6.1 Introduction: p53 gene was initially considered to be an oncogenic protein. However a study disproved this evidence when it was shown that in Friend virus induced mouse erythroeukemia; p53 gene was a target for viral integration resulting in its inactivation. Subsequent studies showed that it was a tumor suppressor gene and recognized as one of the frequently inactivated gens in more than 50% of all human cancers. The human p53 gene is found in chromosome 17p13 and Sothern blot analysis has revealed that there is single p53 gene in the human genome spanning about 20 kb of the genomic DNA. The gene (figure 1) is made of eleven exons; the first exon is a non-coding exon, followed by a large first intro of 10kb in length. There are some highly conserved domains within the exons 5, 7, and 8. The mRNA transcript of p53 is about 2.8kb in length and can be found in most of human cells, with the exception of cell in the GO phase.
Figure 1: Organization of human p53 gene
The gene encodes about 393 amino acids with a molecular weight of 53Da. Based on its structure and function, the gene is divided into three distinct domains: 1. the transcriptional activation domain at the amino or N-terminus, 2. the central sequence specific DNA binding domain, and 3. the multifunctional basic carboxyl or C-terminus. . The p53 pathway is made of hundreds of gene and their product that respond to a number of intrinsic and extrinsic stress signals such as DNA damage, oncogene activation, NO production, hypoxia, etc. These stress signals all impact under the cellular haemostatic mechanism that monitor and control the fidelity of DNA replication, chromosome segregation, and cell division. Among the stresses that activate the p53 protein is damage to the integrity of DNA in a cell. The p53 has number of functions. These includes: 1. Cell cycle checkpoint, 2. Promote apoptosis through transcription-dependent and independent mechanisms, 3. Has transcription-independent pro-apoptotic activities through its ability to modulate the functions of protein involved in the apoptotic machinery, 4. Anti-apoptotic capabilities, 5. Critical regulator of the senescence response, 6. Maintain genetic stability, 7. Has neurogentic ability, and 8. A role in tumorigenesis
6.1 Mutational Analysis of p53: The p53 gene is one of the most often mutated in human cancers. It involves mainly point mutation resulting in amino acid substitution in the central domain of the protein which impairs normal functions. Analysis of the mutational event that targets the gene has shown that both exogenous and endogenous mutational mechanisms are involved. Classes of DNA damage include deletion, insertion, and base substitution; either transition or transversion. Transition predominates in colon, brain, and lymphoid cancers; whereas G: C to T: A transversion are the most common substitution seen in cancer of the lungs and liver. Mutation at A: T base pair are seen more common in esophageal carcinoma than in other solid tumors. Most transition in colorectal, brain tumors, leukemia and lymphomas are at CpG dinucleotide mutational hot spots. G to T transversion in lung, breast, and esophageal carcinoma are dispersed among numerous codons. In liver tumors in geographic areas in which both aflatoxin (AFB) B1 and HBV are cancer risk factors, most mutation are at one nucleotide pair of codon 246. A number of investigation have indicated that the presence of chronic HBV infection and p53 mutation in the same tumors. However, HBV infection alone does not influence the rate of p53 mutation. Aflatoxin is the most influence on the prevalence of mutation. A number of studies have provided evidence albeit inconclusive to support the argument that combination of aflatoxin and HBV infection may be responsible for mutation. In a prospective cohort study of 18,244 people, it was showed that AFB has an etiological role in HCC and indicated a synergy between HBV and AFB. The study showed a statistical significant association in the presence of AFB and its metabolites in urine specimen, serum HBV surface antigen positivity, and HCC risk. In addition, the presence of promutagenic AFB-N7 guanine adduction in the urine provided further evidence that AFB has been activated to its electrophilic carcinogenic metabolite, AFB 8,9-oxide. Another study showed high frequency of AGG→ AGT transversion on the nontranscribed strand at p53 codon 249 in HCCs in some part of China and Mozambique with high incidence of HCC. This was associated possibly with high mutability of the third base codon 249 by AFB and/or selective growth advantage of hepatocyte clones carrying this surface 249sex mutant in liver of chronically infected HBV individuals. A study by Aguiler et al to test the preferential mutability concept showed that in a human liver cell exposed to AFB, the third base in