Restoring p53 Function in Cancer: Novel Therapeutic Approaches for Applying the Brakes to Tumorigenesis
Alessandra Di Cintio, Elena Di Gennaro and Alfredo Budillon1,*
Experimental Pharmacology Unit, National Cancer Institute of Naples 'G Pascale' Naples 80131, Italy
Received: May 28, 2009; Accepted: July 9, 2009; Revised: July 27, 2009
Abstract: p53 Tumor suppressor gene encodes for a critical cellular protein that regulate the integrity of the cell and can
induce cell cycle arrest and/or apoptosis upon cellular stresses of several origins, including chemotherapeutics. Loss of
p53 function occurs in an estimated 50% of all cancers by mutations and deletions while in the presence of wild-type p53
alleles other mechanisms may affect the expression and activity of p53. Alternative mechanisms include methylation of
the promoter of p53, deletion or epigenetic inactivation of the p53-positive regulator p14/ARF, elevated expression of the
p53 regulators murine double minute 2 (MDM2) and MDMX, or alteration of upstream regulators of p53 such as the
kinase ATM. MDM2 is a p53 E3 ubiquitin ligase that mediates the ubiquitin-dependent degradation of p53 while
p14/ARF is a small MDM2-binding protein that controls the activity of MDM2 by displacing p53 and preventing its
degradation. MDMX antagonize p53-dependent transcriptional control by interfering with p53 transactivation function.
The understanding of the key role of p53 inactivation in cancer development generated considerable interest in developing
compounds that are capable of restoring the p53 functions. Several patents have been issued on such compounds.
Adenovirus-based p53 gene therapy as well as small molecules such as PRIMA that can restore the transcriptional
transactivation function to mutant p53, or NUTLIN and RITA that interfere with MDM2-directed p53 degradation, have
tested in a preclinical setting and some of these approaches are currently in clinical development.
Keywords: p53, MDM2, gene therapy, small molecules, molecular-targeted drug.
INTRODUCTION
The p53 protein plays a key role in the regulation of complex intracellular networks by allowing the cell to maintain their genomic integrity and homeostasis. p53 helps the cell to properly modify gene expression profiles in res-ponse to different types of cellular stresses, including hy-poxia, DNA damage, metabolic stress and altered prolife-ration signals, such as oncogene activation. In response to a stress signal, p53 is stabilized and activated by post-trans-lational modifications. In its tetramerized form, p53 acts as a transcriptional factor and regulates the expression of genes involved in important cellular events such as the cell cycle, apoptosis, DNA repair, angiogenesis and cellular senescence. Since alterations in these cellular pathways are among the most important hallmarks of cancer, it is not surprising that the transcriptional activities of p53 are impaired by mutation or deletion in more than 50% of human cancers. In the remaining cancers retaining wild-type p53, the transcrip-tional activities of p53 are often inactivated through an alter-native mechanism. These mechanisms are largely related to the dysregulation of the MDM2 (murine double minute-2) protein, an E3 ubiquitin ligase that regulates p53 degradation and is the primary cellular inhibitor of p53. Moreover, because most cancer therapies target the p53-mediated stress response, the functional inhibition of p53 may significantly reduce the response to cancer treatment.
*Address correspondence to this author at the Experimental Pharmacology
Unit, Departement of Research, Istituto Nazionale Tumori, Via M. Semmola, 80131 Napoli, Italy. Tel: 39-081-5903292; Fax: 39-081-5903813;
The understanding of the key role that p53 inactivation plays in carcinogenesis has provided a rational for the development and patenting of compounds that are capable of restoring p53 function.
p53 REGULATION AND FUNCTION
The human p53 protein has five domains: 1) the trans-activation domain that includes the MDM2 protein binding site; 2) the proline-rich domain; 3) the core sequence-specific DNA-binding domain, which directly binds to sequences in target gene promoters; 4) the tetramerization domain; 5) the C-terminal regulatory domain.
The p53 family members, p63 and p73, share an high sequence homology with p53, but both genes encode multiple protein isoforms via alternative promoters and mRNA splicing. The Transactivation Domain (TA) isoforms that contain the TA domain (Tap63 Tap73) can activate a subset of p53 target genes involved in cell cycle progression and apoptosis. The isoforms, lacking the TA domain, retain their DNA binding and tetramerization competence and thus can act as dominant-negative inhibitors for the members of p53 family. Moreover, both genes can undergo extensive C-terminal splicing, producing different species of TP63 and TP73 with different “tails” which modulate the p53-like function of TA proteins [1].
