer. From these studies, one can define three stages (▶ multistep development) of toxicological carcinogenesis: 1. Initiation is the first stage and likely represents mutations in a single cell. The nature of the initial changes in cells is still uncertain. In experimental toxicological carcinogenesis in the skin, the Harvey-▶ RAS gene has been identified as being frequently mutated. This gene is involved in epidermal proliferation, and when it becomes abnormal, epidermal cells are less responsive to signals that

normally cause terminal differentiation. Only relatively few genes have been identified as being mutated in other animal models of toxicological carcinogenesis. 2. Promotion follows initiation and is characterized by clonal expansion of the initiated cell. Induction of cell proliferation takes place at this stage. The altered cells do not exhibit autonomous growth, but remain dependent on the continued presence of the promoting stimulus, including an exogenous chemical or physical agent or an endogenous mechanism, such as hormonal stimulation. In this phase of carcinogenesis, a promoting agent brings about increased cell proliferation. Promotion is initially reversible if the promoting agent is withdrawn. 3. Progression is the third stage, in which growth becomes autonomous and is independent of the

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carcinogen or promoter. At this stage, additional genomic changes presumably endow cells with a relative growth advantage that, in turn, results in their further clonal expansion. Cancer is the end result of the entire sequence and is established when the cells acquire the capacity to invade and metastasize. If there is persistent cell proliferation, initiated cells acquire secondary genetic abnormalities in oncogenes, which first lead to dysregulation and eventually to autonomous cell growth. The ultimate end point of progression is development of an invasive neoplasm. The various tests that have been applied to identifying agents with carcinogenic potential may be classified into several general areas on the basis of the time involved in the assay: short, medium, and long. These include short-term tests for mutagenicity (e.g., the Ames test), gene mutation assays in vivo (e.g., the LacZ mouse, the LacI mouse, the LacI rat), assay for chromosomal alterations (e.g., ▶ micronucleus assay, sister chromatid exchange), measurement of primary DNA damage in vitro and in vivo, and chronic bioassays for carcinogenicity (e.g., chronic 2-year bioassay, medium-term bioassays-Ito model, multistage models of neoplastic development, transgenic and knockout mice as models of carcinogenesis). History It is widely recognized that exposure to chemicals in the workplace and the environment can contribute to human cancer risk. This was first indicated in 1775 by Dr. Pott, who attributed scrotal skin cancers to prolonged exposure to soot in London chimney sweeps. In 1914, Dr. Boveri first hypothesized that cancer was a genetic disease, prior to the discovery of the genetic material. In 1915, Dr. Yamagiwa and co-workers successfully induced skin cancer in rabbits by painting their ears continuously with benzene solutions of tar. In the 1930s Dr. Kenneway and co-workers demonstrated that pure chemicals isolated from coal tar could also produce tumors in animals. In the 1950s there were parallel discoveries of the structure

Toxicological Carcinogenesis

of the DNA double helix and its establishment as the hereditary material and mutagenic potential of ionizing radiation and certain chemical carcinogens in humans and experimental systems and extensive investigations into the relationship between chemically induced mutations and human cancer. The 1980s saw the elucidation of the first oncogenes that appeared to be responsible for the initiation of cancer as first predicted by Dr. Boveri. This era also saw the development of the Ames Salmonella bacterial mutagenesis assay (the Ames test) and similar genetic toxicology assays. These developments firmly established the basic paradigm for the field of toxicological carcinogenesis: chemicals capable of induction of mutations are presumed to be carcinogens. It was predicted that any chemical or physical agent that can covalently damage DNA could also cause mutations through its DNA-damaging mechanism and hence can be a carcinogen. The data that followed in the 1990s appeared to strongly support this central assumption, as numerous chemicals that were initially tested for DNA damage or mutations were also carcinogens in experimental animals. Since then, our understanding of the molecular basis of cancer has improved substantially. In addition, investigations into the molecular basis of toxicological carcinogenesis, as well as more extensive human cancer epidemiology studies using modem molecular tools, have greatly expanded our knowledge in this area.

Cross-References ▶ Adducts to DNA ▶ Alkylating Agents ▶ Cancer ▶ Carcinogenesis ▶ Cytochrome P450 ▶ Detoxification ▶ DNA Damage ▶ Micronucleus Assay ▶ Multistep Development ▶ Polycyclic Aromatic Hydrocarbons ▶ Repair of DNA

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References

Definition

Clayson DB (2001) Toxicological carcinogenesis. CRC Press LLC, Boca Raton Ito N, Tamano S, Shirai T (2003) A medium-term rat liver bioassay for rapid in vivo detection of carcinogenic potential of chemicals. Cancer Sci 94:3–8 Sugimura T, Ushijima T (2000) Genetic and epigenetic alterations in carcinogenesis. Mutat Res 462:235–246 Tanaka T (1997) Effect of diet on human carcinogenesis. Crit Rev Oncol Hematol 25:73–95 Williams GM, Iatropoulos MJ, Weisburger JH (1996) Chemical carcinogen mechanisms of action and implications for testing methodology. Exp Toxicol Pathol 48:101–111

The TP53 ▶ tumor suppressor gene is located on chromosome 17p13.1 and encodes a ubiquitous phosphoprotein of molecular mass 51,000–53,000, essentially expressed in the nucleus. This gene is frequently inactivated by somatic mutation or by loss of alleles in many common human cancers. More than 25,000 such mutations have been described so far. Inherited, heterozygous mutations have been identified in about 400 families with ▶ Li-Fraumeni syndrome and Li-Fraumeni-like syndromes (LFS and LFL), characterized by the early occurrence of cancers at multiple organ sites. TP53 belongs to a ▶ p53 family that also includes TP73 (1p36) and P63 (3p28). In contrast with TP53, these two genes have a restricted, tissue-specific, and developmental expression pattern and are not frequently mutated in cancer. The p53 protein is a latent transcription factor that is activated in response to multiple forms of physical and chemical stress to exert diverse, complementary effects in the regulation of cell proliferation, genetic integrity, and survival. These effects include:

See Also (2012) Cytotoxic Carcinogen. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1058. doi:10.1007/978-3-642-16483-5_1498 (2012) Direct-Acting Carcinogen. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1124. doi:10.1007/978-3-642-16483-5_1641 (2012) DNA Repair. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1141. doi:10.1007/978-3-642-16483-5_1687 (2012) Genotoxic Carcinogen. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1540. doi:10.1007/978-3-642-16483-5_2394 (2012) Human Cancer Epidemiology. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1752. doi:10.1007/978-3-642-164835_2846 (2012) Mitogenic Carcinogen. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2336. doi:10.1007/978-3-642-16483-5_3772 (2012) Non-Genotoxic Carcinogen. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2537. doi:10.1007/978-3-642-16483-5_4109 (2012) Procarcinogen. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2989. doi:10.1007/978-3-642-16483-5_4746

TP53 Pierre Hainaut International Prevention Research Institute, Lyon, France

Synonyms p53

• Induction of ▶ apoptosis • Control of cell division through regulation of cell cycle progression in G1 and G2, ▶ centrosome duplication, and mitosis • Modulation of DNA replication and ▶ repair of DNA The main function of the p53 protein is to act as an “emergency brake” to prevent the proliferation of cells with damaged genetic material, caused by exposure to genotoxic agents (Fig. 1). In a broader context, the protein acts as an integrator of multiple exogenous and intracellular signals to regulate cell proliferation during replicative senescence, differentiation, and development. Inactivation of TP53 in mice resulted in accelerated development of multiple tumors, while a fraction of p53-deficient embryos displayed a lethal defect in neural tubule closure, resulting in exencephaly.

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4602 TP53, Fig. 1 The p53 pathway. The p53 protein is induced in response to various forms of stress and mediates a set of coordinated, antiproliferative responses including cell cycle arrest, control of replication, transcription, repair, and apoptosis. Blue: factors that bind to p53 and that are regulated by protein interactions. Red: factors that are regulated by p53 at the transcriptional level

TP53

Genotoxic stress γ and X rays UV chemical carcinogens

p14arf

p21

Non-genotoxic stress Hypoxia depletion of ribonucleotides or microtubules

JNK

mdm2

14-3-3σ Gadd45

CDK PCNA

CDC25

G1 G1/S

G2/M

Cell cycle arrest

Characteristics The TP53 gene spans 20 kb and contains 11 exons, the first one being noncoding. The coding sequence contains five regions showing a high degree of conservation in vertebrates, located in 2, 5, 6, 7, and 8. An orthologue has been described in Drosophila. Several gene polymorphisms are identified in the human population, with allele frequencies that vary with ethnic origin. However, there is only limited evidence to prove that these polymorphisms play a role in tumor susceptibility. The TP53 gene does not contain a conventional TATA box but is under the control of several ubiquitous transcription factors, including NFkB, Sp1, and Jun. It is expressed in the form of one major transcript of 2.8 kb and several isoforms generated by alternative splicing or use of an alternative promoter, intron 4.

XPB XPD RPA

Control of DNA replication, transcription and repair

Bax Apol/Fas Killer/DR5 Pig 3, 6, 12 IGF-BP3

Apopstosis

The protein contains 393 residues and is organized in a hydrophobic, central core (residues 110–296, encoded by exons 5–8) flanked by an acidic N-terminus and a basic C-terminus (Fig. 2 top). The N-terminus contains two complementary transcriptional activation domains, with a major one at residues 1–42 and a minor one at residues 55–75, specifically involved in the regulation of several proapoptotic genes. The central core is made of a scaffold of two b-sheets supporting a set of flexible loops and helices stabilized by the binding of an atom of zinc. These loops and helices make direct contact with DNA sequences containing inverted repeats of the motif RRRC(A/T). The C-terminus contains the main nuclear localization signals and oligomerization domains (residues 325–366). The active form of the protein is a tetramer (in fact, a pair of dimers). The extreme C-terminus has multiple regulatory functions and exerts a negative control on

TP53 TP53, Fig. 2 Diagram of the p53 protein structure. c: linear structure, showing the three main structural domains. Codon numbers of the main mutation hotspots are shown as colored boxes. Sites of posttranslational modifications are shown as “P” (phosphorylations), “A” (acetylations), and “Z” (zinc binding sites). bottom: 3-D structure of the central core of p53 in complex with target DNA. Hotspot residues are shown in the same color code as above

4603 N-terminus

Core domain

C-terminus

1-42

96-296

325-393

A

P P P

sequence-specific DNA-binding activities. Both N- and C-terminal regions contain multiple posttranslational modification sites, while only a few have been identified so far in the central core (see Table 1). Upstream of p53: Signaling of DNA Damage The p53 protein is constitutively expressed in most cells and tissues as a latent factor. Due to its rapid turnover (5–20 min), the protein does not accumulate unless it is stabilized in response to a variety of intracellular and extracellular stimuli. Signals that activate p53 include diverse types of ▶ DNA damage (strand breaks, bulky adducts, oxidation of bases), blockage of RNA elongation, ▶ hypoxia, depletion of microtubules, ribonucleotides or growth factors, modulation of cell ▶ adhesion, and alteration of polyamine

A P P

P

Z Z ZZ

175

A

273

245 248

249

282

P

Phosphorylation sites

A

Acetylation sites

Z

Zinc-binding sites

metabolism. Most of the current knowledge of p53 protein activation is derived from studies using DNA strand breaks as inducing signals. The main regulator of p53 protein activity is ▶ MDM-2, a protein which binds p53 in the N-terminus (residues 17–29); it conceals its transcription activation domain, redirects p53 from the nucleus to the cyclasm, and acts as a ubiquitin ligase to target p53 for degradation by the ▶ proteasome. The MDM-2 gene is a transcriptional target of p53, thus defining a regulatory feedback loop in which p53 controls its own stability. The p53/mdm-2 complex is regulated by Arf (alternative reading frame), a 14 kD protein encoded by the p16/CDKN2A gene. The kinetics, extent, and consequences of p53 activation vary according to the nature and intensity of the inducing signals. In response to

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TP53

TP53, Table 1 Factors involved in the activation and posttranslational modification of p53 Factor PARP HMG-1 E6AP Hif-1 14-3-3 s p300/CBP c-abl mdm-2 NO Cdc2/Cdk2Cyclin A/B cdk7-cyclin H CKII

Biochemical function/activated by p53 ADP-ribose polymerase/DNA strand breaks, nucleotide depletion High mobility group 1/? E6 accessory protein/ubiquitin-mediated degradation Hypoxia-inducible factor/hypoxia Cell cycle regulator/ionizing radiations Histone acetyltransferases/co-activators of transcription Tyrosine kinase/irradiation, DNA strand breaks Oncogene/negative control of p53 Nitric oxide/oxidative stress, inflammation, irradiation Cell cycle-dependent kinases Component of TFIIH Kinase/UV

MAPK ATM DNA-PK Chk-2 JNK/p38 PKC CKI

Mitogen-activated protein kinase/UV? Kinase/ionizing radiations Kinase/UV Cell cycle-dependent kinase Stress-activated kinases/UV Protein kinase C Kinase/?

p19arf

Cell cycle inhibitor, alternative product of CDKN2A Redox-repair enzyme/oxidative stress, hypoxia

Ref-1

ionizing radiation, activation of the p53 protein is thought to proceed through several consecutive steps, with first phosphorylation of p53 in the N-terminus by kinases involved in the sensing of DNA damage such as Atm (the product of the ataxia telangiectasia mutated gene) and Chk-2 (a cell cycle regulatory kinase). These phosphorylations contribute to the dissociation of the p53/ mdm-2 complex and stabilize the protein. Second, p53 binds co-activators with acetyltransferase activity such as ▶ P300/CBP co-activators and pCAF. These factors acetylate p53 in the C-terminus. These processes, as well as other coordinated posttranslational modifications of the C-terminus, induce conformational changes that turn the protein into an active form with a

Interaction with p53 ADP-ribose polymers bind to p53 Binding to N-terminus or to DNA-binding domain Binding to p53 Binding to p53 Binding, C-terminus (Ser-376) Binding, N-terminus acetylation, C-terminus Binding, proline-rich region Binding, residues 13–29 Oxidation of cysteines in DNA-binding domain Phosphorylation of Ser-315; forms complexes with p53 Phosphorylation of Ser-33 Phosphorylation of Ser-389; forms complexes with p53 Phosphorylation, Thr-73, and 83 (mouse p53) Phosphorylation, Ser-15 Phosphorylation, Ser-15 and Ser-37 Phosphorylation, Ser-20 Phosphorylation, Ser-34, mouse p53 Phosphorylation, Ser-378 Phosphorylation, several N-terminal serines (including Ser-6 and Ser-9) Prevents p53-mdm-2 interactions Reduction of cysteine in DNA-binding region binding to C-terminus

high affinity for specific DNA-binding sites. The third step involves redox regulation of sensitive cysteines within the DNA-binding domain of the protein. This three-step mechanism may account for p53 induction in response to most forms of DNA damage. Downstream of p53: Cell Cycle Control, Apoptosis, and DNA Repair Once activated, p53 exerts its effects through two major mechanisms: transcriptional control (activation or repression of specific genes) and complex formation with other proteins. Important downstream effectors of p53 (Table 2) include regulators of ▶ cell cycle checkpoints (in G1/S, G2, and during mitosis), factors involved in the

TP53

4605

TP53, Table 2 Some important downstream effectors of p53 functions Factor Apo-1/Fas/CD95

Activity Death signaling receptor

Bax-1

Dominant negative inhibitor of Bcl-2 Repressor of apoptosis Inhibitor of IGF-I Death signaling receptor Regulatory subunit of PI3 kinase Glutathione transferase homologue Quinone oxidase homologue Proline oxidase homologue Growth factor Survival factor Inhibitor of angiogenesis Binding to PCNA Inhibitor of proliferation Inhibitor of CDK2–4 and 6

Bcl-2 IGF-BP3 Killer/DR5 P85 Pig-12 Pig-3 Pig-6 IGF-I IL-6 Thrombospondin-1 Gadd45 BTG2 p21waf-1 Cyclin A Cyclin G GPx NOS2/iNOS COX2 Pig-1 PCNA RPA

Cell cycle regulation, S phase Cell cycle regulation Glutathione peroxidase Inducible nitric oxide synthase Inducible cyclooxygenase Galectin-7 Auxiliary subunit of polymerase d Replication protein A

ERCC2/ERCC3

Helicases, TFIIH complex

P53RR2 TBP

Ribonucleotide reductase homologue TATA box-binding protein

Mdm-2 MDR-1

Oncogene Multidrug resistance

signaling of apoptosis, and components of the transcription, replication, and repair machineries. At the cellular level, activation of p53 most frequently results in either cell cycle arrest (mostly in G1 and/or G2/M) or apoptosis. How a given cell “chooses” between cell cycle arrest and apoptosis in response to specific stimuli may depend upon many factors, such as the nature and intensity of the stress, as well as the cell type. In many tissues, p53 plays a role in drug-induced apoptosis and is thus

Mode of regulation Transcriptional activation? Transcriptional activation

Function Apoptosis

Transcriptional repression Transcriptional activation Transcriptional activation Transcriptional activation Transcriptional activation

Apoptosis Apoptosis Apoptosis Apoptosis Apoptosis

Transcriptional activation Transcriptional activation Transcriptional repression Transcriptional repression Transcriptional activation Transcriptional activation Transcriptional activation Transcriptional activation

Apoptosis Apoptosis Apoptosis? Apoptosis? Apoptosis? Cell cycle arrest? Cell cycle arrest, G1 Cell cycle arrest, G1, and G2/M Cell cycle arrest, G1/S Cell cycle arrest? Control of oxidative stress Control of oxidative stress Control of oxidative stress? Differentiation? DNA repair/replication

Transcriptional repression Transcriptional activation Transcriptional repression Transcriptional repression Transcriptional repression Transcriptional activation Transcriptional activation

Apoptosis

Inhibition by protein binding Activation by protein binding Transcriptional activation

DNA repair/replication

Inhibition by protein binding Transcriptional activation Transcriptional repression

Inhibition of transcription

DNA repair/transcription DNA repair?

Repression of p53 Resistance to chemotherapy

an important effector in the response of cancer cells to chemo- or radiotherapy. In addition, loss of p53 function results in deficient cell cycle arrest, inefficient mitotic spindle checkpoint, aberrant centrosome duplication, premature reentry into S phase, ▶ genomic instability, and ▶ aneuploidy. Clinical Relevance The TP53 gene is often inactivated by missense mutations, in contrast with many other tumor

T

4606

TP53

GC to TA

GC to AT(CpG)

Colon Thyroid Digestive organs Haematopoietic Brain Urinary system Female gential organs Skin Bladder Breast Prostate Connective tissue Rectum Head and neck Kidney Esophagus Liver Bronchus and lung 50

40

30

20

10

5

10

15

20

25

30

35

40

TP53, Fig. 3 Prevalence of two common mutation types: G to T transversions and C to T transitions at dipyrimidine (CpG) repeats in tumors of various organs. Tumors with high prevalence of G to T transversions often have a low

prevalence of transitions and vice versa. G to T transitions are a common molecular signature of many environmental carcinogens, such as tobacco smoke components (lung and esophageal cancers) or dietary mycotoxins (liver cancer)

suppressors such as APC, RB1, ▶ BRCA1, or p16/▶ CDKN2A that are inactivated by gene deletion or truncation. The mutations described to date mostly occur in the region of the gene encoding the DNA-binding domain. Most of these mutations impair DNA binding by disrupting the structure of the domain or crucial contact points between the protein and target DNA. About 30% of missense mutations affect six “hotspot” codons (175, 245, 248, 249, 273, and 282) (Fig. 2 bottom). The other mutations are scattered over 300 different codons. Mutations are very common in the invasive stages of many

epithelial tumors. A database of all published mutations is available at the International Agency for Research on Cancer (▶ IARC TP53 Database, http://www-p53.iarc.fr/). In many cancers, the patterns of mutations show variations, revealing clues about the mechanisms responsible for the formation of the mutations. Specific carcinogen-induced mutations have been identified in ▶ hepatocellular carcinoma (mutations induced by ▶ aflatoxins in sub-Saharan Africa and in Southeast Asia), in skin tumors (double transitions at adjacent cytosines, a typical signature of mutagenesis by UV in

TRA-8

squamous and in basal cell carcinomas), and in lung cancers (G to T transversions associated with exposure to tobacco smoke; ▶ tobacco carcinogenesis; (Fig. 3)). The usefulness of TP53 mutation detection in molecular pathology is still a matter of debate. As mutation often results in the accumulation of the protein, ▶ immunohistochemistry (IHC) has often been used as a criterion to detect TP53 abnormalities. However, positive IHC does not always correlate with mutation as several common missense mutants, as well as most frameshift and nonsense mutants, do not result in protein accumulation. Several well-established methods have been described for the detection of mutations in the TP53 gene, including SSCP (single-stranded conformation polymorphism) analysis, TTGE (temporal temperature gradient electrophoresis), yeast-based functional assays, and microarray hybridization assays. TP53 gene mutations are good markers for the clonality of tumor lesions. In many tissues, mutation correlates with bad prognosis and poor response to therapy, but TP53 mutation has been shown to behave as an independent marker of prognosis only in rare cases such as breast and head and neck cancers. Evidence suggests that the nature and position of the mutation may help to predict poor response to treatment. Detection of circulating anti-p53 antibodies as well as of free plasmatic DNA-containing mutant TP53 may be of interest in the early detection of cancer lesions. TP53 is the target of several experimental therapeutic approaches. Gene transfer of wild-type TP53 into cancer cells has been tested in several human tumors. However, the effects reported to date are limited and, at best, transient. Another approach is based on the use of cytolytic viruses selectively replicating in TP53-deficient cells (ONYX vectors). Several preclinical studies have investigated the use of small lipophilic compounds or peptides to activate TP53 function or to restore the activity of mutant proteins.

4607

References Foster BA, Coffey HA, Morin MJ et al (1999) Pharmacological rescue of mutant p53 conformation and function. Science 286:2507–2510 Levine AJ, Hu W, Feng Z (2006) P53 pathway: what questions remain to be explored. Cell Death Differ 13:1027–1036 Lowe SW, Bodis S, McClatchey A et al (1994) p53 status and the efficacy of cancer therapy in vivo. Science 266:807–810 Olivier M, Hussain SP, Caron de Fromentel C et al (2004) TP53 mutation spectra and load: a tool for generating hypotheses on the etiology of cancer. IARC Sci Publ 157:247–270 Vogelstein B, Lane D, Levine AJ (2000) Surfing the p53 network. Nature 408:307–310

TP63 ▶ p53 Family

TP73 ▶ p53 Family

TP73L ▶ p53 Family

T TPI ▶ Cystatins

Cross-References

TRA-8

▶ p53 Family

▶ TRAIL Receptor Antibodies

4608

Trabectedin

Trabectedin Federico Gago1 and Sergio Moreno2 1 Departamento de Ciencias Biomédicas, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain 2 Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Salamanca, Spain

Synonyms Ecteinascidin Yondelis

743;

ET-743;

NSC-684766;

Definition A potent antitumor tetrahydroisoquinoline alkaloid in clinical development originally derived from a marine tunicate and now obtained by a synthetic process developed by PharmaMar starting from microbially produced cyanosafracin B.

Characteristics Crude aqueous ethanol extracts of the ascidian, or sea squirt, Ecteinascidia turbinata were shown to have powerful immunomodulating and antiproliferative properties as early as 1969, but the active principles were not identified until the early 1990s. The first six alkaloids that were characterized received the names ecteinascidins 729, 743 trabectedin (Fig. 1), 745, 759A, 759B, and 770, in accordance with the molecular masses ascribed to these compounds. They revealed a unique chemical structure consisting of a novel pentacyclic skeleton composed of two fused tetrahydroisoquinoline rings (subunits A and B) linked to a ten-member lactone bridge through a benzylic sulfide linkage and attached through a spiro ring to an additional ring system (subunit C) made up of either tetrahydroisoquinoline (as in trabectedin) or tetrahydro-b-carboline (as in ET-736). The first two subunits bear a clear structural resemblance to

microbially derived safracins and saframycins and also to sponge-derived renieramycins, all of them less potent anticancer agents than ecteinascidins. On the other hand, a reactive a-carbinolamine or hemiaminal (N-C-OH) group is also present in naphthyridinomycins, quinocarcins, and pyrrolo [4]benzodiazepine antibiotics such as anthramycin, sibiromycin, and tomaymycin. By analogy to these related antibiotics, the potent biological activity of trabectedin and other ecteinascidins was rapidly associated with their ability to form covalent adducts to DNA, following in situ dehydration of the carbinolamine group to an iminium intermediate that is covalently attached to the amino group of guanine in the minor groove. Ecteinascidia turbinata was first harvested from the wild and then successfully grown by the Spanish pharmaceutical company, PharmaMar, in aquaculture facilities in Spain (near Formentera Island, in the Mediterranean Sea). Subsequently, several synthetic schemes were developed to produce the multigram quantities required for clinical studies worldwide and overcome the limitation of the very low yield (0.0001%) of trabectedin in its natural source. The first enantioselective total synthesis of trabectedin was achieved in 1996, but industrial manufacturing was made possible through a synthetic route involving the conversion of cyanosafracin B, readily available by fermentation of the bacterium Pseudomonas fluorescens, to trabectedin in a very short and straightforward way developed by PharmaMar. Structural and Biophysical Characterization of Trabectedin-DNA Adducts Direct evidence that trabectedin alkylates duplex DNA at the exocyclic amino group of guanines was provided by a variety of experiments including gel electrophoresis, DNA footprinting, nuclear magnetic resonance (NMR) spectroscopy, and band shift assays, as well as molecular modeling studies. As a result of this work, it was found that trabectedin was protonated on N12 at physiological pH, and a role for hydrogen bonding in sequence recognition and orientation in the DNA minor groove was demonstrated, with TGG, CGG, AGC, GGC, and AGA being established

Trabectedin

a

4609

b

HO C NH MeO

OMe

O HO

O H3C

O

A

S O

H3C

CH3

12

N B

O

CH3

N 21

O

OH

Trabectedin, Fig. 1 Chemical formula and three-dimensional stick representation of the X-ray crystal structure of trabectedin

as the preferred DNA triplets for stable adduct formation and much higher rates of reversibility being measured for site-directed AGT- versus AGC-containing adducts. The proposed mechanism for activation takes advantage of the increased strength of the hydrogen bond between the proton on N12 and the hydroxyl group on C21 as the trabectedin molecule approaches the minor groove and is desolvated. This proton, which is essential for both sequence recognition and adduct stabilization, would then catalyze the dehydration of the carbinolamine, yielding the reactive iminium intermediate that undergoes nucleophilic attack at C21 by the exocyclic amino group of the guanine. Since a similar mechanism operates in the activation of pyrrolo[4]benzodiazepine antibiotics, it appears that nature ensures the reactivity of these carbinolamine-containing molecules by the inclusion of an internal catalytic proton adjacent to the leaving hydroxyl group. As a consequence of trabectedin bonding, the double helical structure is only minimally perturbed except for the widening of the minor groove and a net smooth bending toward the major groove due to the introduction of positive roll. This latter feature was novel among minor groove DNA monoalkylating agents as covalent modification of N3 of adenine in AT-rich regions by (+)-CC-1065 and related compounds is accompanied by the bending of the DNA into the minor rather than the major groove. Furthermore, if

multiple binding sites for trabectedin are properly phased in a relatively short stretch of DNA, the imposed cumulative curvature could bring closer together specified fragments not contiguous in primary sequence, and the drug could be serving a surrogate protein function. On the contrary, binding of three trabectedin molecules in a headto-tail fashion to three adjacent optimal binding sites would result in no net DNA curvature because the localized bends, brought about by the increase in roll at the sites of covalent attachment, would cancel out over virtually one turn of the helix. In fact, the DNA structure in one such complex (containing the sequence TGGCGGCGG) was shown to be intermediate between the canonical A and B forms of DNA, thereby strongly resembling the conformation that DNA adopts when bound to the consecutive C2H2 zinc fingers that are present in transcription factors (transcription factor) such as EGR-1 and Sp-1 (which bind to the major groove of GC-rich regulatory sequences in many gene promoters) or that observed in the hybrid double helix of template DNA paired to nascent RNA in the active site of RNA tumor viruses (RNAPII) elongation complex. The close contacts and the hydrogen-bonding interaction network that are established between trabectedin and DNA on both sides of the covalent adduct involve both DNA strands and therefore give rise to a significant increment in the stability of the resulting drug-DNA complexes. As a consequence, notable increases in the temperature of

T

4610

thermal denaturation of duplex DNA and substantial blockade of the helicase activities of both simian virus (SV40) large tumor antigen (T-antigen) and bacterial UvrABC and RecBCD enzymes have been reported for DNA oligonucleotides containing trabectedin adducts. This hampering or prevention of strand separation is also expected to result in stalled replication and transcription forks, as observed for a variety of conventional interstrand cross-linkers (e.g., nitrogen mustards, mitomycin, or cisplatin). An added advantage in the case of trabectedin would be the minimal distortions inflicted on the normal DNA structure that could help evade some of the recognition and repair mechanisms used for the processing of cross-links produced in the major groove by these other agents. Biological Activity In vitro cytotoxicity studies with trabectedin and other ecteinascidins established subnanomolar potencies against L1210 and P388 mouse leukemia cells, as well as human A549 lung cancer, HT29 colon cancer, MEL-28 melanoma cells, and human tumors explanted from patients. Tumorspecific responses and concentration-dependent relationships were observed when a soft agar cloning assay was used to determine the effects of a continuous exposure of trabectedin at different concentrations. These experiments clearly indicated that the duration of exposure to trabectedin was an important factor in human tumors, thereby pointing to preferential administration schedules in clinical trials. In vivo activity was then evaluated in several mouse tumor models and a variety of human tumors xenografted into nude mice, including melanoma, non-small cell lung carcinoma, and ovarian cancer. Long-lasting, complete, or partial regressions were observed in both chemosensitive and marginally cisplatin-resistant xenografts at the maximum tolerated dose (MTD), but no activity was seen in highly chemoresistant tumors such as MNB-PTX-1, MEXF 514, and LXFA 629. Importantly, the absence or incomplete cross-resistance with cisplatin and the comparable efficacy against the ovarian carcinoma xenografts justified the clinical assessment of trabectedin in ovarian cancer.

Trabectedin

The activity parameters for trabectedin in the panel of 60 human tumor cell lines of the National Cancer Institute (NCI) Anticancer Drug Screen revealed a rather unique profile that encouraged further development as an anticancer agent. The COMPARE algorithm (COX inhibitors) established a very high correlation coefficient (0.96) with chromomycin A3, an aureolic acid derivative shown to give rise to a pattern of distinct bands in human metaphase chromosomes, thus suggesting similarities in their apoptotic mechanisms. Despite the fact that these two compounds display very different modes of binding to DNA (i.e., covalent versus noncovalent, carbinolamine activation versus ion-mediated dimerization, etc.), both share a strong binding affinity for some common DNA sites, such as the self-complementary hexanucleotide TGGCCA, to which two trabectedin molecules can bind in a tail-to-tail fashion, each covalently bonded to a different strand. Furthermore, these two natural products are known to exert at least part of their cytotoxicity by interfering with DNA replication and transcription. Thus, at physiologically relevant concentrations (1–100 nM), trabectedin has been shown to effectively inhibit intracellular DNA synthesis by decreasing replication origin activity and by inducing unusual replication intermediates that may be blocked in fork progression. In addition, trabectedin is able to abrogate the transcriptional activation of a number of genes, including those encoding the multidrug resistance P-glycoprotein (MDR1), heat shock protein 70 (hsp70), the cell cycle inhibitor p21 Cip1 (p21), and collagen a1(I) (COL1A1). Nevertheless, global gene expression profiling of trabectedin-treated cancer cells has revealed rather complex patterns of both up- and downregulation. The reported effects on MDR1 and additional in vitro data showing enhancement by trabectedin of the cytotoxicity exerted by other chemotherapeutic agents that are substrates for P-gp/MDR1 suggest that combination treatment may be valuable in the clinic. The extremely low concentrations of trabectedin that are necessary to cause cell cycle arrest and cell death are suggestive of a trans-acting mechanism that probably operates

Trabectedin

through one or more cellular DNA damage response pathways or checkpoints. In this respect, it is notable that cell sensitivity to trabectedin appears to be somehow dependent on a proficient transcription-coupled ▶ nucleotide excision repair (TC-NER) machinery and more specifically on the presence of selected components that are implicated in ▶ xeroderma pigmentosum and Cockayne syndromes. Thus, the initial observation that hamster cells deficient in XPB, ERCC1, or CSB, as well as human XPA and XPC cells, had reduced sensitivity to trabectedin was followed by the report that a human colon carcinoma cell line selected for increased (20-fold) resistance to trabectedin (following continuous exposure to increasing concentrations of this drug for 1 year) had a truncated and inactive form of the XPG structure-specific endonuclease. Furthermore, drug sensitivity was restored in all cases upon complementation with the respective wild-type protein. These intriguing effects were recapitulated and expanded using yeast as a simpler eukaryotic model system. It was seen that trabectedin activates the G2-M and S-phase DNA damage checkpoints, in good agreement with the G2/M block and S-phase delay reported in human cells. Likewise, cells deficient in the XPG orthologue (rad13 in Schizosaccharomyces pombe) were shown to be much more resistant to trabectedin and underwent much less DNA damage than the corresponding isogenic wild-type strains. However, it became clear that it was not the missing endonuclease activity of this protein that conferred resistance to trabectedin but the lack of part of its DNA-binding domain in the COOHterminal region. Furthermore, on the basis of a homology model suggesting that the rad13/ DNA/trabectedin ternary complex could be stabilized through the direct interaction of subunit C of the drug with a highly conserved arginine residue, an S. pombe strain carrying an Arg961!Ala point mutation in rad13 was generated. This mutant displayed normal endonuclease and NER activity but was found to be strongly resistant to the drug. Haploid yeast mutants with deletions in the RAD52 epistasis group of genes encoding proteins responsible for homologous recombination

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(HR) and hence most double-strand break (DSB) repair in eukaryotic cells (e.g., rad51, rad22 (the fission yeast counterpart of mammalian rad52), and rad54) were found to be extraordinarily sensitive to trabectedin. This result reinforced other indications that the drug is giving rise (directly or indirectly) to DSBs that need to be repaired by homologous recombination, and the fact that the absence of rad13 partially rescued rad51D cells supports the view that a rad13-containing complex is somehow involved in the induction or irreparability of lethal DSBs. These results may have important implications for the optimal use of trabectedin in cancer therapy because patients harboring tumor cells with proficient NER and deficient HR systems would be expected to respond best to the treatment. Clinical Studies Trabectedin was selected for clinical development in preference to other related ecteinascidins because of its outstanding potency and greater relative abundance in the tunicate. Among the criteria that were taken into account for bringing it into clinical trials in early 1996 both in Europe and in the United States, we can summarize the following: (i) a novel chemical entity harboring a potential new mode of action, (ii) evidence for a positive therapeutic index, (iii) lack of complete cross-resistance with conventional chemotherapeutic agents, and (iv) feasibility of supply for clinical development. Toxicity to trabectedin so far has been shown to follow a transientreversible pattern and to be predictable, doserelated, and mostly limited to bone marrow and liver. Following a favorable opinion adopted by the Committee for Orphan Medicinal Products (COMP) of the European Agency for the Evaluation of Medicinal Products (EMEA), trabectedin was granted by the European Commission’s Orphan Medicinal Product Designation for the treatment of soft tissue sarcoma (STS) in April 2001 and for P-glycoprotein family (OC) in October 2003. The US Food and Drug Administration (FDA) awarded Orphan Drug Designation to trabectedin in the indication of STS in October 2004 and in OC in April 2005.

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Extended phase II trials and comparative studies have produced evidence of long-lasting responses and tumor control in advanced pretreated sarcomas, breast carcinoma, ovarian carcinoma, and prostate cancer. In July 2007, Yondelis received a positive opinion from the EMEA for the treatment of metastatic or advanced soft tissue sarcoma after failure to anthracyclines and ifosfamide. In 2009, trabectedin also received a positive opinion from EMEA, in combination with liposomal doxorubicin, for the treament of patients with relapsed platinum-sensitive ovarian cancer. Finally, in 2015, the FDA approved Yondelis for the treament of specific soft tissue sarcomas (liposarcomas and leiomyosarcomas) that are unresectable or metastatic.

Trace Elements

Traditional Chinese Medicine ▶ Chinese Versus Western Medicine

Traffic ATPases ▶ ABC-Transporters

TRAIL ▶ TNF-Related Apoptosis-Inducing Ligand

References

TRAIL Receptor Antibodies Feuerhahn et al (2011) XPF-dependent DNA breaks and RNA polymerase II arrest induced by antitumor DNA strand interstrand crosslinking-mimetic tetrahydroisoquinoline alkaloids. Chem Biol 18:988–999 Gago F, Hurley LH (2002) Devising a structural basis for the potent cytotoxic effects of ecteinascidin 743. In: Demeunynck M, Bailly C, Wilson WD (eds) Small molecule DNA and RNA binders: from synthesis to nucleic acid complexes. Wiley-VCH, Weinheim, pp 643–675 Herrero AB, Martín-Castellanos C, Marco E et al (2006) Cross-talk between nucleotide excision and homologous recombination DNA repair pathways in the mechanism of action of antitumor trabectedin. Cancer Res 66:8155–8162 Manzanares I, Cuevas C, García-Nieto R et al (2001) Advances in the chemistry and pharmacology of ecteinascidins, a promising new class of anticancer agents. Curr Med Chem Anticancer Agents 1:257–276 Martínez et al (2013) Inhibitory effects of marine-derived DNA-binding antitumour tetrahydroisoquinolines on the fanconi anemia pathway. Br J Pharmacol 170:871–882 Rinehart KL (2000) Antitumor compounds from tunicates. Med Res Rev 20:1–27 Sainz-Diaz CI, Manzanares I, Francesch A et al (2003) The potent anticancer compound ecteinascidin-743 (ET-743) as its 2-propanol disolvate. Acta Crystallogr C 59:o197–o198

Trace Elements ▶ Mineral Nutrients

Claus Belka Department of Radiation Oncology, University of Tübingen, Tübingen, Germany

Synonyms DR4 antibodies; DR5 antibodies; Lexatumumab; Mapatumumab; TRA-8

Definition TRAIL induces ▶ apoptosis preferentially in malignant tissues. Therefore, TRAIL is considered to be a potential antineoplastic drug. Agonistic TRAIL receptor antibodies have been developed as alternative pharmacological tool for apoptosis induction via the TRAIL receptors.

Characteristics After having identified TRAIL as a member of the family of cell death-inducing ligands, it became obvious that TRAIL has a strong propensity for transformed or malignant tissues.

