cbt2/2/Suppl. 1

[Cancer Biology & Therapy 2:4:Suppl. 1, S55-S63; July/August 2003]; ©2003 Landes Bioscience

Models of Anti-Cancer Therapy

Restoring p53-Dependent Tumor Suppression ABSTRACT

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*Correspondence to: Wafik S. El-Deiry, M.D., Ph.D.; Laboratory of Molecular Oncology and Cell Cycle Regulation; Howard Hughes Medical Institute; Departments of Medicine, Genetics, Pharmacology, and Cancer Center; University of Pennsylvania School of Medicine; 415 Curie Blvd. CRB 437; Philadelphia, Pensylvania 19104 USA; Tel.: 215.898.9015; Fax: 215.573.9139; Email: [email protected]

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2Cancer Drug Discovery; Pfizer Global Research & Development; Groton, Connecticut USA

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1Laboratory of Molecular Oncology and Cell Cycle Regulation; Howard Hughes Medical Institute; Departments of Medicine, Genetics, Pharmacology, and Cancer Center; University of Pennsylvania School of Medicine; Philadelphia, Pennsylvania USA

p53 represents an ideal target for anti-cancer drug design, because p53 is mutated in more than half of human tumors. Most of the remaining tumors, although carrying wild-type p53, have defects in the p53-mediated apoptotic pathway. Activation of p53 activity by either chemotherapy or radiotherapy induces p53-dependent apoptosis in tumor cells with wild-type p53. Supplying exogenous wild-type p53 in cancer cells by gene delivery is effective in suppressing tumor growth of both mutant and wild-type p53-containing tumors. Blockage of p53 degradation pathways either by overexpression of ARF or interruption of MDM2:p53 interaction is effective in inducing p53 triggered tumor cell death. Since unlike most other tumor suppressor genes, mutant p53 is over expressed in tumor cells, a promising approach involves restoring tumor-suppressing function to mutant p53. The activity of the mutant p53 in tumor cells is restorable based on the fact that PAb421 antibody against the carboxy-terminus of p53 and peptides corresponding to the p53 carboxy-terminus can restore specific DNA-binding ability to some mutant p53 proteins. High throughout screening of chemical libraries has led to the identification of a group of small synthetic molecules such as CP-31398, which can restore p53 function to mutant p53 by stabilizing the active conformation of the protein that is destabilized in many mutants. Subsequent identification of PRIMA-1 provides further evidence to the possibility of developing anti-cancer drugs that may rescue mutant p53. Further understanding of the mechanisms by which CP-31398 and PRIMA-1 restore p53 activity may not only lead to discovery of more potent analogs but may also suggest new strategies for p53-targeting in tumor therapy.

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Wenge Wang1 Farzan Rastinejad2 Wafik S. El-Deiry1,*

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p53, p63, p73, CP-31398, Prima-1, peptides, protein stabilization, post-translational modification, ubiquitination, DNA damage, phosphorylation, transcriptional activation, MDM2, ARF

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KEY WORDS

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Previously published online as a CB&T E-publication at: http://www.landesbioscience.com/journals/cbt/toc.php?volume=2&issue=0

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INTRODUCTION

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The tumor suppressor protein p53 remains one of the most intensively studied genes in cancer biology and therapy.7,9,30,75,123 It was regarded as an oncogene for 10 years after it was discovered more than twenty years ago.123 Subsequent studies revealed that p53 is a tumor suppressor, which binds to a specific DNA sequence and transactivates target genes leading to cell cycle arrest and/or apoptosis. Epidemiological data demonstrated that p53 is mutated in more than a half of the human tumors.53 Most of the remaining tumors, although containing wild-type p53, are defective in the pathway of p53-induced cell cycle arrest or apoptosis due to virus infection, MDM2 overexpression, ARF or ATM deficiency. p53 null mice develop normally, but are prone to the spontaneous development of a variety of neoplasms at an early age,27 mimicking Li-Fraumeni syndrome, a genetic disorder associated with p53 mutation. Deficiency of p53 function also confers a growth advantage under hypoxic conditions, which contributes to tumor progression.101 In the clinic, the functional status of p53 has been related to prognosis, progression and therapeutic response of tumors.29,87,129 Tumor cells containing wild-type p53 are usually more sensitive than those bearing mutant p53. All these characteristics make p53 an ideal molecular target for cancer therapy.13,29,96 Restoring p53 dependent tumor suppression is no doubt one of the most promising strategies in the war against cancer.

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Wafik S. El-Deiry, M.D., Ph.D.