codon 249 is preferentially but not exclusively mutated in comparison to the immediate adjacent codons. This suggests that both preferential mutability and clone selection are involved in the development of HCC. In HPV-associated cervical cancer, HPV type 16 and 18 carcinomas have been linked with the p53 pathway. HPV type 16 and 18 both produces E6 protein which complexes with the cellular protein AP6 and degrades p53 in the cytoplasm. This is a typical example of epigenetic p53 inactivation. Mutation analysis supports this concept if p53 mutant were found more in HPV-negative than in HPV-positive malignancies, and IHC would demonstrate no p53 staining n HPV-positive cases and nuclear staining in HPV-negative cases with missense mutations. Reports has shown that mutation frequency was 100% in HPV-negative and 0% in HPV-positive tumor cell lines but this results have not been validated in further studies in clinical tumors. Other studies confirmed the rarity of mutation in HPV-positive tumors and lines; mutation was however reported in 7 of 39 HPV-negative carcinomas. These studies provide evidence that dysfunction of the p53 protein might have a role in cervical carcinogenesis, but unless there are other alterations in the p53 pathway, it cannot be considered essential. In light of these data, more studies are needed to address the issue of low rate of mutation in HPV-negative tumors by searching for other p53 pathway abnormalities. Other data provides evidence of the involvement of other factors in p53 associated HCC. A study by Chen et al showed that mutation in p53 gene is linked to recurrence of HCC and that ZBP-89 may play a role in the nuclear accumulation of p53 protein in a subset of recurrent HCC. The ZBP-89 is a 4-zinc finger transcription factors that regulates the expression of several genes related to cell growth through binding to GC-rich DNA elements. ZBP- 89 has the ability of stabilizing p53 gene. The study suggested that co-localization of p53 protein with ZBP-89 may define subset of recurrent HCC that is more sensitive to treatment. Regarding the pattern of mutation, the study by Chen et al found that mutation in exon 7 of the p53 gene accounted for 43.8% of all alterations found thereby suggesting that exon 7 should be considered a prevalent site of p53 mutation.
6.2 Anti-p53 Drugs: With the central role played by p53 in cancer prevention and suppression as well as chemo sensitization or radio sensitization, abrogation of p53 is essential during carcinogenesis for most cancers to occur. Therefore targeting directly targeting mutated p53 would be ideal therapeutic strategies in cancer care. This intervention would potentially reduce the deleterious side effect resulting from most of the current treatment which is based on DNA damage. Another additional advantage of such intervention is mutated p53 is frequently over expressed and posttranslationally modified in tumor cells. The cellular environment of tumor cells also favors functional p53-induced apoptosis. Therefore any molecule or peptide chaperones that can stabilize mutated p53 weight conformation will activate the apoptosis pathway in tumor cells. Also most chemotherapeutic agents and radiotherapy require a functional p53 pathway. Therefore p53 chemical activators can increase the sensitivity of chemotherapy or radiotherapy. Below a review is made of the current strategies been explore for pharmacological modulation of p53 protein, gene therapy and utilization of this gene in cancer detection and monitoring.
6.2.1 Gene Therapy using TP53: In 1996, the first gene therapy using p53 was reported. It involved a retroviral vector which comprising the wild-type p53 gene which was under the control of actin promoter. This was injected directly into tumors of nonsmall lung cancer patients. With the development of a replication defective recombination p53 genes (Ad5CMV-p53) a number of clinical trials have been performed with a few reaching phase III but no approval has been granted yet.
6.2.2 Use of PRIMA1 & PhiKa083: One of the concepts is using a class of small molecules that can reactivate the wild-type functions of the mutated p53. The best studied is PRIMA-1, which entered the second phase of clinical trials in 2011. PRIMA-1 is converted to PRIMA-1MET, the methylated form of the drug which works in a number of ways to restore the function of p53. One of the mechanisms is that PRIMA-1MET covalently binds to and modifies the thiol groups in the central domain of the mutated gene. It causes the reactivation of the p53 gene that enables it to regain its ability to induce apoptosis. Another Phikan083, a carboxyl derived from in silico screening of the crystal structure of pp53. By binding to mutated p53, it raises the melting temperature of the mutated p52 thereby resulting in the reactivation of its function.