TRAIL Receptor Antibodies

Therefore, TRAIL is considered to be a candidate anticancer drug. TRAIL exerts its apoptosisinducing activity via the two respective agonistic TRAIL receptors DR4 and DR5. TRAIL itself is able to induce cell death in a wide array of cancer cells in vitro or when grown in xenograft settings. The efficacy of TRAIL is increased whenever the ligand is combined with conventional cytostatic agents or ionizing radiation. Depending on the production process, there was a concern that TRAIL, like CD95-L, could exert considerable hepatotoxicity. Released data from phase I trials suggest that TRAIL can be safely administered in patients up to serum concentrations consistent with those demonstrating efficacy in tumor xenograft models. In parallel to the development of TRAIL as anticancer drug, agonistic antibodies directed against both death-inducing TRAIL receptors were developed. Up to now relevant data on three agonistic TRAIL antibodies are available. The signaling pathways triggered by agonistic antibodies have not been reported to differ from the cascades triggered by TRAIL. Treatment of susceptible cells with agonistic antibodies results in the activation of ▶ caspase-8, caspase-9, and cleavage of PARP. As shown for TRAIL, agonistic TRAIL antibodies also induce cleavage of the antiapoptotic MCL-1 protein. How far key regulatory molecules including FADD, c-FLIP, and caspase-10 are involved in the regulation of cell death induction via agonistic TRAIL antibodies has not been tested in detail. However, the fact that there is a cross-resistance between TRAIL and the agonistic antibodies in such a way that cells being resistant toward TRAIL cannot be killed by either antibody indicates that the signaling pathways are identical or at least highly similar. The agonistic TRA-8 antibody directed against DR5 has been developed by Sankyo together with researchers from the University of Alabama (Birmingham, USA) and was the first agonistic antibody being described. TRA-8 was generated by immunizing BALB/c mice with a fusion protein containing the extracellular domain of DR5 and the Fc proportion of human IgG1. The antibody does not cross-react with murine DR5. The

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Kd values for TRAIL or TRA-8 binding to DR5 were estimated at 59 and 3 nM, respectively. The high specificity of TRA-8 for DR5 was documented by competition assays showing that TRA-8 efficiently competed with TRAIL for binding to DR5 but not for binding to DR4. In addition, these results indicate that TRA-8 potentially recognizes an epitope within the TRAILbinding site on DR5. In general, TRA-8 induces apoptosis in tumor cell systems in vitro as well as in murine xenograft model systems (Jurkat and 1321N1 astrocytoma cells). In contrast to the TRAIL preparation used for comparison, the TRA-8 antibody did not induce any signs of hepatopathy in mice. In subsequent studies, the increased efficacy of multimodal approaches combining either TRA-8 with radiation, chemotherapy, or other response modifiers was documented. In this regard it is important to notice that the efficacy of the tested combinations in terms of growth delay was shown in xenograft models for ▶ cervical cancer, ▶ breast cancer, and pancreal cancer. The overexpression of Bax using an adenoviral vector system increased the efficacy of TRA-8 in a wide array of glioma cells suggesting that Bax is involved in the efficacy of the combined treatment. The increased cell death induction translated into an increased growth delay. Besides the TRA-8 antibody, Sankyo also develops an agonistic DR4 antibody 2E12. However, considerably less data are available regarding the pharmacology and efficacy of this antibody. The second group of antibodies was developed by Cambridge Antibody Technology in conjunction with Human Genome Sciences (Rockville, USA). HGS-ETR1 (mapatumumab) is directed against DR4 and HGS-ETR2 (lexatumumab) is directed against DR5. HGSI also develops a third agonistic TRAIL antibody (HGS-TR2, targeting DR5) that was initially developed by Kirin Brewery Ltd. Up to now only data on mapatumumab and lexatumumab are available. Mapatumumab is a fully humanized monoclonal antibody and was isolated from 102 different antiTRAIL receptor mAbs that were generated by phage display technology. The antibody has a high affinity for the DR4 receptor and exerts

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antitumor activity (EC50 values of 3.4 nM) in diverse preclinical tumor models including breast, gastrointestinal, lymphoma, ovarian carcinoma, and uterine cancers. Mapatumumab was shown to specifically recognize the TRAIL-R1 protein without any relevant interactions with the TRAIL decoy receptors. The efficacy of mapatumumab is increased by combinations with various cytostatic drugs including carboplatin, cisplatin, camptothecin, topotecan, paclitaxel, as well as radiation. Xenograft models for breast cancer, colorectal cancer, non-small cell lung cancer, as well as uterine cancer revealed a high activity of either the drug alone or in combination with other cytotoxic treatment approaches including radiation. Like mapatumumab, lexatumumab is a fully humanized IgG1g antibody. The antibody was also generated using a phage display and screening with 383 short chain fragments for DR5 binding properties. In contrast to mapatumumab, no data on specificity, selectivity, and affinity of the antibody are publicly available. Similar to TRA-8, lexatumumab exert pronounced apoptotic reactions in a wide array of malignant cell systems when used alone. Importantly the drug has proven efficacy on tumor growth in xenograft systems from renal cell carcinoma, non-small cell lung cancer, breast cancer, and glioma. As already shown for TRA-8, the combination of lexatumumab with various chemotherapeutic agents (camptothecin, cisplatin, carboplatin, paclitaxel, doxorubicin, bortezomib) or radiation increased the efficacy in cell lines and xenografts. The underlying mechanisms of sensitization are still not completely understood. However, it seems likely that the presence of the proapoptotic Bax molecule as well as the upregulation of the respective receptor participates in the increased efficacy of the combined approach. Clinical Aspects Mapatumumab (Anti-DR5)

Data from several early clinical trials are available and allow a cautious judgment regarding

TRAIL Receptor Antibodies

pharmacological and toxicological aspects of mapatumumab. An open label phase Ia/b trial was conducted in 39 patients with various advanced solid tumors. During the first phase, dose escalation of mapatumumab was performed (0.01, 0.03, 0.1, 0.3, 1.0, or 3.0 mg/kg). The second phase of the trial involved administration of mapatumumab (10 mg/kg) once every 28 days or once every 14 days. The i.v. administration of mapatumumab produced dose-proportional pharmacokinetics up to a dose of 1.0 mg/kg, with a half-life of 15 days for 1.0 mg/kg. The pharmacokinetic data indicate that distribution and clearance follow a two-compartment model, with first-order elimination from the central compartment. The best clinical responses reported so far are stable diseases in a proportion of the heavily pretreated patients. No data from ongoing trials combining chemotherapy with TRAIL are available at present. In addition to various phase I trials, mapatumumab was tested in a multicenter phase II trial in patients with relapsed or refractory non-Hodgkin lymphoma. Patients (n = 40) received either (3 or 10 mg/kg mapatumumab once every 21 days). Partial responses were observed in three patients, and one patient with relapsed follicular mixed-cell lymphoma demonstrated a more pronounced regression. Two other phase II trials with mapatumumab are ongoing. One trial was conducted in patients with relapsed or refractory colorectal cancer. No data on safety, tolerability, pharmacokinetics, tumor response, time to response, duration of response, and progression-free survival from this trial are available. The second phase II trial including patients with solid tumor was performed in 32 patients with non-small cell lung cancer (median of three previous treatment cycles). These patients received 10 mg/kg mapatumumab every 21 days until disease progression. Mapatumumab was well tolerated with no treatment discontinuations due to drug-related toxicity. In 29% of these patients, a stable disease (median duration of 2.3 months) was observed. The most

Transabdominal Metastasis

common mapatumumab-related adverse events were nausea, fatigue, hypotension, myalgia, pyrexia, peripheral sensory neuropathy, diarrhea, constipation or abdominal pain, rash, hypertension, and thrombocytopenia. A clear maximumtolerated dose had not been achieved. No antibodies to mapatumumab had been observed. The phase II trial in NSCLC patients revealed that mapatumumab administration was generally safe and well tolerated. In 97% of these patients at least one adverse event was reported; however, only 44% of the patients experienced an adverse effect that was considered to be drug related. Again, no immunogenic responses were observed. Lexatumumab (Anti-DR5)

Data from several phase I trials using lexatumumab are available. Results from a US dose escalation trial (0.1, 0.3, 1.0, 3.0, and 10 mg/kg i.v. lexatumumab every 2 weeks) revealed that dose responses were linear up to the 10 mg/kg level with a mean half-life of 11 day at the 10 mg/kg dose level. The analysis of a similar trial performed in the UK (37 patients with advanced cancer of different organs sites treated with doses of 0.1, 0.3, 1.0, 3.0, 10, or 20 mg/kg every 3 weeks) revealed linearity over the whole dose range and a distribution model consistent with a two-compartment model with first-order elimination from the central compartment (reported pharmacological values: mean parameters for the 1 and 10 mg/kg groups were C max = 24.0 and 195.9 mg/ml; AUC = 190.8 and 2,379 mg/days/ml; half-life = 12.0 and 15.3 days; plasma clearance = 5.7 and 4.8 ml/kg/day; V1 = 44.1 and 50.8 ml/kg; and VdSS = 83.7 and 87.1 ml/kg). Clinical results from both trials have been reported with no major toxicity. Of 31 patients entered in the US trial, 10 experienced disease stabilization and 20 had disease progression. Of 37 patients entered in the UK trail, 11 experienced disease stabilization and 26 had disease progression. Data from the studies indicate that lexatumumab was well tolerated at doses up to 10 mg/kg. The most frequently reported

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toxicities were fatigue, nausea, anorexia, constipation, diarrhea, tachycardia, and vomiting. The DLT for lexatumumab was defined as 10 mg/kg. No data from phase II trials are available at present. TRA-8 (CS1008)

Up to now no results from clinical trials have been reported with the humanized version of TRA-8 (CS1008). Perspectives TRAIL receptor-based treatment strategies are currently entering clinical trials. The feared liver toxicity of TRAIL and agonistic compounds has not been documented in any of clinical trials currently available. No judgment on the definitive clinical anticancer activity of agonistic TRAIL receptor antibodies can be made although the phase I and phase II data revealed some clinical activity.

TRAM1 ▶ Steroid Receptor Coactivators

TRAM-1 ▶ Amplified in Breast Cancer 1

T Transabdominal Dissemination ▶ Transcoelomic Metastasis

Transabdominal Metastasis ▶ Transcoelomic Metastasis

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Transcoelomic Metastasis David S. P. Tan1 and Stanley B. Kaye2 1 Department of Medical Oncology, National University Cancer Institute, Singapore (NCIS), National University Hospital, and Cancer Science Institute, National University of Singapore, Singapore, Singapore 2 Drug Development Unit, Institute of Cancer Research, The Royal Marsden Hospital, Sutton, UK

Synonyms Peritoneal dissemination; Transabdominal dissemination; Transabdominal metastasis

Definition Dissemination or spread of malignant tumor throughout the peritoneal (abdominal and pelvic) cavity.

Characteristics Transcoelomic (meaning “across the peritoneal cavity”) ▶ metastasis refers to the dissemination of malignant tumors throughout the surfaces and organs of the abdominal and pelvic cavity covered by \peritoneum. Transcoelomic metastasis can occur as a result of ▶ invasion into the peritoneal cavity by (i) a primary cancer arising from within the abdominal/pelvic cavity, e.g., ▶ ovarian cancer; (ii) as a manifestation of systemic metastasis following hematogenous or lymphatic invasion by a primary cancer, e.g., advanced ▶ breast cancer; or (iii) following intraperitoneal seeding during surgical manipulation, e.g., during surgical resection of a colorectal tumor. The incidence of transcoelomic metastasis is higher with tumors that arise from the peritoneal cavity, e.g., ovarian (up to 70% of patients at presentation) and colorectal (up to 28% of patients at presentation). In contrast, extraperitoneal

Transcoelomic Metastasis

cancers, e.g., breast and lung, are associated with a much lower overall incidence of transcoelomic metastasis, although certain histological subtypes, e.g. infiltrating lobular breast cancers, have demonstrated a greater predilection for metastases to the gastrointestinal tract, gynecological organs, and peritoneum/retroperitoneum. This suggests that while the location of the primary tumor may be a key determinant in the development of transcoelomic metastasis, the tumor phenotype is also an important factor. Hence, it appears that a combination of anatomical and tumor-specific factors is involved in the transcoelomic metastatic process. Transcoelomic metastases contribute considerably to the morbidity associated with carcinomatosis because they have the capacity to affect multiple vital organs within the abdomen. Common examples include bowel obstruction caused by lesions along the gastrointestinal tract and renal failure caused by obstruction of the ureters. In addition, transcoelomic metastases are frequently associated with the formation of malignant ascites resulting in raised intra-abdominal pressure with consequent abdominal distension and discomfort. This results in early satiety, leading to dietary deficiency, impaired circulation of blood and ▶ lymphatic vessels, and respiratory compromise secondary to diaphragmatic splinting. Hence, there are potentially significant therapeutic advantages to be gained in understanding the process of transcoelomic metastasis. Mechanisms of Transcoelomic Metastasis Models of Metastasis

Two models have been hypothesized for the genetic origins of tumor metastases. The first model, often referred to as the seed-and-soil hypothesis, is that tumors are genetically heterogeneous and metastases arise from clones with a genetically acquired metastatic phenotype, which determines the final site of metastasis. The alternative hypothesis, the stochastic model, is that metastatic cells do not represent a genetically selected clone distinct from the primary tumor but arise as a stochastic event from tumor cell clones genetically identical to the primary tumor.

Transcoelomic Metastasis

Studies exploring this question using in vivo models have suggested a combination of both models of metastasis. Regardless of the metastatic model, there are certain observed characteristics that appear to be important for transcoelomic metastatic progression, in which complex cellular adaptations need to occur after cell detachment from the primary tumor mass to ensure survival within the peritoneal cavity. Cell Detachment Anchorage-independent growth and the ability to resist ▶ anoikis is a vital step for the initiation of metastasis. This process appears to involve the increased expression of ▶ survivin and X-linked inhibitor of ▶ apoptosis (XIAP), members of the inhibitor of apoptosis protein (IAP) family, which suppress apoptosis by inhibition of ▶ caspases. Other mediators of anoikis resistance include the family of extracellular matrix (ECM) to cell adhesion molecules known as integrins. Alterations in levels of integrin-mediated ECM-ligand binding have been found in many different tumor types and can result in decreased cell adhesion, changes in cell morphology and increased ▶ migration in vitro, and activation of ECM degrading enzymes including ▶ Matrix Metalloproteinases (MMP). Peritoneal Fluid and Anatomy The peritoneal cavity is normally empty except for a thin film of fluid that keeps surfaces moist. Peritoneal fluid arises primarily from plasma transudate and ovarian exudate. Other sources of peritoneal fluid include fallopian tubal fluid, retrograde menstruation, and macrophage secretions. The volume of peritoneal fluid is usually 5–20 ml and varies widely depending on physiological or pathological conditions. Peritoneal fluid contains a variety of free-floating cells, including ▶ macrophages, natural killer (NK) cells, lymphocytes, eosinophils, mesothelial (peritoneal surface epithelial) cells, and ▶ mast cells, which are all involved in immunological surveillance. Intraperitoneal fluid flow is directed by gravity to its most dependent sites and then drawn via the paracolic gutters to the diaphragm by the generation of negative intra-abdominal pressure in the upper abdomen during respiration. There is

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preferential flow along the right paracolic gutter, liver capsule, and diaphragm. Therefore, a natural flow of peritoneal fluid exists within the abdominal cavity, providing a route for the transcoelomic dissemination of detached tumor cells. As the epithelial surfaces of the female genital tract (i.e., ovaries, fallopian tubes, and endometrium) share a common embryological lineage with the peritoneal epithelium, it has been suggested that transcoelomic metastases from gynecological malignancies, such as fallopian tube and ovarian tumors, are not true metastases but a result of malignant transformation at multiple foci throughout the peritoneum, i.e., peritoneal metaplasia. If the metaplasia hypothesis is correct, then one might expect metastatic lesions to be randomly distributed throughout the peritoneum. Alternatively, if the theory of dissemination via peritoneal/ascitic fluid is true, then one might expect that detached tumor cells would, by virtue of gravity, be more frequently implanted in the floor of the pelvis, e.g., the pouch of Douglas (the space between the rectum and back wall of the uterus), followed by the organs in the paracolic gutters, and finally on the diaphragm, i.e., along the normal route of peritoneal fluid circulation. Studies have shown that a high incidence of metastatic implants for all cancers, including ovarian malignancies, within the peritoneal cavity is found on organs where peritoneal fluid resorption occurs (omentum and omental appendages). In addition, the colon, greater omentum, and pouch of Douglas are most often affected, with a reduced incidence of implants seen on the small bowel and its mesentery, which is free to move by peristalsis, compared to the ileocecal area (the junction between the ileum and cecum), which is fixed to the retroperitoneum. Hence, location and topography with regard to the flow of peritoneal/ascitic fluid appear to be key determinants in the process of transcoelomic dissemination for all cancers. As such, in the case of gynecological cancers, peritoneal metaplasia alone appears unable to fully account for the peritoneal distribution of carcinomatosis. Ascites: A Metastatic Milieu The development of transcoelomic metastasis is often associated with the formation of excess

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peritoneal fluid known as malignant ascites. It is hypothesized that, in addition to hypoalbuminemia (low plasma albumin levels) secondary to dietary deficiency, at least three other pathological events can cause ascites: (i) reduced lymphatic drainage from the peritoneal cavity caused by the obstruction of lymphatic vessels by tumor cells, (ii) increased vascular permeability of the peritoneal cavity, and (iii) tumor neo-▶ angiogenesis. While lymphatic obstruction is a well-recognized cause of ascites, the fact that massive amounts of fluid can accumulate in patients despite relatively little tumor burden suggests the involvement of other nonobstructive factors. These include ▶ Vascular Endothelial Growth Factor (VEGF), a glycoprotein which induces angiogenesis, and increased vascular permeability in response to hypoxia. Other immune modulators, vascular permeability factors, and MMPs secreted by both tumor cells and mesothelial cells also contribute significantly to ascites formation and stimulate tumor growth, invasion, and angiogenesis. Immune Evasion Many immune cells, such as macrophages, are present in peritoneal fluid and accumulate in so-called milky spots within the omentum. These omental macrophages have been found to be cytotoxic against tumor cells ex vivo. Consequently, omental macrophages might play an important role in killing tumor cells, thereby preventing development of transcoelomic metastasis and local peritoneal recurrences. Paradoxically, however, in vivo studies have shown that cancer cells seeded intraperitoneally specifically infiltrate the milky spots in the early stage of peritoneal metastasis. These studies suggest that omental milky spots are insufficient to prevent tumor progression and that intraperitoneal metastasis requires tumor cells to possess or acquire mechanisms for evasion of immunological surveillance. Tumor-infiltrating and malignant ascitesderived lymphocytes, in particular gamma delta T cells, from patients with metastatic ovarian and colorectal cancer, have also been shown to possess antitumor activity. Hence, it appears that metastatic tumor cells have also developed strategies

Transcoelomic Metastasis

to evade T cell-mediated cytotoxicity. Fas ligand (FasL) is a transmembrane protein belonging to the tumor necrosis factor superfamily that can trigger apoptotic cell death following binding to its receptor, Fas. Expression of FasL has been observed in renal, ovarian, colorectal, and head and neck tumors and may be responsible for the immune privilege of tumor cells by inducing apoptosis of antitumor immune effector cells within the tumor microenvironment – the “Fas counterattack.” Studies have also shown that tumor progression and metastasis are associated with increased expression of FasL. Other examples of immune evasion include the recruitment of regulatory T (Treg CD4+CD25+) cells to suppress tumor-specific T-cell immunity; the presence of high concentrations of soluble forms of the complement pathway inhibitors C1 inhibitor, factor H, and FHL-I on isolated metastatic ovarian cancer cells in ascitic fluid; and the phenomenon of spheroid formation observed in breast, colorectal, and ovarian cancer where tumor cells clump together by upregulating cell adhesion molecules, thus resulting in increased complement resistance due to insufficient penetration of antibodies and complement into the spheroids. Tumor Implantation Although topography appears to be a key determinant in the final site of metastatic implantation within the peritoneum, the actual mechanisms behind tumor implantation remain unclear. However, there is evidence to suggest the involvement of a dynamic regulation of the tumor cell’s adhesiveness and its interaction with the underlying peritoneal mesothelium. Potential mechanisms for the attachment of tumor cells to the peritoneal mesothelium include binding to ECM proteins like collagen type I and IV, laminin, and fibronectin via tumor cell surface integrins and to hyaluronan expressed on the surface of human peritoneal mesothelial cells via the ▶ CD44 tumor cell surface protein, of which there are ten alternative exon splice variants (v1–v10). Upregulation of certain CD44 variants have been associated with distant metastasis in breast, colorectal, and ovarian cancer. Tumor antigen/marker CA125, a glycoprotein overexpressed on the cell

Transcoelomic Metastasis

surface and secreted by ovarian tumor cells in the majority of ovarian cancer patients, has been shown to bind to mesothelin, a glycosylphosphatidylinositol-linked cell surface molecule expressed by mesothelial cells. Upregulation of the cell adhesion molecule ▶ Ecadherin may also mediate adhesion of circulating tumor cells to metastatic sites. Adhesion onto the peritoneal surface may be followed by haptotactic migration in which coordinated anti- and pro-migratory signals mediated by ECM proteoglycans confer directionality to tumor cell motility, effectively laying the tracks until a “stop” signal is encountered. Once attached to the peritoneal surface, metastatic cells proliferate and invade into the subjacent epithelium. The MMP family of proteinases and the urokinase-type ▶ plasminogen activator (uPA) system appear to be major contributors to this process. Human peritoneal epithelial cells and their associated immune and stromal cells have been shown to release regulatory ▶ chemokines and cytokines, such as IL-1, IL-6, and IL-8, in response to serosal inflammation and injury induced by tumor implantation, which in turn facilitate tumor angiogenesis and ascites formation (via increased secretion of VEGF), and enhanced tumor migration, attachment, proliferation, and invasion. Finally, just as extraperitoneal tumors can metastasize to the peritoneum, intraperitoneal tumors can also metastasize extraperitoneally. Apart from the rich intraperitoneal network of blood and lymphatic vessels which can be invaded by tumors, peritoneal fluid is also continually being returned to the systemic circulation via the subdiaphragmatic lymphatic network and thoracic duct into the left subclavian vein, thus providing a direct “metastatic expressway” for peritoneal metastases to gain access into the lymphatic and circulatory system. Clinical Aspects Patients with transcoelomic metastasis often present with signs and symptoms of abdominal pain, abdominal distension secondary to an enlarging tumor or ascites, constipation or diarrhea, shortness of breath, fatigue, loss of appetite, and weight loss. A careful clinical history followed by

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thorough clinical examination is required to ascertain the likely source of the primary tumor. Investigations should include routine blood tests, including relevant tumor markers, followed by radiological investigations including ultrasound and computer tomographic (CT) scans of the chest, abdomen, and pelvis to confirm the likely source of tumor and disease stage. In all cases, particularly those in which there is no obvious source of primary tumor (i.e., carcinoma of unknown primary origin), a biopsy of an accessible lesion should be obtained for histopathological and immunohistochemical confirmation and diagnosis. In the past, clinical situations involving transcoelomic metastasis were treated mainly with palliative intent. Increasingly, studies have shown that an aggressive approach to peritoneal surface malignancy involving peritoneal debulking (cytoreductive) procedures, combined with optimal perioperative or postoperative systemic or intraperitoneal ▶ Chemotherapy in carefully selected patients, can result in long-term survival. Clinical assessment parameters that need to be considered include the patient’s performance status; preoperative abdominal and pelvic CT scans to define the extent and operability of disease, including the presence of extraperitoneal metastases; and tumor histopathology. Key prognostic indicators following surgery include the completeness of peritoneal debulking surgery, the presence of intraperitoneal lymph node and visceral metastases, and tumor type. Of the various scoring systems used to assess the extent of peritoneal carcinomatosis, the most frequently quoted is the peritoneal cancer index (based on the intraoperatively observed distribution and size of intraperitoneal metastasis) and the completeness of cytoreduction score (based on the amount of residual disease following peritoneal debulking surgery), which have been found to correlate well with prognosis in colorectal, gastric, and ▶ ovarian cancer. A meta-analysis of studies comparing combined peritoneal debulking surgery and perioperative intraperitoneal chemotherapy with systemic chemotherapy alone for the treatment of peritoneal carcinomatosis from colorectal carcinoma has demonstrated improved survival in the

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combination therapy group. In patients with ovarian cancer and peritoneal metastasis, 2-year survival following radical resection of all macroscopic tumors is 80%, in contrast to less than 22% for the patients with residual lesions larger than 2 cm. Early aggressive treatment of minimal peritoneal surface dissemination appears to confer the most benefit. In patients with inoperable tumors at presentation, primary systemic or intraperitoneal chemotherapy is recommended, following which reassessment for surgical intervention may be possible if a good treatment response is observed. Palliative measures in the management of malignant ascites include repeated paracentesis (drainage of ascites), which provides relief in up to 90% of patients, and permanent percutaneous drains. The creation of a peritoneovenous shunt (which allows ascitic fluid to drain from the peritoneal cavity into the superior vena cava) prevents the need for repeated paracentesis. Promising experimental approaches in the treatment of transcoelomic metastasis include the use of intraoperative hyperthermic intraperitoneal chemotherapy, antiangiogenic agents such as the MMP inhibitors and the VEGF antagonists, as well as ▶ immunotherapy approaches including antibody targeted T-cell therapy and combinations of intraperitoneal immunotherapy and thermochemotherapy.

Cross-References ▶ Angiogenesis ▶ Anoikis ▶ Apoptosis ▶ Breast Cancer ▶ Caspase ▶ CD44 ▶ Chemokines ▶ Chemotherapy ▶ Colorectal Cancer ▶ Colorectal Cancer Premalignant Lesions ▶ E-Cadherin ▶ Gastric Cancer ▶ Immunotherapy ▶ Invasion

Transcoelomic Metastasis

▶ Lymphatic Vessels ▶ Macrophages ▶ Mast Cells ▶ Metastasis ▶ Migration ▶ Ovarian Cancer ▶ Plasminogen-Activating System ▶ Survivin ▶ Vascular Endothelial Growth Factor

References Becker G, Galandi D, Blum HE (2006) Malignant ascites: systematic review and guideline for treatment. Eur J Cancer 42(5):589–597 Baratti D, Kusamura S, Pietrantonio F, Guaglio M, Niger M, Deraco M (2016) Progress in treatments for colorectal cancer peritoneal metastases during the years 2010-2015. A systematic review. Crit Rev Oncol Hematol. pii: S1040-8428(16)30017–30018. doi:10.1016/j.critrevonc.2016.01.017. [Epub ahead of print] Review Fidler IJ (2002) Critical determinants of metastasis. Semin Cancer Biol 12(2):89–96 Koppe MJ, Boerman OC, Oyen WJ et al (2006) Peritoneal carcinomatosis of colorectal origin: incidence and current treatment strategies. Ann Surg 243(2):212–222 Seimetz D1, Lindhofer H, Bokemeyer C (2010) Development and approval of the trifunctional antibody catumaxomab (anti-EpCAM x anti-CD3) as a targeted cancer immunotherapy. Cancer Treat Rev 36(6): 458–467. doi:10.1016/j.ctrv.2010.03.001. [Epub 2010 Mar 27] Tan DS, Agarwal R, Kaye SB (2006) Mechanisms of transcoelomic metastasis in ovarian cancer. Lancet Oncol 7(11):925–934

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) Integrin. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1884. doi:10.1007/978-3-642-16483-5_3084 (2012) Lymphatic System. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2120. doi:10.1007/978-3-642-16483-5_3450 (2012) Metaplasia. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2259. doi:10.1007/978-3-642-16483-5_3670 (2012) Omentum. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2609. doi:10.1007/978-3-642-16483-5_4213 (2012) Palliative. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2759. doi:10.1007/978-3-642-16483-5_4350

Transduction of Oncogenes (2012) Performance Status. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2814. doi:10.1007/978-3-642-16483-5_4444 (2012) Peritoneal Debulking Surgery. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2818. doi:10.1007/978-3-642-16483-5_4459 (2012) Peritoneum. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2819. doi:10.1007/978-3-642-16483-5_4467 (2012) Retroperitoneum. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3292. doi:10.1007/978-3-642-16483-5_5078 (2012) Stage. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3499. doi:10.1007/978-3-642-16483-5_5478

Transdifferentiation ▶ Stem Cell Plasticity

Transduction of Oncogenes Jaquelin P. Dudley Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA

Synonyms Oncogene transduction; Retroviral transduction

Definition Retroviruses are RNA-containing viruses that replicate through a DNA intermediate (provirus) using the enzyme reverse transcriptase. During retroviral replication, which requires integration into the host chromosomal DNA for efficient transcription of viral RNA, some retroviruses have acquired specific cellular ▶ oncogenes, usually with multiple modifications and often with the loss of trans-acting viral functions. Inclusion of one or more oncogenes in the viral genome then imparts transforming activity on the recombinant virus independent of the site of integration in the cellular genome.

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Characteristics Identification of Cellular Oncogenes In 1911, Peyton Rous described the isolation of a virus that caused fibrosarcomas in chickens. The Rous sarcoma virus (RSV) subsequently was shown to transform chicken embryos and the surrounding membranes and formed small tumors on the chorioallantoic membrane in proportion to the number of virus particles. Further quantitative assays were developed when RSV and other retroviruses were shown to transform or induce morphological and growth behavior changes in cultured cells that mimicked tumor formation in the animal. The ability of RSV to transform cells in culture led to the conclusion that the virus encoded a gene responsible for such changes. Isolation of transformation-defective variants of RSV allowed comparisons with wild-type RSV and the discovery of the viral oncogene, v-src. Experiments from the laboratories of Harold Varmus and Michael Bishop revealed that the v-src gene is highly related to a specific cellular gene or proto-oncogene, c-src, which encodes a protein tyrosine kinase. Unlike the v-src gene in RSV, the cellular homologue contained introns, which could be alternatively spliced in various cell types to give different mRNAs. Further characterization showed that the product of the v-src gene, v-Src, had substitutions within several functional domains that prevented the normal regulation of the kinase activity during the process of ▶ signal transduction. Shortly after the discovery of v-src, other transforming viruses were isolated and characterized. Some of these viruses were recovered by treatment of normal cells with halogenated pyrimidines to induce endogenous retrovirus expression, by coculture of primary cells with chemically transformed cells that express non-transforming retroviruses, whereas others were isolated by in vivo passage of nontransforming retroviruses. In each case, the transforming virus appears to be the result of recombination between a non-transforming retrovirus and one or more cellular genes. The ability of retroviruses to acquire cellular sequences in their genome and transmit these genes to other

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cells is known as retroviral transduction or, in the case of proto-oncogenes, oncogene transduction. Examples of transforming viruses and the acquired proto-oncogenes are listed in Table 1. Deregulation can occur at many steps of ▶ signal transduction, leading to oncogenesis. Nevertheless, recombination events leading to the generation of a transforming retrovirus appear to be rare in nature. Most of the resulting viruses are defective for replication because the acquisition of cellular proto-oncogenes is accompanied by the deletion of viral structural genes, which are necessary to produce viral particles. Such defective transforming viruses are not transmissible unless they successfully coinfect a cell with a related retrovirus that provides the missing gene products in trans. Thus, most transforming viruses are more interesting as research tools for the identification and functional characterization of oncogenes and the process of transduction than as a major cause of disease in animals and humans. Mechanism of Oncogene Transduction The majority of transforming retroviruses are defective for viral replication. Since these viruses are isolated after acquisition of transforming activity, the exact steps required to form these retroviruses are unknown nor is it clear whether every transforming virus has been generated by the same mechanism. However, a general model has emerged for the formation of such viruses (Fig. 1). First, a non-transforming retrovirus integrates upstream of a cellular gene, an event known to occur at a reasonable frequency. Many retroviruses integrate preferentially within coding sequences or near sites of active transcription. Non-transforming retroviruses often cause tumors by insertion in or near proto-oncogenes, resulting in the activation of transcription or production of abnormal transcripts. These transcripts arise due to enhancer activation of the cellular promoter or activity of the viral promoter on the cellular gene. If the proviral integration results in cellular transformation, cells containing the integration site will be selected for growth. However, some retroviruses integrate upstream or downstream and in the opposite orientation relative to the

Transduction of Oncogenes Transduction of Oncogenes, Table 1 Oncogenes transduced by retroviruses Transforming virus Abelson murine leukemia virus AKT8 Cas NS-1 virus Avian sarcoma virus CT10 Avian erythroblastosis virus-ES4 Avian erythroblastosis virus-ES4 Avian myeloblastosis virus-E26 Avian retrovirus RPL30 Snyder-Theilen feline sarcoma virus Gardner-Rasheed feline sarcoma virus McDonough feline sarcoma virus Finkel-Biskis-Jinkins murine sarcoma virus Fujinami avian sarcoma virus Avian sarcoma virus 17 Hardy-Zuckerman-4 feline sarcoma virus Avian retrovirus AS42 (sarcoma) Mill-Hill virus 2 (avian myelocytoma virus) Moloney murine sarcoma virus Mouse myeloproliferative leukemia virus Avian myeloblastosis virus-E26 Myelocytomatosis virus 29 Avian retrovirus ASV31 (sarcoma) 3611 murine sarcoma virus Harvey murine sarcoma virus Kirsten murine sarcoma virus Avian reticuloendotheliosis virus T UR2 avian sarcoma virus S13 avian erythroblastosis virus Simian sarcoma virus SKV770 avian sarcoma virus Rous sarcoma virus Y73/Esh avian sarcoma virus

Acquired proto-oncogene abl akt cbl crk erbA erbB ets eyk fesa fgr fms fos fpsa jun kit maf milb mos mpl myb myc qin rafb HRAS KRAS rel ros sea sis ski src yes

a

fes and fps are the same oncogene derived from feline and avian genomes, respectively b mil and raf are the same oncogene derived from avian and murine genomes, respectively

proto-oncogene, and it is believed that the generation of transforming retroviruses requires proviral integration upstream and in the same orientation as the oncogene. Such events can result in cancer induction without transduction of

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4623 U3RU5 5’LTR

3’LTR

Proviral integration

c-onc DNA 3’LTR

5’LTR

RNA Readthrough transcription of truncated provirus

Readthrough transcription of full-length provirus

5’LTR

5’LTR

3’LTR

DNA RNA RU5

RU5

U3RU5

Splicing and co-packaging of normal and hybrid oncogene transcripts RU5

RU5 Retroviral particles

(-) strand DNA RU5

U3RU5

(-) strand DNA RU5

U3R

U3R

Reverse transcription: recombination and mutagenesis

v-onc 5’LTR

3’LTR

Transducing provirus with mutations

Transduction of Oncogenes, Fig. 1 Mechanism of retroviral oncogene transduction (See text for details)

oncogenes. Thus, many proviral insertion events may lead to cancer, but not formation of transforming retroviruses. Second, transcription initiating in the 50 LTR generates a transcript that would read through the normal polyadenylation sequences in the 30 LTR. Evidence suggests that increases in retroviral transcriptional readthrough also result in transductive recombination. Potentially, this readthrough transcript could be packaged into virions, although it is generally believed that transcripts longer than 150% of the genome would be accommodated poorly in the virus capsid. Alternatively, a rare deletion of the cellular DNA or aberrant splicing could provide a truncated provirus or mutant transcripts that may encompass a much greater portion of the cellular transcripts. Third, hybrid oncogene transcripts may be packaged along with normal viral transcripts into

virions. Retroviruses have a diploid genome that includes cis-acting sequences (usually near the 50 end of the viral genome) necessary for packaging into the viral capsid (designated the psi sequence). Thus, hybrid transcripts including the psi sequence would be preferentially packaged with normal retroviral sequences to give an RNA heterodimer instead of the normal RNA homodimer. However, normal cellular RNAs can be packaged into retroviral particles at low frequency, and co-packaging would allow copying by reverse transcriptase, which is not template specific. Fourth, the hybrid oncogene transcript and the wild-type transcript both will be used as templates for reverse transcriptase, which also is incorporated into viral particles. In some cases, incorporation of the proto-oncogene and expression at high levels from the retroviral promoter appears

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to be transforming. Nevertheless, most transduced oncogenes have multiple genetic alterations. Reverse transcriptase has several properties that favor the types of genomic changes observed in transforming retroviruses. These properties include template switching, deletion formation, and the introduction of point mutations. If the resulting recombinants retain all of the cis-acting sequences needed for replication, the transforming virus will be capable of propagation in the presence of replication-competent retroviruses. Supporting the importance of readthrough transcripts for this process, transforming retroviral genomes have been observed that carry poly(A) stretches typical of mRNAs at the junction between the host and 30 viral sequences. Furthermore, the incorporated oncogenes lack introns. Lessons from Retroviral Transduction of Oncogenes Cancer is believed to be a multistep process, where several genetic events contribute to the generation of tumor cells. Some avian retroviruses are known to have transduced two oncogenes, including avian erythroblastosis virus-ES4 and avian erythroblastosis virus-R (AEV-ES4 and AEV-R) (erbB and erbA), Mill-Hill 2 (MH2) avian myelocytoma virus (mil (aka raf) and myc), and avian myeloblastosis virus (AMVE26) (ets and myb). Some viruses may have transduced more than one cellular proto-oncogene and been selected for increased transforming capacity, a process that may be similar to the acquisition of multiple genetic changes in cancer cells during tumor progression. For example, evidence suggests that erbA expression is necessary for the full transforming activity of the erbB oncogene in AEV. Also, the MH2 retrovirus requires expression of both mil/raf and myc to transform neuroretinal cells from 7-day-old chicken embryos, an event that myc-expressing retroviruses cannot induce. However, AMV-E26 contains two oncogenes, but only one of them has been shown to be necessary for full transforming ability. Deletion of the ets oncogene does not diminish the transforming ability of AMV-E26 relative to wild-type virus when injected in newborn chickens. Furthermore, many of the

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transduced oncogenes contain deletions or point mutations that reveal regulatory regions of the encoded gene products, leading to a greater understanding of their normal functions in cellular growth control. Retroviruses, including simple retroviruses that lack regulatory genes as well as lentiviruses, have become common vectors for therapeutic gene delivery. These viruses have been used to deliver genes for treatment of a variety of illnesses, including cancers and genetic disorders. Lentiviral vectors offer the advantage of being able to infect and replicate in non-dividing cell types. However, simple retroviruses may be advantageous for preferential infection of dividing tumor cells and delivery of the therapeutic gene relative to adjacent normal cells. In both cases, viral structural genes in the vector are replaced with a therapeutic gene of interest. Expression of the transduced genes can be controlled by nonviral promoters, internal ribosome entry sites, or splicing. Although these vectors have shown promise as therapeutic agents, the safety of such vectors has been the overriding concern, i.e., preventing the formation of replication-competent viruses and avoiding the ill effects of integration. Limitations to replication and the avoidance of multiple insertions within target cells should minimize insertional activation of oncogenes. Improvements to the retroviral vector system have resulted from removal of the 30 U3 region, thus creating a self-inactivating (SIN) virus after reverse transcription. Another possible safety concern associated with ▶ gene therapy is the generation of new transforming viruses, especially in human patients. These events should be extremely rare because of several precautions included during the construction of these vectors, including the removal of U3 sequences as well as viral structural genes, thus confining these retroviruses to a single round of replication. Furthermore, the absence of homologous endogenous lentiviruses in humans should reduce recombination events that lead to generation of transforming retroviruses. In the early part of this century, a major milestone in human gene therapy was achieved. Early treatments of 20 children with X-linked severe combined immune deficiency (SCID-X1) using a

Transforming Growth Factor-Beta

wild-type Moloney murine leukemia virus-based vector allowed normal expression of the defective gene and corrected the immune defect. Unfortunately, in five of the patients, this vector inserted in close proximity to the LMO2 or CCND2 protooncogene, leading to dysregulation of expression and development of leukemia. A current gene therapy clinical trial suggests that removal of the 3’ LTR enhancer in the gammaretrovirus vector (SIN vector) prevents or delays such adverse events. Additional improvements continue to be made, including prevention of transcriptional readthrough or inclusion of insulator elements to block the formation of hybrid viral-cellular transcripts that may lead to oncogene transduction.