STRUCTURE AND FUNCTION OF P53 The p53 gene, TP53 located at human chromosome 17p13.1, encodes a 393 amino acid nuclear protein. p53 protein contains several well characterized functional domains (Fig. 1). The transactivation (TA) domain is located at the amino-terminus and mediates both transcriptional activity and MDM2-binding.1 A proline-rich domain (PRD) resides adjacent to the TA domain, which is reported to be important for interaction with SH3-containing proteins.126 In the central core region of p53, the DNA-binding domain www.landesbioscience.com

Cancer Biology & Therapy

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RESTORING P53-DEPENDENT TUMOR SUPPRESSION

(DBD), binds DNA in a sequence-specific manner and is frequently mutated in tumor cells. There is a 21 amino acid sequence (aa 92–112) in conjunction with the prolinerich domain and the DNA-binding domain, which was identified to be essential for p53 degradation by both MDM2 and HPV-E6.44,45 The carboxy-terminus of p53 contains an oligomerization domain (OD), nuclear export and nuclear localization signals as well as ubiquitination sites for p53 degradation. Of note, the last 30 amino acids of the carboxy-terminus of p53 possess a negative regulatory function, which inhibits the specific DNA-binding and transcriptional activity of p53.22,134 Of particular interest, there exist two polymorphisms of p53 in humans, the Pro72 polymorphism and the Arg72 polymorphism.86 It has been reported that the Arg72 variant of wild-type of p53 induces apoptosis remarkably better than does the Pro72 variant.28 The Arg72 variant has also been Figure1. Functional domains of p53, post-translational modifications and hot-spot mutation sites. TA, shown in the context of mutated p53, to transactivation domain; PRD., proline-rich domain; OD, oligomerization domain; CRD, carboxy-terminal regulatory domain. interact with p73 and inhibit apoptosis following exposure to chemotherapy.12 p53 functions as a tetrameric transcription factor by binding to mutant-expressing cell lines.18,125 Moreover, some transcriptionally specific DNA sequences and transactivating or repressing a large, inactive p53 mutants, including p53dl21449 and p53 (Gln22/ and increasing, group of target genes.30,122,123 Under cellular stress- Ser23),69 act as potent death triggers in tumor cells. In cell free es, p53 is induced and inhibits cell growth either by arresting cells in models, p53 protein mediates caspase-3 activation,25 which is the G1 or G2 phases of the cell cycle or by inducing apoptosis. In dependent on the activation of caspase-8. Furthermore, p53 protein the case of DNA damage, p53 arrests cells and induces gene expres- was shown to localize to mitochondria rapidly at the onset of p53sion for DNA repair,116 predominantly global genomic repair dependent apoptosis but not during p53-mediated cell cycle (GGR), but not transcription-coupled repair (TCR). If the damage arrest.85 Subsequent experiments showed that p53 binds to BclXL is too severe to recover, apoptosis is induced by p53 to keep homeosta- through its DNA-binding domain and induces cytochrome c release sis. In addition, p53 can induce cell differentiation and senescence by forming inhibitory complexes with the protective BclXL and under certain circumstances.30,88 When p53 is mutated or the Bcl2 proteins.92 It has also been reported that the greater ability of pathways of p53 induced cell cycle arrest or apoptosis are deficient, the Arg72 versus the Pro72 polymorphism of p53 to induce apopcells grow without control, resulting in development of various tosis may be due to the greater ability of the Arg72 p53 to localize to the mitochondria.28 tumors. The transcriptional target genes of p53 have been extensively explored.30 One of the first and most notable targets is the cyclinREGULATION OF p53 ACTIVITY dependent kinase inhibitor p21Waf1/Cip1, which arrests cells in the p53 mRNA is constitutively expressed and regulation of p53 is G1 or G2 phase.31,32 Other p53 target genes involved in the cell cycle arrest include 14-3-3-σ and GADD45. There are additional p53 mainly at the protein level. Under physiological conditions, p53 is targets including KILLER/DR5, Bax, caspase-6, puma and expressed at low or undetectable levels with a half-life of approxip53AIP1, etc., which appear to be involved in both extrinsic and mately 10–20 min in most cells. The ubiquitin-proteasome pathway intrinsic pathways of p53-induced apoptosis. p53 is also responsible following the interaction of MDM2 with the amino-terminus of for maintaining genetic stability by transactivating a series of genes p53, at least in part, mediates this rapid degradation.48,70 MDM2 such as XP-C, DDB2, which sense or recognize DNA damage and binds the TA domain of p53 with its p53-binding domain and catinitiate a response for repair of the damaged DNA.47,83,120 Yet other alyzes p53 ubiquitination through it’s RING domain.33,54 Six p53 target genes are involved in anti-angiogenesis,23 and their acti- lysines (370, 372, 373, 381, 382, and 386) in the carboxy-terminus vation may also contribute to p53-mediated tumor suppression. of p53 were identified as target residues of MDM2-mediated p53 Another category of p53 targets, including MDM2 and the ubiquitination.95,105 MDM2 catalyzes only mono-ubiquitination of recently identified Pirh2,74 regulate p53 stabilization. MDM2 or p53 on these lysine residues.71 Recently, polyubiquitination of p53 Pirh2 directly bind to p53 and mediate p53 degradation and thereby was demonstrated to be mediated by p300.43 p300 bears E3, or so-called E4, ubiquitin ligase activity within the amino-terminus, form negative regulatory feedback loops with p53.1 p53 also functions independently of transcriptional activity. It which is not homologous to any known E3 motifs, such as the had been reported that p53 induced apoptosis can occur independ- RING domain in MDM233,54 or the HECT domain in HPV-E6 ently of new RNA or protein synthesis in temperature-sensitive p53 associated protein.56,58 Of interest, the p300-induced polyubiquitiS56