6.2.3 P53 stabilizer: Stabilizing p53 is a novel strategy that can be utilized for therapeutic intervention. MDM2 is an E3 ubiquitin ligase with the ability of controlling the degradation of p53. Most of tumors over express MDM2. It has been reported that even tumors without p53 mutation over express MDM2. In a study, it was shown that the nutlin which are cis-imidazoline compounds and act as antagonists of MDM2-p53 interaction binds in the pocket of MDM2 to prevent the p53-MDM2 interaction. Nutlin can activate the p53 pathway thereby inducing cancer cells and xenograft tumors in mice to undergo cell cycle arrest, apoptosis, and growth inhibition. M1-219 is also small molecule that can inhibit the p53 pathway in cells with wild-type p53. It was shown that apoptosis and cell cycle arrest were observed in xenograft tumors thereby resulting in tumor regression. But utilizing MDM2 inhibition and p53 in normal tissue can be harmful as reported by Ringshausen et al in a study that showed that p53 is spontaneously activated in many tissues in MDM2-deficient mice. In addition, p53 can induce a number of pathologies. Other classes of p53 stabilizers have been described. These include RITA (reactivation of p53 and induction of tumor cells apoptosis) was discovered by Issaeva et al. It acts by binding to p53 and inhibits the p53-MDM2 interaction both in vivo and in vitro. RITA therefore induces apoptosis in various cancer cells that retain wild type p53. Tenovin also stabilizes p53 by activating the gene. Two compounds have been identified: Tenovin-1 and Tenovin-6. Tenovin-6 is more water soluble than Tenovin-1. It has been established that Tenovin-1 inhibits protein-deacetylating activities of SirT1 and SirT2 of the Sirtuin family. Deacetylation of p53 does not only stabilize p53 but also interfere with MDM2-mediated p53 degradation. This means these compounds can target p53 gene modulation and also activate or increase the activity of p53.
6.2.4 P53 based immunotherapy: Immunotherapy can be used as an adjuvant therapy. Although p53 frequently mutates, the remainder of the molecule keeps its wild-type sequence. Nonmutated peptides can be processed from the altered p53 molecule and presented by tumor cells for T cell recognition. CTLs are the most important effector for antitumor immune response. A study found that adoptive immunotherapy of tumor-bearing mice with p53-specific CTL resulted in eradication of p53-overexpressed tumors in the absence of immunopathological damage to normal tissue. These tumors also eliminated tumors that did not show greatly enhance expression of p53. This indicates that the sensitivity of these tumors for p53-specific CTL is determined by the efficacy by which the p53-derived peptides are processed into Class I MHC not by the steady state level of p53. Tumor-associated antigen specific CTL can mediate immune response of host against cancer in vivo. Targeting the missense mutation form can be candidate of tumor antigen since cancer patients have antibodies against p53; although the frequency and clinical significance are matters of debate. A Speetjens et al reported clinical trials of a p53-specific synthetic long peptide (p53-SLP) vaccine for metastatic colorectal carcinoma. 10 patients were vaccinated with p53-SLP in phase I and II clinical trials. P53-specific T cells were isolated from the vaccination site which were characterized as Th cell that displayed mixed T-helper 1 and 2 cytokine profile with different percent of IFN- and IL-2 producing p53-specific T cells. Six of the patients showed strong proliferative p53- specific T cell response six months after the last vaccination. This vaccine is based in viral vector-based. Menon et al utilizing the concept of same modality performed a phase I/II clinical study in which end-stage colorectal cancer patients were vaccinated with a recombinant canarypox virus (ALVAC) encoding wild-type p53. Patients were immunized intravenously with an increasing dose of ALVAC-p53. The study reported that the vaccine was safe and capable of stimulating p53-specific Th1 (IFN-y) response in most of the patients. Fever was the only vaccine-associated adverse event reported. The conclusion of the authors was repeated immunization would probably be needed for good clinical response. Antivector responses were also observed in all the patients. Dendritic cell –based vaccine have also been tried, although it has been reported that this model of vaccines are laborious to produce and restricted to individual patients. But it has an advantage of being highly efficient as APCs. Svane et al tried this model in phase II study in direct continuation of their phase I study. Only 5 out of the 26 patients completed all the planned immunization scheduled as a result of rapid progression of disease or death. In most of the cases, an increase in the number of p53-specific CTLs were measure but a decline at late point after vaccination was observed. Another concept is peptide-based vaccine in which patients are immunized with a single peptide epitope. However the relatively poor immunogenicity of peptide epitopes means they need to be injected together with adjuvant. Rahma et al utilized this concept when they compared subcutaneous wild-type p53 epitope vaccination with IV peptide-pulsed DC administered in 21 ovarian cancer patients combined with IL-2 adjuvant in a randomized phase II study. IL-2 administered resulted in direct induction of expanded Treg and in grade II/IV adverse event in both arm of the study which were subsequently removed from the regimen of these patients. P53-specific T-cell was observed in about 70% of the patients, irrespective of whether they received short peptide or peptide-pulsed DC. Although limited success has been reported in these models, there is the need for further exploration. Also new vaccine strategies need to be tried, for e.g. Vermeij et al suggested that p53 vaccine can easily be combine with low-dose cyclophosphamide, anti-CTLA-4, chemotherapeutic regimens, or other tumor antigens as immunopotentiation treatment modalities. Future research should analyze whether addition of multiple antigen to p53 vaccine will elicit the required immune response. The best combination of therapy should be developed and identify those most likely to respond to combined anti-p53 therapy need to be established. Only then can we say we are nearer to de ĵăvŭ!