Cross-References ▶ Gene Therapy ▶ Oncogene ▶ Signal Transduction

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Transfer of T Cells ▶ Adoptive T-Cell Transfer

Transforming Gene ▶ Oncogene

Transforming Growth Factor-Beta Jorma Keski-Oja1 and Katri Koli2 1 Departments of Pathology and of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland 2 Translational Cancer Biology Program, University of Helsinki, Helsinki, Finland

References

Definition

An W, Telesnitsky A (2004) Human immunodeficiency virus type 1 transductive recombination can occur frequently and in proportion to polyadenylation signal readthrough. J Virol 78:3419–3428 Hacein-Bey-Abina S et al. (2014) A modified gammaretrovirus vector for X-linked severe combined immunodeficiency. New England J Med 371:1407–1417 Nevins JR (2001) Cell transformation by viruses. In: Knipe DM, Howley PM (eds) Fundamental virology, 4th edn. Lippincott William & Wilkins, Philadelphia, pp 245–283 Swain A, Coffin JM (1992) Mechanism of transduction by retroviruses. Science 255:841–855 Swanstrom R, Parker RC, Varmus HE et al (1983) Transduction of a cellular oncogene: the genesis of Rous Sarcoma Virus. Proc Natl Acad Sci U S A 80:2519–2523

Transforming growth factors were identified on the basis of their ability to induce soft agar growth and morphological changes in nonmalignant cells. The original observation was of an activity, which was named sarcoma growth factor. Soon afterward the term transforming growth factor, TGF, was adopted. Sarcoma growth factor was subsequently found to be composed of an epidermal growth factor like protein, which was named transforming growth factor alpha, TGF-a, and of TGF-b. TGF-a is a member of the epidermal growth factor family and is unrelated to TGF-b. Targeting of TGF-b activity by LTBPs (binding proteins) and different mechanisms of activation from the latent forms is a crucial feature in its biology.

See Also (2012) Endogenous Retrovirus. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1232. doi:10.1007/978-3-642-16483-5_1879 (2012) Retrovirus. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 3296–3297. doi:10.1007/978-3-642-16483-5_5084 (2012) Reverse Transcriptase. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3297. doi:10.1007/978-3-642-16483-5_5086

Characteristics TGF-bs are multifunctional polypeptide growth factors involved in the regulation of cellular growth and differentiation and immune functions. The number of known members of the TGF-b

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superfamily is close to 40 different growthmodulating proteins. Besides TGFbs, these include bone morphogenetic proteins (BMPs), growth and differentiation factors (GDF), nodal, inhibins, and activins. TGF-bs are in many senses unique among growth factors in their potent and widespread actions. Three different mammalian gene products, TGF-bs 1–3, have been molecularly cloned. Almost all types of cells in the body make some form of TGF-b, and nearly all cells have cell surface receptors for it. One of their major effects is inhibition of cell proliferation, a property needed in developmental processes, for instance. In addition, TGF-bs regulate differentiation and cellular plasticity during development and in adult tissue repair processes. TGF-bs have important roles in the control of the pericellular proteolytic balance and in the regulation of the production and structure of the components of the connective tissues and extracellular matrices. TGF-b stimulates the transcription and synthesis of various components of the extracellular matrix like collagens, ▶ fibronectin, vitronectin, tenascin, and proteoglycans. TGF-bs are potent chemotactic factors for many cell types like fibroblasts, eosinophils, and various inflammatory cells at very low concentrations. They also suppress matrix degradation by decreasing the expression of proteinases (▶ serine proteases (type II) spanning the plasma membrane), such as plasminogen activators (▶ plasminogenactivating system) and numerous metalloproteinases, and by inducing proteinase inhibitors, such as plasminogen activator inhibitor-1 and ▶ tissue inhibitors of metalloproteinases (TIMPs). In addition, TGF-bs regulate cellular functions by modulating the expression of matrix receptors, the integrins. For these reasons the activities of TGF-bs must be tightly regulated. TGF-b Receptors and Signaling Mechanisms Members of the TGF-b superfamily have diverse functions in cell-cell signaling. TGF-bs play different roles in tissue homeostasis and at various stages of development. The mechanisms of regulation of TGF-b activity are multifaceted and complex. Three different TGF-b isoforms and the types, affinity, and signaling functions of its

Transforming Growth Factor-Beta

receptors also add complexity to the regulation of their effects. The effects of TGF-bs and the other family members are mediated from the cell membrane to nucleus through distinct combinations of type I and type II serine/threonine kinase receptors and downstream effectors, the Smad proteins. TGF-b signals are mediated by TGFbRII and two different type I receptors, activin-like kinase (ALK) receptors ALK5 and ALK1. Endoglin and betaglycan, also called TGF-b type III receptors, act as coreceptors for the signaling complex. The receptor-regulated Smads become phosphorylated by activated type I receptors, and they form heteromeric complexes with a common partner, Smad4, which gets translocated into the nucleus for gene transcription control. In addition to the signal-transducing Smads, inhibitory Smads also play a role in the outcome of the signaling. They downregulate the activation of receptor-regulated Smads. TGF-b can also elicit non-Smad signaling responses in target cells, such as activation of mitogen-activated protein kinase (MAPK) cascades, PI3K/AKT/mTOR pathway, and RhoA. These play an important role in the tumorpromoting activities of TGF-b. Latency TGF-b TGF-bs are produced by the majority of cells in latent complexes unable to associate with TGF-b signaling receptors. Some primary cells and established cell lines secrete active TGF-b. TGF-bs are secreted from cells as latent dimeric complexes containing the mature C-terminal TGF-b and its N-terminal pro-domain, LAP, the TGF-b latency-associated peptide. The two polypeptide chains of pro-TGF-b associate to form a disulfide-bonded dimer. TGF-b is cleaved from its propeptide by furin-like endoproteinase during secretion at RRXR sequence. The LAP propeptide dimer remains associated with the TGF-b dimer by non-covalent interactions. This complex is referred to as small latent TGF-b. TGF-bs are secreted in most cultured cell lines as large latent complexes, consisting of small latent TGF-b covalently bound to one of the three latent TGF-b binding proteins (LTBP-1, LTBP-3, or LTBP-4) covalently linked to LAP (Fig. 1). The expression and secretion of LTBPs

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Large latent TGF-β complex

LAP Hinge region (Plasmin and MMP cleavage sites) LTBP

Proteinase sensitive region

TGF-β

ECM binding site Latent TGF-β binding site

8-Cys repeat ECM binding sites

Hybrid domain Ca2+ binding EGF-like repeat

Extracellular matrix fibers

Non-Ca2+ binding EGF-like repeat

Transforming Growth Factor-Beta, Fig. 1 Large latent TGF-b complex. The small latent complex contains the C-terminal mature TGF-b and its N-terminal pro-domain, LAP (TGF-b latency-associated peptide). This complex

forms a disulfide-bonded complex with the third 8-Cys repeat of LTBP. LTBP associates with the ECM mainly via the 8-Cys domains and some adjoining regions

and TGF-bs is, in general, coordinately regulated. Interestingly, LTBP-2 is unable to form complexes with any of the small latent TGF-bs and has other functions as a microfibril-associated protein. In addition, LTBP-4 binds only to TGF-b1 and with much lower affinity than LTBP-1 and LTBP-3. LTBPs have a central role in the processing, secretion, and matrix targeting of TGF-bs, but they also have other roles as regulators of the structure and function of the extracellular matrix.

complexes can be a two-step process, the release of the large latent complex from ECM by proteolytic truncation and subsequent activation, which can be achieved by different mechanisms. It appears that integrins can activate TGF-b complexes directly without proteolytic release. Matrix stiffness plays an important role in integrinmediated activation of TGF-b. Since TGF-b regulates the cellular production of ECM components as well as the proteolytic balance, the matrix association and activation of TGF-b complexes form a finely tuned control network for the maintenance of the organization of extracellular structures. Cancer cells produce frequently aberrant amounts of both the matrix components and TGF-b. Malignant cells do also frequently fail to deposit TGF-b complexes to the extracellular matrix, probably due to their perturbed deposition of fibronectin-collagen matrix, as well as altered LTBP production.

Matrix Association and Release of TGF-b LTBPs have a central role in the targeting of TGF-b to extracellular matrix structures. LTBPs are produced in excess to TGF-b, and since TGF-b secretion is very inefficient in the absence of LTBP, most secreted cellular TGF-b is in the large latent complexes (Fig. 1). The release of active TGF-b from matrix-associated latent

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Transforming Growth Factor-Beta

Association of LTBPs with different TGF-β isoforms TGF-β1

TGF-β2

TGF-β3 RGD

RGD

LTBP-4

Latent complexes of TGF-b in the ECM may provide tissues with a readily available storage form of this growth factor. The release and activation of stored growth factors by proteases or migrating cells can generate rapid and highly localized signals like in wound healing or during radiotherapy. Cell movement causes traction of the latent matrix-associated complexes and induces activation. Rapid activation of extracellular signaling mechanisms could be important in the healing of tissues after damage, in the control of cells of the immune system during acute infections, and in the initial stages of angiogenesis. It is unclear how soluble growth factors could form gradients in highly cellular tissues. Matrix-bound growth factors might generate this kind of an immobilized activity gradient. LTBPs: Expression and Functions Data of the functions of LTBPs is accumulating rapidly. Structurally they resemble fibrillins, which are components of the extracellular microfibrils. LTBPs have a typical structure consisting of four eight-cysteine (8-Cys) repeats and several EGF-like repeats. The association of latent TGF-b with the matrix is mediated by LTBPs (Fig. 1). Not only the N-terminal domains but also a region of the C-terminus of LTBP is important in this association. The N-terminus contains transglutaminase

LTBP-1

LTBP-3

LTBP-2

substrate motifs, and transglutaminase is required for the covalent ECM association. In addition, TGF-b2 and TGF-b3 become associated with LTBPs. It is thus likely that LTBPs mediate and target the binding of all three TGF-b isoforms to various extracellular matrices. The TGF-b1 binding region in LTBPs is located close to their C-terminus in the third 8Cys repeat. The association between LTBP-1 and the propeptide part, LAP, is mediated by disulfide bonding. The respective 8-Cys repeats of LTBP-3 and LTBP-4 also bind small latent TGF-bs. Of the numerous known 8-Cys repeats of the LTBPs and fibrillins, only three have been found to have the capacity to associate in a covalent manner with the small latent TGF-bs (modeled in Fig. 2). LTBPs bind fibrillins, which form the extracellular microfibrils. This binding is important also for non-TGF-b-related functions of LTBPs. LTBP-4 is an important regulator of elastogenesis through association with fibulins and elastin. Activation of Soluble and Extracellular Matrix Forms of Latent TGF-b TGF-b can be activated in vitro by multiple mechanisms, including proteolysis, enzymatic deglycosylation, and extremes of pH. Activation of latent TGF-b involves proteolytic disruption of the non-covalent interaction between the

Transforming Growth Factor-Beta

propeptide LAP and TGF-b, which releases biologically active TGF-b capable of binding to its signaling receptors. LAP may also undergo conformational changes in such a manner that TGF-b is released or exposed to its receptors. The existence of different TGF-b isoforms and latent complexes, as well as the number of different LTBPs, suggests that there are variable mechanisms for the activation of TGF-bs. The electrostatic interaction between LAP and TGF-b can be dissociated in vitro by extremes of pH, chaotropic agents, and heat treatment. From the physiological point of view, the acidic environment in the bone (osteoclasts) or during wound healing could induce this kind of TGF-b activation. In vivo analyses of tumor-bearing mice indicated that irradiation causes rapid activation of TGF-b in the tumors. This effect appears to result from the activation of existing, most probably of matrix-bound latent TGF-b. Irradiation produces reactive oxygen species leading to redoxmediated activation of latent TGF-b complexes. Redox-mediated TGF-b activation may be involved in chronic tissue processes, where oxidative stress is implicated, such as carcinogenesis. The processing of pericellular matrixassociated LTBPs and activation of TGF-b are constant events in the ▶ apoptosis or ▶ anoikis of endothelial and epithelial cells, pinpointing the importance of pericellular latent complexes as a physiological source of TGF-b. Thrombospondin-1 (TSP-1), a platelet a-granule and ECM protein, plays a role in the activation of latent TGF-b complexes via a mechanism that does not involve cell surfaces or proteases. Using purified plasma TSP-1 or the recombinant protein, it was found that it is able to activate both small and large latent TGF-b complexes. The activation mechanism is not fully understood but seems to involve the N-terminal end of LAP and the type I repeats of TSP-1, possibly by inducing a change in the conformation of LAP and thus releasing the active TGF-b. TSP-1 interacts with LAP as a part of a biologically active complex, and this may prevent the reassociation of the inactive complex of LAP with TGF-b. The expression of TSP is induced

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during wound healing. TGF-b may thus get focally activated at sites of injury by enhanced TSP synthesis. Accordingly, TSP-deficient mice display many phenotypic features, similar to those detected in TGF-b1-deficient mice. The abnormalities in some tissues of the TSP null animals were even reverted by TSP-derived TGF-b activating peptides, further emphasizing the role for TSP in TGF-b activation. The LAP part of TGF-b contains an RGD-motif, which is recognized by many integrins (▶ Integrin Signaling). Integrin avb6 is able to activate TGF-b. This activation model is particularly interesting, because avb6 integrin is expressed solely on epithelial cells, which are very sensitive to TGF-b-mediated growth inhibition, and also because the overlap of the phenotypes of TGF-b1 and integrin b6 chain-deficient mice. b6 integrin-deficient mice show increased inflammation and decreased fibrosis, processes that are regulated by TGF-b. In addition, other av-containing integrins have been suggested to activate TGF-b directly through a mechanism requiring proteolytic activity or a change in the conformation of the latent complex. Hormonal effectors can also affect TGF-b activation. Originally it was found that antiestrogens could induce the production and secretion of active TGF-b in cultured breast cancer cells. Activation of TGF-b has subsequently been observed in a number of cell culture models using estrogens and antiestrogens, retinoids, and vitamin D derivatives. Steroid hormone superfamily members are efficient regulators of the expression of TGF-b isoforms, and TGF-bs are likely to act as local mediators of the diverse actions of steroids. Estrogens and antiestrogens regulate TGF-b1 formation in different cells and tissues like in mammary carcinoma cells and in fetal fibroblasts. TGF-b functions, for instance, as an autocrine negative growth regulator in breast carcinoma cells. TGF-b and LTBP Knockout Mice The importance of the three different TGF-bs is elucidated in the gene knockout studies. Knockout of Tgf-b1 results in multifocal inflammatory disease leading to the death of the animal. Tgf-b2

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knockout is embryonally lethal. The mice develop severe cardiac, lung, and craniofacial defects. The inner ear and eye are also affected. Tgf-b3 null mice develop cleft palate. Accordingly, null mice unable to produce LTBPs develop serious physiological defects. Ltbp-3 knockout mice develop multiple defects such as growth retardation, emphysema, bone malformations, and abnormalities of thymus and spleen. Their lifespan is, however, normal, and they are able to reproduce. Hypomorphic Ltbp-4 / mice develop early emphysema and colorectal tumors. The mice have also severe defects in elastogenesis. The short and long splice forms of LTBPs have different functions. Ltbp-1L null mice exhibit a cardiac phenotype, which reveals a crucial role for Ltbp-1L and matrix as extracellular regulators of Tgf-b activity in heart organogenesis. Ltbp2 / mice developed lens luxation caused by compromised ciliary zonule formation. Perspective Growth factors of the TGF-b family are important autocrine and paracrine regulators of cell proliferation and differentiation. The regulation levels of their activities include the expression of TGF-b receptors, availability of TGF-bs, their activities, and modulation of the cellular response. Most cells secrete TGF-b in a large latent complex, which associates with the extracellular matrix and is unable to bind to the TGF-b signaling receptors. LTBPs have a central role in TGF-b secretion, extracellular matrix deposition, and activation. In addition, LTBPs have structural and other functions not directly related to TGF-b signaling. Structural diversity in LTBP proteins is tremendous, and the possible functions of the different forms include, among others, the modulation of cell adhesion and the functions of integrins. Focal activation of latent TGF-b in the matrix by physicochemical means offers a rapid way to induce TGF-b signaling. In addition to plasminmediated TGF-b activation, novel mechanisms have been found including other proteases, reactive oxygen species, and thrombospondin- and integrin-mediated activation. The modulation of pericellular proteolytic activity by TGF-b supports a general cascade of

Transforming Growth Factor-Beta

events, where proteinases and latent matrixbound growth factors are components of extracellular signal transduction machinery. This directs tissue construction and remodeling and probably also regulates the activity of infiltrating immune cells. Disturbances in these control systems could participate in the pathogenesis of a variety of disease states like atherosclerosis, cancer, various fibrotic diseases, and chronic inflammation.

Cross-References ▶ Anoikis ▶ Apoptosis ▶ Fibronectin ▶ Integrin Signaling ▶ Plasminogen-Activating System ▶ Serine Proteases (Type II) Spanning the Plasma Membrane ▶ Smad Proteins in TGF-Beta Signaling ▶ Tissue Inhibitors of Metalloproteinases

References Koli K, Saharinen J, Hyytiäinen M, Penttinen, Keski-Oja J (2001) Latency, activation and binding proteins of TGF-b. Microsc Res Tech 52:354–362 Moustakas A, Heldin CH (2009) The regulation of TGF-b signal transduction. Development 136:3699–3714 Munger JS, Sheppard D (2011) Cross talk among TGF-b signaling pathways, integrins, and the extracellular matrix. Cold Spring Harb Perspect Biol 3:a005017 Saharinen J, Keski-Oja J (2000) Specific sequence motif of 8-Cys repeats of TGF-b binding proteins, LTBPs, creates a hydrophobic interaction surface for binding of small latent TGF-b. Mol Biol Cell 11:2691–2704 Sporn MB (2006) The early history of TGF-b, and a brief glimpse of its future. Cytokine Growth Factor Rev 17:3–7 Todorovic V, Rifkin DB (2012) LTBPs, more than just an escort service. J Cell Biochem 113:410–418

See Also (2012) Latent TGF-β Binding Protein. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1986. doi:10.1007/978-3-642-164835_3288 (2012) TGF-β Activation. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3662. doi:10.1007/978-3-642-16483-5_5754

Transgenic Mouse

4631

Characteristics

Transfusion of T Cells ▶ Adoptive T-Cell Transfer

Transgenic Mice ▶ Mouse Models

Transgenic Mouse Aparna Gupta Life Science Research Associate, Department of Gastroenterology and Hepatology, Stanford University School of Medicine, Stanford, CA, USA

Definition A transgenic mouse contains additional, artificially introduced genetic material/DNA in every cell. The foreign DNA that has been purposely inserted into the mouse genome can be a foreign gene (not usually present in mice), a gene from the mouse, or a gene with a mutation (mutation: a change in the DNA code that alters the protein properties). The foreign gene is carefully constructed using recombinant DNA technology. In addition to the target gene itself, the DNA also includes other sequences that ensure: • Incorporation into the genomic DNA of the mouse • Proper expression of the gene by the cells In order to incorporate the foreign DNA into every cell of the mouse, it has to be introduced into very early mouse embryo so that the germ cells (sperm and ovum) also receive the gene and pass it on to the next generation.

Methodology There are two major methods that can be used to generate a transgenic mouse: • Pronuclear microinjection: Foreign DNA is introduced directly into the mouse egg just after fertilization. Using a fine needle under the microscope, the DNA is injected into the large male pronucleus, which is derived from the sperm. The fertilized egg divides actively, and the DNA integrates at a random position in the genome. There can be a single copy of foreign DNA or multiple tandem copies of DNA integrated into the genome. The integration occurs after a few cell divisions; therefore, the resulting mouse is only partially transgenic (some cells lack the transgene, referred to a mosaic). If the transgenic cells contribute to the germ line, then some transgenic eggs or sperm will be produced, and the next generation of mice will be fully transgenic (Fig. 1). • Embryonic stem cell (ES) injection: In this method, DNA is introduced into embryonic stem cells (ES cells). ES cells are derived from the very early mouse embryo and can therefore differentiate into all types of cell when introduced into another embryo. Foreign DNA is introduced into ES cells growing on plates; DNA may integrate randomly, as in the case of pronuclear microinjection. The manipulated ES cells are then selected for cells that express the transgene. These are injected into the inner cell mass (ICM) of a blastocyst (blastocyst is an early stage of embryo formation where the inner layer of cells called ICM gives rise to the embryo and the outer layer, called trophoblast, gives rise to the placenta.) The injected blastocysts are implanted into the uterus of a pseudopregnant (by mating a female mouse with a vasectomized male) female mouse. This triggers changes in the female that facilitate embryo implantation. If the procedure is successful, the implanted embryos will give rise to healthy pups (Fig. 1).

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Transgenic Mouse

Transgenic Mouse, Fig. 1 Methodology for generating a transgenic mouse

Pups are screened for the transgene, only a small percentage of them will be positive. The ones positive for the transgene are bred to each other to generate transgenic animals. Random Versus Targeted Gene Insertion Random insertion of DNA results in multiple copies of the transgene in the genome. It can also insert itself in a coding sequence or could disrupt some regulatory sequences in the genome. Thus, targeted gene insertion is now the preferred method as it ensures that the foreign DNA does not disrupt other genomic sequences. The targeted DNA sequence has (as shown in Fig. 2): • The gene of interest. • Neomycin-resistant gene that inactivates the antibiotic neomycin and allows the cells to grow in the presence of the drug, by which positive cells can be selected. • A gene that encodes thymidine kinase (TK), an enzyme that phosphorylates ganciclovir. Nonfunctional phosphorylated ganciclovir inserts into freshly replicating DNA and kills the cells. So ganciclovir in the growth medium

kills cells that contain the TK gene and have random insertion of the transgene. The foreign DNA with all the right components is given to the cells, and the cells are cultured in the presence of neomycin and ganciclovir in the medium: • The cells that failed to take up the foreign DNA are killed by neomycin. • The cells in which the DNA is inserted randomly are killed by ganciclovir (because they insert nonfunctional nucleotide due to presence of tk gene). • The surviving cells are the ones transformed by homologous recombination where the DNA integrates in the desired genomic site (Fig. 2). These cells are injected into blastocysts, implanted into pseudopregnant females, and pups are analyzed. Knockout Mice As the name suggests, these mice have a complete loss or knockout of the gene of interest. This is achieved by using a recombinant DNA without any sequence for gene G (the target gene to be

Transgenic Mouse

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Transgenic Mouse, Fig. 2 Random and targeted gene insertion

Transgenic Mouse, Fig. 3 Conventional gene knockout

deleted). The foreign DNA will recombine with the genomic locus of gene G and result in replacement of G with neomycin resistance gene (Fig. 3). This is the simplest method to generate knockout mice. Figure 3 shows a knockout strategy that results in gene loss in every cell of the body. Knockout of a critical gene can result in dead mice not allowing further study of the gene of interest. If the scientific question addresses to study the function of a gene in a

particular tissue or at a particular stage of development, then tissue-specific knockout mice will be generated. Tissue-Specific Knockout Mice (The Cre/loxP System) Cre recombinase, abbreviated Cre, is an enzyme from bacteriophage P1 that can recognize a specific DNA sequence (called loxP site) and excise and recombine them. This system involves two mice lines (Fig. 4):

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Transglutaminase Type-2

Transgenic Mouse, Fig. 4 Tissue-specific and conditional gene knockout

• Mice expressing the Cre enzyme: (a) The expression of Cre can be driven by a tissue-specific promoter that allows the gene loss only in a desired tissue/ cell type. (b) The promoter can also be designed such that the Cre is expressed only after a certain drug is given to the mice. This allows the loss of gene at certain desired age and not from birth. • Mice carrying the conditional gene (the target gene between two loxP sites)

Kos CH (2004) Cre/loxP system for generating tissuespecific knockout mouse models. Nutr Rev 62(6 Pt 1):243–246 Kuhn R, Torres RM (2002) Cre/loxP recombination system and gene targeting. Methods Mol Biol 180:175–204

The two transgenic mice are bred, and the pups are analyzed for mice carrying both the Cre and loxP conditional gene. These mice have a loss of the target gene in the tissue/ cell targeted by the promoter used for the Cre-expressing mice.

Transglutaminase-2

References Cameron ER (1997) Recent advances in transgenic technology. Mol Biotechnol 7(3):253–265 Cameron ER, Harvey MJ, Onions DE (1994) Transgenic science. Br Vet J 150(1):9–24

Transglutaminase Type-2 ▶ Transglutaminase-2

Kapil Mehta The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Synonyms Cytosolic transglutaminase; Endothelial transglutaminase; Liver transglutaminase; TG2; TGc; Tissue transglutaminase; Transglutaminase type-2; tTGase

Transglutaminase-2 Integrin 28 kDa fragment) 139 aa1

4635 Catalytic triad

β-sandwich

N

81 Fn 140

520 358 GTP 460

277 335 358

Catalytic core C

H D

EGSEEERE 430 Ca2+ 453

β-barrel1 αIβ-adrenergic receptor

687

β-barrel2

C

LHMGLKLV 657 677 PLCδ1

Transglutaminase-2, Fig. 1 Schematic representation of various functional domains of the TG2 protein. In addition to catalyzing calcium-dependent protein cross-linking function, TG2 can catalyze calcium-independent GTPase, ATPase, protein kinase, and protein disulfide isomerase activities. TG2 can modulate the functions of other proteins by directly interacting or associating with them; examples include phospholipase-d1, members of the

b-integrin family, focal adhesion kinase, fibronectin, osteonectin, RhoA, multilineage kinases, and ▶ retinoblastoma protein. Through these activities, TG2 plays a role in biological processes such as ▶ apoptosis, wound healing, and cataract formation. Recent work suggests that TG2 can also serve as a signaling molecule and promote cell growth, drug resistance, and metastatic functions in tumor cells

Definition

types. An important feature of TG2 is its high binding affinity for ▶ fibronectin; in cancers, membrane-associated TG2 can promote a stable interaction between cell surface integrins and fibronectin and promote cell growth and survival (Fig. 1).

Tissue transglutaminase (TG2; EC 2.3.2.13) is a ubiquitous and most diverse member of the transglutaminase family of enzymes. TG2 catalyzes calcium-dependent posttranslational modification of proteins by inserting highly stable isopeptide bonds between polypeptide chains or by conjugating ▶ polyamines to proteins. In addition, TG2 exhibits ▶ GTPase activity and can serve as a ▶ signal-transduction G protein. Less studied functions of TG2 include its protein disulfide isomerase and ▶ protein kinase activities.

Characteristics TG2 is a multifunctional protein whose expression in some cell types (e.g., endothelial and smooth muscle cells) is constitutively high. In other cell types, TG2s expression is upregulated via discrete signaling pathways, such as those induced by certain stress factors, inflammatory stimuli, differentiation agents, and growth factors. Although predominantly a cytosolic protein, TG2 can translocate to the nucleus by “piggyback riding” other proteins, such as importin-alpha-3, or translocate to membranes in association with integrins. TG2 can also be secreted outside the cell (by an as-yet-unknown mechanism), where it can cross-link extracellular matrix (ECM) proteins and promote ▶ adhesion of several cell

TG2 and Apoptosis A role for TG2 in apoptosis was initially suggested by Dr. Laszlo Fesus and his coworkers in 1987 based on the observation that leadinduced hypertrophy of the liver in rats was associated with cellular expression of increased TG2. Since then, many reports have supported the role of TG2 in apoptosis. In general, the expression of TG2 is markedly increased in cells undergoing apoptosis. Forcibly increasing the expression of TG2 in several cell types results in apoptosis or makes them susceptible to death-inducing stimuli. Conversely, reducing TG2 levels by antisense RNA renders the cells more resistant to apoptosis. It is believed that TG2 promotes apoptosis by cross-linking intracellular proteins, preventing their leakage from cells and induction of an inflammatory response. These observations suggest that cells generally do not tolerate the increased expression of TG2 and that TG2 overexpression leads to apoptotic death. However, some reports have provided paradoxical evidence and suggest that TG2 expression and apoptosis do not always go hand in hand. For example, TG2/ knockout mice (mice lacking

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4636

all TG2 expression) did not show any genetic alterations that are suggestive of perturbed apoptosis. The possibility that some other proteins compensate for the loss of TG2 in these mice cannot be ruled out. Furthermore, various other studies have provided data suggesting that increased expression of TG2 can prolong cell survival by preventing apoptosis. TG2 in Drug Resistance and Metastasis Evidence is accumulating that cancer cells that are resistant to chemotherapeutic drugs or that are isolated from metastatic sites express elevated levels of TG2. Also, there is evidence that drugresistant and metastatic cancer cells share some common pathways. For example, cells from advanced-stage cancers accumulate a large number of genetic alterations that can render them resistant to apoptosis. Resistance to apoptosis can enable cancer cells not only to grow and survive in the stressful environment of distant tissues (i.e., to metastasize) but also to withstand the toxic effects of drugs. Moreover, cell lines selected in vitro for resistance against chemotherapeutic drugs are more metastatic in vivo, while cancer cells isolated from metastatic sites, in general, exhibit higher resistance to chemotherapeutic drugs. Based on these observations we hypothesized that aberrant expression of TG2 in drugresistant and metastatic cancer cells may dysregulate some intrinsic apoptotic pathways in order to protect cells from apoptosis. Indeed, downregulation of endogenous TG2 by antisense RNA, TG2-specific ribozyme, or small interfering RNA (▶ siRNA) could reverse drug resistance in ▶ lung cancer, ▶ ovarian cancer and ▶ breast cancer cells. Similarly, inhibition of TG2 by siRNA in breast cancer and malignant ▶ melanoma cells augmented their response to chemotherapeutic drugs and reduced their invasiveness in laboratory experiments. In pancreatic cancer cells, inhibition of TG2 by siRNA resulted in massive accumulation of lysophagosomes and onset of ▶ autophagy (type II apoptosis). These properties suggest that TG2 expression in cancer cells contributes to the development of drug resistance and ▶ metastasis. Studies to elucidate the mechanisms involved in the development of TG2-mediated drug

Transglutaminase-2

resistance in cancer cells revealed that TG2 expression augments cell survival signaling by promoting a stable interaction between cell surface integrins and the ECM proteins. Depending on the cell type, 20–30% of total TG2 can exist in complex with b-integrins (e.g., b1, b4, and b5). The association of TG2 with integrins occurs primarily at their extracellular domains and promotes their interaction with ECM ligands such as fibronectin, collagen, and vitronectin. Downregulation of TG2 in glioblastoma cells resulted in decreased assembly of fibronectin in the ECM and cell death. Importantly, treatment of mice that had orthotopic glioblastomas with the TG2 inhibitor KIP1 sensitized the tumors to chemotherapy, induced apoptosis of cancer cells, and prolonged survival of the animals. Further, the interaction between TG2 and integrins is independent of the cross-linking activity of TG2 and results in increased cell adhesion, ▶ migration, and activation of downstream survival signaling pathways such as ▶ focal adhesion kinase (FAK). Interestingly, TG2 can also interact directly with focal adhesion kinase and result in its autophosphorylation (pY397) and consequent activation of the downstream PI3K and Akt signaling. Activation of the ▶ nuclear factor-kB (NF-kB), which plays an important role in regulating cell growth, apoptosis, and metastasis, has also been associated with increased TG2 expression in cancer cells. Tumor cells that overexpressed TG2 exhibited increased levels of constitutively active NF-kB. Activation of TG2 led to activation of NF-kB, and conversely, inhibition of TG2 activity inhibited the activation of NF-kB. Similarly, ectopic expression of TG2 caused activation of NF-kB, and inhibition of TG2 expression by siRNA abolished the NF-kB activation and rendered drug-resistant breast cancer cells sensitive to doxorubicin-induced cytotoxicity. Notably, immunohistochemical analysis of pancreatic ductal adenocarcinoma tumor samples further supported a strong correlation between TG2 expression and NF-kB activation. These observations suggest that TG2 induces constitutive activation of NF-kB in tumor cells via a novel pathway. Therefore, TG2 may be an

Transitional Cell Carcinoma

attractive target for inhibiting constitutive NF-kB activation and rendering cancer cells sensitive to anticancer therapies. Clinical Relevance Drug resistance and metastasis are major impediments to the successful treatment of cancer. More than 90% of cancer-related deaths can be attributed to the failure of chemotherapy. On the basis of published results that drug-resistant and metastatic tumors and tumor cell lines express high levels of TG2, that TG2 expression promotes cell survival and invasion, and that downregulation of TG2 results in increased sensitivity of cancer cells to chemotherapeutic drugs and to undergo programmed cell death (apoptosis or autophagy), TG2 may offer an attractive target for treating drug-resistant and metastatic tumors.

4637

Transitional Cell Carcinoma Jun Hyuk Hong1, Seong Jin Kim2 and Isaac Yi Kim1 1 Division of Urologic Oncology, The Cancer Institute of NJ, Robert Wood Johnson Medical School, New Brunswick, NJ, USA 2 Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD, USA

Synonyms TCC; Transitional cell carcinoma of bladder; Transitional cell carcinoma of renal pelvis; Transitional cell carcinoma of ureter; Urothelial carcinoma; Urothelial tumor

References

Definition

Eckert RL, Kaartinen MT, Nurminskaya M, Belkin AM, Colak G, Johnson GV, Mehta K (2014) Transglutaminase regulation of cell function. Physiol Rev 94:383–417 Lorand L, Graham RM (2003) Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol 4:140–156 Mehta K, Eckert R (eds) (2005) Transglutaminases: family of enzymes with diverse functions. Prog Exp Tumor Res 38:125–138 Mehta K, Fok J, Miller FR et al (2004) Prognostic significance of tissue transglutaminase in drug resistant and metastatic breast cancer. Clin Cancer Res 10:8068–8076 Mehta K (2005) Mammalian transglutaminases: a family portrait. In: Transglutaminases: family of enzymes with diverse functions. Karger Publ pp 1–18 Mehta K, Fok JY, Mangala LS (2006) Tissue transglutaminase: from biological glue to cell survival cues. Front Biosci 11:173–185 Verma A, Wang H, Manavathi B et al (2006) Increased expression of tissue transglutaminase in pancreatic ductal adenocarcinoma and its implications in drug resistance and metastasis. Cancer Res 66:10525–10533

Transitional cell carcinoma (TCC) arises in the urothelium that covers the lining of the renal calyx, renal pelvis, ureter, bladder, and part of the urethra. Although the WHO/ISUP consensus conference has determined that the term urothelial cancer is preferable to the term transitional cell cancer, the latter remains in widespread use. “Urothelial cancer” may also be confusing because cancers of other histological types, such as squamous cancers and adenocarcinoma, also arise in the urothelium.

Transin-1 ▶ Stromelysin-1

Characteristics Epidemiology It was estimated that in 2007, 67,160 new cases of bladder cancer would be diagnosed and 13,750 patients would die of invasive bladder cancer in the United States. ▶ Bladder cancer is nearly three times more common in men than in women, and more than 90% of bladder cancers are TCCs. The median ages at diagnosis for TCC are 69 years in males and 71 years in females.

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Upper urinary tract urothelial tumors involving the renal pelvis or ureter are relatively uncommon, accounting for about 5–7% of all renal tumors and about 5% of all urothelial tumors. Etiology One of the genetic changes that must occur for malignant transformation is the induction of oncogene. Oncogenes associated with TCC include those of the RAS gene family, including ▶ P21 RAS oncogene, and up to 50% of TCCs have been claimed to have RAS mutations. Another important molecular mechanism in the process of carcinogenesis is the inactivation of tumor suppressor genes. These include that of P53, the most frequently altered gene in human cancers, the retinoblastoma (RB) gene (▶ Retinoblastoma Protein, Biological and Clinical Functions), and genes on chromosome 9. Overexpression of normal genes including those for EGF receptor (ERBB1) and ERBB2 (epidermal growth factor receptor ligands) occurs in most TCCs. Cigarette smokers have a fourfold higher incidence of TCC than do people who have never smoked (▶ tobacco-related cancers). Ex-smokers have a reduced incidence of TCC, but the reduction of this risk down to baseline takes nearly 20 years. Nitrosamines, 2-naphthylamine, and 4aminobiphenyl are suggested as being responsible for TCC in cigarette smoke. Women treated with radiation for carcinoma of the uterine cervix or ovary have a two- to fourfold increased risk of developing bladder cancer. Patients treated with cyclophosphamide have up to a ninefold increased risk of developing bladder cancer. Signs and Symptoms Microscopic or gross hematuria is the most common presenting symptom. Patients with gross hematuria have reported rates of bladder cancer of 13–35%. With microscopic hematuria, the rates decreased to 0.5–10.5%. So, if a patient has unexplained hematuria, either microscopic or gross, cystoscopic examination is usually warranted, especially in individuals older than age 60 or younger people with a smoking history. The second most common presentation is the constellation of lower urinary tract irritative

Transitional Cell Carcinoma

symptoms such as urinary frequency, urgency, and dysuria. These irritative symptoms usually occur with hematuria. In fact, the risk of TCC may be doubled in patients with irritative voiding symptoms that coexist with hematuria. Evaluations In all patients with signs and symptoms suggestive of bladder cancer, excretory urography (IVU) is indicated. It is useful in examining the upper urinary tracts for associated urothelial tumors. Large bladder tumors may appear as filling defects in the bladder, but small ones may not be detected. Computed tomography (CT) has replaced IVU in the evaluation of hematuria. After imaging studies, all patients suspected of having bladder cancer should have cystoscopy. Retrograde pyelography should be done if the upper tracts are not visualized on IVU or CT. CT can help to assess the extent of the primary tumor and provides information about the presence of pelvic and para-aortic lymphadenopathy and visceral metastases. But CT fails to detect nodal metastases in up to 40–70% of patients who have them. MRI is not much more helpful than CT. Pelvic lymphadenectomy, which can be done with cystectomy, is the most accurate means of determining regional node involvement. The primary regions of lymphatic drainage of the bladder are the perivesical, hypogastric, obturator, external iliac, and presacral nodes. As some patients with limited nodal metastases can be benefited by lymphadenectomy, bilateral node dissection should be done. The usually recommended metastatic evaluation for invasive bladder cancer includes a chest radiograph, abdominal-pelvic CT, bone scan, and liver function tests. A flexible cystoscope is often used for the initial diagnosis and follow-up of patients with bladder tumors. It has much less discomfort than a rigid cystoscope. Bugbee electrode devices can be inserted through a flexible cystoscope to allow destruction of small, noninvasive papillary tumors. Malignant urothelial cells have large nuclei with irregular, coarsely textured chromatin and can be observed on microscopic examination of

Transitional Cell Carcinoma

the urinary sediment. This microscopic cytology is more sensitive in patients with high-grade tumors or carcinoma in situ (CIS). The specificity and positive predictive value of cytology are quite high. Staging (1997 AJCC-UICC, TNM Staging) As tumor stage forms the foundation for determining therapy, accurate staging is critical. The first treatment decision based on tumor stage is the presence or absence of muscle invasion. Because metastases are very rare with a superficial (non-muscle-invasive) tumor, treatment strategy can be grouped into superficial (Ta, T1, and Tis), muscle-invasive, and metastatic tumors. About 70% of bladder tumors are superficial at presentation. Of these, 70% present as stage Ta, 20% as T1, and 10% as CIS. Ta Tis T1 T2a T2b T3a T3b T4a T4b N0 N1 N2 N3 M0 M1

Papillary, epithelium confined Flat carcinoma in situ Lamina propria invasion Superficial muscularis propria invasion Deep muscularis propria invasion Microscopic extension into perivesical fat Macroscopic extension into perivesical fat Cancer invading pelvic viscera Extension to pelvic sidewalls, abdominal walls, or bony pelvis No histological pelvic node metastasis Single positive node 2 cm in diameter, below common iliacs Single positive node 2–5 cm in greatest diameter or multiple positive nodes Positive nodes >5 cm in diameter No distant metastases Distant metastases documented

Treatment of Superficial Bladder Cancer Local resection of a bladder tumor usually enables complete removal of the tumor and provides diagnostic information about the depth and the grade of the tumor. For this, first the bulk of the tumor and then the deep portion with some underlying bladder muscle should be resected. To detect dysplasia or CIS elsewhere in the bladder, selected site mucosal biopsies from areas adjacent to the tumor, bladder dome, trigone, and prostatic urethra have been recommended.