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any evidence of ubiquitination.6 Most recently, another p53 negative regulator, Pirh2, was identified.74 Pirh2, which contains a RING-H2 domain with E3 ubiquitin ligase activity, binds to the DNAbinding domain of p53 and promotes p53 degradation. Interestingly, besides these p53 degrading proteins, Parc appears to be another kind of p53 regulator, which anchors p53 in the cytoplasm and therefore hinders p53 from entering into the nucleus to transactivate target genes.67,97 Although more and more proteins are found to potentially regulate p53 stability, the predominant role of MDM2 in p53 protein stabilization is unchallenged based on the fact that removal of MDM2 from ES cells leads to embryonic lethality while MDM2/p53 double knockout mice develop normally.63,94 While the MDMX knockout has a similar phenotype as the MDM2 knockout, this MDM2 homologue binds and inhibits the transcriptional activity of p53 but does not ubiquitinate or target p53 for degradation.61,119 Figure 2 illustrates the pathways regulating p53 stability. Multiple post-translational modificaFigure 2. Pathways of p53 stabilization and degradation. MDM2 binds to p53 and ubiquitinates p53 tions (Fig. 1) regulate p53 function.2,90 through its RING domain that possesses E3 ligase activity. The ubiquitinated p53 is then degraded by Phosphorylation is the most common 26S proteasomes or further polyubiquitinated by p300 for more efficient degradation. DNA-damage modification of p53. At present, at least stabilizes p53 by inducing p53 phosphorylation, which prevents the binding of MDM2. The stabilized fifteen phosphorylation sites of p53 have p53 in the form of a tetramer transactivates target genes, which include MDM2. MDM2 therefore forms been reported, including seven serines (6, a negative feedback loop with p53 and regulates p53 stabilization. Another feedback loop regulating p53 stabilization is the recently identified Pirh2 pathway. Activation of oncogenes stabilizes p53 by the 9, 15, 20, 33, 37 and 46) and three threARF pathway, in which ARF binds to MDM2 and releases p53 from MDM2-mediated degradation. onines (18, 55 and 81) within the aminoHAUSP, which contains deubiquitinating enzyme activity, acts as a potent p53 stabilizer, and can terminal region and five serines (315,371, rescue p53 from degradation. Parc, a large molecular weight cytosolic protein, also participates in 376, 378 and 392) within the carboxy-terregulating p53 by anchoring p53 in the cytoplasm. In case of DNA damage, p53 is heavily phosphorylated by ATM/Chk2 or ATR/Chk1 etc. Phosphorylation of p53 at the amino-terminus prevents MDM2minal region. Under conditions of cellular binding and p53 is stabilized. stress, p53 is highly phosphorylated by a number of specific protein kinases includnation is dependent on MDM2-induced mono-ubiquitination.43 The ing ATM, ATR, Chk2, DNA-PK and HIPK2.20,62,73,141 However, ubiquitinated p53 is then rapidly degraded by the 26S proteasomes none of the stimuli can induce all the phosphorylation pathways and either in the cytoplasm or in the nucleus.41,136 The recent finding of the phosphorylation pattern of p53 is different in response to differa p53 deubiquitinating enzyme, HAUSP, which rescues ubiquitinated ent stimuli. Different types of cells also respond differentially to the p53 from degradation and serves as a potent p53 stabilizer, adds further same stimulus. For example, phosphorylation at serine 6, 9 and 15 knowledge to the tight and complex control of p53 stability.76,108,135 can be detected in 30 minutes after ionizing irradiation, while in DNA damage, caused by either UV light, ionizing radiation or response to UV light exposure, phosphorylation at these sites is less DNA damage-inducing drugs, results in stabilization of endogenous rapid but lasts longer.51 Phosphorylation of serine 15 occurs in p53 through a series of physiological responses, including response to various DNA-damaging chemicals such as cisplatin and ATM/ATR activation, phosphorylation of p53 and blockage of the camptothecin, but not in response to actinomycin D.5 It has become binding of MDM2 to the p53 N-terminus.10,15,21,72,114 Activation well accepted that phosphorylation of p53 protein contributes to of some oncogenes such as c-myc, ras, or E2F1 also stabilizes p53 by p53 stabilization and transcriptional activity under stressful condithe ARF-MDM2 pathway, in which case, p14ARF, or p19ARF in tions.2,3,141 It is equally worthy to note that dephosphorylation may mice, binds to MDM2 and releases p53 from MDM2 association.55,65 play an important role in regulation of p53 function. In unstressed Many other factors are also involved in the regulation of p53 cells, serine 376, 378 and threonine 55 are commonly phosphorystability. JNK is reported to target p53 ubiquitination and degradation lated.40,128 After irradiation, dephosphorylation at serine 376 occurs in nonstressed cells.38 It was also reported that NAD(P)H quinone and presents a consensus binding site for 14-3-3 proteins.128 oxidoreductase 1 (NQO1) plays a role in p53 stabilization. Inhibition Phosphorylation of some specific sites may affect p53 transcriptional of NQO1 activity by the specific inhibitor dicoumarol induces target selection. For example, phosphorylation at ser46 by HIPK2 proteasomal degradation of p53 and its family member p73 without appears to selectively transactivate p53AIP1, an apoptotic target www.landesbioscience.com