The stability and transcriptional activities of p53 are regulated through multiple post-translational modifications, such as phosphorylation, acetylation, methylation, and ubiquitylation [2-5]. These covalent modifications in res-ponse to a wide range of signals indicate that p53 activity can be modulated in response to many changes in the cellular
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environment. In unstressed mammalian cells, p53 is inactive and maintained at low levels by continuous ubiquitylation and subsequent degradation by the proteasome. When cellular stress occurs, p53 ubiquitylation is suppressed and p53 is transiently stabilized and accumulates into the nucleus, where it induces or represses the transcription of downstream target genes. It is well documented that a p53-MDM2 auto-regulatory loop exists because, in unstressed cells, low levels of MDM2 induce monoubiquitylation of p53, which is not sufficient for degradation and leads to cytoplasmic translocation of the protein. When DNA damage occurs, p53 becomes stabilized and activated, induces MDM2 transcription, promoting polyubiquitylation and nuclear degradation of p53, and thus rendering it unable to access its target genes. MDMX, a homologue of MDM2 with an independent and complementary role, inhibits p53-dependent transcription by binding the p53 transactivation domain. Notably, MDM2 also degrades itself as well as MDMX in response to stress signals, thus completing this complex regulatory loop [6]. Another model, according to the regulation of p53, proposes that the formation of a MDM2-MDMX heterodimer complex is the most efficient
means to inhibit p53 transactivation and/or enhance p53 turnover. This suggests that MDM2 homodimers, when asso-ciated with p53, form a suboptimal structure for ubiqui-tylation [7]. The MDM2-p53 interaction is inhibited by p14ARF, a sensor of hyper-proliferative stimuli that is nor-mally expressed at low levels. Upon activation of oncogenes such as Ras, Myc, or v-Abl, p14
ARF expression is induced
and interfering with MDM2 activates p53-dependent trans-cription [8, 9]. Other activators of p53 include the kinase mutated in the ataxia telangectasia syndrome (ATM) and/or the ATM and RAD3-related kinase (ATR). In res-ponse to many cellular stresses that produce DNA damage, these kinases phosphorylate p53 enhancing its transcriptional activity through the CHK kinases, [10-13] Fig. (1).
The p53 protein activation in response to DNA damage leads to the induction of several downstream cellular pro-cesses including DNA repair, cell cycle checkpoint acti-vation and apoptosis. The mechanisms involved in DNA repair include both transcriptional and post-translational regulation by p53 of the DDB2 and XPC (xeroderma pig-mentosum group C) gene products, which are two critical DNA damage-recognition proteins required for genomic
Fig. (1). Cellular events regulated by p53 (Modified by Shangary et al. Clin Cancer Res 2008).
p53 as Anticancer Therapeutical Target Recent Patents on Anti-Cancer Drug Discovery, 2010, Vol. 5, No. 1 3
nucleotide excision repair as well as the transcription of the gene encoding the p53-inducible small subunit of ribonuc-leotide reductase (P52R2) [14]. Moreover, activated p53 can induce cell cycle arrest in both the G1 and G2M phases [15-17] as well as extrinsic and intrinsic caspase-dependent pro-apoptotic pathways [18, 19] Fig. (1).
The other two member of p53 family, p63 and p73, are also implicated in proapoptotic processes despite N iso-forms may have antiapoptotic effect. The induction of apop-tosis by p53 can require the presence of p73 as showed by Flores et al. [20-22].
Interestingly, in addition to its role in the response to cellular stresses described above, p53 is also involved in the regulation of angiogenesis. The notion that p53 plays a role in limiting tumor vascularization comes from a number of clinical studies that demonstrated that tumors carrying a mutation in p53 are more vascularized than tumors harboring wt p53. p53 Inhibits angiogenesis through three different mechanisms: the inhibition of hypoxia-sensing systems, the downregulation of pro-angiogenic factors such as VEGF and the upregulation of antiangiogenic factors such as Thrombospondin-1 (TSP-1). Hypoxia inducible factor (HIF-1) is the main regulatory component in the cellular responce to oxygen deprivation (hypoxia) activating the transcription of several proangiogenic genes including VEGF. p53 Inhibits HIF-1 activity by targeting the HIF-1 subunit for MDM2-mediated ubiquitylation and proteosomal degra-dation [23]. Conversely, HIF-1 protects p53 from MDM2-mediated degradation and can abrogate transcriptional repression of p53 by MDM2 [8]. p53 Inactivation, caused by expression of a dominant-negative p53 mutant or by p53 gene knockout, has been found to be accompanied by a notable increase in both HIF-1 levels and the expression of VEGF mRNA [24]. In response to VEGF, TSP-1 protein is secreted into the extracellular environment where it inhibits angiogenesis by suppressing migration and proliferation of endothelial cell. In addition, the activation of transforming growth factor beta (TGF-beta) by TSP-1 plays a crucial role in the regulation of tumor progression [25]. The loss of p53 leads to a deficiency in TSP-1 expression and the subsequent inability to shut off angiogenesis. Restoration of p53 expression in tumors re-establishes TSP-1 expression [26], Fig. (1). Interestingly, a recent paper by Adorno et al. reported a critical opposite role of p63 and mutant p53 in the regulation of metastasis [27]. Cell migration, invasion and metastasis, induced by TGF , can be potentiated by mutant p53 and opposed by p63. While a gain of such new molecular properties of mutant p53 appears to be crucially regulated by oncogenic Ras, p63 protective role is unveiled by the observation that for metastatic progression it should be functionally inactivated by trapping into a TGF -induced ternary complex with mutant-p53 and Smad. In details, Smad would act as a critical bridge for the formation of the complex between p53 mutated protein and p63, in which the p63 trascriptional function are antagonized.