4639

If 5-aminolevulinic acid (ALA) is administered into the bladder in conjunction with fluorescent cystoscopy, lesions invisible with normal cystoscopy can be detected. The most important issue in tumor biology of superficial tumors is recurrence and progression to higher stages. Low-grade Ta tumors recur at a rate of 50–70% and progress in about 5%. High-grade T1 lesions recur in 80% and progress in 50% of patients. The most important risk factor for progression in superficial bladder tumors is grade, not stage. Prognosis also correlates with the presence of CIS, tumor size, multiplicity, and lymphovascular invasion and the configuration of the tumor (papillary vs. sessile). Of patients with CIS, 40–83% will develop muscle invasion if untreated. For T1 tumors, the depth of lamina propria invasion determined by the muscularis mucosa invasion or the extent of invasion below the urothelial surface has been known to be correlated with prognosis. CT and MRI appear to be inaccurate in determining the microscopic muscle infiltration and the minimal extravesical spread, which can also be aggravated by post-tumor recurrence (TUR) changes. To prevent recurrence and progression of bladder tumors, intravesical immunotherapy using BCG (Bacillus Calmette-Guerin) has been used. Treatments are generally begun 2–4 weeks after TUR and a 6-week course is usually administered. With BCG, tumor recurrence was reduced by 20–65% and progression was reduced by 23–27%. Intravesical chemotherapy using ▶ mitomycin C, doxorubicin, thiotepa, epirubicin, and ▶ gemcitabine also has been administered. Treatment of Invasive Bladder Cancer The standard surgical approaches to muscleinvasive bladder cancer are radical cystoprostatectomy in the male patient and anterior exenteration in the female patient, with bilateral pelvic lymphadenectomy. Anterior exenteration in the female requires removal of the uterus, fallopian tubes, ovaries, bladder, urethra, and a segment of the anterior vaginal wall. A nervesparing modification has been proposed in the

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male patient and results in improved postoperative return of erectile function. The more prevalent the orthotopic reconstruction becomes, the stricter indications for urethrectomy have been applied. The most significant factor for the anterior urethral recurrence and local/distant failure in the male patient has been identified as prostatic urethral involvement. The estimated 5-year probability of urethral recurrence is 5% without any prostate involvement and 12–18% with prostate involvement. CIS of the bladder neck and trigone was also significantly associated with prostatic urethral involvement. In the female patient, overt cancer at the bladder neck and urethra, diffuse CIS, or positive margin at surgery should be treated by en bloc urethrectomy as a part of the radical cystectomy. The mortality rate for radical cystectomy is 1–2% and the overall complication rate is about 25%. After urinary tract diversion, bowel obstruction rate is 4–10%. Stricture of anastomosis between ureter and bowel is found in less than 3%. Depending on the type of neobladder, metabolic disorders, vitamin deficiency, and urinary tract infection can occur. As for the neoadjuvant chemotherapy, results suggested improvement in overall survival of 5–6% among patients with locally advanced disease (stages T3–T4a). Some reports suggest that for patients with locally advanced disease and lymph node involvement, adjuvant chemotherapy may also provide a survival advantage. Due to the small numbers of patients, these results are as yet insufficient for the routine use of adjuvant therapy. Treatment of Metastatic Bladder Cancer These patients are routinely treated with systemic chemotherapy. The most commonly used agents are methotrexate, vinblastine, doxorubicin, and ▶ cisplatin (MVAC). MVAC chemotherapy produces a complete response in about 20% of patients, although long-term disease-free survival is rare. The combination of cisplatin and a newer agent, ▶ gemcitabine (GC), has produced similar survival outcomes with less toxicity compared with MVAC. ▶ Paclitaxel and ▶ docetaxel have also been used in clinical trials and demonstrate response rates of 25–83%.

Transitional Cell Carcinoma of Bladder

Cross-References ▶ Urothelial Carcinoma, Clinical Oncology

References Herr HW, Dotan Z, Donat SM et al (2007) Defining optimal therapy for muscle invasive bladder cancer. J Urol 177:437–443 Messing EM (2007) Urothelial tumors of the bladder. In: Wein AJ, Kavoussi LR, Novick AC, Partin AW, Peters CA (eds) Campbell-Walsh urology, 9th edn. SaundersElsevier, Philadelphia, pp 2407–2446 Sengupta S, Blute ML (2006) The management of superficial transitional cell carcinoma of the bladder. Urology 67:48–54 Sternberg CN, Donat SM, Bellmunt J et al (2007) Chemotherapy for bladder cancer: treatment guidelines for neoadjuvant chemotherapy, bladder preservation, adjuvant chemotherapy, and metastatic cancer. Urology 69:62–79

Transitional Cell Carcinoma of Bladder ▶ Transitional Cell Carcinoma

Transitional Cell Carcinoma of Renal Pelvis ▶ Transitional Cell Carcinoma

Transitional Cell Carcinoma of the Urinary Bladder ▶ Urothelial Carcinoma

Transitional Cell Carcinoma of Ureter ▶ Transitional Cell Carcinoma

Translesion DNA Polymerases

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Pol K

Translesion DNA Polymerases

Pol I

W. Glenn McGregor University of Louisville, Louisville, KY, USA

REV1 3⬘

6 18

TT

REV7 Pol ␨ REV3

Definition Translesion DNA synthesis (TLS) is a highly conserved mechanism for the completion of replication of damaged genomes. Analogous pathways exist in bacteria, and homologs with remarkable similarity exist in all eukaryotic cells, including postmitotic organisms such as D. melanogaster. Advances in elucidating the molecular mechanisms of carcinogen-induced mutagenesis indicate that replication of DNA templates that contain replication-blocking adducts is accomplished with error-prone DNA polymerases. These polymerases have relaxed base-pairing requirements and can insert bases across from adducted templates, but with potentially mutagenic consequences.

Characteristics Most mutations induced by genotoxic carcinogens occur when a DNA template that contains residual (unrepaired) damage is replicated during S-phase of the cell cycle. Presumably, the replication complex is blocked by bulky adducts in the DNA such as those induced by ultraviolet light (UV) or a variety of chemical carcinogens (▶ Adducts to DNA). As diagrammed in Fig. 1, advances indicate that error-prone translesion synthesis (TLS) is responsible for the majority of base substitutions induced in the DNA. TLS is defined as the incorporation of a nucleotide across from DNA damage followed by extension of the potentially mispaired primer-template. This process is undertaken by at least five accessory DNA polymerases, several of which have been purified and studied in vitro (▶ DNA damage responses). The properties of these polymerases have been extensively reviewed. Based on structural homology, these

Pol ␩ XPV/hRAD30A 5⬘

Pol ␦ TT GA

Translesion DNA Polymerases, Fig. 1 Model for translesion replication. The replicative polymerase complex stalls at sites of helical distortion induced by DNA damage, such as UV-induced photoproducts. The presumed ubiquitin ligase RAD18 targets a ubiquitinconjugating enzyme, RAD6, to the site of damage. There are two closely related homologs of RAD6 in higher eukaryotic cells, termed RAD6A and RAD6B. One of the targets of ubiquitination appears to be PCNA, which signals accessory polymerases in ways that are not fully understood, although at least one TLS polymerase, pol Z, has a higher affinity for monoubiquitinated PCNA. Current thinking is that one of the Y-family polymerases (pol Z, pol ι, or pol k) may insert a base directly across from the lesion, but pol z is required to extend the resulting primer such that the pol d can continue processive DNA replication. REV1 is required for mutagenesis, but this role is probably separate from its dCMP transferase activity (data indicate that REV1 may tether pol z to the other accessory polymerases)

polymerases fit into one of two families: the Y-family (REV1, pol Z, ι, and k) or the B-family (pol z). The cellular roles of this universe of polymerases are not known. In particular, the extent to which each of these polymerases participates in TLS most likely depends on the structure of a particular adduct and on the sequence context. As shown in Fig. 1, it has been suggested that pol Z, ι, and/or k inserts a base directly across from a lesion and that pol z extends the mispair to form a template-primer that can be extended by pol d. Although REV1 is a DNA polymerase, its role in mutagenesis is thought to be structural rather than catalytic. The unrestrained activity of error-prone polymerases would lead to widespread mutagenesis and genomic instability, so there are signaling mechanisms that tightly control polymerase

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Translesion DNA Polymerases

PCNA

MMS2Ubc13; Rad5

Rad6 Rad18

K-63 Damage avoidance

K-164-Ubc Translesion synthesis

PCNA

PCNA Ubc9 siz1

K-164-SUMO PCNA

RAD52dependent recombination

Translesion DNA Polymerases, Fig. 2 Regulation of lesion bypass in the budding yeast Saccharomyces cerevisiae is thought to be signaled by modification of PCNA at lysine 164 (K-164). The presence of a stalled DNA replication fork recruits Rad18, which is a presumed ubiquitin ligase, and Rad6, a ubiquitin conjugase, to the site of the replication-blocking lesion. Monoubiquitination at K-164 leads to recruitment of TLS polymerases with potentially mutagenic consequences. Polyubiquitination at lysine 63 (K-63) of ubiquitin by MMS2-Ubc13 leads to damage avoidance. A proposed mechanism for damage

avoidance is uncoupling of the replication fork, such that the undamaged strand is replicated for some distance beyond the blocked replication complex. The nascent strand, which has the same sequence as the damaged strand, then acts as a template for replication. The damage is thereby avoided in an error-free manner. A competing reaction is sumoylation at K-164 by the SUMO (small ubiquitin modifier)-specific ligase Siz1 and conjugase Ubc9. This reaction is thought to suppress Rad52dependent recombination and damage-induced genomic instability

switching events. Although not fully understood, the mechanisms used by cells to accomplish polymerase switching events at blocked primer termini have been studied most intensively in the budding yeast, Saccharomyces cerevisiae. In this organism, replication-blocking lesions in the template strand can be bypassed by proteins in the Rad6dependent DNA damage tolerance pathway. This process prevents the collapse of stalled replication forks, and replication of the damaged template is completed by TLS with potentially mutagenic consequences or by damage avoidance mechanisms mediated by recombination that are largely error-free. As diagrammed in Fig. 2, the ubiquitinconjugating enzyme encoded by Rad6 and the presumed ubiquitin ligase encoded by Rad18 are central to this process, since mutants cannot bypass replication-blocking lesions in the template and are sensitive to many DNA-damaging

agents. Insights into the biochemical function of this complex were gained when the Rad18/Rad6 complex was found to be responsible for the monoubiquitination of PCNA at K-164. PCNA modified in this fashion is thought to signal translesion synthesis and further ubiquitination is thought to signal damage avoidance (Fig. 2). Although the molecular details of the signaling pathways downstream of monoubiquitination are unknown, at least one TLS polymerase, pol Z, has been shown to have enhanced affinity for monoubiquitinated PCNA. The strategies used by yeast cells to complete the replication of damaged genomes appear to have been conserved in higher eukaryotes, but with additional layers of complexity. For example, higher eukaryotic cells have at least two Y-family polymerases that are not found in yeast and one of these (pol k) appears to be independent of RAD18. Human RAD18 was cloned and the

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protein was purified. It is a 56 kDa protein that shares 26% identity and 59% similarity with its yeast counterpart. The protein interacts with the two RAD6 homologs found in higher eukaryotes (RAD6A and RAD6B) with equal affinity and is ubiquitously expressed in all human tissues. Among the conserved regions are a RING finger motif found in the N-terminus that is required for the interaction with RAD6A/B and a zinc finger that is presumably required for interaction with DNA. In principle, the accessory DNA polymerases and associated proteins described herein represent potential targets for antimutagenesis strategies. However, deficiency of individual polymerases may result in enhanced carcinogenesis. The most well-studied example of this is the human syndrome xeroderma pigmentosum variant, which is a skin cancer-prone condition that results from an inherited deficiency of DNA polymerase Z. This enzyme is posited to be specialized for the error-free bypass of cyclobutane dimers between adjacent thymidine bases. In its absence, data indicate that polymerase iota assumes its function and is error-prone when doing so. Unexpectedly, however, when both polymerases are deficient in mouse models, UV-induced skin cancer is accelerated despite reduced UV-induced mutant frequencies in the double knockout. These data support a role for polymerase iota as a tumor suppressor separate from its role in TLS.

References Dumstorf CA, Clark AB, Lin Q et al (2006) Participation of mouse DNA polymerase iota in strand-biased mutagenic bypass of UV photoproducts and suppression of skin cancer. Proc Natl Acad Sci U S A 103:18083–18088 Friedberg EC, Lehmann AR, Fuchs RP (2005) Trading places: how do DNA polymerases switch during translesion DNA synthesis? Mol Cell 18:499–505 Wang Y, Woodgate R, McManus TP et al (2007) Evidence that in xeroderma pigmentosum variant cells, which lack DNA polymerase eta, DNA polymerase iota causes the very high frequency and unique spectrum of UV-induced mutations. Cancer Res 67: 3018–3026

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Transmembrane 4 Superfamily Protein ▶ Metastasis Suppressor KAI1/CD82

Transmembrane Protease Serine 1–13: Hepsin (TMPRSS 1) ▶ Serine Proteases (Type II) Spanning the Plasma Membrane

Transplacental Carcinogenesis Mark Steven Miller Department of Cancer Biology, Comprehensive Cancer Center, Wake Forest School of Medicine, Winston-Salem, NC, USA

Synonyms Fetal exposure to carcinogenic agents; In utero exposure to carcinogenic agents

Definition Transplacental carcinogenesis is a subfield of cancer research that looks at the effects of exposure of the fetus to chemical and physical agents that may cause cancer. The best known example of a chemical administered during pregnancy that resulted in tumors in the children of the treated mothers is diethylstilbestrol (DES). Young women of mothers who were treated during pregnancy with DES exhibited an increased incidence of vaginal cancer (Miyagawa et al. 2011). The best known example of a physical agent that resulted in cancer in the offspring of exposed mothers is radiation. Studies from the survivors of the atomic bomb blasts in Japan showed that exposure of the fetus to radiation caused increased incidences of cancer at several organ sites (Pierce and Preston 2000).

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Characteristics Types of Transplacental Carcinogens Studies conducted in the field of transplacental carcinogenesis use a variety of experimental techniques to demonstrate the effects of different agents on the developing fetus. These include statistical analysis of incidences of cancer following exposure of pregnant women to chemical or physical carcinogens (such as the DES and atomic bomb studies discussed in the previous section), studies in animal models, and cell culture studies using embryonic cells. Transplacentally active chemical carcinogens can come from a number of different sources. These can include: 1. Pharmaceutical agents such as DES, which are administered for medical reasons but have unintended side effects. Because of concerns over the potential for new pharmaceutical agents to cause harm to the fetus, the Food and Drug Administration requires specific testing in animal models before the agency will allow any testing in people. Since this is often very expensive, many companies will not test new pharmaceutical candidates in younger age-groups, and many drugs are thus not approved for use during pregnancy in the United States. 2. Environmental contaminants. Many chemicals found in the environment are the result of industrial processes. Some of these are chemicals that have been manufactured for a specific use and later determined to be a threat to human health, such as pesticides. Other chemicals are industrial by-products that have leaked into the environment and can be found in water, air, or in the ground. Agents such as pesticides, cigarette smoke, and benzene, as well as a variety of other chemicals, have been linked to increased incidences of both childhood- and adult-onset cancers (Anderson et al. 2000; Alexander et al. 2001). 3. Diet. Many chemicals can be found in the diet as a result of exposure of fish, chicken, cattle, and food animals to environmentally

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prevalent chemicals. While it is unlikely that one can consume sufficient quantities of transplacental carcinogens from a normal diet to affect their developing fetus, the high levels of mercury found in certain fish would be an example where dietary moderation would be advisable. In addition, the safety of a variety of food supplements and nutriceuticals for consumption during pregnancy is not known. Mechanisms of Cancer Causation It is now known that many chemicals that the pregnant mother is exposed to have the potential to cross through the placental circulation into the developing fetus. A number of studies using carefully controlled animal experiments have demonstrated that the developing embryo and fetus exhibit very different sensitivity to many environmental chemical and physical agents than does an adult. Exposure during fetal development can thus pose significant risk to the developing organism at doses that would not affect an adult, including the pregnant mother. For example, treatment of pregnant mice with polycyclic aromatic hydrocarbons (chemicals that are found in cigarette smoke and diesel exhaust) causes a high incidence of lung tumors in the offspring at doses of the chemical that do not affect the pregnant mother (Miller 2004). On the other hand, with some environmental agents, the fetus may be less sensitive than the adult organism to the toxic effects of these agents (Rice 1979). A number of physiological mechanisms have been proposed to account for the differential sensitivity of the fetus to chemical and physical agents. It is likely that this differential sensitivity to cancer induction is the result of combinations of a number of different biological factors. The developing fetus is not a “little adult” and exhibits significant differences in the way they respond to chemical, physical, pharmaceutical, and dietary agents. In addition, the developing fetus is a remarkably adaptable organism, presenting a constantly changing picture of gene expression as a variety of gene systems are turned on and off at different stages of development. As such, the ability of the fetus to mount a defensive response to

Transplacental Carcinogenesis

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Transplacental Carcinogenesis, Fig. 1 Conversion of nonpolar benzene to either water soluble, sugar conjugated, inactive polar metabolites that are readily excreted in the urine and feces (Eqs. 1 and 3); or DNA-bound benzene adduct that can cause gene mutations and disease (Eqs. 1 and 2)

environmental insults can be markedly different at different stages of fetal development (Miller 2004; Shorey et al. 2011). Most living organisms respond to potentially toxic environmental chemicals by producing enzymes that can eliminate the environmental toxin. Many environmental chemicals are actually fairly inert. When they enter the body, these compounds tend to be poorly soluble in water (hence they are referred to as “nonpolar” compounds) and prefer to remain in fattytissue deposits. In order to get rid of these agents, the cells in our bodies increase the production of drug-metabolizing enzymes. Phase I enzymes are the first step in this process. These enzymes catalyze the conversion of the parent nonpolar compound to slightly more watersoluble compounds by adding oxygen to the parent compound. The presence of the oxygen molecule makes the parent compound more reactive, thus creating a target site for Phase II enzymes. These enzymes can then add very water-soluble molecules, such as sugar molecules, to the parent compound. This converts the nonpolar compounds to water-soluble compounds (also called polar compounds) that can be readily excreted in either the urine or feces, as shown in Eq. 1 and schematically in Fig. 1 using benzene metabolism as an example.

Equation 1 Phase I metabolism, the addition of oxygen to a nonpolar compound (R) RH þ O2 ! ROH þ H2 O The initial conversion step carried out by Phase I enzymes can also result in converting the original parent compound into a highly reactive molecule that can bind to DNA, cause gene mutations, and thus initiate the carcinogenic process. Equation 2 Binding of reactive molecules to DNA ROH þ DNA ! R  DNA þ H2 O ! genetic damage Phase II enzymes will bind to these toxic metabolites and aid in their elimination from the body before they can do any harm. Equation 3 Phase II metabolism, inactivation of toxic compounds ROH þ sugar ! R  sugar þ H2 O In the fetus, the levels of both Phase I and Phase II enzymes can differ drastically from

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Trask (Transmembrane and Associated with Src Kinases)

those found in the adult. The fetus lacks many of the enzymes that are normally expressed in adult tissues and thus contain only a subset of these enzymes. In addition, the levels at which these enzymes are expressed can differ by the gestational age of the fetus. In general, both Phase I and Phase II enzymes are poorly expressed during the first trimester, making the fetus at early stages of development less susceptible to chemicals that require enzymatic conversion to become toxic. However, once these enzyme systems start to become prevalent in fetal tissues, the Phase I enzymes become more responsive to chemical toxins than do the Phase II enzymes. This can result in a greater production of toxic metabolites and an enhanced sensitivity of the fetus to certain chemicals relative to the adult organism, which has a greater ability to carry out the second phase of metabolism and eliminate these chemicals (Miller 2004). In addition to drug metabolic enzymes, living organisms have other defensive mechanisms to prevent genetic damage. These include DNA repair enzymes which can fix the damage caused by chemical binding to DNA as shown in Eq. 2 and Fig. 1. Because the fetus is constantly growing and developing, fetal tissue cells exhibit a high rate of cell proliferation. As a result, when genetic damage occurs as a result of exposure to chemical or physical carcinogenic agents, the cells may synthesize new DNA before the cell has time to repair the damage. In this case, the bound chemical will cause gene mutations that are then genetically inherited and passed on to new cells. Although the levels of DNA repair enzymes differ between fetal and adult organisms, their role in the relative protection of fetal DNA is still uncertain. Scientists are continuing to identify new compounds that can cause cancer following in utero exposures, the mechanisms by which transplacental carcinogens exert their effects, and are also looking for preventive measures that can be taken to protect the fetus from known environmental exposures. In addition to regulating environmental release of

potentially harmful chemical and physical transplacental carcinogens, one promising area is chemoprevention. This would allow a pregnant woman to take a relatively harmless drug that would protect her fetus from environmental toxins. As our understanding of transplacental carcinogens increases, new strategies will be developed to protect this vulnerable population.

References Alexander FE, Patheal SL, Biondi A, Brandalise S, Cabrera ME, Chan LC, Chen Z, Cimino G, Cordoba JC, Gu LJ, Hussein H, Ishii E, Kamel AM, Labra S, Magalhaes IQ, Mizutani S, Petridou E, de Oliveira MP, Yuen P, Wiemels JL, Greaves MF (2001) Transplacental chemical exposure and risk of infant leukemia with MLL gene fusion. Cancer Res 61:2542–2546 Anderson LM, Diwan BA, Fear NT, Roman E (2000) Critical windows of exposure for children’s health: cancer in human epidemiological studies and neoplasms in experimental animal models. Environ Health Perspect 108(Suppl 3):573–594 Miller MS (2004) Transplacental lung carcinogenesis: molecular mechanisms and pathogenesis. Toxicol Appl Pharmacol 198:95–110 Miyagawa S, Sato M, Iguchi T (2011) Molecular mechanisms of induction of persistent changes by estrogenic chemicals on female reproductive tracts and external genitalia. J Steroid Biochem Mol Biol 127:51–57 Pierce DA, Preston DL (2000) Radiation-related cancer risks at low doses among atomic bomb survivors. Radiat Res 154:178–186 Rice JM (1979) Perinatal period and pregnancy: intervals of high risk for chemical carcinogens. Environ Health Perspect 29:23–27 Shorey LE, Castro DJ, Baird WM, Siddens LK, Lohr CV, Matzke MM, Waters KM, Corley RA, Williams DE (2011) Transplacental carcinogenesis with dibenzo [def,p]chrysene (DBC): timing of maternal exposures determines target tissue response in offspring. Cancer Lett

Trask (Transmembrane and Associated with Src Kinases) ▶ CDCP1

Trastuzumab

Trastuzumab Wen Jin Wu and Milos Dokmanovic Division of Monoclonal Antibodies, Office of Biotechnology Products, Office of Pharmaceutical Science, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD, USA

Synonyms Anti-c-erB-2; Anti-c-erbB2 monoclonal antibody; Anti-ERB-2; Anti-erbB-2; Anti-erbB2 monoclonal antibody; Anti-HER2/c-erbB2 monoclonal antibody; Anti-p185-HER2; MOAB HER2; Monoclonal antibody c-erb-2; Monoclonal antibody HER2; rhuMabHER2

Definition Trastuzumab (US brand name: Herceptin ®) is a humanized monoclonal antibody IgG1 directed against the human epidermal growth factor receptor 2 (HER2) and is approved for the treatment of HER2-positive breast cancer and HER2-positive metastatic adenocarcinomas of the stomach or gastroesophageal junction (GEJ).

Characteristics Antibody Antibodies, also known as immunoglobulins, are large protein molecules produced by the body’s immune system in response to antigenic stimuli that are either infectious agents, such as a bacteria, fungi, viruses, or parasites, or other molecular components recognized by the body’s immune system as foreign. Antibodies consist of two

The entry “Trastuzumab” appears under the copyright Springer-Verlag Berlin Heidelberg (outside the USA) both in the print and the online version of this Encyclopedia.

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identical pairs of polypeptide chains, comprised of a heavy chain and a light chain. The type of heavy chain determines the immunoglobulin isotype (IgA, IgD, IgG, IgE, or IgM). These polypeptide chains are arranged in a large Y-shaped protein. Both heavy and light chains have constant and variable regions. Variable regions are contained within the amino (NH2) terminus of the polypeptide chain where complementarity determining regions (CDRs) are found. CDRs serve to recognize and bind specifically to antigen. Monoclonal antibodies are antibody molecules with identical amino acid sequences expressed by a single clone of cells. Molecular Target of Trastuzumab HER2 (also known as neu and ErbB2) is encoded by ERBB2/neu gene and is a member of the HER family of receptor tyrosine kinases. HER family receptors are composed of four type I receptors: EGFR/HER1/ErbB1, HER2/ErbB2, HER3/ ErbB3, and HER4/ErbB4. All receptors share a similar structure composed of an extracellular ligand-binding region, a single transmembrane lipophilic segment, and a cytoplasmic tyrosine kinase-containing domain. The extracellular ligand-binding region of HER family receptors is composed of four domains (I–IV). Domains I and III are important for ligand binding. Domain II mediates receptor dimerization. Domain IV forms intramolecular interactions with domain II and thus blocks dimerization. Ligand binding to the extracellular domain of HER family members disrupts the autoinhibition conformation. This results in receptor homo- or heterodimerization and transphosphorylation followed by the activation of the downstream signaling pathways. A ligand for HER2 has not been identified yet. However, the HER2 extracellular domain adopts a fixed conformation that resembles a ligand-activated state that permits it to form a dimer in the absence of a ligand. This conformation of HER2 probably explains why HER2 is the preferred dimerization partner for the other HER family members. Moreover, although none of the ligands for the HER family receptors directly binds to HER2, activation of EGFR, HER3, or HER4 by their ligands

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can facilitate transactivation of HER2 through ligand-induced heterodimerization. Overexpression of HER2 as a result of amplification of the HER2 gene (ERBB2/neu) appears to mediate the initiation, progression, and metastasis of many types of human cancer, including breast and gastric cancers. HER2 is overexpressed in approximately 20–25% of breast cancers and is associated with poor disease-free survival and poor response to chemotherapy. Gene amplification is the most common mechanism resulting in HER2 overexpression in breast cancer. HER2positive expression was also observed in 22.1% metastatic gastric or gastroesophageal junction (GEJ) patients. Murine Anti-HER2 Monoclonal Antibody (mAb) 4D5 The murine anti-HER2 monoclonal antibody, 4D5, was shown to recognize human HER2 and to suppress the growth of HER2-overexpressing tumor cells, as well as to enhance the sensitivity of tumor cell killing by the host immune system. Further studies demonstrated that radiolabeled 4D5 localized to HER2-overexpressing tumors in patients. In order to make a therapeutic monoclonal antibody that would maintain the half-life and effector functions of human antibodies and not be recognized as foreign by a patient’s immune system, 4D5 was humanized by engineering the 4D5 CDR heavy and light chain sequences into the framework of a consensus human monoclonal antibody IgG1 isotype. The humanized version of 4D5 (also known as rhuMabHER2; later named trastuzumab) alone or in combination with other chemotherapy agents showed significant inhibitory effects in HER2-overexpressing breast cancer cells and in mouse xenograft models of HER2-overexpressing breast cancer. Clinical Indications For the past 20 years, the development of monoclonal antibodies targeting HER family receptor tyrosine kinases has been intensely pursued as an important cancer therapeutic strategy. Based on results from a phase III investigational clinical trial of trastuzumab, which showed that trastuzumab in combination with chemotherapy increased time to

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disease progression and response rates compared to chemotherapy alone, trastuzumab received FDA approval in September 1998 for use in women with HER2-overexpressing metastatic breast cancer. Trastuzumab is indicated for treatment of patients both as first-line therapy in combination with paclitaxel chemotherapy and as a single agent for those who have received one or more chemotherapy regimens. Trastuzumab was the first HER2 targeted treatment for metastatic breast cancer. Dako’s HercepTest™, which is a semi-quantitative immunohistochemical (IHC) assay for determination of HER2 protein overexpression in breast cancer tissues, was approved simultaneously to aid in the identification of patients eligible for trastuzumab treatment. On November 16, 2006, the FDA granted approval to trastuzumab as part of a treatment regimen containing doxorubicin, cyclophosphamide, and paclitaxel for the adjuvant treatment of women with early-stage HER2-positive and node-positive breast cancer. This approval was based on evidence of a significant prolongation in disease-free survival in women receiving trastuzumab and chemotherapy compared to those receiving chemotherapy alone. On October 20, 2010, the FDA granted approval for trastuzumab in combination with cisplatin and a fluoropyrimidine (either capecitabine or 5-fluorouracil) for the treatment of patients with HER2-overexpressing metastatic gastric or GEJ adenocarcinoma who have not received prior treatment for metastatic disease. This approval is based on results of a single international multicenter openlabel randomized clinical trial BO18255 (ToGA trial), which enrolled 594 patients with locally advanced or metastatic HER2-overexpressing adenocarcinoma of the stomach or GEJ. HER2 Status Trastuzumab is used for the treatment of breast or gastric/GEJ cancers where HER2 is overexpressed. Two testing methodologies can be used clinically to determine the HER2 status of tumor samples: immunocytochemistry (IHC) and fluorescence in situ hybridization (FISH). IHC detects the level of HER2 protein in cancer samples, whereas FISH detects the level of HER2 gene amplification. The American Society of

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Clinical Oncology/College of American Pathologists guideline recommends the use of both IHC and FISH testing to determine the HER2 status of human breast cancer. According to this guideline, a positive HER2 result is the IHC staining of 3+ and a FISH result of more than six HER2 gene copies per nucleus or a FISH ratio [HER2/chromosome 17 (CEP17)] of more than 2.2. In the ToGA trial, patients were eligible for trastuzumab treatment if their tumor samples were scored as 3+ on ICH or if they were FISH positive (HER2:CEP17 ratio 2). HER2 heterogeneity in gastric/GEJ tissue is greater than in breast cancer tissue, which may cause discordance between FISH and IHC. Mechanisms of Action of Trastuzumab Trastuzumab directly binds to the extracellular domain IV of HER2 to mediate an inhibitory effect on cancer cells. While the mechanisms by which trastuzumab induces regression of HER2positive breast cancers are still being investigated, it is currently believed that the binding of trastuzumab to HER2 contributes to its therapeutic effect either by direct modulation of proliferative and pro-survival signaling downstream of HER2 or by its effect on angiogenesis and immune cell recruitment. Binding of trastuzumab to the extracellular domain of HER2 directly inhibits HER2 signaling by (a) prevention of the cleavage of HER2 extracellular domain by metalloproteinase ADAM10, (b) inhibition of either HER2 homodimerization or heterodimerization. and (c) induction of HER2 endocytosis followed by receptor degradation. Taken together, binding of trastuzumab to HER2 leads to the inhibition of pro-survival and proliferative pathways, such as the phosphatidylinositol 3kinase (PI3K) pathway, mitogen-activated protein kinase (MAPK) pathway, and cell cycle progression (Fig. 1). Trastuzumab has also been shown to inhibit tumor angiogenesis, resulting in the decreased microvessel density of tumor in vivo and reduced endothelial cell migration in vitro. Trastuzumab has been demonstrated to kill tumor cells not only by its direct action on tumor cell signaling but also through antibodydependent cell-mediated cytotoxicity (ADCC). More specifically, trastuzumab is an IgG1 isotype

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and its constant region is capable of binding to Fc receptors presented on certain immune cells, which activates antibody effector functions. These immune cells release enzymes and factors that kill the tumor cells. Studies have demonstrated that when trastuzumab is present, immune cells preferentially target HER2-overexpressing cancer cells compared to cancer cells that do not overexpress HER2. Mechanisms of Trastuzumab Resistance Treatment with trastuzumab has significantly improved the outcome in women with HER2positive breast cancer. However, tumor resistance to trastuzumab poses a significant hurdle in breast cancer therapy. Clinical data has shown that approximately two thirds of HER2-positive metastatic breast cancer patients demonstrated primary resistance to single-agent trastuzumab and that the majority of patients with HER2-positive breast cancer who achieve an initial response to trastuzumab acquire resistance within 1 year. While the mechanisms of trastuzumab resistance are still being investigated, the following are proposed mechanisms based on clinical and preclinical studies: (1) overexpression of membraneassociated glycoprotein mucin-4 (MUC4), which may mask the epitope of HER2 recognized by trastuzumab; (2) formation of homodimers and heterodimers among EGFR, HER2, and HER3 due to overexpression of HER family ligands, which interferes with trastuzumab-mediated growth inhibition; (3) upregulation of Rac1, a member of Rho family small GTPases, which may impair trastuzumab-induced HER2 endocytic downregulation; (4) increased heterodimerization between HER2 and IGF-1R, which may interfere with trastuzumab-mediated induction of the cell cycle progression inhibitor, cyclindependent kinase p27 (kip1); (5) activation of PI3K either by constitutive activation, such as PIK3CA mutant, or by loss of PTEN which results in activation of PI3K signaling; (6) cyclin E amplification/overexpression; (7) increased cleavage of HER2 extracellular domain, which results in the formation p95HER2, a hyperactive membrane anchored fragment that drives breast cancer progression in vivo; and (8) loss of HER2

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Trastuzumab, Fig. 1 Signal transduction by the HER family and potential mechanisms of action of trastuzumab (Reproduced from Hudis (2007)). As shown in Panel A, the four members of the HER family are HER1, HER2, HER3, and HER4. There are receptor-specific ligands for HER1, HER3, and HER4. An intracellular tyrosine kinase domain exists for HER1, HER2, and HER4. Phosphorylation of the tyrosine kinase domain by means of

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homodimerization or heterodimerization induces both cell proliferation and survival signaling. HER2 is the preferred dimerization partner for the other HER family members. The phosphorylated (activated) tyrosine residues on the intracellular domain of HER2 activate the lipid kinase phosphoinositide 3-kinase (PI3-K), which phosphorylates a phosphatidylinositol that in turn binds and phosphorylates the enzyme Akt, driving cell survival. In parallel,

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expression in HER2-overexpressing breast cancer cells. Trastuzumab-Induced Cardiotoxicity HER2 signaling in the heart is essential for cardiac development and function, as well as for the prevention of dilated cardiomyopathy. Deletion of HER2 gene in a mouse model has been shown to result in early death of the animal. In the adult heart, HER2 may continue to have an important function in modifying the cardiac response to stress. It is possible that trastuzumab treatment results in a loss of HER2-mediated signaling in cardiomyocytes and that this interferes with the heart’s ability to respond to stress. Molecular mechanisms by which trastuzumab induces cardiac dysfunction still remain elusive. Trastuzumab cardiotoxicity in the metastatic clinical trial: Cardiotoxicity was initially reported in a phase III trial, which tested the efficacy of combining chemotherapy with trastuzumab versus chemotherapy alone in metastatic breast cancer disease. This study found that the combination of anthracyclines and cyclophosphamide (AC) alone was associated with a rate of overall cardiac dysfunction (CD) of 8% and a New York Heart Association (NYHA) class III/IV rate of 4%. However, when trastuzumab was added to AC, the overall CD rate was 27% with a rate of NYHA class III/IVCHF of 16%. Paclitaxel alone was associated with a CD rate of 1% and a NYHA class III/IV rate of 1%. The addition of trastuzumab to paclitaxel resulted in a CD rate of 13% with a NYHA class III/IV rate of 2%. Trastuzumab associated CD is manifested as severe congestive heart failure

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(CHF) and a significant decrease in left ventricular ejection fraction (LVEF). Trastuzumab cardiotoxicity in the adjuvant trials: Adjuvant trastuzumab prolonged survival, including both overall survival (OS) and diseasefree survival (DFS), among women with HER2positive early breast cancer, but the benefits were accompanied by the risks of cardiac toxicity. Based on the assessment of several major adjuvant trastuzumab clinical trials, including the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-31, North Central Cancer Treatment Group N9831, Herceptin Adjuvant, Breast Cancer International Research Group 006, and Finland Herceptin trials, up to 4% of patients experienced severe CHF during treatment. However, a large number of patients on these trials experienced some form of cardiotoxicity that ultimately required discontinuation of trastuzumab. Approximately 14% of patients in the NSABP B-31 trial were reported to discontinue trastuzumab treatment due to asymptomatic decrease in LVEF. A systematic review of eight clinical trials, which involved 11,991 women with HER2-positive operable breast cancer who were treated with trastuzumab or the standard therapy (with no trastuzumab), found that breast cancer mortality was reduced by one third, but the risk of cardiac toxicity (i.e., CHF and LVEF) was five times more likely for women receiving trastuzumab than women receiving standard therapy alone. The incidence of cardiac toxicities may be associated with duration of trastuzumab administration, such that the longer treatment (1 year) may involve a greater

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ä Trastuzumab, Fig. 1 (continued) a guanine nucleotide exchange factor, the mammalian homologue of the son of sevenless (SOS), activates the rat sarcoma (RAS) enzyme that, in turn, activates receptor activation factor (RAF) and then the mitogen-activated protein kinase (MAPK) and mitogen extracellular signal kinase (MEK). MEK phosphorylates, among others, the MAPK, driving cellular proliferation. One of many other downstream effects of HER2 signaling is the production of vascular endothelial growth factor (VEGF) supporting angiogenesis. The most well-documented potential mechanisms of action are shown in Panels C through F. Cleavage of the extracellular domain of HER2 leaves a membrane-bound

phosphorylated p95, which can activate signal transduction pathways (Panel C). Binding of trastuzumab to a juxtamembrane domain of HER2 reduces shedding of the extracellular domain, thereby reducing p95 (Panel C). Trastuzumab may reduce HER2 signaling by physically inhibiting either homodimerization, as shown, or heterodimerization (Panel D). Trastuzumab may recruit Fc-competent immune effector cells and the other components of antibody-dependent cell-mediated cytotoxicity, leading to tumor cell death (Panel E). Additional mechanisms such as receptor downregulation through endocytosis have been postulated (Panel F)

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risk of severe heart toxicities than shorter treatment (6 months or less). Trastuzumab-associated cardiotoxicity is believed to be reversible upon stopping the treatment. However, the concept that trastuzumabrelated cardiotoxicity is reversible has been challenged due to the lack of sufficient long-term follow-up cardiac data. Additionally, in NSABP B-31trial among those diagnosed with a cardiac event, two thirds of patients continued to receive cardiac medications and 71% had a decrease in LVEF relative to baseline on follow-up, suggesting persistent cardiac dysfunction. Disclaimer The information presented in this entry represents publically available information. Any opinions expressed reflect the views of the authors and do not represent the policy of the US Food and Drug Administration.