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gene.24,98 Phosphorylation of p53 at ser392, in particular, activates specific DNA binding functions by stabilizing p53 tetramer formation.107 The carboxy-terminus of p53 can be modified not only by phosphorylation, but also by ubiquitination, acetylation and other modifications.3,89 Six lysine residues within the carboxy-terminal region are targets for ubiquitination,95,105 including lysine 370, 372, 373, 381, 382 and 386. Four of them, lysine 372, 373, 381 and 383, as well as lysine 320, are also targets for acetylation.2 Lysine 386 can also be conjugated to a small ubiquitin-like protein, SUMO-1.42,106 Although the ubiquitination of p53 is regarded as a clear signal for degradation, it was also reported that the carboxy-terminus ubiquitination of p53 contributes to its nuclear export.81 It has been well documented that acetylation of p53 may contribute to p53 specific DNA-binding and transcriptional activity, but the exact physiological role of p53 acetylation still remains to be further investigated.2,102 Because acetylation and ubiquitination occur mostly at the same sites, it has been suggested that acetylation of p53 may inhibit its ubiquitination.77 Besides the modifications described above, it has been reported that p53 may also be modified by O-linked glycosylation113 and ribosylation.131 p53 Family Members. Nearly twenty years after the discovery of p53, the first homologue, p73 was cloned.64 p73 is located at chromosome 1p36, a region frequently deleted in neuroblastoma and other tumors and thought to contain multiple tumor suppressor genes. Similar to p53, p73 contains an acidic amino-terminal transactivation (TA) domain, a highly conserved core DNA-binding domain and a cyboxy-terminal oligomerization domain. Further experiments showed that p73 can bind to the p53-specific DNAbinding sequence and transactivate transcription of p53 target genes, including p21, MDM2, Bax and GADD45 etc. Soon after the discovery of p73, several other proteins were reported to be homologous to p53.8,99,138 These proved to be different transcriptional products of the p63 gene, located at chromosome 3q27-29. Like p73, p63 shares structural similarity with p53 and can transactivate p53 targets and induces cell cycle arrest and apoptosis. Unlike p53, which encodes essentially only one known alternative splice variant, both p73 and p63 genes possess two major separate transcriptional start points, which in turn produce two structurally different proteins. The product from the first transcriptional start site contains the amino-terminal transactivation domain, namely TAp73, or TAp63 respectively. The second product is a truncated isoform lacking the amino-terminal transactivation domain known as ∆Np73, or ∆Np63. Alternative splicing adds additional complexity at the coboxy-terminus and gives rise to at least 6 isoforms of p73 (α, β and γ isoforms of both TAp73 and ∆Np73) and 6 isforms of p63 (α, β and γ isoforms of both TAp63 and ∆Np63), some of which possess a sterile α motif (SAM), believed to be involved in protein-protein interaction.4 One major question in this field has been whether p73 and p63 are involved in tumorigenesis and cancer progression. Although p73 and p63 are structurally and functionally similar to p53, genetic studies failed to link appreciable mutations of either p73 or p63 to tumorigenesis. In addition, unlike p53-/- mice, p63-/- and p73-/-mice are not tumor prone but instead manifest various developmental abnormalities.93,139,140 All these findings did not support the idea that p73 and p63 may be tumor suppressors. However, overexpression of either p73 or p63 in tumor cells does induce apoptosis.34,35 Moreover, DNA damage caused by ionizing radiation and some DNA damage-inducing drugs activates p73 in tumor cells.132 S58