Finally, an increasing body of evidence, including a variety of in vivo models, suggests that p53 as well as its related protein p63 and p73, drives also organismal ageing [28, 29]. In details it has been shown that loss of function of p53, although not sufficient, is necessary for spontaneous
cellular immortalization, a critical step in the tumorigenic transformation [30]. Several genes, including those regulated by p53, have been identified as mediator of cellular senescence, a state of permanent growth arrest and altered morphology that is activated in human cells after extended doubling passages because of their limited proliferative potential [31]. A mechanism associated with replicative senescence is the progressive telomere shortening, which can be bypassed by inactivating p53 [32].
THE ROLE OF p53 IN CANCER
The p53 pathway is considered an hallmark of human tumors. More than 50% of human cancers contain a mutation in p53 and over 90% of these mutations occur in solid tu-mors [33]. The loss of properly functioning p53 is also asso-ciated with an unfavorable prognosis in some types of cancer.
In the IARC (The International Agency for Research on Cancer) p53 mutations database, three distinct sets of sequence alterations in the p53 gene are described: p53 germ-line mutations in familial cancers, common p53 polymorphisms identified in human populations and somatic mutations of p53 in sporadic cancers Fig. (2). p53 germ-line mutations have been found in individuals with Li-Fraumeni syndrome, but although several criteria have been developed to identify families at risk for germ-line p53 mutations, recognition of these families remains a daunting task given the wide variety of cancer types associated with Li-Fraumeni syndrome. In addition, although p53 mutations are widely researched, there are few studies including a large number of patients in order to gain a broader understanding of the spectrum of tumors associated with germ-line p53 mutations. Results from a recent report from Gonzalez et al. analyzing 525 families will probably help in the identification of high risk families [34]. On the other hand, p53 polymorphisms, unlikely somatic mutations and mutations associated with Li-Fraumeni syndrome, are expected to be phenotypically silent. Most of these variations are intronic and are presumed to be silent; only a small fraction occur in exons and result in the alteration of a protein product. Traditional case-control studies with limited enrolments have not yielded definitive answers, and only high-throughput technologies studying a large number of individuals will provide information on the real impact of p53 polymorphisms on cancer development.
The tumor-associated mutations that occur in sporadic cancers arise in somatic cells, both spontaneously or cones-quence of DNA damaging agent exposure, and represent the majority of p53 alterations associated with human cancers. Most are missense mutations localized in the DNA-binding domain of the protein, and 80% of these mutations exert a dominant negative effect within the tetramer and lead to altered transcriptional activities. Moreover, mutated p53 protein has a longer half-life than wt p53 and can accumulate in high levels in cancer cells by evading the tight regulation to which wt p53 protein is subjected [35, 36]. Because of these mutations, the ability of the cell to induce cell cycle arrest and trigger apoptosis is abolished, and the cell does not react appropriately to stresses associated with the accumu-lation of DNA damage. These conditions act synergistically to promote cell transformation. Some p53 mutant proteins
4 Recent Patents on Anti-Cancer Drug Discovery, 2010, Vol. 5, No. 1 Budillon et al.
are only partially dysfunctional; for example, R175P and R181C mutant proteins are able to transactivate p21, but not BAX, thus failing to induce apoptosis. This effect can be explained by the different affinity of p53 for the p21 and BAX promoters [37, 38]. Conversely, some mutations result in a specific gain of function of the mutated p53 protein, and cause the transactivation, or potentiation of the transacti-vation, of genes such as MDR1, EGFR, c-myc, PCNA, IGF-II or VEGF, which are not transactivated by wt p53 protein and do not necessarily contain a p53 binding site in their regulatory regions. These new gain-of-function p53 mutants have oncogenic properties and are responsible for the resistance to anti-cancer drugs [39].
Tumors that retain wt p53 often acquire mutations that lead to alternative mechanism of p53 inactivation, such as the deletion or epigenetic inactivation of the positive regulator of p53 p14ARF; methylation of the p53 promoter; or elevated expression of MDM2 and MDMX, which regulate p53. Gene amplification or transcriptional mecha-nisms leading to the overexpression of MDM2 and subse-quent increased degradation of the p53 protein represent the main mechanisms of p53 dysregulation in the absence of p53 mutations [40]. In hematological malignancies, p53 is muta-ted in only a minority of cases [41], but it is functionally impaired through alternative mechanisms including, but not exclusively limited to, MDM2 deregulation. In this regard, a proportion of patients with chronic lymphocytic leukemia (CLL) have mutations in ATM kinase in their tumor cells that impair the radiation-induced p53 response, and have poor clinical prognosis similar to those of patients carrying p53 mutations [42].
p53 AND CHEMOTHERAPY
Fluoropyrimidines and platinum-based drugs are among the most widely used conventional anticancer chemothera-peutic drugs, and their efficacy is influenced by tumor p53 status. Moreover, breaks in double-stranded DNA induced by DNA-damaging drugs, such as topoisomerase I and II inhibitors, rapidly trigger the elevation of p53 protein levels in cells that possess wt p53 alleles, which in turn leads to apoptotic cell death [43].
It has been demonstrated that a novel gain of function conferred by certain p53 mutants is linked to fluoro-pyrimidine chemoresistance [44, 45]. Several clinical studies have revealed higher levels of resistance to fluoropyrimidine therapy in tumors with p53 mutations [46]. Additionally, it was recently reported, in colon cancer cells transfected with mut-p53, an increased mRNA and protein expression as well as activity of thymidilate synthase (TS), the target of 5-fluorouracil (5-FU), leading to decreased sensitivity to 5-FU and antifolates, compared to the wild-type parental cells [47].