Cross-References ▶ Drug Design ▶ Herceptin

Treatment-Refractory Germ Cell Tumors

Trefoil Factors Christian Gespach Laboratory of Molecular and Clinical Oncology of Solid tumors, Faculté de Médecine, Université Pierre et Marie Curie-Paris 6, Paris, France INSERM U. 673, Paris, France

Synonyms Spasmolytic polypeptide SP/TFF2; Trefoil peptides pS2/TFF1

Keywords Angiogenesis; Epithelial tumors; Gastric ulcers; Inflammation; Intestinal trefoil factor (ITF/TFF3); Invasion; Metastasis; Migration; Mucosal protection and regeneration

Definition References Dokmanovic M, Wu WJ (2011) Trastuzumab: resistance and breast cancer. In: Gunduz M, Gunduz E (eds) Breast cancer: carcinogenesis, cell growth and signaling pathways. InTech, Rijeka, pp 171–204 Hudis CA (2007) Trastuzumab: mechanism of action and use in clinical practice. N Engl J Med 357:39–51 http://www.cancer.gov/cancertopics/druginfo/trastuzumab Moja L, Tagliabue L, Balduzzi S, Parmelli E, Pistotti V, Guarneri V, D’Amico R (2012) Trastuzumab containing regimens for early breast cancer. Cochrane Database Syst Rev 4:CD006243 Telli ML, Hunt SA, Carlson RW, Guardino AE (2007) Trastuzumab-related cardiotoxicity: calling into question the concept of reversibility. J Clin Oncol 25: 3525–3533

Treatment-Refractory Germ Cell Tumors ▶ Platinum-Refractory Testicular Germ Cell Tumors

Trefoil factors (TFF) belong to a family of heat, acid, and protease-resistant regulatory peptides ubiquitously expressed in the brain, blood, and peripheral organs. In inflammatory conditions and generation of cancer lesions, they are induced, lost, or modified by gene silencing and somatic mutations. Thus, TFF overexpression or invalidation is either the consequence or the causal origin of human solid tumors and their ▶ progression to metastatic situations. While the TFF receptors or recognition systems are not still clearly identified, TFF are involved in mucosal and epithelial cell cytoprotection, wound healing, cancer cell survival and ▶ invasion, and ▶ angiogenesis, through several oncogenic pathways involved in neoplasia. Finally, TFF are now considered as multifaceted factors with beneficial and pejorative functions on inflammatory and cancer diseases, according to their dual and divergent impacts at early and late stages of these pathological states.

Trefoil Factors

Characteristics TFF Discovery and Expression Since the discovery and molecular annotation of the trefoil factor pS2 (TFF1) in human breast cancer, much attention has been devoted on TFF1 and its structurally related protease and acid-resistant factors spasmolytic polypeptide (SP-TFF2) and intestinal trefoil factor (ITF-TFF3). These TFF contain either one (TFF1 and TFF3) or two (TFF2) trefoil domains delimited by three disulfide bridges. TFF are involved in the stabilization of the mucus layers secreted by mucosal epithelial cells. The three human trefoil genes are located in a cluster region of 55 kb on chromosome 21q22.3. A novel two trefoil domains Bm-TFF2 protein-activating platelet aggregation has been purified from the frog Bombina maxima skin secretions. TFF are widely expressed in the brain, the urogenital system (breast, kidney, prostate), the lymphoid tissue, the respiratory and the digestive tract (esophagus, stomach, intestine, exocrine and endocrine pancreas, and liver), and in conjunctival goblet cells and pterygium. TFF1 and TFF2 are predominantly detected in the normal stomach, whereas TFF3 is found mainly in the small and large intestine. TFF are regulated via genetic, ▶ epigenetic, and tissue-specific mechanism including amplification of the chromosomal region 21q22 harboring TFF family genes in ▶ cholangiocarcinoma, promoter methylation, chromatin modification, histone H3 acetylation, and transcription factor downstream signaling pathways involved in cellular ▶ stress responses, ▶ inflammation, and cancer. These pathways include gastrin and bFGF growth factors, the ▶ interleukin 6 family cytokine receptor gp130/ STAT1–3 and SHP-2/ERK cascades, ras, the ▶ hypoxia-induced HIF-1a transcription factor, allergens in the lungs, nuclear ▶ estrogen receptors and GEFs, NF-kB, peroxisome proliferatoractivated receptor gamma (PPAR-g), hepatocyte nuclear factor 3 (HNF3), the homeodomain transcription factor CDX2, and the activator protein ▶ AP-1 via the negative control of COBRA1, the cofactor of BRCA1 (breast cancer-associated protein 1) involved in DNA damage repair.

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TFF are secreted by the gastrointestinal mucosa in a mucus, inflammation, and ulcerassociated cell lineage (UACL)-dependent manner. Chronic inflammation and ulceration in the gastrointestinal tract is associated with the development of the reparative UACL from mucosal stem cells. UACL was originally described as pyloric metaplasia in the ileum, reflux esophagitis associated with Barrett esophagus, peptic ulcer in the stomach, and chronic cholecystitis. TFF display multifaceted roles in mucosal repair and during cancer progression. TFF in Mucosal Repair and Protection It is now well accepted that TFF are involved in maintenance and repair of the mucosal barrier, wound healing, and cytoprotection during hypoxia and transient inflammatory situations in experimental ulcerative colitis. Local administration of recombinant TFF and ectopic expression of TFF3 in cellular models and transgenic animals supported this general idea of a cytoprotective role for TFF in mucosal repair. Both epithelial and stromal cells contribute to wound healing and mucosal repair. Consistent with a signaling role of TFF in mucosal protection, the tetraspanin family member Vangl1 is involved in the migratory response to TFF3 through Ser/Thr phosphorylation in intestinal epithelial cells. In addition TFF3 improved intestinal crypt stem cells survival following combined radiation and ▶ chemotherapy in both wild-type and TFF3/ knockout transgenic mice. TFF in Chronic Inflammation and Cancer Progression Although some studies argue for a therapeutic potential of TFF in mucosal injury and wound healing, advances in the field support their adverse effects during chronic inflammation and cancer progression. Persistent inflammatory situations initiate several genetic, molecular, and cellular dysfunctions associated with tumor promotion and cancer progression. Notably, selfinduction and cross-talk between TFF at their regulatory sequences have been described as a molecular signature of chronic inflammatory situations and neoplasia.

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Trefoil Factors Adenoma - edenocarcinoma transitions: hypoxia and angiogenesis

Cin-loh tumors Normal mucosa

Premalignant ACF APC, Ki-ras, c-myc, src

Colon crypt

Msi tumors

MUC-5AC APC, COX-2 PAR-1, IGF p21, p16, cyclin D1

ADENOMA (FAP) Wnt-2, DCC SMAD-2/-4

TGFβ-RII, IGF-RII, ß-catenin/ TCF4 PTEN,E2F, BAX c-kit, EGF-R, Axin

ADK: Local Invasion, Metastasis

Invasion Angiogenesis Survival

TFF-1

Duke’s stages TP53 - MET - src A B C D

Target organs

hMSH2, hMLH1, hMSH6, hPMS1, hPMS2 Mutations, Hypo-hypermethylations (HNPCC)

Trefoil Factors, Fig. 1 Genetic and molecular alterations linked to the multistep progression of familial and sporadic human colorectal cancers

For example, TFF1 exerts divergent functions in the digestive mucosa. In the stomach, gastric TFF1-deficient mice develop antropyloric adenomas and carcinomas, suggesting that TFF1 is a candidate gastric-specific ▶ tumor suppressor gene to protect the mucosa against repetitive injury from digestive secretions, ulceration, and chronic inflammation induced by acid, proteases, and pathogens, such as ▶ Helicobacter pylori. Somatic mutations and loss of heterozygosity (LOH) of the TFF1 gene is observed in human gastric cancers, in association with TFF3 overexpression. In coherence with these observations, ▶ cyclooxygenase (COX)-2 was strongly induced in pyloric adenomas induced by genetic ablation of the TFF1 gene in mice. Similarly, COX-derived products are reported to exert beneficial roles in mucosal protection and wound healing, but deleterious functions during chronic inflammation and neoplasia. Conversely, in the normal human colon, TFF1 is absent but is induced at high levels in Crohn disease, colitis, and colorectal cancers. It is therefore likely that TFF1 is a cancer progression factor in the human colon, according to its aberrant expression and

transforming functions at the adenoma and carcinoma transitions (Fig. 1). Thus, TFF exert opposing functions, one counteracting transient inflammatory situations and the other linked to pejorative functions, in cooperation with other dominant genetic and molecular alterations during the neoplastic progression in the human colon. These include the cancer predisposition pathways controlled by Wnt/APC/b-catenin, TGF-b/ SMAD-4, ras, ▶ src, and deleted in colon cancer (DCC). Validation of this model can be explored in transgenic animals harboring selectively these oncogenic alterations, in cooperation with forced expression of TFF1 in intestinal stem cells, through intestinal promoters that are functional in this cellular compartment, such as the villin promoter (pVIL) and the carcinoembryonic antigen (pCEA) regulatory regions. The emergence of colorectal adenocarcinomas (ADK) is a complex multistep process linked to genomic and ▶ chromosomal instability (CIN) and LOH, ▶ microsatellite instability (MSI), DNA ▶ aneuploidy, and generalized deregulation of gene expression and signal transduction pathways. The first mechanism, which accounts for

Trefoil Factors

80% of sporadic cases, is connected with CIN and LOH targeting the tumor suppressors APC (5q), ▶ TP53 (17p), DCC (18q), and TGFb pathway signaling elements SMAD-2 and SMAD-4 (SMAD-4/DPC4) at 18q. Sporadic MSI tumors are frequently mucinous, predominantly localized in the right colon, and generally diploid. In MSI patients, alterations in TGFb-RII, IGF receptors IGF-RII, b-catenin, TCF-4, and E2F transcription factors, as well as loss of the PTEN tumor suppressor, and the ▶ apoptosis regulator BAX are frequently reported. Sporadic cancers are also driven by epigenetic mechanisms, hypo- and hypermethylation of promoter genes encoding cancer markers and/or effectors, such as TFF, MUC-5 AC, COX-2, protease-activated receptors PAR-1, IGF, and ▶ p21/p16/cyclin D1. Dominant activation of proto-oncogenes by point mutations or constitutive activation by other oncogenic pathways is also frequently observed at early stages (ACF, polyps: src, ras, c-myc) and late stages concomitant with cellular invasion, angiogenesis, and ▶ metastasis (c-Kit, EGF-R, VEGF, the hepatocyte growth factor receptor MET, src, and many others). Familial adenomatous polyposis (FAP) is induced by mutations of the APC gene, a defect that contributes to CIN and appearance of more than 100 colorectal adenomas. Molecular alterations in other elements of the Wnt pathways are also concerned in sporadic colon cancers, including Wnt-2, axin, b-catenin, and TCF4 transcription factor. Nonpolyposis form of the hereditary colon cancer (HNPCC) is more frequent than FAP and is caused by germ cell mutations that invalidate the DNA repair systems. DNA mismatch repair is deficient in 90% of the HNPCC patients. The mutations concern mostly the hMSH2 and hMLH1 DNA repair enzymes, less frequently hPMS1 and hPMS2. Such genetic and molecular changes lead to the formation of aberrant crypt foci (ACF), which precede the appearance of premalignant adenomas anchored in the colon mucosal wall. The next stage is the evolution of the adenoma toward more aggressive lesions (ADK) and irreversible acquisition of dominant and anarchic functions, chronic inflammation, oxidative ▶ DNA damage,

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autocrine and paracrine regulatory loops linked to IGF-R, VEGF-R, EGF-R ligands, induction of thrombin protease-activated receptors PAR-1, trefoil factor pS2 (TFF-1), and the immediate response gene COX-2. Aberrant Expression of TFF as Clinical Markers of the Neoplasia TFF are involved in the neoplastic progression in human epithelial tumors according to their ability to confer several transforming functions including resistance to apoptosis, induction of cellular scattering, anchorage-independent growth in soft agar, and proinvasive and proangiogenic activities in vitro and in vivo. Both TFF1 and TFF3 reduced apoptosis induced by serum privation and loss of cellular adhesion (▶ Anoikis), a major response linked to cancer cell transformation, survival, and dissemination. Accordingly TFF are connected with several oncogenic and tumor suppressor elements such as ▶ E-cadherins, EGF-R, the RhoAROCK axis, PI3-kinase (PI3K), phospholipase C, COX-2, nitric oxide synthase 2, NF-kB, STAT3, and Cdc25. The nuclear phosphatases Cdc25A and B are associated with hypergrowth activity and control of the G2/M checkpoint in response to DNA damage and repair. This is supported by clinical investigations on aberrant expression of TFF in human solid tumors of the prostate (TFF3), premalignant changes and neuroendocrine differentiation in human prostate cancer (TFF1), human hepatocellular carcinomas (promoter hypomethylation of the TFF3 gene), hepatolithiasis, and cholangiocarcinomas (TFF1–MUC5AC), primary mucinous carcinomas of the skin (TFF1–TFF3), and ulcerating Barrett esophagus, a precancerous lesion considered as a gastric-type metaplasia. Barrett esophagus is characterized by the specific expression of the gastric-type markers TFF1 and MUC5AC with high levels and strong colocalization in the surface epithelium. In contrast, TFF3, MUC6, and MUC5B were found in the deeper glandular structures. Similarly, gastric metaplasia of the duodenum (GMD) is characterized by replacement of the intestinal epithelium with gastric-type mucus cells, villus damage, and atrophy and is frequently found in association with inflammation and

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gastritis induced by H. p. GMD expressed TFF1, TFF2, and the colon cancer -associated ▶ mucin MUC5AC, a marker of ACF now considered as precancerous lesions in the large bowel. Progressive loss of TFF1 and TFF2 associated with reciprocal induction of TFF3 is likely to be involved in the early stages of gastric ▶ carcinogenesis. In the normal gastric mucosa, TFF2 is expressed in surface mucus neck cells. Decreased TFF2 expression in chronic atrophic gastritis possibly attributes to the decrease in the number of surface epithelial cells expressing TFF2. Reexpression of TFF2 in gastric epithelial dysplasia implies that TFF2 possibly contributes to the progression of gastric carcinoma. It has been reported that TFF3 induction in gastric tumors correlated with an aggressive phenotype with advanced stages, infiltrative growth pattern, and positive lymph nodes. TFF3 is now considered as a marker of poor prognosis in human gastric carcinomas and is associated with aggressive behavior and lethality of colon cancer cells in rats. Ectopic expression of TFF3 promoted the invasive phenotype in Rat-2 fibroblast cells associated with upregulation of b-catenin, MMP-9 matrix metalloproteinase, and downregulation of the tumor suppressor gene product ▶ E-cadherin involved in b-catenin-associated ▶ adherens junctions. In fact, several reports suggest that TFF participate to morphogenesis and differentiation programs for epithelial cells in breast, gastrointestinal tract, and lungs. Conversely, depletion of TFF3 in the human gastric cancer cell line SNU-1 that expresses TFF3 resulted in decreased ability to form colonies in soft agar and in a marked increase in apoptosis and chemosensitivity to anticancer agents. The situation is probably more complicated since both TFF2 and TFF3 are induced in advanced gastric carcinomas and linked with neoangiogenesis, thus having a negative impact on patient survival, and are an independent predictor of disease recurrence. Both TFF1 and TFF2 are strongly expressed in diffuse-type gastric cancers, suggesting that the academic definition of TFF1 as a gastric-specific tumor suppressor gene should be applied in relation with the corresponding status of TFF2/TFF3, and the clinicopathologic context and oncogenic

Trefoil Factors

status of human gastric tumors. For example, TFF1 can be considered as a bona fide gastric tumor suppressor acting against ulcerative and procarcinogenic inflammarory situations. Exocrine gastric secretions and digestive functions as well as stress conditions are known to induce predisposition to the neoplasia. In this scenario, TFF1 mediates gastric mucosal protection and epithelial cell reconstruction of the gastric gland units. However, when the TFF1 gene is silenced by loss of heterozygosity in man, experimental knockdown in transgenic mice, or methylated at the TFF1 promoter region (epigenetic silencing), the non-expressing TFF1 gastric mucosa is now subjected to the agressive secretions of acid and pepsinogen by the stomach. In the Tff1-KO transgenic mice, a direct link between TFF1 loss and bcatenin signaling to its target genes c-Myc and Ccnd1 during gastric oncogenesis. This experimental data was validated in clinical samples from human gastric tumors showing increased nuclear localisation of b-catenin linked to TFF1 deficiency. In contrast, TFFs are found overexpressed during the progression of several human epithelial tumors, including colorectal and breast cancers. Thus, TFFs are now usually considered as critical growth, migratory and invasion-promoting factors in epithelial cancers through direct and indirect mechanisms inducing tumor angiogenesis and metastasis. Several reports indicate that TFF2 is a gastric marker of tumor metastasis frequently upregulated in diffuse gastric cancers in correlation with decreased survival. TFF2-expressing cells are upregulated in the stomach of Helicobacter-infected mice and seem to give rise to invasive cancerous lesions. Consequently, both TFF2 and COX-2 are overexpressed in patients with H. pylori-induced chronic fundic gastritis in association with dysplasia. In the established H. felis/C57BL/6 mouse model of gastric cancer induced by chronic infection with Helicobacter felis, bone marrow-derived mesenchymal progenitor cells, but not hematopoietic stem cells, are recruited to the site of gastric mucosa injury and inflammation. This proliferative zone gives rise to spasmolytic-expressing metaplasia (SPEM) and differentiation toward an epithelial phenotype, evidenced by positive

Trefoil Factors

staining for TFF2 and the epithelial cell cytokeratin KRT1–19 in deep antral and fundic glands. Thus, experimental Helicobacter infection can give rise to a new mucosal microenvironment in the infected gastric mucosa following upregulation of the stem cell factor SCF-1 (the ligand of the c-kit tyrosine kinase) and the ▶ chemokine SDF-1 binding the ▶ G-protein-coupled receptor CXCR4, two key factors involved in the mobilization of bone marrow progenitors and cancer metastasis. It remains to be elucidated whether subpopulations of human gastric cancers may originate from the neoplastic transformation of bone marrow progenitors with gastric mucosal cell gene expression pattern. In addition, SPEM was suppressed by invalidation of the TNF-a gene in Tnf / K19-C2mE transgenic animals expressing simultaneously COX-1/-2 in gastric mucosa via the cytokeratin 19 gene promoter. Finally, we cannot exclude the possibility that illegitimate and constitutive expression of TFF2 by mesenchymal bone marrow stem cells may also target the gastric progenitor niche for metaplasia and dysplasia. Notably, SPEM is associated with gastric H. pylori infection, aberrant expression of the mucin 6 (MUC-6) gene, and progression of human gastric adenocarcinoma. Therefore, the combined loss of the gastric-specific tumor suppressor gene TFF1 with induction of TFF2 and TFF3 provides insights into the complex mechanisms underlying the biological significance and versatility of TFF in gastric cancer progression and neoplasia. In breast cancer, TFF1 and TFF3 but not TFF2 were identified as informative markers for the detection of ▶ micrometastases in the axillary lymph nodes and blood. Significance analysis of microarrays identified a positive correlation between TFF1 overexpression and breast cancer metastasis to the bone in a cohort of 107 patients with primary breast tumors who were all lymph node negative at the time of diagnosis. The involvement of the FGF signaling pathway was also incriminated in preference of tumor cells that relapse to the bone. The fact that TFF1 may contribute to tumor relapse to the bone is underscored by its abundant presence in breast cancer micrometastases.

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In addition, morphogenetic effects have been attributed to TFF2 in human breast cancer cells following the induction of highly complex branched ductular structures typical of migratory, invasive, and survival functions, in a TFF1dependent manner. TFF3 was also expressed in breast ductal and lobular breast carcinomas in situ and invasive lobular carcinomas. While TFF1 is a surrogate indicator for the response to antihormonal therapy and favorable outcome in estrogen receptor-a (ERa)-positive and welldifferentiated breast cancers, its deregulated expression is now considered to contribute to the progression of both ER-a-positive and -negative human breast cancers. Of note, plasma levels of TFF were found elevated in patients with advanced prostate cancer. Conclusions TFF are now considered as valuable therapeutic tools for the treatment of injured mucosal epithelial cells and protection against mucosal and tissular damages following transient injury and other damages caused by radiation therapy and chemotherapy. Selective loss, induction, and overexpression of TFF observed during inflammatory processes and neoplasia deregulate TFF signaling cross-talks and signals and compensatory functions linked to TFF. The molecular complexity of TFF is further illustrated by their ability to form covalent disulfide-linked dimers in vitro and in vivo. The possibility that TFF could form heterodimers adds further complexity for their relevance in receptor and signal transduction. Despite increasing interest on TFF in molecular research and clinical applications, TFF are still orphan signaling peptides facing unknown receptors in the classical definition of receptormediated signal transduction pathways from the plasma membrane, cytoplasmic and nuclear domains, and vice versa. Advances in the field pointed the discovery of new TFF-binding proteins apparently linked to mucosal protection, such as MUC-5 AC and the gastrokine-1-like peptide blottin. It is conceivable that TFF are not released via normal secretory pathways in inflammatory situations and neoplasia. The epithelial cell polarity and its normal microenvironment

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with stromal and vascular cells, immune cells, and extracellular matrix components and their receptors are lost during cancer progression. In this case, it is tempting to assume that TFF are in the abnormal situation interacting with key signal transducers and cancer-associated stromal cell lineages during cancer progression, via illegitimate, pejorative, and persistent mechanisms. In this scenario, TFF were shown to signal through distinct transduction pathways following external addition of the peptides versus ectopic expression. Future attempts and new strategies to identify the sensu stricto TFF receptors and direct transducers are therefore expected in order to exploit the positive facets of TFF for therapeutic purposes and to fight against their deleterious functions in pathological states.

Trefoil Peptides pS2/TFF1

TRF1-Interacting, Ankyrin-Related ADP-Ribose Polymerases ▶ Tankyrases

TR-FRET ▶ Time-Resolved Fluorescence Resonance Energy Transfer Technology in Drug Discovery

Trident ▶ Forkhead Box M1

References Emami S, Rodrigues S, Rodrigue CM et al (2004) Trefoil factors (TFFs) and cancer progression. Peptides 25:885–898 (Review) Lefebvre O, Chenard MP, Masson R et al (1996) Gastric mucosa abnormalities and tumorigenesis in mice lacking the pS2 trefoil protein. Science 274:259–262 Rodrigues S, Rodrigue C, Attoub S et al (2006) Induction of the adenoma-adenocarcinoma progression and Cdc25A-B phosphatases by the trefoil factor TFF1 in human colon epithelial cells. Oncogene 25:6628–6636 Soutto M, Peng D, Katsha A et al. (2015) Activation of βcatenin signalling by TFF1 loss promotes cell proliferation and gastric tumorigenesis. Gut 64(7):1028–39 Taupin D, Podolsky DK (2003) Trefoil factors: initiators of mucosal healing. Nat Rev Mol Cell Biol 4:721–732 Thim L, May FE (2005) Structure of mammalian trefoil factors and functional insights. Cell Mol Life Sci 62(24):2956–2973 (Review)

Trefoil Peptides pS2/TFF1 ▶ Trefoil Factors

40 ,5,7-Trihydroxyisoflavone ▶ Genistein

3,40 ,5-Trihydroxystilbene ▶ Resveratrol

Triple-Negative Breast Cancer Benoit Paquette Department of Nuclear Medicine and Radiobiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada

Synonyms

Treg ▶ Regulatory T Cells

Estrogen receptor, progesterone receptor, and HER-2 negative; TNBC

Triple-Negative Breast Cancer

Definition Triple-negative breast cancer (TNBC) is a subgroup that accounts for 10–20% of all breast carcinomas. TNBC is characterized by the absence of three receptors: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2).

Characteristics Breast cancer is a heterogeneous disease, encompassing a number of distinct biological entities associated with specific morphological and clinical behavior. Technological developments have allowed to sequence the complete genome of breast cancer and compared a large number of tumor samples. From these, studies have emerged five subtypes: luminal A, luminal B, HER2-enriched, basal-like, and claudin-low. TNBC is sometime referred to as basal-like tumors, as a majority of basal-like cancers are TNBC and approximately 80% of TNBC are also basal-like breast cancers. TNBC is also associated with younger patients and high-grade tumors. Whereas hormone receptor-positive and HER2-positive breast cancers have had favorable outcomes with chemotherapy and treatment targeting the ER or HER-2, TNBC on the contrary has got mixed result and still lacking a targeted therapy. For some TNBC patients, the prognosis is poor when compared to other subtypes of breast cancer because distant recurrence appears between the first and third years after treatment. However, 5 years after diagnosis, in TNBC patients whose cancer didn’t reappear early, the probability to develop metastasis is not significantly different compared to non-TNBC patients. This suggests that TNBC patients can be divided in two groups, the good responders to treatment and the poor responders where an early recurrence is observed. However, there is presently no biomarker to identify them. Breast Cancer Treatment by Radiotherapy Microfoci of cancer cells are often dispersed throughout the breast and may be present at few

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cm from the edge of the primary tumor. Conservative surgery removes the primary tumor, while microfoci of cancer cells are targeted by radiotherapy. The treated area frequently covers the whole breast and the axillary and subclavian lymph nodes. Unfortunately, the radiation dose used in clinic does not always eradicate all cancer cells scattered in the breast because it is rather aimed at optimizing long-term results with minimal adverse effects. Many cancer cells are effectively killed since radiotherapy leads to a measurable reduction of local recurrence and distant metastases. A moderate hypofractionated schedule is frequently used in postoperative breast irradiation (2.66 Gy/day, 16 fractions, boost of 2.5 Gy/day, 4 fractions in the tumor bed, 52.6 Gy). More aggressive hypofractionated treatments are under study. For example, whole-breast irradiation of 3 Gy/day in 13 fractions for 39 Gy, followed by a tumor bed boost of 9 Gy in 3 fractions, and even more aggressively with 5 weekly fractions of 6.0 Gy for a total of 30 Gy. The aim is to further improve the local recurrence and overall survival, with acceptable side effects to normal tissues. Radiation and Metastases Development Radiotherapy induces an inflammatory response in all patients treated. The level of inflammation induces by the radiotherapy can vary greatly from one patient to another. Dry skin after breast cancer radiation treatment is fairly common. In some women, a significant inflammation of the skin can occur that looks like a sunburn in treated area (dermatitis). Rarely, this dermatitis is accompanied by severe pain that will require to stop the radiation treatment. Accumulating studies suggest that the burst of inflammatory cytokines induced by radiation may stimulate the development of new metastases. Supporting the observations made in clinic, it has been shown in a mouse model that pre-irradiation of mammary gland before implantation of TNBC D2A1 cells stimulated the migration of cancer cells in the mammary gland, increased the number of circulating tumor cell, and favored the development of

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lung metastases. A similar stimulation of metastases development was also observed after irradiating a TNBC D2A1 tumor already implanted in the mammary gland of the mouse. The inflammatory pathways involved still need to be investigated in details. Nevertheless, these adverse effects of radiation were associated with the cytokine interleukin-6 (IL-6) and the inflammatory enzyme cyclooxygenase-2 (COX-2). These observations raise some concerns about the hypofractionated radiation protocols for breast cancer. On one hand, a higher radiation dose per fraction may be a promising alternative to standard radiotherapy since a greater number of cancer cells would be eliminated by fraction. However, a more important inflammation could be induced. Therefore, the balance between the removal of a large number of cancer cells and the deleterious effects caused by significant inflammation must be determined. The hypofractionated protocols may also not be appropriate for patients who are at higher risk of early recurrence.

Biomarkers of TNBC Poor Responders Radiation oncologists are aware that the risk of recurrence is non-negligible in TNBC patients. As recurrence may appear shortly after radiotherapy, it is possible that inflammatory cytokines induced by radiation might stimulate the development of metastases. On the other hand, physicians also know that TNBC good responders will benefit from radiotherapy. Identification of the TNBC poor responders before treatment is therefore crucial. Some commonly applied approaches for biomarker discovery, such as gene expression profiling, have not yet succeeded in identifying highly sensitive and specific markers related to disease progression of TNBC. A protein signature has been reported to identify the TNBC poor prognosis (recurrence within 5 years). However, their analysis was restricted to patients who did not receive adjuvant therapy. Studies are therefore required to determine

Tripterine

whether TNBC poor responders can be identified based on a specific pattern of gene expression in the tumors or by an inflammatory response characterized by an exaggerated intensity or involving specific cytokines.

Cross-References ▶ Breast Cancer Immunotherapy

References Bouchard G, Bouvette G, Therriault H, Bujold R, Saucier C, Paquette B (2013) Pre-irradiation of mouse mammary gland stimulates breast cancer cell migration and development of lung metastases. Br J Cancer 109:1829–1838 Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, Lickley LA, Rawlinson E, Sun P, Narod SA (2007) Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res 13:4429–4434 Holland R, Veling SH, Mravunac M, Hendriks JH (1985) Histologic multifocality of Tis, T1-2 breast carcinomas. Implications for clinical trials of breastconserving surgery. Cancer 56:979–990 Liu NQ, Stingl C, Look MP, Smid M, Braakman RBH, De Marchi T, Sieuwerts AM, Span PN, Sweep FCGJ, Linderholm BK et al (2013) Comparative proteome analysis revealing an 11-protein signature for aggressive triple-negative breast cancer. J Natl Cancer Inst 106:djt376 Pogoda K, Niwinska A, Murawska M, Pienkowski T (2013) Analysis of pattern, time and risk factors influencing recurrence in triple-negative breast cancer patients. Med Oncol 30:388

Tripterine ▶ Celastrol

TROP-1 ▶ EpCAM

Tuberous Sclerosis Complex

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Trophectoderm

TSC

Isabelle Gross INSERM U1113, Université de Strasbourg, Strasbourg, France

▶ Tuberous Sclerosis Complex

TSC1 Definition The fertilized egg of mammals cleaves several times to generate a 16-cell morula consisting of small inner cells enclosed within larger outer cells. Most of the outer cells are then epithelialized (▶ epithelium) and form the trophectoderm, whereas the inner cells go on to generate the inner cell mass in blastocysts. Cell-fate analyses revealed that the inner cell mass gives rise to all of the embryonic cells and the extraembryonic endoderm, whereas the trophectoderm forms the embryonic portion of the placenta and represents the first differentiated cell lineage of mammalian embryogenesis.

Cross-References ▶ Epithelium

▶ Hamartin

tTGase ▶ Transglutaminase-2

Tuberous Sclerosis Complex Andrew R. Tee1, Julian R. Sampson1, Jeremy P. Cheadle1 and David Mark Davies2 1 Institute of Medical Genetics, Cardiff University, Heath Park, Cardiff, UK 2 Department of Oncology, South West Wales Cancer Centre, Swansea, UK

Synonyms TSC

Trp63 ▶ p53 Family

Trp73

Definition Tuberous sclerosis complex (TSC): An autosomal dominant disorder caused by a mutation in either the TSC1 or TSC2 genes and characterized by the development of benign tumors in multiple organs, seizures, and neurocognitive impairment.

▶ p53 Family

Characteristics

Trx ▶ Thioredoxin System

Clinical Aspects Tuberous sclerosis complex (TSC) is a genetic, multisystem disorder caused by mutations in

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Tuberous Sclerosis Complex

Tuberous Sclerosis Complex, Fig. 1 Tuberous sclerosis complex is characterized by the development of hamartomas in a variety of tissues and organs. (a) A facial angiofibroma, (b) an abdominal MRI scan showing a renal angiomyolipoma (arrowed) (c) a cranial MRI scan showing a partly intraventricular subependymal giant cell astrocytoma (arrowed), and, (d) a renal ultrasound scan showing a renal cell carcinoma (within a cyst) (arrowed)

either of the TSC1 or TSC2 genes. TSC occurs in up to 1 in 6,000 live births. The condition has a variable phenotype, both in terms of type and severity of clinical features. Neurological problems are often the most disabling feature of TSC. Approximately, 75–90% of people with TSC have epilepsy. The seizures usually begin in the 1st year of life, although they can also occur for the first time in later childhood or in adulthood. There are correlations between the early onset and severity of seizures and neurodevelopmental and cognitive problems. A wide variety of seizure types occur. Infantile spasms are particularly associated with an increased risk of neurodevelopmental impairments and occur in about one third of infants with TSC. Rapid recognition and treatment is imperative. Infantile spasms are typically characterized by clustered repetitive flexion of the limbs. However, infantile spasms can also present with rapid extension of the limbs, a mixture of flexion and extension, or more subtle signs such as head nods, upward eye deviation, or shoulder

elevation. Onset peaks at 4–6 months. There is characteristic chaotic interictal electroencephalography (EEG) pattern, which, when typical, is termed hypsarrhythmia. TSC is associated with several types of brain lesions. Cortical tubers are focal developmental abnormalities of the cerebral cortex and occur in 80–90% of patients. Subependymal giant cell astrocytomas (SEGAs) are low grade tumors occurring in approximately 10% of patients with TSC (Fig. 1). Growth, typically occurring in childhood or adolescence, may lead to hydrocephalus and present with a deterioration in epilepsy and/or behavior as well as with symptoms such as headache, vomiting, and visual problems due to raised intracranial pressure. SEGAs are thought to arise from subependymal nodules, asymptomatic hamartomas found on the walls of the lateral and third ventricles in most patients with TSC. A minority of individuals with TSC are profoundly intellectually impaired; many more have a slight reduction in IQ, while others haven normal intellectual ability. Specific cognitive deficits

Tuberous Sclerosis Complex

in areas such as memory, attention, and executive function are very common. Developmental disorders such as autistic spectrum disorder and attention deficit hyperactivity disorder are also common in TSC as are depression and anxiety. TSC is associated with a range of behavioral disorders such as aggressive outbursts, sleep problems, and poor social interaction. Whilst behavioral problems are more often seen in patients with intellectual impairment, they also occur in those with normal intelligence. Renal angiomyolipomas (AMLs) are benign tumors found in approximately 80% of patients (Fig. 1). They often develop during later childhood and adolescence. They are usually multiple, bilateral, and asymptomatic but can cause flank pain, impair renal function, and bleed causing hematuria and occasionally major hemorrhage. Renal cysts are also common in TSC and are usually asymptomatic, but patients with a contiguous deletion of the TSC2 and adjacent PKD1 genes develop polycystic kidney disease that often progresses to renal failure in early adult life. The incidence of renal cancer in TSC is similar to the general population, but it tends to occur in younger patients and may be bilateral (Fig. 1). Lymphangioleiomyomatosis (LAM) is a disorder of the lungs and lymphatics, which can occur sporadically or in association with TSC. LAM occurs almost exclusively in females, and radiographic studies suggest 40% of female patients with TSC are affected, although many are asymptomatic. LAM is characterized by proliferation of abnormal “LAM” cells and cystic changes within the lung parenchyma that can lead to respiratory failure. LAM usually presents with dyspnea or recurrent pneumothorax. The sporadic form is caused by acquired mutations in TSC2. It is thought that “LAM” cells arise from renal angiomyolipomas or other remote sites and migrate to the lung in a process that has been termed “benign metastasis.” The skin is affected in approximately 90% of individuals with TSC. The main skin manifestations are facial angiofibromas, shagreen patches, periungual fibromas, and hypopigmented macules. Hypomelanotic macules are often present at birth or develop during infancy and are best seen

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under ultraviolet light using a Wood’s lamp. They are, however, one of the least reliable diagnostic signs. Facial angiofibromas are red/reddish brown papules, typically occur over the nose, cheeks, and chin, often first developing at around 3–5 years of age (Fig. 1). Shagreen patches are elevated irregular light brown or flesh colored plaques often present in the lumbar sacral area that often develop during childhood. Ungual fibromas are fleshy nodules that occur on the finger- or toe-nail beds. A linear depression in the nail can suggest the presence of a subungual fibroma. Fibrous plaques can develop on the forehead, and some individuals with TSC develop “confetti-like” hypopigmentation on the limbs. Cardiac rhabdomyomas are an early manifestation of TSC, appearing at 22–28 weeks of gestation. They usually regress during childhood and are normally asymptomatic but can be associated with obstructive heart failure or arrhythmias. They are often detected on antenatal ultrasound and be sporadic but are associated with a substantial risk of tuberous sclerosis, especially if the lesions are multiple. The Genetic Basis of TSC

TSC is caused by mutation in either TSC1, located on chromosome 9 or TSC2, located on chromosome 16, adjacent to PKD1, one of the genes responsible for autosomal dominant polycystic kidney disease. TSC is an autosomal dominant condition, but about two thirds of cases result from a new mutation with neither parent being affected. Each child of a person with TSC has a 50% chance of inheriting the mutation. All those who inherit the mutation develop TSC (i.e., the disorder shows full penetrance). TSC1 mutations are associated with less severe disease manifestations than TSC2 mutations, but it is not possible currently to predict severity at an individual level on the basis of genotype. Parents of a child with TSC who are apparently unaffected should be evaluated for subtle signs of the disease. If neither parent has any features of TSC nor a TSC-causing mutation in DNA is extracted from leucocytes, the risk of each future child having TSC is 1–2% because of germline mosaicism

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where two or more genetically distinct cell lines are present in the testis or ovary. In addition to an inherited TSC1 or TSC2 mutation an acquired “second hit” mutation in the second TSC1 or TSC2 allele in a cell appears to be required for the formation of most lesions in TSC, consistent with the two-hit tumor suppressor gene model of Knudson. However, mutation of just one allele may contribute to some of the manifestations of TSC, such a cognitive impairment, through a gene dosage effect.

Tuberous Sclerosis Complex 1

angiomyolipomas have been successful, leading to regulatory approval in many countries. Sirolimus has been demonstrated to slow the deterioration in lung function in patients affected by sporadic or TSC associated LAM.

References Kwiatkowski DJ, Whittemore VH, Thiele EA (eds) (2010) Tuberous sclerosis complex: genes, clinical features and therapeutics. Willey Blackwell, Weinheim

The Molecular Pathology of TSC

The TSC1 and TSC2 tumor suppressor proteins together with TBC1D7 form a complex, which regulates multiple cellular processes and functions to inhibit mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). mTORC1, in turn, regulates many processes such as protein synthesis and the biosynthesis of ribosomes, lipid and glucose metabolism, nucleotide synthesis, mitochondrial biogenesis, and autophagy. Rheb (Ras homolog enriched in brain) is a GTPase complex that functionally links TSC1/ TSC2 to mTORC1. The TSC1-TSC2-TBC1D7 complex (termed the TSC-TBC complex) has GTPase-activating protein activity converting Rheb from an active GTP-bound state, which in turn potently activates mTORC1, to an inactive GDP-bound state which turns off mTORC1. Functional TSC1-TSC2-TBC1D7 allows the integration of multiple stimuli such as the levels of cellular oxygen, energy, and nutrients as well as growth factor signaling into the nuanced control of the processes regulated by mTORC1. Loss-offunction mutations in either TSC1 or TSC2 cause aberrant signal transduction through mTORC1 leading to many of the pathological features of tuberous sclerosis.

Tuberous Sclerosis Complex 1 ▶ Hamartin

Tubulin-Interacting Proteins ▶ Microtubule-Associated Proteins

Tumor Antigens Roberto Bei Department of Clinical Sciences and Translational Medicine, Faculty of Medicine, University of Rome “Tor Vergata”, Rome, Italy

Definition Tumor antigens (TAs) are antigens recognized by specific immune effector cells and/or antibodies and differentially expressed (qualitatively and/or quantitatively) in the tumor tissue as compared to the normal counterpart tissue.