Further experiments demonstrate that p73 is phosphorylated and may participate in mismatch DNA repair. Most recently, p73 was demonstrated to be induced by a variety of chemotherapeutic agents and appears to be involved in chemosensitivity of mutant p53containing tumor cells.12,60,117 p63 appears more sensitive to UV irradiation with a result of accumulation of TAp63, and a decrease of ∆Np63.79 The expression levels of these two p53 family members are frequently higher in mutant p53-containing tumor cells.118,142 A reason may simply be a compensatory feedback due to the deficiency of p53 function. Of note, some mutant p53 proteins, especially the mutants that also contain the Arg72 polymorphism,86 can form hetero-tetramers with full-length p73 and p63 and lead to inactivation of their transcriptional activities. A conformational change of the DNA-binding domain appears to influence the ability of mutant p53 protein to interact with p73.11 On the other hand, ∆Np73 and ∆Np63 can also act as dominant negative inhibitors not only towards their full-length counterparts, but also towards p53.59,80,137,138 Thus, p53 members form a complicated regulatory network in tumor cells.

RESTORING AND ENHANCING p53 FUNCTION FOR CANCER THERAPY Since p53 mutation, as well as wild-type p53 impairment, is observed in most human malignancies, restoring p53 function in tumors has been pursued as a promising strategy for cancer therapy. Many approaches have been employed to achieve this purpose as illustrated in Figure 3. Supplying exogenous wild-type p53 into cancer cells, either by gene delivery or by direct protein delivery, has been explored in recent years. Another strategy in targeting p53 for cancer therapy is activating or enhancing the activity of endogenous p53, as well as p53 family members, in tumor cells. At least in part through this mechanism the currently used chemotherapy and radiotherapy in the clinic suppress tumor growth. Another approach in preclinical development involves restoring tumor-suppressing function to mutant p53. Evidence has shown that the conformation of the DNA-binding domain is flexible and that conformational changes in mutant p53 are reversible. It has long been established that carboxy-terminal peptides and antibodies against the carboxyterminus can restore a wild-type activity to mutant p53 at least in some cases.111,134 Unlike wild-type p53, mutant p53 is normally expressed at high levels in tumor cells, most likely due to the inability to induce components of the p53 negative feedback loops such as MDM2 and Pirh2. Thus, it can be imagined that restoration of p53 wild-type function to the highly accumulated mutant p53 in tumor cells, even partly, could trigger tremendous therapeutic responses at least in principle. The identification of the mutant p53 conformation-modifying drug, CP-31398, demonstrates the feasibility of this strategy (Fig. 4). The finding of another small molecule PRIMA-1 provides further evidence that there exist compounds that can selectively kill cancer cells with mutant p53. p53 Gene Therapy. Reconstitution of p53 function in cancer cells by introduction of exogenous wild-type p53 genes has been investigated as a strategy for molecular cancer therapy. Overexpression of p53 is sufficient to induce apoptosis in most cancer cells with somewhat reduced efficacy in tumor cells containing wild-type p53.14 Various p53 gene therapy protocols have been proposed for both experimental cancer models and clinical trials. Among them, replication-deficient adenovirus-mediated p53 gene delivery represents a common approach due to a high amount virus generation




Figure 3. p53 activation and suppression in tumorigenesis and tumor therapy. Cellular stress, such as DNA damage, oncogene activation, hypoxia or deprivation of nutrients, activates p53 and induces cell cycle arrest and/or apoptosis by activating p53 targets. Cells proliferate without control and become malignant when they lose p53 function. Genetic mutation or deficiency of p53 occurs in more that half of all human tumors. In the majority of the remaining human tumors, p53-mediated pathways of cell cycle arrest or apoptosis are defective due to virus infection, MDM2 overexpression or ARF deficiency. The currently used chemotherapy and radiotherapy inhibit tumor growth at least partly due to activation of p53-induced tumor suppressing pathways. p53 gene delivery can induce cell death of both wild-type and mutant p53-expressing tumors. Restoring transcriptional function to mutant p53 by specific peptides, CP-31398 or PRIMA-1 is promising in cancer therapy.

and a broad spectrum of target cells.39,133 The exogenous p53 functions both in ex vivo cell death induction and in vivo tumor suppression. In clinical trials,124,130 adenovirus-mediated p53 gene therapy has been carried out in patients with head and neck, lung or ovarian cancers. Intratumoral or intraperitoneal administration of high titer adenoviruses carrying p53 proved to be safe with no significant toxicity observed even in repeated subsequent injections. Exogenous p53 expression and p53 target gene activation were detected in clinical trials even in the presence of high titer of antiadenovirus antibodies in the circulation. Therapeutic response was achieved in some cases with reduced tumor mass or stabilized tumor progression. Induction of immune responses by the virus infection may also contribute more or less to the tumor shrinkage by adenovirus-mediated p53 delivery.19 Inactivation of the exogenous p53 in the presence of predominant mutant p53 and the rapid degradation of exogenous p53 by MDM2 may limit the therapeutic effect of p53 gene delivery. Efforts have been made to combine p53-mediated gene therapy with chemotherapy109 and radiotherapy.52,57 DNAdamage induced by chemotherapy or radiotherapy may create a microenvironment favorable for exogenous p53 stabilization and activation. Development of more stable or active p53 variants are www.landesbioscience.com