Inactivation of the p53 gene has been reported to enhance tumor cell resistance to a number of additional chemothera-peutic agents, including cisplatin and other DNA-damaging agents. This is a consequence of the tumor cells’ reduced ability to activate the apoptotic response to DNA damage. Consistently with this hypothesis, missense mutations are associated with cisplatin resistance in the clinical setting [48]. Moreover, some reports have shown that p53 mutations in ovarian cancer are correlated with resistance to platinum-based chemotherapy, resulting in early relapse and a short
Fig. (2). p53 mutation in tumors (Modified by IARC TP53 mutation database).
p53 as Anticancer Therapeutical Target Recent Patents on Anti-Cancer Drug Discovery, 2010, Vol. 5, No. 1 5
survival [49, 50]. Furthermore, it has been reported that the level of Np63 in primary head and neck squamous cells carcinomas correlates with the clinical response of patients to cisplatin chemotherapy [51] while the presence of an active Np63/TAp73 pathway may be a useful clinical pre-dictor of cisplatin sensitivity in treatment-refractory breast cancers [52].
Conversely, taxanes, used in combination with platinum compounds because of their ability to overcome cisplatin resistance, do not require the presence of a functional p53 gene for the induction of apoptotic cell death. Lavarino et al. suggest that p53 dysfunction may indeed be advantageous for patients undergoing therapy based on taxane combination with platinum compounds and demonstrate that patients with mutant p53 tumors, which are expected to be relatively resistant to platinum compounds, seem to be responsive to paclitaxel in combination with platinum compounds [53]. In contrast, Kupryjanczyk et al. underscore the importance of the accumulation of p53 protein in tumor tissue as the main determinant of the increased benefits associated with taxane-platinum therapy [54].
Another drug that can overcome platinum resistance when used in combination with a platinum-based agent is gemcitabine. Gemcitabine is not easily detected or excised by proofreading exonucleases [55, 56], but gemcitabine-induced DNA damage can be recognized by p53, resulting in apoptosis. Based on this finding, Cascallò et al. tested different combination-chemotherapy approaches by utilizing wt p53 gene reintroduction and the genotoxic action of gemcitabine and cisplatin combination therapy. Their find-ings suggest that exogenous p53 can efficiently recognize the DNA damage caused by even low doses of cytotoxic drugs and can commit the affected cells to apoptosis. This apop-totic effect occurs only if p53 is introduced after treatment with the drugs because the cell cycle arrest produced by p53 precludes the action of these drugs, and thus induces chemoresistence. Therefore, the authors propose that the introduction of the wt p53 gene via Ad5CMV-p53 (see below) under an appropriate administration schedule may improve the efficiency of chemotherapeutic drugs [57].
TARGETING p53
The key role of p53 dysfunction in cancer development and the characterization of the different mechanisms of p53 deregulation described above, suggested several potential therapeutical approaches finalized to restore p53 functions in cancer cells Table 1.
Replacement of p53 Expression by Gene Therapy
One of the first approaches tested to restore p53 function in tumor cells was the use of gene therapy. A retrovirus vector containing the wt p53 gene is an attractive candidate for cancer gene therapy applications because it can be integrated into the genome of infected cells in a stable form and requires cell division to be expressed. However, this approach proved to have too many drawbacks to be used as a cancer therapy [58, 59]. A second strategy for p53 gene replacement therapy involves the use of adenovirus vectors.
Contusegene adenovec or Ad5CMV-p53 (Advexin, Introgen Therapeutics Inc) is a non-integrating vector derived from adenovirus serotype 5 (Ad5) in which the p53 gene is under the control of the CMV promoter. The genomic region encoding the envelope glycoprotein (E)1, and part of the E3 region of the parental Ad5 DNA, is deleted so that the adenovirus replication is defective [60]. Ad5CMV-p53 has demonstrated efficiency in expressing high levels of functional wt p53 protein both in vitro, in Ad5CMV-p53-infected cells, and in clinical studies, in which intratumoral injection of AdCMV-p53 resulted in vector-specific wt p53 RNA expression in patients’ tumor tissues [58]. Ad5CMV-p53 has some clinical activity described either as mono-therapy or in combination with chemotherapy and radiation in a variety of tumor types, including squamous cell carcinoma of the head and neck (SCCHN), non-small cell lung cancer (NSCLC), and glioma as well as breast, prostate, colorectal, and hepatocellular carcinoma [60]. However, since AdCMV-p53 is a replication-impaired adenovirus, heterogeneity or lack of expression of the Coxsackie-Ad receptor (CAR) or its co-receptors (which are used by the virus for to attach to the surface of the tumor cells) as well as the presence of adenovirus-neutralizing antibodies, can lead to a poor efficiency of infectivity by this vector. This shortcoming should be addressed for future applications of this approach. A recombinant human adenovirus–p53 injec-tion (trademarked as Gendicine, SiBiono; Shenzhen, China) was approved by the State Food and Drug Administration of China (SFDA) in 2003 for the treatment of HNSCC, but few studies based on the preclinical and clinical experiences with this vector have been published up to this point [61].