Targeted Therapy for TSC

The direct link between deregulated mTORC1 signaling and the pathogenesis of TSC provided a strong rational for the use of mTORC1 inhibitors, such as everolimus and sirolimus, as a targeted therapy. Clinical trials of everolimus for the treatment of TSC-related subependymal giant cell astrocytoma (SEGA) and renal

Characteristics Tumor Antigens Classification, Expression, and Immune Response TAs can be divided into tumor-specific antigens (TSAs) that are unique antigens expressed by

Tumor Antigens

tumor cells and not by normal cells and tumorassociated antigens (TAAs), which are also expressed by normal cells although at lower levels than in tumor cells (see also Yang and Yang 2005). TAs include mutated antigens, oncofetal antigens (OFs), cancer–testis antigens (CTs), aberrantly glycosylated and expressed antigens, tissue-lineage antigens, overexpressed antigens, and virally encoded antigens. Classification and examples of tumor antigens are shown in Table 1 (see also Khodadoust and Alizadeh 2014). Mutated antigens are antigens which display structural mutations, including point mutations and chromosomal translocations, the latter leading to the generation of chimeric fusion proteins (see also Khodadoust and Alizadeh 2014). Mutations in genes which encode for proteins involved in normal regulation of growth (proto-oncogenes) induce their activation to oncogenes, whose products contribute to cellular transformation. The prototype of a tumor antigen carrying a point mutation is the product of the RAS protooncogene, which encodes for a protein (p21) which induces growth factor-mediated signal transduction and whose point mutation makes it always active. Missense p21 mutations were detected in several types of cancer. Antibody responses to wild-type and mutated p21 ras were detected in a high portion of patients with colon cancer. In addition, in vitro stimulation of human lymphocytes from cancer patients with mutant ras peptides induced the expansion of CD4+ and CD8+ T-cell precursors. Mutations associated with the development of papillary thyroid cancer involve the B-type Raf kinase (BRAF). Mutations of the BRAF gene are the most common genetic alterations in melanoma. Mutated B-Raf/B-Raf (V599E)-specific antibodies and CD8+ T cells were found in melanoma patients. The t(9;22) chromosomal translocation (Philadelphia chromosome, Ph) results in the formation of the Bcr-abl fusion protein which has deregulated protein kinase activity as compared to the normal kinase abl and contributes to the pathogenesis of chronic myelogenous leukemia (CML). The Bcr-abl chimeric protein represents

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a target for therapy in CML, in Ph+ acute lymphoblastic leukemia (ALL), and in some acute myelogenous leukemias (AMLs). Bcr-ablspecific T cells were detected in CML patients. Autoantibodies to p210 Bcr–Abl were found in both Ph+ and Ph- leukemias. A mutated cyclin-dependent kinase 4 (CDK4) and mutated b-catenin were found in melanoma and shown to induce specific CD8+ T cells generated from tumor-infiltrating lymphocytes. Tumor suppressor genes, i.e., those genes that negatively regulate cell growth, are also modified by mutations that make them inactive during tumor development. The prototype of a tumor suppressor gene that is mutated in the majority of tumors is the p53 protein which is capable of blocking the cell cycle and induces apoptosis in cells with DNA damage. An altered activity of p53 leads to accumulation of DNA damage and contributes to neoplastic transformation. Antibodies to p53 were shown to be elicited in patients with tumor mutated p53, and human cytotoxic T lymphocytes (CTLs) that preferentially recognize tumor cells bearing a conformational p53 mutant could also be isolated. Wilms’ tumor 1 (WT1) is a transcription factor expressed in embryonic kidney cells and hematopoietic stem cells. WT1 is mutated in patient with Wilms’ tumor and in most AMLs and CMLs. Patients with AML show antibodies reactive with full-length or NH2-terminal WT1 protein. Direct recognition and lysis of leukemia cells by WT1-specific T lymphocytes were also reported. OF antigens are expressed in fetal tissues, partially repressed in adult tissues and expressed at high levels by cancer cells (see also Reuschenbach et al. 2009). OF antigens include carcinoembryonic antigen (CEA), alphafetoprotein (AFP), oncofetal antigen/immature or precursor laminin receptor protein (OFA/iLRP), oncofetal antigen 5T4, glypican-3 (GPC-3), insulin-like growth factor II mRNA-binding protein (IMP) 3 (IMP-3) and the identical KOC (KH domain-containing protein overexpressed in cancer), human chorionic gonadotropin-b (hCGb), and pancreatic oncofetal antigen (POA) (see also Canevari et al. 1996).

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Tumor Antigens

Tumor Antigens, Table 1 Classification and examples of tumor antigens Antigen group Mutated

Name RAS BRAF BCR/ABL b-catenin CDK4 p53

Oncofetal

WT1 CEA

AFP

OFA/iLRP 5T4 oncofetal antigen GPC-3 IMP-3 hCGb

Cancer testis

Aberrantly glycosylated and expressed

MAGE-A1, MAGE-A3, MAGE-A10, NY-ESO-1, SSX-2, GAGE1, KP-OVA-52, XAGE-1b MUC1

Thomsen–Friedenreich (TF or T) antigen LeY

Tissue lineage

Stage-specific embryonic antigen-1 (SSEA-1) (LeX) Gangliosides (GM1, GM2, GD1, GD2, GD3) FucGM1 Melanoma differentiation antigens (tyrosinase, TYRP1/gp75, Melan-A/ MART-1) Prostate-specific antigen (PSA) Idiotype

Site/type of the tumor Colon, lung, pancreas, prostate, leukemias, bladder, etc. Thyroid, melanoma CML, ALL, AML Melanoma Melanoma Breast, colon, lung, colon rectum, ovary, thyroid, bladder, pancreas, B-cell lymphoma, etc. AML, CML, Wilms’ tumor Colorectal, stomach, pancreas, lung, breast, gallbladder, ovary, endometrium Testicular, liver, pancreas, lung, embryonal cell carcinoma, genitourinary tract, yolk sac (ovary) Hematological, breast, mesenchymal tissue, kidney Breast, kidney, colorectal, prostate, ovary Liver, melanoma, yolk sac, stomach Pancreas, lung, stomach, esophagus, colon, kidney, soft tissue Colon, lung, pancreas, esophagus, breast, bladder, cervix, stomach, prostate, trophoblast, testis Bladder, brain, colon, head and neck, lung, liver, esophagus, melanoma, myeloma, neuroblastoma, prostate, thyroid, ovary, NHL, lung Breast, pancreas, ovary, endometrium, lung, prostate, bladder, gastrointestinal tract, multiple myeloma, T-cell and some B-cell lymphomas Colon, breast, bladder, prostate, liver, ovary, stomach Ovary, prostate, colon, breast, pancreas, lung, embryonal tissues, yolk sac, testis Colon, stomach, breast, ovary, kidney, bladder Neuroblastoma, melanoma, lung

Spontaneous immune response in cancer patients Humoral and cell mediated Humoral and cell mediated Humoral and cell mediated Cell mediated Cell mediated Humoral and cell mediated

Humoral and cell mediated Humoral and cell mediated

Humoral and cell mediated

Humoral and cell mediated Cell mediated Cell mediated Cell mediated Cell mediated

Humoral and cell mediated

Humoral and cell mediated

Humoral Humoral

Humoral Humoral

Lung Melanoma

Humoral Cell mediated and humoral (TYRP1/gp75)

Prostate

Humoral and cell mediated

B-cell malignancies

Humoral (continued)

Tumor Antigens

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Tumor Antigens, Table 1 (continued) Antigen group Overexpressed

Name ErbB receptors (EGFR, ErbB2, ErbB3, ErbB4) Ribosomal P0 protein Ribosomal protein S6 Ribosomal protein L19 HSPs MYC

OPN MDM2 Mesothelin Survivin

Virally encoded

HPV (E6, E7) EBV (EBV-encoded nuclear antigens (EBNAs), BZLF1 protein)

Site/type of the tumor Breast, colon, head and neck, lung, pancreas, prostate, bladder Colon, liver, head and neck, breast NHL, breast, colon, kidney Lung Breast, ovary, lung, pancreas, colon, prostate, urinary tract, AML Neuroblastoma, small cell lung cancer, alveolar rhabdomyosarcoma, retinoblastoma Prostate CLL Mesothelium, pancreas, ovary Breast, ovary, lung, pancreas, colon, liver, prostate, glioma, esophagus meningioma, urinary tract Cervix, oral cavity B-cell malignancies, nasopharyngeal carcinoma

CEA is present in normal epithelial cells of several tissues. CEA expression in these organs normally begins during the early fetal period (week 9–14) and appears to continue throughout life. High levels of CEA are found on a wide range of human carcinomas. The presence of anti-CEA antibodies was observed in the serum of patients with gastrointestinal malignancies, and CEA-specific CTLs could be generated using CEA peptide-pulsed dendritic cells. AFP is synthesized in the fetal yolk sac at 9week gestation and later in the fetal liver and gastrointestinal tract. In adult life, AFP is reexpressed in multiple tumors of endodermal and mixed mesodermal/endodermal origin. There are different AFP isoforms and lectin glycan-associated forms demonstrable by electrophoretic and chromatographic procedures. Autoantibodies and T-cell immune responses to AFP have been reported to occur in patients with hepatocellular carcinoma (HCC) or with liver diseases. OFA/iLRP is widely expressed in many types of human tumors, while it is absent in normal adult differentiated tissues. Gained expression of the OFA/iLRP facilitates cancer cells to penetrate tissue and vessel barriers expressing laminin.

Spontaneous immune response in cancer patients Humoral and cell mediated Humoral Humoral Cell mediated Humoral Humoral

Humoral Cell mediated Humoral and cell mediated Humoral and cell mediated

Humoral and cell mediated Humoral and cell mediated

A spontaneous tumor-specific humoral immune response against OFA/iLRP was detected in a significant proportion of chronic lymphocytic leukemia (CLL) patients, while several OFA/iLRPspecific T-cell clones were established in breast cancer patients. The oncofetal antigen 5T4 is highly expressed in several carcinomas but has limited expression in normal tissues. This antigen has been isolated from the term placenta. 5T4 was found to be expressed in tumor-initiating cells and associated with worse clinical outcome in non-small cell lung cancer. The presence of a CD8+ T-cell repertoire specific for 5T4 was demonstrated in an apparently healthy donor. In addition, CTLs specific for a 5T4 epitope were induced in colorectal cancer patients following vaccination with a recombinant modified vaccinia Ankara (MVA) virus expressing 5T4. GPC-3 is a membrane-anchored heparin sulfate proteoglycan which is normally expressed in fetal liver and placenta, but not in normal adult liver. GPC-3 is overexpressed in HCC and melanoma. In HCC patients, GPC-3 peptide-reactive CTLs could be established from PBMCs by in vitro stimulation with these peptides.

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IMP-3 is produced in developing epithelia, muscle, and placenta during early stages of human and mouse embryogenesis. The expression of IMP-3/KOC is also observed in malignant tumors, but it is weakly or not detectable in adjacent benign tissues. The existence of specific T-cell responses to HLA-A24-restricted IMP-3 epitopes in esophageal squamous cell carcinoma patients was established. hCG is glycoprotein hormone secreted by trophoblastic cells during normal gestation. The b-subunit of hCG (hCGb) was found to be overexpressed in several tumors. Peripheral blood mononuclear cells (PBMCs) from patients with hCGb-productive bladder and testis tumors displayed an hCGb-specific proliferative response. CT antigens are aberrantly expressed by tumors of different histological origins and normal adult reproductive tissues such as testis and placenta. CT antigens comprise the melanomaassociated antigen family (MAGE-A1, MAGEA2, MAGE-A3, etc.), the GAGE/PAGE/XAGE superfamily, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), synovial sarcoma X (SSX), synaptonemal complex protein 1 (SCP-1) and BAGE, etc (see also Van den Eynde and van der Bruggen 1997). Melanomareactive CTLs could be induced from the peripheral blood lymphocytes (PBLs) of melanoma patients or normal donors by stimulation with the HLA-Al-binding epitope of MAGE-1 or MAGE-3. Spontaneous MAGE-A10- and/or SSX-2-specific CD8+ T cells were detected in HCC patients (see also Yang and Yang 2005). In addition, humoral immune responses to NY-ESO-1 were detected in patients with ovarian cancer, and a CTL epitope capable of inducing NY-ESO-1-specific CTLs in vitro was identified from PBMCs of healthy donors. GAGE1 was found to elicit autoantibodies in 6% of patients with thyroid cancer but not with benign nodules. A novel cancer/testis antigen KP-OVA-52 was discovered by SEREX in ovarian cancer and was found to be regulated by DNA methylation. 11.3% of non-Hodgkin lymphoma (NHL) samples were found to express at least 1 CT antigen including MAGE-A family (6.6%), GAGE (5.7%), and NY-ESO-1 (4.7%). Humoral and

Tumor Antigens

T-cell responses against XAGE-1b (GAGED2a) were observed in non-small cell lung carcinoma (NSCLC) and in adenocarcinoma lung patients (see also Van den Eynde and van der Bruggen 1997). Altered glycosylations frequently occurring in tumors lead to the creation of aberrantly glycosylated and expressed antigens. Glycosyltransferases are the key enzymes for the biosynthesis of carbohydrate chains. In cancer cells, glycosylation often is similar to that performed in fetal or immature cells. Changes in patterns of glycosylation may involve incomplete synthesis and variation of normally existing carbohydrates or changes in the backbone or in the inner core structures of the carbohydrates (see also Heimburg-Molinaro et al. 2011). Mucins are high molecular weight glycoproteins composed of 20 amino acid residues repeated in tandem, always heavily glycosylated with N-acetylgalactosamine O-linked to serine and threonine residues. Cancer tissues show increased levels of mucin mRNA and an aberrant glycosylation as compared to normal tissues, which lead to unusual expression of the core of the protein. Mucins exist as transmembrane (MUC1, MUC3, MUC4, MUC10-18) or soluble (gel forming) (MUC2, MUC5AC, MUC5B, MUC6-9, and MUC19) glycoproteins. MUC1 is present on normal ductal epithelial cells as a heavily glycosylated protein. However, it was found that cancer cells have reduced or no activity of the b1–6GlcNAc transferase, while they have increased activity of a2-3sialyltransferase which competes for the same galactose substrate by the addition of sialic acid, thus precluding additional carbohydrates insertion. In cancer cells, this phenomenon leads to the uncovering of novel peptide antigenic determinants and the production of new oligosaccharide epitopes. MUC1 expression is upregulated in the majority of adenocarcinomas as well as in hematological malignancies. Autoantibodies to MUC1 were found in breast, ovarian, pancreas, and non-small cell lung cancers. In addition, MUC1 peptide-stimulated CTLs could be isolated from patients with different adenocarcinomas (see also Heimburg-Molinaro et al. 2011).

Tumor Antigens

Similarly, aberrant glycosylation in cancer cells often results in the exposure of tumorassociated carbohydrate structures and in enhanced expression of GalNAca1–Ser/Thr (Tn antigen), Neu5Aca2–6GalNAc (sialyl-Tn antigen), and Galb1–3GalNAca1–Ser/Thr (Thomsen–Friedenreich, TF or T, antigen). The TF or T antigen represents the core 1 structure of O-linked mucin-type glycans. The TF antigen is hidden in normal epithelium by sialic acids and sulfates or by the addition of other sugar chains to produce branched and complex O-glycans. However, unsubstituted Galb1–3GalNAc antigen frequently occurs in a high percentage of tumors. Immunoglobulins G to TF, Tn, and aGal were detected in patients with breast cancer. Blood group antigens related to the ABO and Lewis system are found in the peripheral structure of glycoproteins and glycolipids. The expression of Lewis Y antigen structure (LeY) appears to be low in normal tissues, while it was found high in several tumors. LeY is also expressed on granulocytes. LeY circulating immune complexes were found in serum of breast and gastric cancer patients. Stage-specific embryonic antigen-1 (SSEA-1) (LeX) was found to be expressed on normal epithelial cells, leukocytes, bone marrow, macrophages, spleen cells, and in some areas of the central nervous system. Increased SSEA-1 expression was found in several tumors. Antibodies to LeX were detected in patients with gastric cancer. Gangliosides (G) are complex glycosphingolipids mono- (M), di- (D), or tri-sialylated; the number (1, 2, 3) in their nomenclature represents the order of their distance of migration in thinlayer chromatography. Aberrant expression of gangliosides has been found in neural crestderived tumors. As a result of neoplastic transformation, normal melanocytes expressing GM3 begin to produce large amounts of GD3. GD3 and GM2 were also found in small cell lung cancers (SCLCs). Antibodies to GD1a/b and GM1 were found in patients with lung and gastric cancer, melanoma, and B-cell lymphoma. In addition, antibodies against GM1 gangliosides were associated with metastatic melanoma (see also Reuschenbach et al. 2009).

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Fucosyl GM1 (FucGM1) appears to be a particular ganglioside form expressed in SCLC and with a very low expression in normal tissues. Autoantibodies against FucGM1 were found at low titer in few SCLC, renal carcinoma patients, and in healthy controls. Tissue-lineage antigens are expressed in a tumor of a certain histotype and in the normal tissue from which the tumor is derived. This group of antigens contains melanoma differentiation antigens such as gp100, Melan-A, tyrosinase and tyrosinase-related protein 1 (TYRP1/gp75), prostate-specific antigen (PSA), and idiotypic antigens. Melanoma differentiation antigens are abundantly expressed in most primary and metastatic melanomas. The in vitro stimulation of peripheral PBMCs with peptides from Melan-A/ MART-1 (MART-127–35), gp100, tyrosinase, and TYRP1/TYRP2 was demonstrated to induce CTLs in melanoma patients. In addition, serum antibodies from a melanoma patient were able to immunoprecipitate TYRP1/gp75 (see also Yang and Yang 2005). PSA is a 33- to 34-kDa serine proteinase. The serum PSA test is still the most significant biomarker for the detection and follow-up of prostate cancer; the usefulness may be improved by determination of PSA isoforms in conjunction with free PSA. Circulating autoantibodies to PSA were found in the serum of benign prostatic hyperplasia and prostate cancer patients. In addition, recognition of PSA-derived peptide antigens by T cells from prostate cancer patients was demonstrated. An idiotype represents a unique and characteristic antigenic determinant of an immunoglobulin (Ig) or T-cell receptor. A unique Ig is expressed on the surface of a B lymphocyte. The variable regions of the heavy and light chains of the Ig contain unique determinants called idiotype. Accordingly, a B-cell lymphoma following clonal proliferation of a B cell will express an antigen which cannot only be regarded as a tissue-lineage antigen but has to be considered also as a tumorspecific antigen. It has been demonstrated that CD5-positive B-cell malignancies frequently express cross-reactive idiotypes associated with IgM autoantibodies.

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Overexpressed antigens represent antigens that even if present in normal tissues are present at higher levels in tumor tissues. According to this definition, several of the antigens listed above can be grouped into this category (see also Khodadoust and Alizadeh 2014). Certainly in this category can be included several receptors with tyrosine kinase activity (RTKs) and whose overexpression contributes to the development of the tumor. Among other RTKs, members of the epidermal growth factor receptor (EGFR) family, including EGFR, ErbB2, ErbB3, and ErbB4, have frequently been implicated in human neoplasia by overexpression in the presence or absence of gene amplification. Figure 1 shows overexpression of EGFR and ErbB2 in well-differentiated head and neck squamous cell carcinomas. Humoral response to all ErbB family receptors and T-cellmediated immunity to ErbB2 were demonstrated. Another example of molecules overexpressed by cancer cells is ribosomal proteins which were also demonstrated to induce immune responses in cancer patients. Among ribosomal proteins, the ribosomal P0 protein is overexpressed in colon cancer, hepatocarcinoma, head and neck cancer, and in breast cancer. Overexpression of the 22 amino acid C-terminal epitope of P0 in a well-differentiated head and neck squamous cell carcinoma is provided in Fig. 1. Humoral response to the 22 amino acid C-terminal peptide of P0 has been demonstrated in breast cancer and head and neck cancer patients. Ribosomal protein S6 was found to be overexpressed in non-Hodgkin lymphoma, breast, colon, and renal cell carcinomas. Autoantibodies to S6 were detected in patients with breast cancer. Similarly, ribosomal protein L19 (RPL19) was found to be overexpressed in NSCL cancer tissues and to induce CTL. Overexpression of heat shock proteins (Hsp27, Hsp70, Hsp90) has been found in several tumors. Several HSPs were shown to elicit antibodies in breast and ovarian cancer patients. Patients with AML showed significantly higher anti-Hsp70 antibody concentrations compared to the control group. The transcription factor MYC is involved in cell growth, transformation, angiogenesis, and

Tumor Antigens

cell-cycle control. MYCN amplification has been reported in several tumors. Humoral responses to MYC have been detected in serum of breast, lung, hepatocellular, and colorectal cancer patients. Osteopontin (OPN) expression is deregulated in prostate neoplastic lesions. The frequency of anti-OPN antibodies was found higher in prostate cancer (66%) patients as compared to benign prostate hyperplasia patients (33%) and healthy donors (10%). The human homolog of the murine doubleminute 2 oncoprotein (MDM2) is a putative TAA because it is overexpressed in several malignancies, including CLL cells. MDM2specific T cells were generated in 7/12 CLL patients. Mesothelin is a glycoprotein expressed on normal mesothelial cells and highly expressed on mesothelioma, ovarian, and pancreatic cancer. Autoantibodies to mesothelin were detected in serum of mesothelioma and ovarian cancer patients. In addition, specific mesothelin T cells were generated at high frequency from PBLs of pancreatic cancer patients. Survivin, a member of the inhibitor of apoptosis protein family, is frequently expressed in cancers. Anti-survivin antibodies were observed in patient with brain tumors. Survivin-specific T-cell responses in healthy donors and cancer patients were also demonstrated. Virus-induced tumors express virally encoded proteins. Examples of virally encoded proteins are those produced after human papillomavirus (HPV) and Epstein–Barr virus (EBV) infection. Infection with HPV is a risk factor for the development of cervical cancer. Oral HPV infection has been associated with some cases of oropharyngeal cancer. Lymphoproliferative responses to specific HPV16 E6 and E7 peptides and antibodies to E6 and E7 proteins were detected in HPV-infected individuals (see also Yang and Yang 2005). EBV is a gamma herpes with potent B-cell transforming activity associated with various B-cell malignant diseases and nasopharyngeal carcinoma (NPC). T lymphocytes can discriminate EBV-infected or EBV-transformed B cells.

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Tumor Antigens, Fig. 1 EGFR, ErbB2, and P0 overexpression in head and neck squamous cell carcinoma. Immunohistochemical detection. Immunoperoxidase counterstained with hematoxylin, original magnification 200

Elevated titers of IgA antibodies to EBV replicative antigens frequently precede the appearance of NPC.

Cross-References ▶ Alpha-Fetoprotein ▶ BCR-ABL1 ▶ Cancer ▶ Carcinoembryonic Antigen

▶ Cyclin-Dependent Kinases ▶ Epidermal Growth Factor Receptor ▶ Gangliosides ▶ MDM2 ▶ Mesothelin ▶ Mesothelioma ▶ Mucins ▶ MYC Oncogene ▶ Oncofetal Antigen ▶ Osteopontin ▶ Prostate-Specific Antigen

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▶ Retinoblastoma ▶ Survivin ▶ Wilms’ Tumor

References Canevari S, Pupa SM, Ménard S (1996) 1975–1995 revised anti-cancer serological response: biological significance and clinical implications. Ann Oncol 7:227–232 Heimburg-Molinaro J, Lum M, Vijay G, Jain M, Almogren A, Rittenhouse-Olson K (2011) Cancer vaccines and carbohydrate epitopes. Vaccine 29:8802–8826 Khodadoust MS, Alizadeh AA (2014) Tumor antigen discovery through translation of the cancer genome. Immunol Res 58:292–299 Reuschenbach M, von Knebel Doeberitz M, Wentzensen N (2009) A systematic review of humoral immune responses against tumor antigens. Cancer Immunol Immunother 58:1535–1544 Van den Eynde BJ, van der Bruggen P (1997) T cell defined tumor antigens. Curr Opin Immunol 9:684–693 Yang F, Yang XF (2005) New concepts in tumor antigens: their significance in future immunotherapies for tumors. Cell Mol Immunol 2:331–341

Tumor Cell Invasion ▶ Invasion

Tumor Cell-Induced Platelet Aggregation Mei-Chi Chang1 and Jiiang-Huei Jeng2 1 Biomedical Science Team, Chang Gung Institute of Technology, Taoyuan, Taiwan 2 Laboratory of Pharmacology and Toxicology, School of Dentistry, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan

Synonyms Cancer cell-platelet microemboli; Pathological tumor cell-platelet interaction; Tumor cell-platelet aggregate; Tumor cell-platelet interaction; Tumor-induced platelet aggregation

Tumor Cell Invasion

Definition Tumor cell-induced platelet aggregation (TCIPA) is the ability of cancer cells to generate crucial molecules or surface receptor molecules that mediate platelet aggregation and accelerate the survival advantages of tumor cells in the vasculature, which is important for distant ▶ metastasis of cancer.

Characteristics Hematogenous metastasis of cancer cells to critical distant organs is one of the major reasons for death in most cancer patients. After ▶ invasion into blood vessels, circulating cancer cells may interact with various vascular cells such as leukocytes, platelets, and endothelial cells that may affect the survival and metastasis of tumor cells. Although normal platelet functions are important to control vascular hemostasis and thrombosis, cancer cells may express many factors which modulate the platelet activities. Clinically, the interactions between circulating cancer cells and platelets are crucial for tumor metastasis in patients with cancers. Cancer cells may induce the morphological changes and aggregation of platelets and subsequently trap the cancer cells in capillaries and enhance the adhesion of cancer cells to capillary endothelial walls. These events facilitate further tumor cell invasion and metastasis into distant organs. During these processes, the activated platelets release various factors (like serine proteases factor VIIa, factor Xa, ▶ plateletderived growth factor (PDGF), ▶ vascular endothelial growth factor (VEGF), fibrinogen, and thrombospondin), which increase vascular permeability and promote the growth, survival, motility, and the extravasation of metastatic cancer cells to neighboring organs. Expression of PDGFreceptor in breast cancer tissues has been reported to increase the risk of lymph node metastasis. Similarly, the expression of von Willebrand factor (vWF) in tumor tissues and serum may also affect the metastasis of ▶ osteosarcoma patients. A number of clinical studies further suggest the increased risk of thrombosis in patients with

Tumor Cell-Induced Platelet Aggregation

various cancers, indicating the roles of TCIPA and activation of the coagulation system in the progression of cancer. Mechanisms Induction of TCIPA may form platelet-cancer cell aggregates to prevent cancer cells from killing by human cellular and humoral immunities and from the damage by sheer force of blood flow. Platelet activation further facilitates ▶ angiogenesis, tumor cell adhesion to endothelium, and subsequent invasion into distant organs. Various cancer cells may produce differential amounts of critical factors such as ADP, thromboxane A (TXA), thrombin, cathepsins, ▶ matrix metalloproteinases (MMPs), membrane-type matrix metalloproteinases (MT-MMP), cancer procoagulant, mucin, tissue factors (TF), aggrus/ ▶ podoplanin, etc. These molecules may activate various receptors (P2Y12 purinergic receptor, thromboxane receptors, ▶ protease-activated receptors (PARs), integrin receptors, etc.) and downstream signaling cascades to initiate platelet aggregation and coagulation disorders. TF-bearing cancer cells can stimulate platelet aggregation via generation of thrombin and signaling by glycoprotein IIb/IIIa and VEGF release. The localized production of thrombin and fibrin may increase endothelial cell motility as well as platelet activation. Tumor cells have the capacity to convert fibrinogen into fibrin in vitro, and histologically the deposition of fibrin is popularly noted in the connective tissue surrounding tumor cells, an event being critical for tumor angiogenesis and metastasis. Generation of MMPs by tumor cells may activate platelet integrin receptors followed by induction of platelet aggregation. This event can be inhibited by anti-MMP antibodies and phenanthroline, an MMP inhibitor. MMPs generated by tumor cells and platelets are further shown to degrade the basement membrane and facilitate metastasis. Cancer cells may also activate platelets to release ADP, which binds further to platelet P2Y12 receptors, leading to full platelet aggregation. The ADP-mediated platelet aggregation by tumor cells can be attenuated through degradation of ADP by apyrase (APT102). Moreover, the

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interaction between platelets and tumor cells can be mediated by cell adhesion proteins, e.g., P-selectin in platelets and P-selectin glycoprotein ligand-1/CD162, heparin sulfate proteoglycan, and sialyl-Leqis A/X in cancer cells. This is followed by b3 integrin-mediated processes with concomitant activation of thrombospondin and fibrinogen. Using venous blood from cancer patients for testing, current techniques can detect the presence of circulating tumor cells in the bloodstream. Interestingly, podoplanin (Aggrus), a mucin-line glycoprotein, has been shown to induce platelet aggregation and affect the invasion/metastasis of cancer. The expression of podoplanin in astrocytic tumors has been linked to the malignancy of astrocytic tumors. This is possibly due to early interactions by surface mucins and other selectin ligands of tumor cells with the platelet P-selectin and L-selectin. Heparin as an antithrombotic agent is able to inhibit metastasis further by blocking the tumor cell mucin P-selectin ligands and thus also the platelet-tumor cell interaction. Clinical Aspects and Therapy Higher platelet numbers have been found in many kinds of cancers including lung, gastric, colorectal, and breast cancers. Clinically, cancer patients also have a higher risk for development of vascular thromboembolism. This can be attributed to abnormalities in platelet functions as well as in the thrombotic and hemostatic systems by tumor cellreleased factors. Understanding the mechanisms for TCIPA is helpful for the clinical design of therapeutic agents to control tumor metastasis. A number of clinical studies have found higher levels of thromboxane and lower levels of prostacyclin in plasma and tumor tissues from lung, bone, and breast. Moreover reports have also observed the overexpression of cyclooxygenase2 (COX-2) and thromboxane synthase in the tumors of colon, breast, prostate, brain, and endocrine. Some tumors may generate excessive amounts of TXA2 to enhance tumor growth and metastasis. An elevation of urinary 11-dehydroTXB2 (a major enzymatic metabolite of TXB2) in patients with colorectal cancers has been noted, suggesting the beneficial use of a low-dose

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COX-1 inhibitor to reduce platelet activity in some cancer patients. BM-567, a thromboxane synthase inhibitor and TXA2 receptor antagonist, can inhibit the MG-63 osteosarcoma cell-induced platelet aggregation. Administration of SQ29548, the other TXA2 receptor antagonist, also reduces chondrosarcoma tissue TXB2 levels, vascular permeability, and tumor size in experimental animals in vivo. Whether TXA2 receptor antagonists can be effectively used clinically to prevent tumor metastasis should be further addressed. ▶ Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen may reduce the risk of cancer in colon, esophagus, stomach, prostate, and lung as well as the metastasis of prostate cancer. Aspirin as a COX-1 inhibitor and antiplatelet agent is also tested for its anti-metastatic effects. In experimental animals, aspirin may reduce the metastasis of injected ▶ hepatocellular carcinoma cells to lung. However, a multicenter study shows little effect of aspirin in combination with chemotherapy to improve survival of 303 patients with small cell ▶ lung cancer. More studies on the dosage, duration, and timing of aspirin or other therapeutic administrations are needed. In experimental animals, heparin administration seems effective in the suppression of tumor metastasis, but not primary tumor growth. This can be due to its inhibition of blood coagulation, tumor cell-platelets, and ▶ tumor-endothelial cross talks. Accordingly, clinical administration of aspirin and heparin to humans may suppress platelet aggregation and therefore promote survival in cancer patients. In addition, heparin, warfarin (a vitamin K antagonist), and antiplatelet agents (prostacyclin and dipyridamole) are able to inhibit the metastasis of ▶ pancreatic cancer cells and melanoma cells to the liver and lung. Intriguingly, studies have found that low molecular weight heparin (LMWH) successfully prevents the progression of cancer and improves the survival of patients with advanced ▶ non-small cell lung cancer, colon, pancreatic, breast, and pelvic cancers. Since tumor cells may also express PARs, glycoprotein IIb/IIIa, and integrin avb3, inhibition of these receptors and activation and signaling by agents such as hirudin, PPACK,

Tumor Cell-Induced Platelet Aggregation

antithrombin III, and receptor neutralizing antibodies are potentially useful methods for inhibition of TCIPA and metastasis by some tumor cells. XV454, a glycoprotein IIb/IIIa antagonist, has been shown to suppress the metastasis of Lewis lung carcinoma cells in experimental mouse metastasis models. A combination of LMWH and XV454 is even more effective in reducing cancer-induced thrombosis in vitro. Currently, some new reagents are being tested for their inhibition of TCIPA. Administration of antiplatelet agents (such as antiplatelet antibody, dansylarginine N-(3-ethyl-1, 5-pentanediyl) amide) before inoculation of tumor cells has been shown to effectively reduce tumor metastasis to lung and bone tissue. Integrilin, a plateletspecific integrin inhibitor, was able to effectively suppress TCIPA, but showed only partial inhibitory effects on physiological platelet functions. An elevated expression of aggrus/podoplanin, a new platelet aggregation-inducing factor in tumor tissues, has been found and promotes pulmonary metastasis in experimental animals. Administration of anti-podoplanin antibody is also shown to inhibit the neuroglioblastoma cell-induced platelet aggregation. More clinical trials by inhibition of TCIPA using antiplatelet and anticlotting agents are now in progress. These results will be helpful in developing effective clinical strategy and regimens for antimetastatic therapy of cancer in the near future.

Cross-References ▶ Angiogenesis ▶ Cyclooxygenase ▶ Hepatocellular Carcinoma ▶ Invasion ▶ Lung Cancer ▶ Matrix Metalloproteinases ▶ Metastasis ▶ Non-Small-Cell Lung Cancer ▶ Nonsteroidal Anti-Inflammatory Drugs ▶ Osteosarcoma ▶ Pancreatic Cancer ▶ Platelet-Derived Growth Factor

Tumor Microenvironment

▶ Podoplanin ▶ Protease-Activated Receptors ▶ Tumor–Endothelial Cross-Talk ▶ Vascular Endothelial Growth Factor

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Tumor Grading ▶ Grading of Tumors

References Gupta GP, Massague J (2004) Platelets and metastasis revisited: a novel fatty link. J Clin Invest 114:1691–1693 Hejna M, Raderer M, Zielinski CC (1999) Inhibition of metastasis by anticoagulants. J Natl Cancer Inst 91:22–36 Jurasz P, Alonso-Escolano D, Radomski MW (2004) Platelet-cancer interactions: mechanisms and pharmacology of tumor cell-induced platelet aggregation. Br J Pharmacol 143:819–826 Medina C, Jurasz P, Santos-Martinez MJ et al (2006) Platelet aggregation-induced by caco-2 cells: regulation by matrix metalloproteinase-2 and adenosine diphosphate. J Pharmacol Exp Ther 317:739–745 Timar J, Tovari J, Raso E et al (2005) Platelet-mimicry of cancer cells: epiphenomenon with clinical significance. Oncology 69:185–201

See Also (2012) Cyclooxygenase-2. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1035. doi:10.1007/978-3-642-16483-5_1435 (2012) Glycoprotein. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1570. doi:10.1007/978-3-642-16483-5_2451 (2012) Thromboxane. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3682. doi:10.1007/978-3-642-16483-5_5798

Tumor Markers ▶ Alpha-Fetoprotein ▶ Clinical Cancer Biomarkers

Tumor Metabolism ▶ Antiglycolytics and Cancer

Tumor Microenvironment Isaac P. Witz Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv, Israel

Synonyms Cancer (or tumor) stroma

Definition

Tumor Cell-Platelet Aggregate ▶ Tumor Cell-Induced Platelet Aggregation

The specific conditions existing in the tumor tissue, the nonmalignant cells and the molecules present in proximity to the tumor cells.