future directions for effective restoration of p53 functions. Combination of p53 stabilizing or activating agents, such as CP31398 may also enhance p53-mediated gene therapy.127 It is important to mention the cytolytic adenovirus ONYX015.84,103 ONYX-015 is a recombinant chimeric adenovirus with the 55 KD E1B coding sequence disrupted. This modification makes the virus unable to replicate in p53 wild-type cells but allows replication in p53 deficient cells due to p53 mutation, p53 deletion, loss of p14ARF or MDM2 overexpression, representing the majority of tumor cells. Clinical trials using ONYX-015 have thus far shown that administration of ONYX-015 is safe and well tolerated and showed a therapeutic effect in some cases. Peptides Enhancing or Restoring p53 Activity. Due to their potency and specificity, small specific peptides have been widely used for interruption of protein function. Both amino- and carboxyterminal peptides have been developed for the purpose of enhancing or restoring p53 function. A series of carboxy-terminal peptides were used to interfere with the carboxy-terminal negative transcriptional regulatory element of p53. Among them, a 22 amino acid peptide, corresponding to a p53 fragment from amino acids 361 to 382, manifested high potential for restoring DNA-binding and transcriptional activity to mutated p53 in some mutant p53-containing cell lines.110,112 The effect was dependent on expression of mutant p53 and the peptide was not toxic to p53 wild-type or p53-null cells. Further experiments showed that the peptide binds not only to the carboxy-terminus but also to the central core domain and induces Fas/APO-1-mediated apoptosis.68 Another report showed that a peptide that binds and stabilizes the p53 core domain can serve as a shaperone to rescue the wild-type conformation of mutant p53.37 p53 amino-terminal peptides have also been developed in an attempt to block MDM2-binding to p53 and therefore release p53 from the MDM2-mediated proteasomal degradation.26,66 It was reported that two novel peptides resembling the p53 amino-terminus bind to MDM2 with an affinity 100 times higher than p53. The binding of the peptides to MDM2 blocks the interaction of MDM2 to p53 and p53 is stabilized. After treatment with the peptides, cells with wild-type p53 undergo apoptosis. Most recently, another report showed that several peptides corresponding to the amino-terminal MDM2-binding site of p53 induce cytotoxicity in a variety of tumor cell lines. Surprisingly and interestingly, these peptides induced necrosis, a kind of non-apoptotic tumor cell death, in a variety of tumor cells.26 Furthermore, these peptides induced tumor cell death with no discrimination of p53 status, but appeared less toxic to nonmalignant cells. Another peptide from a series of overlapping synthetic peptides derived from the p14ARF protein sequence was reported to activate p53-mediated transcription by inhibiting p53 degradation.91This peptide, corresponding to the first 20 amino acids of ARF, could bind MDM2 and blocks both MDM2 autoubiquitination and p53 ubiquitination. Although there is no detailed information available on whether this p14ARF peptide can suppress tumor growth, the finding offers further evidence that it may be feasible to achieve an anticancer effect by interfering with protein-protein association. Although peptide therapies are potent, specific and non-toxic, the progress has been limited by the cost of large-scale production. Another obstacle for peptide application is the difficulty to introduce them into tumor cells. Microinjection or lipotransfection was previously used to deliver peptides. More recently cell membrane penetrating signals as a leader, such as Ant, a 17 amino acid sequence derived from Drosophila Antennapedia homeodomain protein, and TAT, an 11 amino acid sequence from HIV TAT protein, are being