Another promising gene therapy approach is the use of replicating modified viruses that specifically target cance-rous cells, such as the modified ONYX-015 (ONYX pharma-cological) adenovirus. ONYX-015 has an 827-bp deletion in the E1B region, that, in wild-type viruses, encodes a 55kDa protein that binds and inactivates wt p53 protein, which usually inhibits viral replication. Consequently, ONYX-015 can replicate only in cells, such as cancer cells, that lack functional p53, without any damage to normal tissue. While the ability of ONYX-015 to kill in vitro cervical, colon, glioblastoma and pancreatic carcinoma cells lacking functional p53 has been previously shown, the mechanism of action and selectivity of this vector remain controversial [58]. ONYX-015 has been found to be able to replicate in several tumor cell lines that retain wt p53 [62]. Additionally, Ries et al. observed that tumor cells that possess genetic lesions affecting molecular factors within the p53 pathway, other than mutations in p53 itself, can render cells per-missive for ONYX-015 replication [63]. Specifically, tumor cells that lack p14ARF expression but possess wt p53 have high levels of uncontrolled MDM2 protein activity and subsequent disruption of p53 expression, therefore facili-tating ONYX-015 replication [63]. Moreover, Geoerger et al. demonstrated that the treatment of malignant human glioma xenografts by the adenovirus ONYX-015 was effective independent of the cellular p53 status, confirming that wt p53 does not prevent lysis of tumor cells by ONYX-015 [64]. The selectivity of ONYX-015 for tumor cells has therefore been called into question and, while highly
6 Recent Patents on Anti-Cancer Drug Discovery, 2010, Vol. 5, No. 1 Budillon et al.
CBS9106 Non-peptidic small molecule Stabilizes p53 by a mechanism that remains to be
determined Pre-clinical studies [91]
PNC-27 Peptide that contains the p53-
MDM2 binding domain
Binds to MDM2 preventing p53-MDM2
interaction Pre-clinical studies [2]
RITA Non-peptidic small molecule Binds to p53 and prevents ubiquitination by
MDM2 Pre-clinical studies
[94,95,97
,99]
PARTHENOLIDE
Natural molecule (extracted from
flowers of the Astaraceae family
of plants)
Induces ubiquitination of MDM2 and results in
the activation of p53 Pre-clinical studies [104]
p53 as Anticancer Therapeutical Target Recent Patents on Anti-Cancer Drug Discovery, 2010, Vol. 5, No. 1 7
promising, it remains to be determined whether if may cause toxicity to normal tissues. So far, ONYX-015 has been shown to have a good safety record in cancer treated patients, but its efficacy has been hampered. In details, different phase I and II trials of ONYX-015 monotherapy, using both intratumoral and peritumoral injections, have been conducted in multiple tumor types showing that is well-tolerability with only flu-like symptoms occurring, however, in most cases, no significant antitumoral response was observed [58, 65]. The most promising data have emerged from studies combining ONYX-015 with cytotoxic chemo-therapy. The response rate of ONYX-015 treatment in combination with systemic chemotherapy for head and neck tumors is very encouraging and shows that the combination of ONYX-015, cisplatin and 5-FU is a good method of local control for this tumor [58, 62, 65]. Furthermore, clinical activity has been demonstrated in patients with pancreatic cancer in several trials [58, 62].
Another gene therapy approach, patented many years ago, is the use of mutant proto-oncogenes expressed in target cells in order to inhibit growth and/or the transformed pheno-type. Among these approaches a mutant MDM2 polynuc-leotide sequence has been shown to block MDM2-mediated p53 inhibition [66]. A recent patent reported several approa-ches combining gene therapy and proteosome modulation, in order to increase the expression of therapeutic proteins including p53 [67].
Reactivation of Mutant p53 Function by Small Molecules
Screening studies have identified several small molecules that reactivate mutant p53 [68].
PRIMA-1 (p53 Reactivation and Induction of Massive Apoptosis) is a molecule that was identified by screening a library of low-molecular-weight compounds using Saos-2-His-273, a p53-null human osteosarcoma cell line engineered to stably express a His-273 mutant of p53 under the control of a tetracycline-regulated promoter [69, 70]. PRIMA-1 showed a significant preference for the growth inhibition of tumor cell lines expressing mutant p53 as opposed to tumor cell lines expressing wt p53 [70, 71]. Multiple different types of p53 mutants can be functionally restored by PRIMA-1. The restoration of DNA-specific binding as well as the restoration of the transcriptional transactivation function of mutant p53 is critical for the induction of apoptosis by this agent [70]. PRIMA-1 selectively induces cell cycle G2-arrest by up-regulating p21 and GADD45 as well as transcription-independent apoptosis in cells with mutant p53 [71, 72]. In 2005, Bykov et al. identified a more specific and active methylated form of PRIMA-1 (PRIMA-1
MET ) [73]. PRIMA-
1MET
has shown synergistic antitumor effect in combination with cytotoxic drugs such as cisplatin and 5-FU only in cells expressing mutant p53 [73] Table 2.