Tumor Cell-Platelet Interaction

Characteristics

▶ Tumor Cell-Induced Platelet Aggregation

It is now widely accepted that “Although abnormalities of cancer genes (▶ Oncogene; ▶ tumor suppressor genes) are essential contributors to cancer, most abnormalities in these genes occur relatively early in the disease process and none of them is known to be associated with the metastatic stage. It is this final stage – the seeding and growth

Tumor Glucose Metabolism ▶ Warburg Effect

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of satellite lesions in other organs – that is ultimately responsible for the great majority of neoplastic deaths.” It is the tumor microenvironment that determines and shapes the malignancy phenotype of cancer cells, in other words its metastatic behavior (▶ Metastasis). The tumor tissue can be viewed as an ecosystem composed of two compartments being intimately associated with each other. The first compartment constitutes the malignant cells. The second is the tumor microenvironment composed of resident cells such as fibroblasts, endothelial cells (▶ Tumor-Endothelial Cross-talk), and other nonmalignant cells; of infiltrating cells such as lymphocytes or macrophages (▶ Tumorassociated macrophages) and of numerous molecules released by the tumor cells as well as by the non malignant cells. These molecules may be in complex with other molecules, for example, in the extracellular matrix. Other molecules such as growth factors, cytokines, ▶ chemokines, antibodies, proteases, other types of enzymes, various metabolites, or drugs may be present in soluble form. The microenvironment of many solid tumors may be characterized by ▶ hypoxia (Hypoxia and Tumor Physiology; ▶ hypoxia inducible factor-1); low extracellular pH and by low glucose concentration. Cellular products released from necrotic tumor cells are also present. Although the term tumor microenvironment is used most often with respect to solid tumors, other types of malignancies have also their specific microenvironments. The bone marrow serving as a microenvironment for certain leukemias and for ▶ multiple myeloma is a case in point. Stephen Paget, over 100 years ago, is credited with being the first to postulate the important role played by the microenvironment in metastasis formation. The concept of his “Seed and Soil” theory, explaining site specific metastasis in ▶ breast cancer, has been supported and confirmed. However, numerous studies published in the last three decades demonstrate very clearly that the “soil” functions also, or even primarily, as an active “educational/inductive” venue in which cancer cells are directed, by interacting with microenvironmental factors, into one of

Tumor Microenvironment

several molecular evolution pathways. In other words, by exerting regulatory functions and selective pressures, the tumor microenvironment determines and shapes the malignancy phenotype of cancer cells. The tumor microenvironment is an interaction arena between microenvironmental components and tumor cells and between different microenvironmental components. This arena is characterized by four major hallmarks: complex regulatory circuits; a yin-yang (double edged sword) interplay; plethora of vicious cycles; abnormality of its “normal,” nonmalignant compartment. Complex Regulatory Circuits The major function of the tumor microenvironment is a regulatory one. Many genes in the tumor cells and in nontumor cells residing in or infiltrating to the tumor microenvironment are regulated by microenvironmental components. Several 100 proteins were identified in the microenvironment of breast cancer. An extremely large number of signaling cascades (▶ Signal transducers and activators of transcription in oncogenesis; ▶ signal transduction) would operate in this microenvironment even if only a small portion of these proteins would interact with tumor cells or with nontumor cells. It is safe to predict that a similar number of proteins will be detected in the microenvironment of other types of solid tumors. These signaling cascades take part in the regulation of genes in tumor and in nontumor cells thereby shaping the phenotype of cancer cells and drive their ▶ progression. The regulatory power of the microenvironment can be amplified by the agonistic or antagonistic cross-talk (▶ Receptor cross-talk) between different signaling cascades. Furthermore, several signaling cascades in cancer cells are aberrant. This may well increase the number of combinatorial signaling pathways, augment their complexity, and decrease the capacity of physiological feedback mechanisms to confront these malignancyassociated processes. Another factor contributing to the complexity of the interactions taking place in the tumor microenvironment is tumor

Tumor Microenvironment

heterogeneity. It is thus to be expected that different tumor variants, expressing different profiles of signaling receptors (▶ Receptor tyrosine kinases), would respond differentially to microenvironment-derived signals. The Yin-yang (Double Edged Sword) Interplay in the Tumor Microenvironment The cross-talk between tumor cells and microenvironmental factors may result in diametrically opposed effects which could either enhance or block tumor formation or progression. There are several examples of such a yin-yang interaction. The activity of ▶ transforming growth factorbeta (TGFb) is an example for a microenvironmental molecule manifesting a “love–hate relationship” with tumor cells. Whereas TGFb is a potent inhibitor of normal mammary epithelial cells, it enhances tumor cell ▶ invasion and metastasis of advanced breast cancer cells (Epithelial Tumors). Moreover, cancer cells may secrete TGFb which augments ▶ angiogenesis and is capable of suppressing antitumor immune responses of the host. On the other side of the coin, it was demonstrated that the progression of pancreatic and of intestinal tumors is enhanced by the inactivation of the TGF signaling cascade. Another prominent example for yin-yang interplay in the tumor microenvironment is ▶ inflammation versus protective tumor immunity. Cells and molecules of the immune system may, under certain circumstances, inhibit tumor growth and under different circumstances promote it. Vicious Cycles in the Tumor microenvironment A vicious cycle may be described as an input event that drives and amplifies other events which, in turn, promote tumor progression. Among such activities, the input event may also augment itself (positive feedback). A well studied vicious cycle in the tumor microenvironment is the cross-talk between osteoblasts, osteoclasts and other microenvironmental factors on the one hand and breast, prostate (▶ Prostate cancer clinical oncology) and lung (▶ Lung cancer) tumor cells on the other hand. This

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cross-talk promotes bone metastasis (▶ Bone tropism). Tumor-derived molecules such as cytokines cause either an osteoblastic or an osteolytic response. Such molecules feedback on the tumor and on various cells in the microenvironment causing the release of factors driving tumor progression. Another example of a vicious cycle in the tumor microenvironment is the cross-talk between chemokines (▶ Chemoattraction; ▶ chemokine) such as CCL2 and CCL5 secreted from mammary tumors of mice or from breast cancer cells of humans and cytokines such as TNFa secreted from macrophages infiltrating into these tumors. The tumor-derived chemokines attract monocytes to the microenvironment. These monocytes differentiate into macrophages which secrete TNFa. This cytokine up regulates the secretion of CCL2 and CCL5 from the tumor cells. CCL2 and CCL5, in turn, promote the secretion of TNFa from the tumor-associated macrophages. In this vicious cycle, the tumor cells and the macrophages promote each other’s ability to express and secrete pro malignancy factors. Hypoxia (Hypoxia and tumor physiology; hypoxia inducible factor-1) characterizes the microenvironment of solid tumors. Hypoxiainduced changes in the proteome (▶ Proteomics) may lead to either impairment of tumor growth and spread or, alternatively, to tumor propagation and progression. In the later case, a vicious cycle is created in which tumor cells surviving and propagating under hypoxia will aggravate the state of tumor hypoxia which in turn promotes genomic instability and further progression. The Nontumor Cells in the Tumor Microenvironment may Express a Different Phenotype than Their Counterparts at Distant Sites The conditions in the tumor microenvironment, for example, hypoxia, may induce or promote genetic instability and cause mutations and alterations in gene expression profiles of cancer cells (Genetic polymorphisms). It is not unlikely that such conditions may induce genetic alterations also in nontumor cells present in the microenvironment. The question, if the phenotype and

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functions of nontumor cells in the tumor microenvironment are similar or different from those of their counterparts in normal microenvironments, is by and large open. Several studies clearly demonstrate that at least some of the nontumor cells in the tumor microenvironment may not represent faithfully the characteristics of their counterparts in other sites of the body. Cancer-associated fibroblasts and endothelial cells are two prominent examples that illustrate the abnormality of tumor-associated nontumor cells. Cancer-associated fibroblasts have genetic changes both at the DNA level as well as at the expression level. For example, fibroblasts in human carcinomas have tumor suppressor gene mutations. DNA microarrays (▶ Microarray (cDNA) technology) identified over 100 genes differentially expressed by prostate carcinoma (Prostate cancer, clinical oncology)-derived fibroblasts and by systemically derived ones. These alterations may well manifest themselves by altered functions of such tumor-associated cells. Similar findings were also reported for endothelial cells. Cytogenetic abnormalities have been shown to occur in tumor endothelium and such cells may also express proteins that are not expressed by endothelial cells of the corresponding normal tissue. Site Specific Metastasis and the Metastatic Microenvironment Paget already realized that there is a predilection of tumors to metastasize to specific organ sites and that the metastatic capacity of a certain tumor is not restricted to a single organ site. Each tumor type has therefore several different potential metastatic microenvironments. Since the tumor and its microenvironment regulate and shape each other’s phenotype, it is to be expected that the metastases arising in one organ site be different from metastases derived from the very same tumor developing in a different organ site. It is also assumed that different reciprocal signaling cascades take place between metastases and nontumor microenvironmental cells in different metastatic microenvironments. These assumptions are indeed supported by experimental evidence.

Tumor Microenvironment

Dormant Micrometastasis and Microenvironmental Control Many organs including those of healthy people harbor solitary tumor cells or very small clusters of such cells. These cells do not proliferate either due to a balance between proliferation and apoptosis or due to cell cycle arrest. There is strong experimental support that such cells are precursors for metastasis and that microenvironmental control mechanisms keep these micrometastatic cells under check, i.e., in a state of dormancy. It is not unlikely that breakdown of these control mechanisms is responsible for the awakening of dormant micrometastases and their progression towards frank metastasis. The Tumor Microenvironment as Target in Cancer Therapy Tumor autonomous factors as well as the tumor microenvironment cooperate in the formation of primary tumors and of metastasis. It is therefore not surprising that cells and molecules that originate in the microenvironment and that drive tumor progression do serve as candidates for cancer therapy. Moreover the fact that nontumor cells in the tumor microenvironment express a different phenotype than that expressed by the corresponding cells in the normal microenvironment (see above) might also be exploited in cancer therapy modalities, targeting these differentially expressed molecules. Numerous clinical trials targeting various components of the tumor microenvironment are in progress.

Cross-References ▶ Angiogenesis ▶ Breast Cancer ▶ Chemoattraction ▶ Chemokines ▶ Desmoplasia ▶ Epithelial Tumorigenesis ▶ Hypoxia ▶ Hypoxia-Inducible Factor-1 ▶ Invasion ▶ Lung Cancer

Tumor Necrosis Factor

▶ Metastasis ▶ Microarray (cDNA) Technology ▶ Multiple Myeloma ▶ Oncogene ▶ Progression ▶ Proteomics ▶ Receptor Cross-Talk ▶ Receptor Tyrosine Kinases ▶ “Seed and Soil” Theory of Metastasis ▶ Signal Transducers and Activators of Transcription in Oncogenesis ▶ Signal Transduction ▶ Tissue Inhibitors of Metalloproteinases ▶ Transforming Growth Factor-Beta ▶ Tumor-Associated Macrophages ▶ Tumor–Endothelial Cross-Talk ▶ Tumor Suppressor Genes

References Baglole CJ, Ray DM, Bernstein SH et al (2006) More than structural cells, fibroblasts create and orchestrate the tumor microenvironment. Immunol Invest 35:297–325 Bierie B, Moses HL (2006) Tumour microenvironment: TGFbeta: the molecular Jekyll and Hyde of cancer. Nat Rev Cancer 6:506–520 Fidler IJ (2002) Critical determinants of metastasis. Semin Cancer Biol 12:89–96 Goss PE, Chambers AF (2010) Does tumour dormancy offer a therapeutic target? Nat Rev Cancer 10:871–877 Klein-Goldberg A, Maman S, Witz IP (2014). The role played by the microenvironment in site-specific metastasis. Cancer Lett 352:54–58 Maman S, Witz IP (2013) The metastatic microenvironment. In: Shurin MR et al (eds) The tumor microenvironment. Springer Science+Business Media, pp 15–38 Vogelstein B, Kinzler KW (2004) Cancer genes and the pathways they control. Nat Med 10:789–799 Witz IP, Levy-Nissenbaum O (2006) The tumor microenvironment in the post-PAGET era. Cancer Lett 242:1–10 Witz IP (2008) Tumor-microenvironment interactions: dangerous liaisons. Adv Cancer Res 100:203–229 Witz IP (2008) The selectin-selectin ligand axis in tumor progression. Cancer Metastasis Rev 27:19–30 Witz IP (2008) Yin-yang activities and vicious cycles in the tumor microenvironment. Cancer Res 68:9–13 Witz IP (2009) The tumor microenvironment: the making of a paradigm. Cancer Microenviron Suppl 1:9–17

See Also (2012) Genetic Polymorphism. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1528. doi:10.1007/978-3-642-16483-5_2382

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Tumor Necrosis Factor Wen-Ming Chu Cancer Biology Program, University of Hawaii Cancer Center, Honolulu, HI, USA

Synonyms Cachectin; Differentiation-inducing Endotoxin-induced factor in serum

factor;

Definition The tumor necrosis factor (TNF) family includes TNF alpha (TNFa) and TNF beta (TNFb), which are among the most important cytokines involved in systemic inflammation, tumor lysis, and initiation of the acute-phase reaction.

Characteristics Discovery More than 100 years ago, Dr. Williams B. Coley used crude bacterial extracts to treat tumor patients. He found that the bacterial extracts had an ability to induce tumor necrosis. While tumors were regressive, patients receiving bacterial extracts also suffered from a severe systematic inflammatory reaction. One of the major inflammatory stimulators now known to cause this reaction was identified in 1975, when a protein factor in the serum of endotoxin-treated animals was found to cause lysis in tumor cells and was therefore named “tumor necrosis factor.” In 1984, TNF gene was isolated and characterized. About the same time, another gene encoding a protein, which was purified from T lymphocytes and named “T lymphotoxin alpha (TLa)” in 1968, was also isolated and characterized. It was found that these two genes were in the same family. Thus, TNF was named “TNFa,” while TLa was called “TNFb.” In 1985, Nobel Prize winner Bruce Beutler and his colleagues purified a protein called cachectin from the supernatant of

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endotoxin-treated macrophages. This protein induced wasting (cachexia) and septic shock in murine recipients. Cachectin and TNF were later revealed to be the same protein. Sources TNFa is mainly produced by macrophages, whereas TNFb is mainly produced by T lymphocytes. Other cells can also express TNFa and TNFb at low levels. Genes and Proteins The human and murine TNFa genes are located on chromosome 6 and chromosome 17, respectively, and are preceded by the TNFb gene. Both genes are present in a single copy, are approximately 3 kb in size, and contain four exons. Several DNA-binding sites for the transcription factor nuclear factor kappa B (NF-kB) have been identified within the promoter region of the TNFa gene; therefore, it appears that the expression of TNFa is NF-kB dependent. A DNA-binding site for the high-mobility group 1 (HMG1) protein is located in the promoter region of the TNFb gene. Two forms of TNFa exist: the membranebound form (mTNFa) and the soluble form (sTNFa). Human mTNFa contains 157 amino acids (aa) and a 76 aa leader sequence, while mouse mTNFa contains 156 aa plus a 79 aa leader sequence. During synthesis, TNFa translocates to the cell membrane where the TNFa converting enzyme (TACE) sheds the mTNFa into sTNFa. In contrast to TNFa, TNFb only exists in the soluble form (sTNFb). TNF is conserved among species. For example, human TNF is 80% homologous to mouse TNF. The homology between TNFa and TNFb in both species is approximately 30%. Biological Functions TNF exerts many important physiological and pathological actions. TNF causes tumor cell necrosis (a process that involves cell swelling, organelle destruction, and finally cell lysis) and apoptosis (a process that involves cell shrinking, the formation of condensed bodies, and DNA fragmentation). Furthermore, studies in

Tumor Necrosis Factor

TNFa- or TNFR-deficient mice have revealed that TNF plays an important role in the regulation of embryo development and sleep-wake cycle and that TNF is vitally important for host defense against bacterial and viral infection. TNF has been shown to be an endogenous pyrogen that causes fever. Additionally, chronic exposure to a low dose of TNF may cause cachexia, wasting syndrome, and depression. It has been suggested that TNF might exhibit antitumor activities. The most phenomenal and critical biological function of TNF is its stimulation of systematic inflammatory reactions. TNF is a vital player in animal models of endotoxin-induced septic shock and in chemotherapy-induced septic shock in latestage lung cancer patients. TNF plays a central role in rheumatoid arthritis (RA) and inflammatory bowel diseases including Crohn disease and ulcerative colitis, as well as other autoimmune diseases. Anti-TNF Therapy Although TNF induces lysis of tumor cells, it has never been considered as an anticancer drug. Instead, due to its crucial role in the pathogenesis of immune disorders as mentioned above, antiTNF treatment has been developed as a therapy for RA and Crohn disease patients. The strategies developed for blockade of TNF either utilize anti-TNF antibodies (Remicade/infliximab, adalimumab/Humira, and golimumab) to neutralize sTNF or utilize Fc fragments of human immunoglobulin 1 fused with extracellular domains of TNFR1 or TNFR2 (certolizumab/Cimzia and etanercept/Enbrel) to block the interaction of TNF with TNFRs. Overall, anti-TNF therapy has been very successful in ameliorating symptoms of immune disorders, although some side effects have been identified. For example, anti-TNF therapy may enhance the risk of tuberculosis infection and the development of lymphoma. Molecular Mechanisms The molecular mechanisms behind TNF’s actions have been extensively investigated. Particularly, scientists have been keen to elucidate how TNF

Tumor Necrosis Factor

triggers activation of the IKK/NF-kB and the MAPK/AP-1 pathways, which are essential for the expression of pro-inflammatory cytokines, and to understand how TNF induces apoptosis. TNF binds to the TNFR1 and TNFR2 receptors, which can be either membrane bound or soluble. TNFR1 and TNFR2 each interact with both mTNFa and sTNFa. However, TNFR1 signaling can be strongly activated by both mTNFa and sTNFa, while TNFR2 signaling can only be efficiently activated by mTNFa. TNFR1 is ubiquitously expressed, while TNFR2 is mainly expressed on lymphocytes and endoepithelial cells. Upon ligation, both TNFR1 and TNFR2 form homotrimers, but interestingly they do not form a TNFR1/TNFR2 heterodimer. TNFR1 has a death domain, which allows it to interact with other death domain-containing adaptor proteins, whereas TNFR2 lacks a death domain. Activation of the IKK/NF-kB and the MAPK/AP-1 Pathways

When TNFR1 binds to TNF, its conformation is changed such that its death domain can interact with TNFR-associated factor containing death domains (TRADD), which in turn recruits TNFR-associated factors (TRAFs) including TRAF2 and TRAF5, as well as the cellular inhibitor of apoptosis proteins 1 and 2 (c-IAP1/ 2) to form the TNF receptor signaling complex (TNF-RSC). Next, the linear ubiquitin assembly complex (LUBAC), comprised of HOIL-1, HOIP, and SHARPIN, is recruited to the TNF-RSC by TRADD, TRAF2/5, and c-IAP1/ 2. LUBAC is not only required for the stabilization of TNF-RSC, but it also adds linear ubiquitin chains [e.g., methionine 1 (M1)-linked ubiquitin chain] to both RIP1 and the regulatory subunit of the IkappaB (IkB) kinase (IKK), IKKg (also called NEMO), thus bringing both RIP1 and IKKg to TNF-RSC. This results in the formation of the IKKg/IKK complex and the TAK1 (TGFb-activated kinase 1)/TAB1/2 (TAK1-binding proteins 1 and 2) complex. Intriguingly, c-IAP1/2, but not TRAF2, are E3 ligases that catalyze lysine (K) 11-, 48-, or 63-linked polyubiquitination of receptorinteracting protein (RIP1) and c-IAP1/2

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themselves, and this ligase activity is required for the recruitment of LUBAC to c-IAP-generated ubiquitin chains. The polyubiquitinated RIP1 triggers activation of TAK1, which in turn activates the catalytic subunits of IKK, IKKa, and IKKb. Although both IKKa and IKKb phosphorylate IkBa, IKKb is the major kinase leading to ubiquitination and degradation of IkBa and subsequently leading to NF-kB translocation to the nucleus where it initiates transcription of more than 200 NF-kBdependent genes, including cell survival genes, pro-inflammatory cytokines, chemokines, growth factors, and TNFa itself. TAK1 also activates mitogen-activated protein kinases (MAPKs), including JNK and p38. JNK phosphorylates both c-Jun and ATF2, and p38 phosphorylates ATF2 leading to the formation of a c-Jun/ATF2 heterodimer called activating protein 1 (AP-1). AP-1 is another critical transcription factor and has similar functions to NF-kB. Induction of Apoptosis

TNFR1 also uses TRADD to recruit the Fas-associated protein-containing death domain (FADD) and deubiquitinated RIP1. Both FADD and RIP1 interact with pro-caspase 8, resulting in its cleavage and activation. Activated caspase 8 in turn mediates the cleavage of pro-apoptotic protein Bid generating a truncated form tBid, which translocates to the mitochondria and decreases mitochondrial membrane potential resulting in cytochrome c release. Cytochrome c, together with the apoptotic protease-activating factor 1 (Apaf1), binds to the initiator pro-caspase 9 forming an apoptosome complex, which activates other caspases including 3 and 7 resulting in cell apoptosis. c-IAPs are able to interact with caspases 3 and 7 and inhibit TNF-induced apoptosis (Fig. 1). Conclusion TNF is one of the most important proinflammatory cytokines and plays a pivotal role in the pathogenesis of immune disorders. Further understanding of TNF’s actions and the mechanisms underlying TNF pathology will allow for the development of a new generation of anti-TNF

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Tumor Necrosis Factor

Tumor Necrosis Factor, Fig. 1 A model of TNFa signaling in inflammation and apoptosis. TNF binds to TNFR1 and TNRF2. TNFR1 interacts with TRADD using its death domain (DD), which in turn recruits TRAF2, TRAF5, and c-AIP1/2 to form the TNF-RSC. RIP1 and LUBAC are then recruited to the TNF-RSC, leading to ubiquitination of RIP1 and IKKg and formation of the IKKg/IKK and TAK1/TAB1/TAB2 complexes. Polyubiquitinated RIP1 triggers TAK1 activation, which in turn activates IKKa/b, JNK, and p38 leading to NF-kB and AP-1 activation, respectively. AP-1 and NF-kB bind to DNA-binding sites located in the promoter regions of their target genes and initiate gene expression. TNFR1 also uses

TRADD to recruit FADD and deubiquitinated RIP1. Both FADD and RIP1 use their DD domains to interact with the DD domain in the initiator pre-caspase 8, leading to its cleavage and activation. Activated caspase 8 triggers cleavage of Bid into tBid, which then enters mitochondria leading to a decrease in mitochondrial membrane potential and subsequent cytochrome C release. Cytochrome C, together with Aparf1 and caspase 9, forms an apoptosome complex that triggers apoptosis. Interestingly, TNFR2 does not have a DD domain, but it is able to form a complex with TRAF2 and TRAF5, leading to activation of NF-kB and AP-1 upon stimulation with TNF

therapies that will cause fewer side effects, yet still maintain high efficacy in the treatment of immune disorders.

complex and is required for TNF-mediated gene induction. Mol Cell 36(5):831–844 Ikeda F, Deribe YL, Skånland SS, Stieglitz B, Grabbe C, Franz-Wachtel M, van Wijk SJ, Goswami P, Nagy V, Terzic J, Tokunaga F, Androulidaki A, Nakagawa T, Pasparakis M, Iwai K, Sundberg JP, Schaefer L, Rittinger K, Macek B, Dikic I (2011) SHARPIN forms a linear ubiquitin ligase complex regulating NF-kB activity and apoptosis. Nature 471(7340):637–641 Tokunaga F, Nakagawa T, Nakahara M, Saeki Y, Taniguchi M, Sakata S, Tanaka K, Nakano H, Iwai K (2011) SHARPIN is a component of the NF-kBactivating linear ubiquitin chain assembly complex. Nature 471(7340):633–636

References Haas TL, Emmerich CH, Gerlach B, Schmukle AC, Cordier SM, Rieser E, Feltham R, Vince J, Warnken U, Wenger T, Koschny R, Komander D, Silke J, Walczak H (2009) Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling

Tumor Suppression

Tumor Necrosis Factor-Alpha Converting Enzyme ▶ ADAM17

Tumor Pathology ▶ Pathology

Tumor Progenitors ▶ Stem-Like Cancer Cells

Tumor Rejection of Non-Irradiated Tumor Areas ▶ Abscopal Effects

Tumor Spread ▶ Metastasis

Tumor Staging ▶ Staging of Tumors

Tumor Suppression Eric Stanbridge Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA

Definition Tumor suppression is the consequence of the functional presence and activity of ▶ tumor

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suppressor genes (TSGs). TSGs are recessive genes whose protein products appear to directly or indirectly negatively regulate cell proliferation, promote ▶ apoptosis, and maintain in vivo homeostatic growth and differentiation potential.

Characteristics Three major classes of genes are involved in cancer causation and progression: • Dominantly acting oncogenes, whose proteins serve to stimulate cell growth and survival • Recessive genes involved in ▶ repair of DNA • Recessive TSGs The recessive TSGs and DNA repair genes are often included under the rubric of tumor suppressor genes. The importance of TSGs in the genesis of cancer became apparent when individuals predisposed to early onset cancer were found to contain a mutated allele of a certain TSG in their germline. This condition predisposes the individual to earlier onset cancer at a significantly higher probability than individuals who possess a sporadic cancer of the same histologic type. This is best illustrated by the prototypic TSG, RB1, that predisposes to childhood ▶ retinoblastoma. The incidence of sporadic retinoblastoma is 1:40,000 and is often unilateral, whereas the incidence of heritable retinoblastoma within an affected family is 40% and often involves both eyes (bilateral retinoblastoma). The affected proband inherits the mutant allele from one of the parents. Thus, the classic presentation of a heritable recessive TSG is one of dominant autosomal inheritance. In the classic two-hit model, the affected individual inherits one mutant RB1 allele, and the second allele is eliminated somatically in the retinoblastoma tumor. In the case of sporadic retinoblastoma, both alleles are eliminated somatically. Since the identification of the RB1 TSG, a large number of cancer-predisposing germline mutations in TSGs have been found (Table 1), and they include DNA repair genes. Many, but not all (e.g., BRCA1 and BRCA2), are commonly

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Tumor Suppression

Tumor Suppression, Table 1 Predisposing germline mutations in tumor suppressor genes Associated cancer syndrome Familial retinoblastoma

Tumor suppressor gene RB1

Human chromosomal location 13q14

Wilms’ tumor Li-Fraumeni syndrome

Wt1 p53

11p13 17q11

Von Recklinghausen disease Neurofibromatosis type 2

NF1

17q11

NF2

22q12

Von Hippel-Lindau disease

VHL

3p25

Familial adenomatous polyposis Familial melanoma

APC

5q21

INK4a

9p21

Gorlin syndrome

PTC

9q22.3

Binds/regulates b-catenin activity p16lnk4a cdki for cyclinD/cdk4/ 6; p19ARF binds mdm2, stabilizes p53 Receptor for sonic hedgehog

Juvenile polyposis

DPC4

18q21.1

Transduces TGF-b signals

Cowden syndrome BZS, LDD Tuberous sclerosis complex Familial prostate carcinoma Peutz-Jeghers syndrome

PTEN

10q23

Dual specificity phosphatase

TSC2

16

Cell-cycle regulator

Basal cell carcinoma, medulloblastoma Pancreatic, colon, hamartomas Glioblastoma, prostate, breast Renal, brain tumor

NKX3.1

8p21

Homeobox protein

Prostate

LKB1

19p13

Serine/threonine kinase

Familial gastric cancer

E-cadherin

16q22.1

Cell adhesion regulator

Ataxia telangiectasia

ATM

11q23

P13K-like kinase

HNPCC

MSH2

2p22

HNPCC

MLH1

3p21

HNPCC HNPCC HNPCC Bloom syndrome Fanconi anemia Complementation Gr A

PMS1 PMS2 MSH6 BLM

2q31 7p22 2p16 15q26.1

Mut S homologue, mismatch repair Mut L homologue, mismatch repair Mismatch repair Mismatch repair Mismatch repair DNA helicase

Hamartomas, colorectal, breast Breast, colon, skin, lung carcinoma Leukemias, lymphomas Colorectal cancer

FAA

16q24.3

Complementation Gr C

FAC

9q22.3

Xeroderma pigmentosum (seven complementation groups)

XPA XPB

9q34.1 2q21

Gene function Transcriptional regulator of cell cycle Transcriptional regulator Transcriptional regulator/ growth arrest/apoptosis Ras-GAP activity ERM protein/cytoskeletal regulator Regulates proteolysis

Involved in DNA cross-link repair Involved in DNA cross-link repair Binds damaged DNA Helicase, part of TFIIH

Cancer type Retinoblastoma, osteosarcoma Nephroblastoma Sarcomas, breast/ brain tumors Neurofibromas, sarcomas, gliomas Schwannomas, meningiomas Hemangiomas, renal, pheochromocytoma Colorectal cancer Melanoma, pancreatic

Colorectal cancer Colorectal cancer Colorectal cancer Colorectal cancer Multiple Leukemia Leukemia Skin

(continued)

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Tumor Suppression, Table 1 (continued) Associated cancer syndrome

Nijmegen breakage syndrome Familial breast cancer Familial breast cancer

Tumor suppressor gene XPC XPD XPE XPF XPG NBS1

Human chromosomal location 3p 19q12.3 ?11 16p13 13q23-33 8q21

BRCA1

17q21

BRCA2

13q12

Gene function ? Helicase, part of TFIIH Binds damaged DNA Structure-specific endonuclease Structure-specific endonuclease Involved in DNA double-strand break repair Transcriptional regulator/DNA repair Transcriptional regulator/DNA repair

Cancer type

Lymphomas Breast/ovarian tumors Breast/ovarian tumors

Metastasis

APC HNPCC hMSH2 hMLH1 hPMS1 hPMS2

KRAS

P53 DPC4

Tumor Suppression, Fig. 1 Multistep progression model for colorectal cancer. Tumorigenesis proceeds through a series of cellular alterations, including hyperplasia, benign polyps of increasing size and disordered growth, and carcinoma in situ with localized invasion.

Genetic alterations associated with this progression include inactivation of TSGs, e.g., APC, p53, and DPC4, mutational activation of the KRAS oncogene, and inactivation of one of the DNA mismatch repair genes

found to be mutated in sporadic cancers of the same histologic type as those seen in the relevant familial cancer cases. Progression to the cancerous condition is a multistep phenomenon. This is best illustrated by the colorectal cancer model (▶ multistep development; Fig. 1). Tumorigenesis proceeds through a series of cellular alterations, including hyperplasia of the colonic epithelium, benign polyps of increasing size and disordered growth, and ▶ carcinoma in situ with localized invasion. Distant metastases may occur. Accompanying these cellular alterations are genetic alterations, including activating mutation of the KRAS proto-oncogene

and loss-of-function mutations in multiple TSGs. In the case of TSGs, mutations in both alleles must occur for complete loss of function. Loss of function of multiple TSGs is a hallmark feature of most, if not all, cancers. TSGs span a broad range of functions (Table 1). These include kinases, phosphatases, cyclin-dependent kinase inhibitors, transcription factors, cell adhesion molecules, proteins involved in specific protein degradation pathways, and a variety of DNA repair processes. There is increasing evidence that tumor suppressor proteins and DNA repair proteins interact in functional networks (an example is given

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Tumor Suppressor Genes DNA damage (double-strand breaks)

ATM

p53

Apoptosis

p21 ↑

CHK2

14−3−3 ↑

BRCA1

NBS1

Recombinational repair

G1 G2 (Cell-cycle checkpoint)

Tumor Suppression, Fig. 2 Networking of tumor suppressor and DNA repair proteins. In response to DNA damage, ATM phosphorylates and activates p53 and the cell-cycle kinase CHK2. This kinase also enhances the activation of p53 via phosphorylation. Activated p53 upregulates expression of the cyclin-dependent kinase inhibitor, p21, and 14–3–3 proteins, resulting in cellcycle arrest at the G1 and G2 checkpoints, respectively.

The activated p53 protein may also induce apoptosis. Transient cell-cycle arrest allows for repair of damaged DNA by preventing the duplication and propagation of damaged DNA. ATM also plays an important role in DNA repair by phosphorylating and activating BRCA1 and NBS1 proteins. See Table 1 for identification of the genes. ! = activation; ┤ = inhibition

in Fig. 2). The ATM kinase phosphorylates and activates the tumor suppressor p53 and CDSI/ CHK2 proteins and the DNA repair proteins, BRCA1 and NBS1. These activations lead to cell-cycle checkpoint arrest and repair of DNA damage. Loss of function of any one of these factors compromises DNA repair, culminating in genomic instability and increased probability of progression to the cancer phenotype.

References

Clinical Aspects Identification of specific germline mutations in TSGs in affected families allows the recognition of those members who are carrying the mutant allele and, therefore, are at a significantly higher risk of getting cancer. Detection of mutant tumor suppressor proteins, e.g., p53, or loss of expression of such proteins may aid in cancer diagnosis and prognosis. Experimental investigations have established that restoration of TSG function in cancer cells that are defective for that function results in the suppression of tumor growth or death of the cancer cells. Clinical trials are in progress using ▶ gene therapy and pharmacologic approaches that aim to apply these procedures to the treatment of human cancers.

Eeles RA, Ponder BAJ, Easton DF et al (1996) Genetic predisposition to cancer. Chapman and Hall Medical, New York Harris CC (1996) Structure and function of the p53 tumor suppressor gene: Clues for rational cancer therapeutic strategies. J Natl Cancer Inst 88:1442–1455 Lengauer C, Vogelstein B (1998) Genetic instabilities in human cancers. Nature 396:643–649 Stanbridge EJ (1990) Human tumor suppressor genes. Annu Rev Genet 24:615–657 Weinert T (1998) DNA damage and checkpoint pathways: molecular anatomy and interactions with repair. Cell 94:555–558

Tumor Suppressor Genes Webster K. Cavenee Ludwig Institute for Cancer Research, UCSD, La Jolla, CA, USA

Synonyms Recessive oncogenes

Tumor Suppressor Genes

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Recombination Inherited genetic mutation

Point mutation or deletion CpG island methylation

Sporadic somatic mutation

Predisposed CH3

Tumor Suppressor Genes, Fig. 1 Chromosomal mechanisms for tumor suppressor gene inactivation. Left side, the first mutation (*) can occur in a single somatic cell and results in sporadic disease. Alternatively, it can occur in a germ cell (de novo mutation) or be inherited from an affected parent and results in heritable disease. Right side,

the first mutation can become completely inactivated by (from top to bottom) physical deletion or recombination of the wild-type chromosome, by a targeted second mutation or deletion of the remaining wild-type gene, or by methylation of the promoter of the wild-type gene, leading to the loss of expression

Definition

occurring mutant strains or those constructed by in vivo homologous recombination “gene knockout” techniques.

Tumor suppressor genes are genes whose products normally negatively regulate cell growth or cell behavior (Fig. 1).

Characteristics The hallmark of a tumor suppressor gene is that its function is lost during tumor initiation or progression. This typically occurs by one of a set of chromosomal processes called loss of heterozygosity but, in some cases, can occur by forming dominant negative forms of the tumor suppressor gene product. Their presence is usually inferred through the cytogenetic or molecular detection of subchromosomal loss. Upon molecular isolation, the genetic inference can be confirmed and dissected by demonstrating a restoration of growth regulation upon ectopic expression of the gene and/or by the formation of tumors or growth abnormalities in animals lacking the functional gene, either naturally

What Was the Evidence for Tumor Suppressors? The primary lines of evidence are genetic: One is that specific kinds of cancer can cluster in families. In most cases, the inheritance pattern is autosomal dominant which means that it is not sex linked, may be transmitted from either parent, and involves the transmission of a gene whose presence is sufficient to cause disease. In addition to familial clustering of the common cancers, two additional clinical observations provide strong epidemiological support for the contention that cancer has a genetic etiology: • First, some individuals and their families have an autosomal dominant transmission of cancer predisposition, not to a single tumor but to multiple tumors occurring independently at different body sites.

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• Second, individuals with a variety of multiorgan developmental defects often also develop specific rare tumors. A statistical argument can thus be made that the combined occurrence of multiple independent tumors or the routine association of developmental defects with tumors which are very rare in the general population is so unlikely as to suggest an etiologic relationship. The apparent dominant transmission of cancer traits is paradoxical in light of three observations. First, hybrid cells formed from the experimental fusion of highly malignant tumor cells with normal cells are not usually tumorigenic, suggesting that the normal phenotype is dominant in the presence of tumorigenic mutations. Furthermore, the occasional hybrid cell that regains tumorigenicity in these experiments has lost specific chromosomes originally contributed by the normal cell, implying that it is not gain of a dominant cancer trait but specific chromosomal loss that is responsible for the tumor phenotype. Second, if a single mutation was sufficient in itself to elicit a tumor, then families segregating for autosomal dominant forms of cancer would be expected to have no normal tissue in the diseased organ. This expectation is in direct contrast to the clinical description of these tumors as focal lesions surrounded by normal, functioning tissue of the same organ. Finally, epidemiological analyses of sporadic and familial forms of several human cancers have indicated that the conversion of a normal cell to a tumor cell requires multiple events. Retinoblastoma: The First Suppressor ▶ Retinoblastoma is a relatively rare tumor (1 in 20,000 births) of young children and occurs in both a sporadic and autosomal dominant inherited form. Based entirely on statistical data from epidemiology and clinical observations, several remarkable conclusions were made regarding the nature of events, leading to retinoblastoma tumor formation. First, the inherited mutation alone was not sufficient to cause the disease, since there are at least 107 retinoblast cells which are potential targets for retinoblastomas, each carrying the inherited mutation, yet on average, only three

Tumor Suppressor Genes

independent tumors form per affected individual. This also suggested that at a genetic level, mutations leading to retinoblastoma may be recessive, rather than dominant as suggested by the inheritance pattern. The hereditary tumors were proposed to arise through an initial germline mutation followed by a second mutation in a somatic cell. The rate at which somatic mutations occurred was similar in hereditary and sporadic cases, although sporadic tumors required two somatic mutations, each in the same retinoblast for tumor formation. Entirely consistent with this was the observation that hereditary cases usually occurred at an earlier age, were often bilateral, and had multiple tumors, whereas the sporadic cases were invariably unilateral and single tumors. Because of the small possibility that a second somatic mutation may never occur in hereditary cases, 5% of carriers do not develop any tumor. The nature of the two mutational targets in the genome was unknown at the time of these clinical observations, but cytogenetics and molecular genetics eventually led to the answer as well as to a general approach to other human cancers. Analysis of the chromosome band patterns from hereditary and sporadic retinoblastoma patients revealed a deletion of chromosome 13q14 (chromosome 13, q or long arm, band 1–4), suggesting that the gene for retinoblastoma (Rb) resided somewhere within this region. DNA from hereditary tumors was then analyzed with cloned DNA probes (termed “DNA markers”) that could distinguish the two copies, or alleles, of chromosome 13 within each cell. It was found that, in tumors of affected individuals, the region containing the suspected Rb gene on chromosome 13 was present in a mutant only state. This conversion from a heterozygous state to homozygosity for the mutation was termed loss of heterozygosity (LOH) and constituted the second hit required for tumor formation in hereditary cases. Furthermore, LOH on chromosome 13q14 also occurred in sporadic retinoblastoma. These data lent strong support to the idea that retinoblastoma tumor formation occurs by the unmasking of a recessive genetic defect. The discovery that LOH occurs in other hereditary and most sporadic cancers in humans marked

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Tumor Suppressor Genes, Table 1 Tumor suppressor genes, their primary biological functions and the types of tumors in which they have been found to be altered Gene RB1 p53 p16

Chromosomal location 13q14.2 17p13.1 9p21

p15

9p21

p18

1p32

▶ p21

6p21

E2F BRCA1 BRCA2 WT1 VHL PTCH TGFbR1 TGFbR2 DPC4 CDH1 APC MCC NF1 NF2 MSH2 MLH1 DCC PTEN

20q11 17q21 13q12–13 11p13 3p25–26 9q22.3 9q33–34 3p21.3 18q21.1 16q22.1 5q21 5q21 17q11.2 22q12 2p22 3p21.3 18q21 10q23.3

Function Cell-cycle regulator Genome-stability regulator Cyclin-dependent kinase inhibitor Cyclin-dependent kinase inhibitor Cyclin-dependent kinase inhibitor Cyclin-dependent kinase inhibitor Transcription factor Transcription factor Transcription factor Transcription factor Modulator of RNA polymerase Transcription repressor TGF-b receptor TGF-b receptor TGF-b pathway growth inhibitor Intercellular adhesion Cell signaling Cell Adhesion Cell signaling Cell signaling Mismatch repair protein Mismatch repair protein Differentiation factor Protein/lipid phosphatase

the simultaneous emergence of somatic cell cancer genetics and its coupling to the genetics of hereditary cancer. By identifying the region of chromosome 13q14 with the most consistent LOH in tumor DNA, the gene responsible for retinoblastoma was eventually isolated and its functionality assessed. Most importantly, the gene was shown to be mutationally inactivated in retinoblastoma tumors. When a normal copy of the gene was transferred to tumor cells, their growth and tumorigenic behavior was reduced. Thus, the conjoint application of epidemiology, cytogenetics, molecular genetics, and molecular biology allows the identification of a gene with tumor-suppressing function.

Cancer sites Retina, bone, bladder, breast, pancreas Brain, breast, leukemia, soft tissue Brain, melanocyte Leukemia Esophagus, lung, bladder, pancreas Prostate, lung Erythroleukemia Breast, ovary Breast, ovary Kidney Kidney, central nervous system Skin Colon, retina, liver, stomach Colon, retina, liver, stomach Pancreas, colon, bladder, liver Breast, ovary, liver, skin, endometrium Colon Colon Peripheral nervous system, skin Central nervous system Colon Colon Colon Brain, melanocytes, prostate, thyroid, breast

Are There Other Tumor Suppressors and What Cellular Role Do They Normally Play? Since the first suppressor was isolated, many others have been molecularly identified. As might be expected, these represent genes whose products are involved in many different aspects of cell growth and behavior. These include regulators of the cell cycle, growth and transcriptional regulators, DNA repair enzymes, differentiation factors, elements of cell ▶ motility, and regulators of cellular signaling. Thus, elucidation of the function and nature of tumor suppressors is not only of importance for understanding cancer etiology but also useful for dissecting normal cellular function.

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Clinical Relevance The intimate involvement of tumor suppressor genes in the etiology of most human cancers places them at the center of cancer research. Such knowledge has been exploited for the first prenatal and premorbid predictions of cancer occurrence, for molecular pathology approaches to tumor subtyping, for ▶ gene therapy approaches toward gene replacement, and as targets for agonist/antagonist development in rational drug design. Just as in research, the continued exploitation of tumor suppressor genes for clinical benefit to cancer patients is likely to assume a central role in modern therapies (Table 1).