6.2.5 P53 Activation pathway: The p53 protein family comprises p53, p63, and p73. They share the same protein structure and biological function. These are also isoforms of each gene and can heterodimerize each other. Since p53 is the most mutated in human cancer but not p63 or p73, these two (i.e. p63 and p73) could be used to step up their suppressive function in p53 defective cancers. Therefore treatment that can increase p63 or p73 protein levels will deliver a p53-like function. One such compound that could activate the p53 pathway is NSC17627 which is an ellipticine derivative. Treatment of NSC176327 increased p53 target genes DR5 and DR1 expression. NSC176327 is however less effective in inhibiting cell growth when p73 was knockdown in HCT16 p53 -/-cells meaning p73 plays important role in NSC176327-induced-p53-like activity. Another compound with similar activity is reactivation of transcriptional reporter activity (RETRA), which was found to enhance p53 reporter activity in a mutated p53-dependent manner. Treatment with RETRA increased p73 expression and prevented the inhibition of p53 and p73, which produced a p53-like tumor suppression effect. However the exact mechanism of how RETRA interfere with mutated p53 and p73 interaction is yet to be elucidated.
6.2.6 Replacement gene therapy: As outlined earlier, the function of p53 is lost in most of cancers through mutation or loss of alleles. Therefore restoring the function of p53 id a novel therapeutic strategy that can be utilized. This can be achieved by replacing mutant gene with a functional wild type version. A requirement for treatment of cancers with replacement gene therapies is the need for highly efficient delivery of the wild type p53 into the tumor cells in vivo. Also the function of p53 protein should be sufficiently expressed to mediate tumor suppression through either mechanism which involves cell death or growth arrest, or by increasing the sensitivity to conventional anti-tumor agents. Low level toxicity towards the normal cells should be considered in order not to develop certain pathologies. Gene delivery system is divided into two categories: viral and non-viral.
6.2.7 Other Molecules: Other compounds with various mechanism actions have been described. Benzodiapinedione (BDA) which interacts with the p53-binding pocket of Mdm2. This compound increase the p53 transcriptional activity, inhibits the proliferation of cancer cells in the wild type p53-dependent manner, and synergized with doxorubicin to inhibit tumor cell growth in vivo and in vitro. Others are CP-31398, CDB3, etc. With the advent of improved screening and molecular technology, more compounds will be identified on continuous basis due to the occurrence of new mutations.
6.2.8 Challenges & Future Perspective: Reactivating the function of p53 is an ideal strategy to control and/or conquer cancer. But because the p53 malfunctioning varies among patients, developing a novel therapeutic interventions are still far reaching currently. A number of challenges would be encountered. Although targeting p53 looks promising, p53 is not an ideal drug target because it is not a receptor or enzyme. Also it is heterotetrameric nuclear transcription factor which is important in keeping the genome stable and guard normal cells growth and physiological function. However advances accrued over the years makes it encouraging researching further on developing tumor-specific p53 restoration therapies. We need to elucidate other roles than its function as a nuclear factor. The data so far show that cytoplasmic p53 can activate a transcriptional –independent apoptotic program. The next generation of p53-based therapies should therefore target this cytosolic function. Mammary stem cells with targeted p53 mutation have been reported. It showed the same properties as cancer stem cell. The reactivation of p53 restored the asymmetric cell division of cancer stem cells and induced tumor growth inhibition. Further studies are needed to elucidate the links between p53 function and cancer stem cells.
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