Using a similar method (screening a low-molecular-weight compound library using Saos-2-His-273 engineered cells), Bykov et al. identified a novel class of molecules that are structurally distinct from PRIMA-1. MIRA-1, the first of these molecules to be tested, contains a maleimide group with a saturated 3-4 double bonds, which allows it to form adducts with thiol and amino groups. This structure probably also allows the alkylation of cysteine and/or lysine residues
of p53, stabilizing the native folding of the protein. The alkylation status of p53 depends on the accessibility of thiol groups, thus unfolded mutant p53 proteins are more extensively modified by MIRA-1 than correctly folded wt p53 proteins [74]. MIRA-1 induces cell death only in cells containing mutant-p53 with even higher potency than PRIMA-1. The reactivation of mutant p53 by MIRA-1 has been demonstrated in vitro by studies revealing the induction of expression p53-target genes such as p21, MDM2 and PUMA. Therefore MIRA-1 and its structural analogs are postulated to act by shifting the equilibrium between the native and unfolded conformations of p53 toward the native conformation, leading to the restoration of p53-mediated transactivation of target genes and the induction of p53-dependent apoptosis. MIRA-3, a MIRA-1 analog, has also been tested in vivo and was found to have observable anti-cancer activity. However, toxicity at the higher doses used have hampered the therapeutic application of this compound [74] Table 2.
It has recently been demonstrated that PRIMA-1 can also be converted into compounds that modify thiol groups, sug-gesting a common mechanism for mutant p53 reactivation among all these molecules and thereby facilitating the design of more potent, more efficient and potentially more clinically active anticancer compounds to target mutant p53 [75].
STIMA-1 is a novel, recently identified low molecular weight compound that induces mutant p53 reactivation. STIMA-1 has demonstrated the in vitro capacity to stimulate DNA-specific p53 binding and to induce the transcription of p53 target genes, triggering apoptosis in mutant p53-con-taining human tumor cells [76].
Finally, phenethyl isothiocyanate (PEITC) is a naturally occurring compound, recently described, that is able to dep-lete mutant p53 levels, thus restoring wt p53 function [77].
Regulation of p53 Expression by Targeting MDM2
The well-defined interaction between MDM2 and p53 has provided the basis for the design of non-peptide, drug-like small-molecules. NUTLIN-3 is a cis imidazoline com-pound that, by binding MDM2 at the p53 binding pocket, disrupts the physical interaction between p53 and MDM2 in highly selective manner, inducing the accumulation of p53 and thus the activation of the p53 pathway [5, 78] Table 2. Notably, serine phosphorylation in the amino-terminal domain of p53, an essential requirement for genotoxic drugs-induced p53-dependent apoptotic activity, is not required for the transcriptional activation by NUTLIN-3 of most p53 target genes [78]. Cell-cycle arrest in the G1 and G2 phases induced by NUTLIN-3 is always observed in tumor models with wt p53. In contrast, the induction of apoptosis is more complex because defective apoptotic signaling is common in solid tumors, and the highest apoptotic index induced by NUTLIN-3 is observed in cell lines overexpressing MDM2, probably because they possess intact p53 signaling [79]. A study by Shangary et al. showed NUTLIN-3-induced antitu-mor activity in several xenograft models of human cancer with wt p53, including osteosarcoma and prostate cancer, and underlined the lack of NUTLIN-3 activity against tumors deficient in wt p53 [79-81].
8 Recent Patents on Anti-Cancer Drug Discovery, 2010, Vol. 5, No. 1 Budillon et al.
Table 2. Small Molecules Used to Target Mutant or Wild-Type p53
Mechanism of Action Chemical Compound Pharmacological Improvments
p53 as Anticancer Therapeutical Target Recent Patents on Anti-Cancer Drug Discovery, 2010, Vol. 5, No. 1 9
(Table 2) Contd….
Mechanism of Action Chemical Compound Pharmacological Improvments
RITA
2,5bis(5-Hydroxymethyl-2-thienyl)furan
PARTHENOLIDE p53 stabilization through the inhibition of
the interaction of MDM2 and p53
Sesquiterpene lactone
Interestingly, several studies have reported the ability of NUTLIN-3 to inhibit angiogenesis in both p53-dependent and p53-independent manners. In details, NUTLIN-3 can favor hypoxic-stress-induced stabilization of p53 which in turn directly binds to HIF-1 and targets the protein for degradation, thus inhibiting angiogenesis [82] (see also Fig. (1)). Additionally, NUTLIN-3 inhibits the transcriptional activity of HIF-1 , thereby preventing its association with MDM2, a process that is necessary to increase the induction of the vascular endothelial growth factor under conditions of normoxia or hypoxia [5, 83]. Of note, because NUTLIN-3 is unable to block MDMX-p53 binding, its antitumor activity could be compromised in certain human tumors that overexpress MDMX [84].