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widely used. Both Ant and TAT are able to cross the cell membrane and lead other conjugated polypeptides into mammalian cells.26,78 CP-31398. Upon screening a library of more than three hundred thousand synthetic compounds, multiple classes of small molecules (300 to 500 Dalton) were identified with the ability to stabilize a wild-type-associated epitope (mAb1620) of the p53 DNA-binding domain in vitro.36,100,115 Among them, CP-31398, a styrylquinazoline, emerged as the first compound reported with the ability to alter a mutant p53 to a wild-type conformation and rescue p53 function in some tumor cell lines and xenografts. Tumor cells with hot-spot p53 mutations were found to be sensitive to CP-31398 induced cell cycle arrest or apoptosis, including p53 mutations at positions 173, 241 and 249.36,121 After several hours of CP-31398 treatment, significant p53 activity was detected either by immunoblotting p53 targets or a p53 reporter assay. Administration of CP-31398 suppressed the growth of p53 mutated tumor xenografts including the DLD-1 colon carcinoma (mutation at 241) and the A375 melanoma (mutation at 249) in mice without obvious toxicity.36 It has been reported that in some cell systems the ability of CP-31398 to promote the expression of the mAb1620 epitope does not correlate with the enhancement of p53-dependent transcription.104 Since even wild-type p53 requires appropriate activation signals such as hypoxia, telomere shortening, or DNA damage to become transcriptionally active, the lack of such signals or the inability to sense them in certain cells may prevent the transcriptional activity of p53 even when the conformation and function of mutant p53 is restored by CP-31398. Indeed, Herbert et al. have recently demonstrated that continuous exposure to CP-31398 does not induce p21 induction or cell death in cultured Li-Fraumeni cells until late in the crisis period where telomere shortening leads to DNA damage.50 The ability of conformation-stabilizing agents to restore mutant p53 without concomitant activation of p53 may provide a safety advantage during therapy, where a restored mutant p53 can be specifically induced within tumor cells by appropriate, tumor-specific activation signals. By promoting the active conformation of p53, CP-31398 not only can restore p53 function in mutant p53-expressing cells but also significantly increases the protein level and promotes the activity of wild-type p53 in multiple human tumor cell lines including ATM-null cells.121,127 Cells treated with CP-31398 undergo either cell cycle arrest or apoptosis. Further experiments showed that CP-31398 blocks the ubiquitination and degradation of p53. Of particular interest, unlike the DNA damaging agent adriamycin which induces strong phosphorylation of p53 on serines 15 and 20, CP-31398 exposure leads to no detectable phosphorylation on these sites. As a consequence, CP-31398 does not block the physical association between p53 and MDM2 in vivo. Moreover, CP-31398 can stabilize exogenous p53 in p53-mutant, wild-type or p53-null cells, even in MDM2/p53 double knockout mouse embryonic fibroblasts. These phenomena suggest a model wherein the conformational effect of CP-31398 can rescue p53 from ubiquitination and cause the accumulation of high levels of transcriptionally active p53. It has been suggested that CP-31398 may incorporate in newly synthesized p53 protein and stabilize its wild-type conformation.36 Yet, to date, there is no physical evidence for a direct interaction between CP-31398 and p53. Efforts that used a p53 DNA binding domain generated from bacteria failed to show any evidence of association.104 Since bacterially generated p53 is mainly in a conformation that is DNA-binding, but unable to display the 1620 epitope, the appropriateness of using this protein source to evaluate binding S60

Figure 4. Structures of CP-31398 and PRIMA-1.

remains a question. In contrast to bacterially generated p53, physiologically relevant, non-activated p53 in mammalian cells displays the 1620 epitope. The expression of the 1620 epitope and the ability of p53 to bind to DNA appear to be mutually exclusive, suggesting that p53 can exist in multiple conformations.46 As CP-31398 was identified based on its ability to stabilize the 1620 epitope, it may interact only with the protein that is in the 1620-reactive conformation. Further studies are needed to establish whether CP-31398 is capable of physically interacting with forms of p53 that exist in a conformation other that the DNA binding conformation. Although many of the cellular effects of CP-31398 require the presence of p53, the evidence of some p53-independent effects suggest that p53 may not be its only target. The global alteration of gene expression profile rather than merely p53 targets following treatment of CP-31398 suggests other pathways may exist in CP-31398 induced cell cycle arrest and apoptosis.121 There is also evidence that CP-31398 can stabilize p53 family members and this may be part of its mechanism of action in mutant p53-expressing tumors.127 Still, when used at lower concentrations, continued exposure to CP-31398 does not induce apoptosis or growth arrest in cells until appropriate DNA-damage signals are relayed through the p53 pathway.50 Of note, CP-31398 induces a significantly high expression of KILLER/DR5, a receptor for TRAIL regulated by p53, in wild-type p53-containing tumor cells, but not in p53 null cells, and therefore sensitizes to the treatment with TRAIL.127 Bax, a cytosolic apoptosis mediator, can also be induced in a p53-dependent manner. Release of cytochrome c and activation of caspase-9 after CP-31398 treatment support the idea that CP-31398 also activates the intrinsic pathway of apoptosis.82 The discovery of CP-31398 reveals a unique pathway different from the well-known DNA-damage induced p53 pathway with no obvious phosphorylation at the amino-terminus of p53.127 Further understanding of the mechanism may lead to novel strategies for p53 stabilization and tumor suppression in cancers, even those with no ARF or high MDM2 expression. PRIMA-1. PRIMA-1 (p53 reactivation and induction of massive apoptosis) is the second class of compound reported to have the capability of restoring tumor suppressor function to mutant p53.17 By screening a chemical library using Saos-2, p53-null cell line stably expressing a His-273 mutant p53 under the control of a tetracycline-regulatory promoter, PRIMA-1 was identified to induce apoptosis dependent on mutant p53 expression. Further experiments showed that PRIMA-1 induced apoptosis in a broad range of human tumor cell lines. Statistical analysis revealed that tumor cell lines carrying mutant p53 were more sensitive to PRIMA-1 than those carrying wild-type p53. Moreover, the sensitivity was related to the expression levels of mutant p53. Unlike CP-31398, which