Cross-References ▶ Allele Imbalance ▶ BRCA1/BRCA2 Germline Mutations and Breast Cancer Risk ▶ CCCTC-Binding Factor ▶ CDKN2A ▶ Deleted in Pancreatic Carcinoma Locus 4 ▶ Gene Therapy ▶ Motility ▶ Neurofibromatosis 1 ▶ Neurofibromatosis 2 ▶ p21 ▶ Repair of DNA ▶ Retinoblastoma ▶ Von Hippel-Lindau Tumor Suppressor Gene

References Cavenee WK, White RL (1995) The genetic basis of cancer. Sci Am 272:50–57 Newsham I, Hadjistilianou D, Cavenee WK (1999) Retinoblastoma. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Vogelstein B (eds) The metabolic and molecular basis of inherited disease, 8th edn. McGraw-Hill, New York Perkins AS, Stern DF (1997) Molecular biology of cancer: oncogenes. In: DeVita VT, Hellman S, Rosenberg SA (eds) Cancer: principles and practice of oncology, 5th edn. Lippincott-Raven Publishers, Philadelphia

Tumor Typing

See Also (2012) Autosomal Dominant. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 323. doi:10.1007/978-3-642-16483-5_489 (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) Chromosome Band. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 848. doi:10.1007/978-3-642-16483-5_1147 (2012) DCC. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 1063–1064. doi:10.1007/978-3-642-16483-5_1524 (2012) DNA Repair. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1141. doi:10.1007/978-3-642-16483-5_1687 (2012) Gene Knockout. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 1523. doi:10.1007/978-3-642-16483-5_2370 (2012) Loss of Heterozygosity. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, pp 2075–2076. doi:10.1007/978-3-642-164835_3415 (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

Tumor Typing ▶ Pathology

Tumor-Associated Macrophages Antonio Sica1, Alessandra Mancino1, Paola Larghi1, Luca Rubino2, Graziella Solinas1, Paola Allavena1 and Alberto Mantovani1 1 Department of Immunology, Fondazione Humanitas per la Ricerca, Rozzano, Milan, Italy 2 Department of Oncology, Humanitas Research Hospital, Humanitas Cancer Center, Rozzano, Milan, Italy

Definition Tumor-associated ▶ macrophages (TAMs) define a subset of myeloid cells that highly infiltrate solid tumors. Accumulating evidence clearly demonstrates, in various mouse and human

Tumor-Associated Macrophages

malignancies, including colon, breast, lung, and prostate cancer, a strict correlation between increased numbers and/or density of TAM and poor prognosis. Based on this, recruitment and activation of TAM are regarded as pivotal steps of tumor progression, and TAM are putative targets for therapeutic intervention.

Characteristics Experimental and clinical studies have revealed that chronic ▶ inflammation predisposes to different forms of cancer, including colon cancer, ▶ prostate cancer, and ▶ liver cancer, and that usage of ▶ nonsteroidal anti-inflammatory drugs can protect against the emergence of various tumors. In the late 1970s, it was found that a major leukocyte population present in tumors, the so-called TAM, promotes tumor growth. Over the years it has become increasingly clear that TAMs are active players in the process of tumor progression and invasion. In several experimental tumor models, the activation of an inflammatory response (most frequently mediated by macrophages) is essential for full neoplastic transformation and progression. This evidence strongly supports the idea that cancers originate at sites of chronic inflammation and suggests that the inflammatory circuits activated at the tumor microenvironment may represent suitable targets of novel anticancer therapies. Macrophages Macrophages (Mj) play an indispensable role in the immune system with decisive functions in both ▶ innate immunity and acquired immunity. In innate immunity, resident Mj provide immediate defense against foreign pathogens and coordinate leukocyte infiltration. Mj contribute to the balance between antigen availability and clearance through phagocytosis and subsequent degradation of senescent or apoptotic cells, microbes, and possibly neoplastic cells. Their role is essential for triggering, instructing, and terminating the adaptive immune response. Mj collaborate with T and B cells, through both cell–cell interactions

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and fluid phase-mediated mechanisms, based on the release of cytokines, chemokines, enzymes, arachidonic acid metabolites, and reactive radicals. Mj activation can be either proinflammatory or anti-inflammatory, thus contributing to tissue–cell destruction or to tissue regeneration and wound healing. These polar phenotypes are not expressed simultaneously but regulated in such a manner that Mj display a balanced, harmonious pattern of functions. Mj are critical effector cells in the acute innate response, for delayed-type hypersensitivity reactions and T cell-mediated immunity. In 1986 Mosmann et al. described two polarized sets of mouse T helper (Th) cells – Th1 and Th2 – with distinct cytokine secretion patterns. Th1 cells secreted interleukin 2 (IL-2), interferon-g (IFN-g), and lymphotoxin (LT, TNF-b). Th2 cells secreted IL-4, IL-5, and IL-6 and promoted B-cell proliferation and antibody secretion. Moreover, additional studies clarified that Th1 and Th2 cells may play opposite roles in pathological conditions, including infections and cancers. Although excluded from the original “type I–type II” paradigm, Mj’s role in the balance of polarized immune responses is being increasingly appreciated. Mj are able to secrete either IL-12 or IL-10, cross-regulatory cytokines crucial for the elicitation of IFN-g production and development of Th1 cells and IL-4/IL-13 secretion and Th2 cells proliferation correspondingly. The preferential production of IL-12 and IL-10 sets the basis for the M1/M2 Mj polarization paradigm, elsewhere defined as the elicitation of functionally distinct Mj populations, in response to the factors that dominate the inflammatory scene. In analogy with the Th1 and Th2 dichotomy, macrophages can be phenotypically polarized by the microenvironment to mount specific M1 or M2 functional programs. Chronic infections can tightly regulate the immune responses, being able to trigger highly polarized type I or type II inflammation and immunity. Classical or M1 macrophage activation in response to microbial products or IFN-g is characterized by high capacity to present antigen; high expression of proinflammatory cytokines, such as interleukin 12 (IL-12) and tumor necrosis factor-a (TNF-a) and consequent

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Tumor-Associated Macrophages Monocytes

M-CSF IL-4, IL-13, IL-10 Corticosteroids PGE VitD3 IG+TLR/IL-1R ligands IL-1ra decoy IL-1RII IL-10 CCL17 CCL18 CCL22 Polyamine Scavenger R Mannose R

GM-CSF LPS, IFNγ bacterial products

M2 macrophage

M1 macrophage

Scavenging matrix remodeling tissue repair angiogenesis immunesuppression

Bactericydal activity inflammatory cytokines immuno-stimulation

Tumor promotion

Tumor suppression

IL-1 TNF IL-12 CXCL9 CXCL10 CXCL11 RNI ROI

Tumor-Associated Macrophages, Fig. 1 Monocytes differentiate into polarized macrophage subsets when exposed to different cytokine milieu. In the presence of GM-CSF, IFN-g, LPS, and other microbial products, monocytes differentiate into M1 macrophages. In the presence of M-CSF, IL-4, IL-13, IL-10, and immunosuppressive agents (corticosteroids, vitamin D3, prostaglandins, and immunocomplexes (IG) in combination with IL-1 or

TLR ligands), monocytes differentiate into M2 macrophages. M1 and M2 subsets differ in terms of phenotype and functions. M1 cells have high microbicidal activity, immunostimulatory functions, and tumor cytotoxicity. M2 cells have high scavenging ability, promote tissue repair and angiogenesis and immunosuppression, and favor tumor progression

activation of a polarized type I response; and high production of toxic intermediates (▶ nitric oxide (NO), ▶ reactive oxygen species). Thus, M1 macrophages are generally considered potent effector cells that kill microorganisms and tumor cells and produce copious amounts of proinflammatory cytokines. In contrast, various signals (e.g., IL-4, IL-13, glucocorticoids, IL-10, immunoglobulin complexes/TLR ligands) elicit different M2 forms, able to tune inflammatory responses and adaptive Th2 immunity, scavenge debris, and promote ▶ angiogenesis, tissue remodeling, and repair. Figure 1 shows selected functions of M1 and M2 polarized macrophages. Microenvironmental signals expressed at the tumor microenvironment have the capacity to pilot recruitment,

maturation, and differentiation of infiltrating leukocytes and play a central role in the activation of specific transcriptional programs expressed by TAM. Tumor-Associated Macrophages To the extent that they have been investigated, differentiated mature TAM has a phenotype and function similar to type II or M2 macrophages. TAM has been shown to exert a negative effect on antitumor immune responses. TAM Recruitment TAMs are protumoral cells that are derived from circulating monocytes (Fig. 2) and are recruited to the tumor by a tumor-derived chemotactic factors,

Tumor-Associated Macrophages

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Blood Monocytes

Tumor cell Growth and survival factors

CC-Chemokines M-CSE VEGF

Promotion of metastasis

Chemotactic and survival factors TAM

Taming adaptive immunity

Matrix remodelling

Neoangiogenesis

Fibroblast

Vessel

Soild Tumor

Tumor-Associated Macrophages, Fig. 2 Tumorderived chemotactic factors (CC-chemokines, e.g., CCL2, macrophage colony-stimulating factor (M-CSF), and vascular endothelial growth factor (VEGF)) actively recruit circulating blood monocytes at the tumor site. In the tumor microenvironment, monocytes differentiate into tumor-associated macrophages (TAMs) that establish a

symbiotic relationship with tumor cells. The above tumor-derived factors positively modulate TAM survival. From their own, TAM secretes growth factors that promote tumor cell proliferation and survival; regulates matrix deposition and remodeling, thus favoring metastasis formation; activates neoangiogenesis; and promotes immunosuppression

originally identified as CC-chemokine ligand 2 (CCL2; also known as MCP-1). Following this observation, other chemokines active on TAM were detected in neoplastic tissues as products of either tumor cells or stromal elements. These molecules have an important role in tumor progression by directly stimulating neoplastic growth, promoting inflammation, and inducing angiogenesis. Evidence supporting a pivotal role for chemokines, in addition to CCL2, in the recruitment of monocytes to neoplastic tissues includes a direct correlation between chemokine production and monocyte infiltration in mouse and human tumors. Molecules other than chemokines can also promote TAM recruitment. In particular, tumor-derived cytokines such as ▶ vascular endothelial growth factor (VEGF) and macrophage colony-stimulating factor (M-CSF) promote

macrophage recruitment, as well as macrophage survival and proliferation, and their expression correlates with tumor growth. TAM Express Selected M2 Protumoral Functions The cytokine network expressed at the tumor site plays a central role in the orientation and differentiation of recruited mononuclear phagocytes, thus contributing to direct the local immune system away from antitumor functions. Immunosuppressive cytokines IL-10 and tumor growth factor-b (TGF-b) are produced by both cancer cells (ovary) and TAM. IL-10 promotes the differentiation of monocytes to mature macrophages and blocks their differentiation to dendritic cells (DC). Thus, a gradient of tumor-derived IL-10 may account for differentiation along the DC

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versus the macrophage pathway in tumors, resulting in tumor promotion. IL-10 promotes the M2c alternative pathway of macrophage activation and induces TAM to express M2-related functions. Under many aspects, TAM summarizes a number of functions expressed by M2 macrophages involved in tuning inflammatory responses and adaptive immunity, scavenges debris, and promotes angiogenesis, tissue remodeling, and repair. The production of IL-10, TGF-b, and PGE2 by cancer cells and TAM contributes to a general suppression of antitumor activities. TAMs are poor producers of nitric oxide (NO), and, in situ in ▶ ovarian cancer, only a minority of tumors and, in these, a minority of macrophages localized at the periphery scored positive for iNOS. Moreover, in contrast to M1-polarized macrophages, TAM has been shown to be poor producers of ROI, consistent with the hypothesis that these cells represent a skewed M2 population. Moreover, TAM was reported to express low levels of inflammatory cytokines (e.g., IL-12, IL-1b, TNF-a, IL-6). Activation of the transcriptional factor NF-kB is a necessary event promoting transcription of several proinflammatory genes. TAM displays defective NF-kB activation in response to M1-polarizing signals lipopolysaccharide (LPS) and TNF-a. Thus, in terms of cytotoxicity and expression of inflammatory cytokines, TAM resembles the M2 macrophages. Angiogenesis is an M2-associated function that represents a key event in tumor growth and progression. In several studies in human cancer, TAM accumulation has been associated with angiogenesis and with the production of angiogenic factors such as VEGF and platelet-derived endothelial cell growth factor. Additionally, TAM participates to the proangiogenic process by producing the angiogenic factor thymidine phosphorylase (TP), which promotes endothelial cell migration in vitro and whose levels of expression are associated with tumor neovascularization. Moreover, TAM accumulates in the hypoxic regions of tumors, and ▶ hypoxia triggers a proangiogenic program in these cells. Therefore, macrophages recruited in situ represent an indirect pathway of amplification of angiogenesis, in

Tumor-Associated Macrophages

concert with angiogenic molecules directly produced by tumor cells. On the antiangiogenic side, in a murine model, GM-CSF released from a primary tumor upregulated TAM-derived metalloelastase and angiostatin production, thus suppressing tumor growth of metastases. Finally, TAM expresses molecules that affect tumor cell proliferation, angiogenesis, and dissolution of connective tissues. These include epidermal growth factor (EGF), members of the FGF family, TGF-b, VEGF, and chemokines. In lung cancer, TAM may favor tumor progression by contributing to stroma formation and angiogenesis through their release of PDGF, in conjunction with TGF-b1 production by cancer cells. Macrophages can produce enzymes and inhibitors that regulate the digestion of the extracellular matrix, such as MMPs, plasmin, ▶ urokinase-type plasminogen activator (uPA), and the uPA receptor. Direct evidence has been presented that MMP-9 derived from hematopoietic cells of host origin contributes to ▶ skin carcinogenesis. Chemokines have been shown to induce gene expression of various MMPs and, in particular, MMP-9 production, along with the uPA receptor. Evidence suggests that MMP-9 has complex effects beyond matrix degradation including the promotion of the angiogenesis switch and release of growth factors. Modulation of Adaptive Immunity by TAM It has long been known that TAM have poor antigen-presenting capacity and can actually suppress T cell activation and proliferation. The suppressive mediators produced by TAM include ▶ prostaglandins, IL-10, TGF-b, and indoleamine dioxygenase (IDO) metabolites. Moreover, TAM is unable to produce IL-12, even upon stimulation by IFN-g and LPS. With this cytokine profile, which is characteristic of M2 macrophages, TAM is unable to trigger Th1-polarized immune responses but rather induce ▶ T regulatory cells (Treg). Treg cells possess a characteristic anergic phenotype and strongly suppress the activity of effector T cells and other inflammatory cells, such as monocytes. Suppression of T cell-mediated antitumor activity by Treg cells is associated with increased tumor growth and hence, decreased survival. For instance, in patients with

Tumor-Associated Stromal Progression

advanced ovarian cancer, an increase in the number of functionally active Treg cells present in the ascites was predictive of reduced survival. The complex network of chemokines present at the tumor site can play a role also in the induction of the adaptive immunity. Chemokines also regulate the amplification of polarized T cell responses. Some chemokines may enhance specific host immunity against tumors, but on the other hand, other chemokines may contribute to escape from the immune system, by recruiting Th2 effectors and Treg cells. Figure 2 summarizes symbiotic relationship between TAM and cancer cells. Conclusion Though the presence of TAM has been long considered as evidence for a host response against the growing tumor, it has become increasingly clear that TAMs are active players in the process of tumor progression and invasion. Molecular and biological studies have been supported by a large number of clinical studies that found a significant correlation between the high macrophage content of tumors and poor patient prognosis. TAM shares many similarities with prototypic polarized M2 mononuclear phagocyte population, in terms of gene expression and functions. In line with the known properties of M2 macrophage populations, several lines of evidence suggest that TAM promotes tumor progression and ▶ metastasis by activating circuits that regulate tumor growth, ▶ adaptive immunity, stroma formation, and ▶ angiogenesis. This hypothesis is now receiving new supporting evidence indicating that in vivo functional switching of infiltrating M2 macrophages toward an M1 phenotype provides therapeutic benefit in mice-bearing tumor xenograft. Identification of mechanisms promoting functional diversion of macrophages toward an M2 direction may disclose new valuable therapeutic targets against tumors.

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▶ Hypoxia ▶ Inflammation ▶ Innate Immunity ▶ Macrophages ▶ Macrophage-Stimulating Protein ▶ Matrix Metalloproteinases ▶ Metastasis ▶ Nitric Oxide ▶ Nonsteroidal Anti-Inflammatory Drugs ▶ Ovarian Cancer ▶ Prostaglandins ▶ Prostate Cancer ▶ Reactive Oxygen Species ▶ Regulatory T Cells ▶ Skin Carcinogenesis ▶ Urokinase-Type Plasminogen Activator ▶ Vascular Endothelial Growth Factor

References Balkwill F, Mantovani A (2001) Inflammation and cancer: back to Virchow? Lancet 357:539–545 Mantovani A, Sozzani S, Locati M et al (2002) Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 23:549–555 Sica A, Bronte V (2007) Altered macrophage differentiation and immune dysfunction in tumour development. J Clin Invest 117:1155–1166

See Also (2012) Arachidonic Acid. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 260. doi:10.1007/978-3-642-16483-5_379 (2012) Liver Cancer. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2063. doi:10.1007/978-3-642-16483-5_3393 (2012) Microenvironment. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 2296. doi:10.1007/978-3-642-16483-5_3720 (2012) Xenograft. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Springer Berlin Heidelberg, p 3967. doi:10.1007/978-3-642-16483-5_6278

Cross-References ▶ Adaptive Immunity ▶ Angiogenesis ▶ Endothelins

Tumor-Associated Stromal Progression ▶ Stromagenesis

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Tumor–Endothelial Communication

Characteristics

Tumor–Endothelial Communication ▶ Tumor–Endothelial Cross-Talk

Tumor–Endothelial Cross-Talk Tobias Görge1, Anke Rattenholl2, Martin Steinhoff3,4 and Stefan W. Schneider5 1 Department of Dermatology, University of Münster, Münster, Germany 2 Applied Biotechnology Division, Department of Engineering and Mathematics, University of Applied Sciences Bielefeld, Bielefeld, Germany 3 UCD Charles Institute of Dermatology, University College Dublin, Belfield, Ireland 4 Department of Dermatology School of Medicine and Medical Sciences, University College Dublin, Dublin, Ireland 5 Hauttumorzentrum Mannheim (HTZM), Universitätsmedizn Mannheim, Mannheim, Germany

Synonyms Tumor–endothelial communication

Definition Research in the field of tumor–endothelial communication focuses on specific interactions and pathways that allow circulating tumor cells to interact with the endothelium – the inner lining of the vascular wall. These interactions lead to modifications of the endothelium that eventually facilitate both tumor cell adhesion and extravasation. These processes are frequently accompanied by microthrombotic events, and tumor–endothelial communication relies in many aspects on mechanisms known from the inflammatory response.

Neoplastic growth and survival of tumor cells in the host environment critically depends on effective mechanisms of tumor–host interactions. This dependency of tumor cell survival in the microenvironment is not only limited to the early phase of primary tumor growth, where obviously, e.g., nutritive supplies are needed, but persists throughout the entire life span of tumor presence that is actually characterized by continuous multiple tumor–host interactions. A characteristic hallmark of malignant tumor cells is their ability to spread via the host vascular system, settling up colonies distant from their place of origin, a process generally referred to as “▶ metastasis.” There is general agreement that in this process of metastasis via the vascular system – which is the most fearsome aspect of cancer as to the survival prognosis – tumor cells that have managed to leave their site of origin need to apply escape mechanisms, allowing avoidance of the host immunosurveillance and an eventual extravasation from the blood stream. The transmigration of circulating tumor cells into the host tissue is a highly regulated process, which up-to-date has not been completely understood. Circulating tumor cells extravasating to the hoststromal tissue will have to cross the natural barrier between blood and tissue: the endothelium. This interaction is a complex process that requires molecular mechanisms similar to those known from the proinflammatory and prothrombotic response. This encyclopedic entry will display the mechanisms that have coined the expression of tumor–endothelial communication. On the one hand, it summarizes the findings on tumor cell–endothelial interactions facilitating the transmigration of tumor cells; on the other hand, it will highlight the molecular mechanisms that are currently known for describing how tumor cells modify endothelial functions that eventually may lead to tumor-induced thrombotic events. It goes without saying that the current trend of linking thrombotic events to mechanisms of the inflammatory response also holds true for the understanding of tumor–endothelial communication.

Tumor–Endothelial Cross-Talk

There are multiple evidences that neoplastic cells from a variety of tumor entities actively modify the vascular endothelium. Tumor cells are capable of transforming the endothelial physiological quiescent disposition into a prothrombotic, proinflammatory, and proadhesive endothelium. One of the oldest documented observations of this phenomenon is generally accepted to date back to works by Trousseau in the late nineteenth century (1865). These studies showed that tumor cells are associated with thrombi of platelets and neutrophils. In addition, there are multiple clinical reports that a thromboembolic event of so far unknown origin might occasionally be the first hint to an ongoing metastatic tumor disease of a patient. So evidently, these observations reflect tumor–host cross-talk. Adhesion Molecules in Tumor–Endothelial Cross-Talk Mediating Extravasation The extravasation from the flowing blood stream is a highly regulated process that relies on a specific interaction of ligands on the circulating cell and their receptors on the vascular endothelium. Much of the current knowledge in this field derives from the studies on thrombosis and inflammation, where platelets and leukocytes are specifically directed to bind to the activated endothelium and vascular wall. So-called selectins are a family of adhesion molecules that are crucial in this process. There are L-, P-, and E-selectins that all recognize carbohydrate structures. While L-selectin is constantly present on leukocytes, Pand E-selectin become upregulated on activated endothelium. Absence of selectins severely delays or even prevents the adhesion of circulating cells. The lectin domain of selectins recognizes sialylated, fucosylated structures displayed mostly on mucin-type glycoproteins containing a terminal tetrasaccharide sLex and sLea. Next to platelets and neutrophils, selectin-ligands have been shown to be expressed by several tumor entities and thus support tumor–endothelial communication. In different mouse models of metastasis, it could be shown that P- and E-selectin deficiency reduces the amount of extravasation of tumor cells. It was also reported that heparin, apart from its properties as an anticoagulant,

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interferes with binding ligands for selectins and thus prevents metastatic tumor spread. The use of heparin for prevention of tumor metastasis has been shown to be beneficial in many human studies and has become an accepted option for the clinical treatment of cancer patients. In continuation of the initial selectin-mediated interaction of tumor cell and endothelium, integrins are recognized to play an important role in cell adhesion. Integrins are heterodimeric transmembrane proteins that consist of noncovalently bound alpha and beta chains. They play a crucial role in cellular processes such as cell adhesion and migration as well as in the control of cell differentiation, proliferation, and survival. Several binding partners for integrins on and below endothelial cells have been characterized so far, such as VCAM-1, ICAM-1, and extracellular matrix proteins such as fibronectin, vitronectin, and laminin. There is also a well-documented expression of integrins on several tumor entities. During the malignant transformation, the expression of integrins has been reported to alter and leads to different binding avidity. Research on integrin-mediated tumor extravasation currently focuses on alphavbeta3 integrin and alpha5beta1 integrin, as their inhibition could decrease metastasis in several studies. In summary, crossing the endothelial barrier, tumor cells rely on mechanisms that resemble those of cell arrest in the inflammatory and thrombotic response. Tumor–Endothelial Cross-Talk Leading to Prothrombotic Endothelial Activation Apart from modifying the vascular endothelium toward a proadhesive state, tumor–endothelial cross-talk also activates the prothrombotic activities of the vascular wall. The clinical observation is that of the so-called Trousseau’s syndrome, where especially visceral cancers lead to venous thrombotic events. In addition, there are several reports showing that progressive cancer is associated with elevated levels of circulating von Willebrand factor (VWF), a plasma surrogate marker for endothelial activation. The pathogenesis of the prothrombotic state in cancer is complex and probably multifactorial; however, some

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studies could elucidate molecular mechanisms critically involved in tumor-induced prothrombotic endothelial activation. A well-studied protein involved in endothelial activation is tissue factor (TF). Several tumor cells can directly release TF or stimulate circulating leukocytes to increase TF release, e.g., by the generation of prothrombotic TF-rich microparticles. Once released, TF activates coagulation factor VII, which in turn activates factorX, thus activating prothrombin to thrombin. On the endothelium, via activation of the thrombin receptor (proteinase-activated receptor 1/PAR-1), thrombin leads to an immediate release of VWF and P-selectin promoting thrombosis and inflammation. A similar mechanism has been described for cancer procoagulant (CP). CP is a single chain cysteine protease that has been identified in a variety of tumors. It can directly activate factorX and thus contribute to the generation of thrombin. Another direct mechanism of endothelial cells by metastatic tumor cells could be demonstrated. It was shown for human ▶ melanoma and ▶ colon cancer that metastatic tumor cell lines of these cancers are capable of generating and releasing matrixmetalloproteinase-1 (MMP-1). MMP-1 has been demonstrated to directly activate the thrombin-receptor on endothelial cells followed by VWF and IL-8 release. This mechanism does not rely on the presence of coagulation factors and is therefore proof of immediate tumor–endothelial communication. Conclusion The prothrombotic reaction of the endothelium upon contact with malignant circulating cells might be understood as a mechanism for prevention of further dissemination of the tumor, making trapped tumor cells more accessible to the host defense system. Indeed, it could be shown that in the absence of surrounding platelets, tumor cells were more prone to ▶ natural killer cell attacks than when protected in platelet emboli. However, it can also be speculated that tumor cell arrest in a thrombus might facilitate tumor cell extravasation. As antithrombotic treatment and depletion of circulating platelets has been demonstrated to positively influence tumor metastasis, specific

Tumor-Induced Platelet Aggregation

strategies of interference with tumor–endothelial cross-talk might eventually prove beneficial for the prevention of tumor cell metastasis.

References Borsig L (2004) Selectins facilitate carcinoma metastasis and heparin can prevent them. News Physiol Sci 19:16–21 Coussens LM, Werb Z (2002) Inflammation and cancer. Nature 420(6917):860–867 Goerge T, Barg A, Schnaeker EM et al (2006) Tumorderived matrix metalloproteinase-1 targets endothelial proteinase-activated receptor 1 promoting endothelial cell activation. Cancer Res 66(15):7766–7774 Smorenburg SM, Van Noorden JCF (2001) The complex effects of heparins on cancer progression and metastasis in experimental studies. Pharmacol Rev 53:93–105

Tumor-Induced Platelet Aggregation ▶ Tumor Cell-Induced Platelet Aggregation

Tumor-Initiating Cells ▶ Cancer Stem-Like Cells ▶ Stem-Like Cancer Cells

Tumor-Reinitiating Cells ▶ Stem-Like Cancer Cells

Tumor-Repopulating Cells ▶ Stem-Like Cancer Cells

Tumor-Transforming 1 ▶ Securin

Tunicamycin

Tunicamycin Tatsushi Yoshida and Toshiyuki Sakai Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan

Definition Tunicamycin is an antibiotic identified and isolated from the fermentation broth of Streptomyces lysosuperificus. Since the antibiotic interferes with the formation of viral and cellular surface coats, it was termed “tunicamycin” after the Latin word “tunica” for coats.

Characteristics Structure and Function Tunicamycin is a nucleoside antibiotic composed of uracil, a fatty acid, and two glycosidically linked sugars. The sugars are N-acetylglucosamine and an unusual 11-carbon aminodeoxydialdose, which has been named tunicamine. Tunicamycin is a white crystalline powder which is soluble in alkaline water, pyridine, and hot methanol; slightly soluble in ethanol and n-butanol; and insoluble in acetone, ethylacetate, chloroform, benzene, and acidic water. High-performance liquid chromatography (HPLC) of tunicamycin shows that the antibiotic is separated into ten different components that have molecular weights ranging from 802 to 858. These differences in molecular mass are attributable to differences in the acyl chain length of C13–C17 (Fig. 1). Tunicamycin inhibits the first step in the lipid-linked saccharide pathway. Tunicamycin specifically inhibits the formation of dolichyl (pyro)-phosphate N-acetylglucosamine (dolichylPP-GlcNAc) in the step of the transfer of GlcNAc1-P from UDP-GlcNAc to dolichyl-P. As a result of inhibition in the lipid-linked saccharide pathway, tunicamycin consequently inhibits the

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N-linked oligosaccharide formation of glycoproteins in the endoplasmic reticulum (ER) (Fig. 2). Antiviral Effect of Tunicamycin Tunicamycin strongly inhibits the multiplication of enveloped RNA and DNA viruses such as Newcastle disease virus, vesicular stomatitis virus, Semliki Forest virus, fowl plague virus, Sindbis virus, measles virus, influenza virus, Rous sarcoma virus, Rauscher murine sarcoma virus, and herpes simplex virus. Hemagglutinin and neuraminidase are glycoprotein components of the viral envelope. Tunicamycin specifically inhibits the biosynthesis of viral envelope glycoproteins. Tunicamycin possesses cytotoxic activity toward transformed mammalian cells infected with virus such as mouse 3T3 cells transformed by ▶ SV40 virus, Moloney sarcoma virus or polyomavirus, and human WI38 fibroblasts transformed by SV40. Parental cell lines are resistant to tunicamycin cytotoxicity prior to neoplastic transformation. Apoptosis Induction by Tunicamycin Tunicamycin induces ▶ apoptosis in various types of human cancer cells containing ▶ neuroblastoma, ▶ breast cancer, ▶ hepatocellular carcinoma, and ▶ cervical cancer. Studies have revealed the mechanism of tunicamycin-induced apoptosis. In eukaryotic cells, ER provides an environment for the synthesis and modification of membrane proteins and secreted proteins. These co- and posttranslational modifications including N-linked ▶ glycosylation are involved in subsequent protein folding and assembly. The accumulation of unfolded proteins causes ▶ endoplasmic reticulum stress, the term given to an imbalance between the cellular demand for ER function and ER capacity. Cells respond to ER stress by activation of the unfolded protein response (UPR). The UPR is mediated through three ER transmembrane receptors, activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and pancreatic ER kinase (PKR)-like ER kinase (PERK). Under unstressed conditions, these sensor proteins are maintained in an inactivated state by an association with ER chaperone protein, glucose-regulated protein

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Tunicamycin

Tunicamycin, Fig. 1 Structure of tunicamycin

(GRP78) (also called Bip). The UPR is a cytoprotective response to reduce the accumulation of unfolded proteins and restore normal ER function; however, prolonged ER stress switches from pro-survival to proapoptotic signaling. Inhibition of N-linked glycosylation by tunicamycin leads to ER stress. Tunicamycin is a representative ER stressor used to elucidate the mechanism of ER stress and UPR followed by apoptosis. The accumulation of unfolded proteins in ER results in the release of GRP78 from IRE1, PERK, and ATF6. PERK phosphorylates the eukaryotic translation initiation factor-2a (eIF-2a) and allows the activating transcription factor 4 (ATF4). ATF4 transactivates C/EBPhomologous protein (CHOP). CHOP is an important factor in the induction of apoptosis by tunicamycin. CHOP-deficient mouse embryonic fibroblasts (MEF) provide partial resistance to apoptosis induced by tunicamycin treatment. MEF are not cells derived from cancer tissues but normal cells. MEF is often used to elucidate the mechanism of tunicamycin-induced apoptosis, since MEF leads to apoptosis by tunicamycin treatment and specific gene deficiency is easily provided in MEF. CHOP is a transcription factor and transactivates various genes. Tribbles-related protein 3 (TRB3) is a CHOP-downstream gene induced by CHOP at the transcriptional level. Knockdown of ATF4 and CHOP expression

represses tunicamycin-induced TRB3 upregulation. Furthermore, downregulation of TRB3 by small interfering RNA (▶ siRNA) partly blocks the apoptosis induced by tunicamycin treatment in human cervical cancer HeLa cells. Tunicamycin treatment also induces a p53-upregulated modulator of apoptosis (▶ PUMA) and NOXA in a tumor suppressor p53 gene-dependent manner. Both PUMAdeficient MEF and NOXA-deficient MEF partially reduce tunicamycin-induced apoptosis. These factors are key molecules of tunicamycininduced apoptosis. Tunicamycin Increases Effects of Anticancer Agents Many glycoproteins exist on the plasma membrane of tumor cells, including the efflux pump of anticancer agents. ▶ P-glycoprotein is a product of the MDR1 gene and protects cells against a broad spectrum of anticancer agents by functioning as an energy-dependent drug efflux pump. Inhibition of N-linked glycosylation of P-glycoprotein results in a reduced multidrugresistance phenotype of cancer cells. Tunicamycin treatment increases the cytotoxicity of doxorubicin, epirubicin, ▶ cisplatin, and vincristine against NIH-3T3 cells into which an exogenesis MDR gene had been introduced. The human epidermoid carcinoma KB-8-5-11 cell line contains the

Tunicamycin Tunicamycin, Fig. 2 Oligosaccharide formation and the action site of tunicamycin. (a) Lipidlinked oligosaccharide formation. Oligosaccharide is assembled onto the carrier lipid dolichol in the ER membrane. Tunicamycin inhibits the formation of dolichyl PP-GlcNAc. (b) N-linked oligosaccharide formation. Synthesized oligosaccharide (Glc)3(Man)9(GlcNAc)2 is transferred from the lipid dolichol to the asparagine side chain of a nascent polypeptide on the ER membrane. GlcNAc N-acetylglucosamine, Man mannose, Glc glucose

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amplified MDR1 gene. The sensitivity of this cell line to doxorubicin, epirubicin, cisplatin, and vincristine also increases by treatment with tunicamycin. Tunicamycin does not influence the uptake of these anticancer agents but reduces efflux of the agents in cells with a multidrugresistance phenotype. Human colon cancer cells with a strong multidrug-resistant phenotype mediated by high constitutive levels of the expression of P-glycoprotein increase daunorubicin accumulation by incubation with tunicamycin. Tunicamycin exposure reduces P-glycoprotein expression on the surface of the cell membrane. Tunicamycin also decreases the 50% inhibitory

concentration (IC50) of cisplatin in human pharyngeal carcinoma KB cells and human maxillary squamous cell carcinoma IMC-3 cells. Combined administration of tunicamycin with cisplatin injected by s.c. around the tumor inhibits tumor growth in C3H/He mice bearing cisplatinresistant squamous cell carcinoma in vivo and increases the in vivo apoptosis of tumor cells. Tunicamycin Enhances Death Ligand TRAILInduced Apoptosis TNF-related apoptosis-inducing ligand (TRAIL) is a type II membrane protein belonging to the TNF family, which preferentially induces

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apoptosis in a variety of tumor cells but not in normal cells in vitro and in vivo. Since some tumor cell lines are resistant to TRAIL, an agent that can overcome resistance to TRAIL has been sought. Tunicamycin sensitizes hormonerefractory prostate cancer cells to TRAIL-induced apoptosis. The combination of tunicamycin and TRAIL activates caspases under the condition in which a single agent hardly activates caspases. Tunicamycin enhances TRAIL activity through the induction of a TRAIL receptor, TRAIL-R2 (also called death receptor 5 (DR5)). Tunicamycin upregulates the transcription of TRAIL-R2 through a transcription factor, CHOP. Normal Cellular Toxicity of Tunicamycin The hepatotoxicity of tunicamycin has been reported in guinea pigs given a single dose of 400 mg/kg of tunicamycin. A periportal pattern of hepatocellular damage was observed with the death of many hepatocytes up to 72 h postinjection. Swollen hepatocyte cytoplasm protruded into many hepatic blood vessels and detached portions of hepatocytes producing emboli in pulmonary and cerebral capillaries. The toxic effects of tunicamycin have been examined in rats at gestation day 15. At 16 h postdosing, all pregnant rats had moderate to extensive vaginal bleeding and one fourth died. The other rats had free blood in the uterus and large decreases in red cell counts, hemoglobin, and packed cell volume. Tunicamycin toxicity has been reported in 6- to 8-week-old mice given a single injection of tunicamycin at a dose of 3 mg/g body weight intraperitoneally (i.p.) After 2 days, characteristic renal lesions were detected in tunicamycin-treated mice. Apoptosis was caused in the renal tubular epithelium. Moreover, tunicamycin treatment inhibited mammalian embryogenesis. Although early cleavage was normal, mammalian embryos did not undergo normal compaction and blastocyst formation. Trophoblast cell adhesion may be disrupted since tunicamycin is cytotoxic to these cells. In later development, tunicamycin inhibits kidney tubule formation when present during the embryonic induction of these structures.

Turban Tumor Syndrome

Tunicamycin induces apoptosis in a variety of malignant tumor cells and possesses cytotoxic activity to virus-transformed cells compared with parental normal cells. Moreover, tunicamycin enhances the effects of anticancer agents; however, tunicamycin also has cytotoxicity against normal cells and causes crucial damage during in vivo administration. The mechanism of tunicamycin-induced apoptosis through ER stress has been revealed, but it has not been completely elucidated yet. If tunicamycin is applied to cancer treatment, its usage should be restricted to local administration, but further studies are needed.

Cross-References ▶ Doxorubicin ▶ ER ▶ p53 ▶ TRAIL

References Eibein AD (1987) Inhibition of the biosynthesis and processing of N-linked oligosaccharide chains. Ann Rev Biochem 56:497–534 Ma Y, Hendershot LM (2004) The role of the unfolded protein response in tumour development: friend or foe? Nat Rev Cancer 4:966–977 Noda I, Fujieda S, Seki M et al (1999) Inhibition of N-linked glycosylation by tunicamycin enhances sensitivity to cisplatin in human head-and-neck carcinoma cells. Int J Cancer 80:279–284 Shiraishi T, Yoshida T, Nakata S et al (2005) Tunicamycin enhances tumor necrosis factor-related apoptosisinducing ligand-induced apoptosis in human prostate cancer cells. Cancer Res 65:6364–6370 Takatsuki A, Tamura G (1982) Inhibition of glycoconjugate biosynthesis by tunicamycin. In: Tamura G (ed) Tunicamycin. Japan Scientific Societies Press, Tokyo, pp 35–70

Turban Tumor Syndrome ▶ Cylindromatosis

Turcot Syndrome

Turcot Syndrome Paola Izzo Department of Molecular Medicine and Medical Biotechnology, School of Medicine and Surgery, University of Naples Federico II, Naples, Italy

Definition Turcot syndrome (TS) is a rare inherited neoplastic disease characterized by the association of primary malignant neuroepithelial tumors of the central nervous system and colon cancers and/or multiple colorectal adenomas.

Characteristics Clinical Criteria The 130 or so Turcot syndrome (TS) cases described to date include various histopathologic types of ▶ brain tumors, e.g., glioma, medulloblastoma, and astrocytoma, associated with a broad spectrum of colorectal findings, from a single adenoma to typical adenomatous polyposis. Usually, polyps are fewer in number than in familial adenomatous polyposis (FAP [▶ APC gene in familial adenomatous polyposis]) but are larger in size, and multiple adenomas or colorectal cancers occur at an early age and undergo an earlier malignant transformation than in FAP or in hereditary nonpolyposis colorectal cancer (HNPCC). The clinical definition and the mode of inheritance of Turcot syndrome are controversial; some authors propose that TS is an allelic variant of FAP and support an autosomal dominant inheritance, while others postulate that TS is a disease independent of FAP with an autosomal recessive pattern of inheritance. Genetics Dominantly inherited cases have been associated with germ line mutations in either the tumor suppressor adenomatous polyposis coli gene (APC), usually mutated in FAP, or in the DNA

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mismatch repair (MMR) genes, which are usually mutated in HNPCC. Few recessive cases have been reported with the causative mutations found to be within the PMS2 gene, a minor MMR gene that is only rarely involved in HNPCC. In the first recessive case described, a germ line nonsense mutation (PMS2134) that was inherited from the healthy mother was found in one allele. The second recessive case was found to be a compound heterozygote for two frameshift germ line mutations within the PMS2 gene: a G deletion (1221delG) in exon 11 and a four-base pair deletion (2361delCTTC) in exon 14, both of which were inherited from the patient’s unaffected parents. This was the first evidence of recessive dominance of TS because two germ line mutations in PMS2 are not individually pathogenic, but become so when occurring together in a compound heterozygote. Interestingly, homozygous splice site mutation in the PMS2 gene, the c.9891G