MI-219 is a spiro-oxindole compound that was designed on the base of the crystal structure of the MDM2-p53 complex Table 2. This small molecule binds to MDM2 with a high affinity, activates the p53 pathway and selectively inhibits cell growth in cancer cell lines with wt p53. MI-219 is characterized by a good pharmacokinetic compared to other compounds (e.g. MI-43 and MI-63) designed using the same structure-based approach [85, 86]. Computational modeling suggests that MI-219 mimics the key binding residues of p53 and interacts with MDM2 with high affinity and selectivity. Via this mechanism, MI-219 determines p53 accumulation and the upregulation of p53 target genes such as MDM2, p21 and PUMA. MI-219, inducing cell cycle arrest in both cancer cells and normal cells, but apoptosis only in cancer cells. Since MI-219 achieved an excellent oral bioavailability, it was tested in mouse xenograft models of human cancer where stimulate a rapid but transient p53 activation in tumor cells resulting in inhibition of cell proliferation, induction of apoptosis, and complete tumor growth inhibition. These results, in addition to the low toxicity demonstrated in normal mouse tissues, suggest that MI-219 should be considered as a promising cancer therapy with a possible future clinical applications [5, 86]. However, like NUTLIN-3, MI-219 also binds to MDMX with a very
weak affinity, so it may not be able to fully activate p53 in cells expressing high levels of the MDMX protein.
Other MDM2-targeting molecules recently selected for preclinical studies are benzodiazepinediones (including TDP521252 and TDP665759) [87], a member of the poly-comb group of proteins (RYBP) [88], non-peptidic small molecules such as Tenovin 6 [89], NXN [90], CBS9106 [91], as well as p53-mimetic peptides [2, 92] Table 1.
Interestingly, the recently selected small molecule JNJ-268554165, unlike other known MDM2 antagonists, pre-vents the association of MDM2/client complexes with the proteasome, showing potent induction of p53, apoptosis and tumor regression in either wt or mutant p53 human xenografts cancer models [93] Table 2.
RITA (REACTIVATION OF p53 AND INDUCTION OF TUMOR CELL APOPTOSIS)
RITA is a small-molecule that can reactivate the tumor suppressor function of wt p53 Table 1. RITA was identified through a screening assay based on a library of compounds that took advantage of two isogenic cell lines differing only in their p53 status: the HCT116 colon carcinoma line (which expresses wt p53) and its derivative HCT116 p53
-/- cell line
(which does not express p53). RITA suppresses the growth of HCT116 cells in a dose-dependent manner, but only slightly inhibits the growth of HCT116 p53
-/-. Although the
IC50 values for RITA vary depending on tumor cell type, growth inhibition is clearly more effective in wt p53-expressing cells. Differently from MDM2-targeting mole-cules such as NUTLIN-3, RITA binds to the amino-terminal domain of p53, inducing a conformational change of the protein and increasing its half life and its accumulation in tumor cells. Specifically, RITA is thought to rapidly bind to p53 during a first step, then, during a slower second step, p53 would undergo a conformational change that in turn prevent MDM-2 binding [94]. It is possible that, due to the polar nature of the compound, the binding of RITA to the
10 Recent Patents on Anti-Cancer Drug Discovery, 2010, Vol. 5, No. 1 Budillon et al.
amino-terminus of p53 affects hydrogen bonds within the MDM2-binding site, thus preventing the formation of the -helix required for MDM2 binding. However, studies examining NMR structures of MDM2 showed that RITA can also bind to the p53-binding cleft of MDM2, suggesting that it may function as a multi-target molecule [95]. However, another study investigating the in vitro binding of RITA to p53 and MDM2, did not find any affinity between RITA and p53 or MDM2, nor was the compound found to be able to dissociate the p53-MDM2 complex [96]. Nevertheless, even though the biological activity of RITA may be derived from additional, still unknown, biochemical mechanisms, this compound has been found to be able to reactivate the transcriptional function of p53, thereby inducing expression of p53 target genes and massive apoptosis in various tumor cell lines expressing wt p53. RITA was also tested in vivo, by intraperitoneal administration in tumor-xenografted mice, showing well tolerability and p53-dependent antitumor effects [94]. Enge et al., in a recent report, confirmed that RITA induces profound changes in gene expression profiles in a p53-dependent manner, and that it activates the intrinsic and extrinsic p53-dependent apoptotic pathways through the induction of genes such as Fas, NOXA and PUMA [97]. Moreover, the authors suggested a model in which functional MDM2 released from p53 after treatment with RITA contributes to the apoptotic response by promoting proteo-somal degradation of the p53 transcriptional cofactor hnRNPK, which is important for the induction of growth-arrest genes, such as p21. The induction of these growth-arrest genes subsequently switch the balance towards the induction of apoptosis [97]. Another recent report showed that reactivation of p53 by RITA inhibits several oncogenes, and consequently several critical survival pathways such as the AKT pathway, both in in vitro and in vivo preclinical models [98]. Notably, the authors also showed that the threshold for p53-mediated repression of survival genes is higher than that for the transactivation of proapoptotic targets [98]. RITA may also affect tumor-induced angio-genesis by blocking HIF-1 and VEGF induction in response to hypoxia by a p53-dependent mechanism [99]. Interes-tingly, the inhibition of HIF-1 expression induced by RITA is not due to an effect mediated on transcription or protein degradation, rather, it depends on the inhibition of HIF-1 protein synthesis mediated by eIF-2 phosphorylation. In addition, HIF-1 status in cells contributes to RITA’s apoptotic outcome: in cells with low basal expression of HIF-1 , RITA has a less potent effect on the inhibition of tumor cell growth than in cells with a normal level of expression. Moreover, RITA may elicit a cellular DNA damage response by inducing amino-terminal phosphory-lation of p53 at Ser-15, while the small molecule NUTLIN-3, described above, was unable to mediate p53 phosphorylation [99].