functions on both mutant p53 and wild-type p53, PRIMA-1 does not appear to have an effect on wild-type p53. This makes PRIMA-1 unique in contrast to drugs currently used in the clinic.16 However, the mechanism by which PRIMA-1 rescues mutant p53 and induces apoptosis has not been well elucidated. No information is available on whether PRIMA-1 directly associates with p53, and if so, whether the binding is sensitive to the conformation of p53. The fact that PRIMA-1 also kills p53-null tumor cells suggests that a p53-independent pathway may exist in PRIMA-1-induced apoptosis. To what extent the PRIMA-1-elevated DNA-binding and transcriptional activity of p53 contributes to PRIMA-1-induced apoptosis remains unclear.

CONCLUSION AND FUTURE PERSPECTIVES Knowledge about the defect in p53-induced cell cycle arrest and apoptosis in human tumors is accumulating and attempts to target p53 for feasible cancer therapy are ever increasing. p53 abnormality occurs in the majority of malignancies and distinguishes tumor cells from normal cells. Thus p53 provides an attractive molecular target for anti-cancer agent development. In normal cells, factors favorable and unfavorable for proliferation are orchestrated coordinately to keep cells alive and under control. In malignant cells, the coordination is out of order, and in most cases, p53 is inactivated. Reconstitution of p53-mediated apoptosis by introducing either an exogenous p53 gene or genes coding for p53 upstream or downstream factors may be effective in reversing the malignancy of both wild-type and mutant p53-expressing tumor cells. The conformation of mutant p53 protein itself, in many cases, is flexible and restorable. Restoration of tumor suppressing function by carboxyterminal peptides, and the subsequent discovery of smaller molecules such as CP-31398 and PRIMA-1 provides evidence for the practicality of targeting mutant p53. Further understanding of mechanisms by which CP-31398 and PRIMA-1 restore p53 activity may not only lead to discovery of even more potent analogs, but may also provide new insights for p53-targeting in tumor therapy. Moreover, combination of the known cytotoxic agents that activate p53 with those that restore mutant p53 function may synergize their anticancer potency. Finally, releasing or activating p53 family members from the negatively regulated network in tumor cells may also contribute to the p53-mediated tumor suppression. References 1. Alarcon-Vargas D, Ronai Z. p53-Mdm2--the affair that never ends. Carcinogenesis 2002; 23:541-7. 2. Appella E, Anderson CW. Post-translational modifications and activation of p53 by genotoxic stresses. Eur J Biochem 2001; 268:2764-72. 3. Appella E, Anderson CW. Signaling to p53: breaking the posttranslational modification code. Pathol Biol (Paris) 2000; 48:227-45. 4. Arrowsmith CH. Structure and function in the p53 family. Cell Death Differ 1999; 6:1169-73. 5. Ashcroft M, Taya Y, Vousden KH. Stress signals utilize multiple pathways to stabilize p53. Mol Cell Biol 2000; 20:3224-33. 6. Asher G, Lotem J, Sachs L, Kahana C, Shaul Y. Mdm-2 and ubiquitin-independent p53 proteasomal degradation regulated by NQO1. Proc Natl Acad Sci USA 2002; 99:13125-30. 7. Asker C, Wiman KG, Selivanova G. p53-induced apoptosis as a safeguard against cancer. Biochem Biophys Res Commun 1999; 265:1-6. 8. Augustin M, Bamberger C, Paul D, Schmale H. Cloning and chromosomal mapping of the human p53-related KET gene to chromosome 3q27 and its murine homolog Ket to mouse chromosome 16. Mamm Genome 1998; 9:899-902. 9. Balint EE, Vousden KH. Activation and activities of the p53 tumour suppressor protein. Br J Cancer 2001; 85:1813-23. 10. Bean LJ, Stark GR. Phosphorylation of serines 15 and 37 is necessary for efficient accumulation of p53 following irradiation with UV. Oncogene 2001; 20:1076-84. 11. Bensaad K, Le Bras M, Unsal K, Strano S, Blandino G, Tominaga O, et al. Change of Conformation of the DNA-binding Domain of p53 Is the Only Key Element for Binding of and Interference with p73. J Biol Chem 2003; 278:10546-55.

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