OTHER APPROACHES REGULATING p53 EXPRESSION AND FUNCTION
The potential use of molecules able to increase wt p53 levels was patented many years ago with the demonstration that the 2-methoxyestradiol induced p53-mediated apoptosis associated with accumulation of cyclin dependent kinase inhibitor p21 [100]. More recently, it has been reported that
the induction of p53 protein acetylation upon the inhibition of histone deacetylase (HDAC) enzymes by HDAC
inhibitors is essential for preventing p53 degradation and for leading to an open p53 folding conformation that allows the protein to bind DNA [101, 102]. It was also demonstrated that HDAC inhibitors may deplete mutant p53 protein levels, suggesting that these agents, currently in advanced clinical development, should be considered as a p53 regulators. We have recently reported that p53 protein, whose functional wild-type expression is critical for sensitivity to fluro-pyrimidines drug such as 5-FU [47], is upregulated by the HDAC inhibitor vorinostat in wt p53 cells but down-regulated in mutant p53 cells. These effects lead to the synergistic antitumor interaction of vorinostat with 5-FU in both wt and mutant p53 cancer cells [103].
Parthenolide is a hydrophobic natural compound that is commonly extracted from the European feverfew herb and is used in traditional remedies for arthritis, headaches, fever and local skin irritation. It is also being studied for potential use as an anti-inflammatory and anticancer agent (Table 2). Recently, Gopal et al. showed that parthenolide induces the ubiquitylation resulting in the activation of p53 and of other MDM2-regulated tumor-suppressor proteins [104]. There-fore, parthenolide, like NUTLIN-3, activates p53 by preventing its interaction with MDM2. However, while NUTLIN-3 increases the levels of unmodified MDM2 in the cell, parthenolide increases cellular levels of ubiquitinated MDM2 and promotes its proteasomal degradation. In addition, besides its pharmacological effect on MDM2, parthenolide may activate p53 by depleting cellular HDAC-1 level through ubiquitylation and proteasomal degradation via an ATM-dependent mechanism. In details, it was proposed that parthenolide may chemically modify a reactive cysteine residue in a thiol-responsive protein, thereby initiating a signaling cascade that leads to ATM activation and subse-quent downstream effects, including the ubiquitylation of the lysine-rich C-terminal region of HDAC1 and its proteasomal degradation along with an increase in p53 acetylation and stabilization. In addition, ATM directly activates p53 by Ser phosphorylation, leading to its dissociation from MDM2 and inducing an increase in its transcriptional activity. Thus, parthenolide may represent an alternative approach for controlling the actions of MDM2 and p53 by regulating several critical cancer-related cellular pathways [104].
CURRENT & FUTURE DEVELOPMENTS
p53 is emerging as an increasingly attractive target for anticancer therapy. Several approaches to restore or increase p53 expression and/or function in cancer cells are under-going preclinically trying to find their way into clinical practice. However, the only extended clinical experiences so far are the gene therapy clinical trials that used viral vectors carrying wt p53. Intratumoral injection of such vectors for localized tumors showed clinical activity as well as tolerability, while intravenous delivery for advanced tumors showed limited activity. New generations of viral vectors as well as more informations about the safety of such approa-ches are critical to obtain, prior to their inclusion in clinical practice. Clinical testing of small molecules that interfere with the interaction between MDM2 and p53 should provide clinical proof for this strategy. However, it should pointed
p53 as Anticancer Therapeutical Target Recent Patents on Anti-Cancer Drug Discovery, 2010, Vol. 5, No. 1 11
out that the high concentrations required for efficacy in preclinical studies of several of these agents will limit their clinical use unless new and more stable generations of compounds are developed. The only reported clinical trial with a small molecule that interferes with p53 stability is the Phase I study with the JNJ-268541645 compound, adminis-tered orally on a continuous schedule in patients with advanced solid tumors that are refractory to standard therapy. Tolerability and some pharmacodynamic data were recently reported in abstract form [93]. A clinical trial with PRIMA-1
met is planned to start in the coming months and should
confirm the feasibility of an antitumor targeting mutant p53. It remains to be seen whether all of these approaches can be combined with conventional as well as molecular-targeted therapy. Only few studies have investigated combination therapy approaches including agents targeting p53, and additional data on mechanistic-based rational combinations are needed for the translation of such approaches to clinical trials. Recent data have added new insights into the understanding of the molecular mechanisms of the antitumor effect of the small molecule RITA and have suggested new approaches to combination therapy [97, 98]. In conclusion p53-therapeutical strategies must consider the complete identification and characterization of the p53-p63-p73 network and its role in tumorigenesis. The determination of the repertoire of p53 family members isoforms expressed in tumor cell type is actually under investigation. Notably, a diagnostic test kit for measuring level and status (wt or mut) of p53, p63 and p73 proteins or mRNAs in cells isolated from patients has been recently patented [92].
AKNOWLEDGEMENTS
This study was partially supported by Italian Ministry of Health FSN 2005 and 2007.
CONFLICT OF INTEREST
No financial interest in any of the compounds or patents reviewed in this article.
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