P53 In Human Tumors Research Articles

Virtual screening of p53 mutants reveals Y220S as an additional rescue drug target for PhiKan083 with higher binding characteristics

Pharmacological intervention to reactivate p53 in human tumors holds great promise for cancer patients. A number of small molecules that reactivate p53 mutants that are either specific to certain mutation or more broadly on various mutants of p53 are discovered by rational design and screening methods. One of the most remarkable among small molecules for the rescue of specific mutant p53, Y220C is PhiKan083 (1-(9-ethyl-9H-carbazole-3-yl)-N-methylmethanamine) that have been demonstrated effective in advanced pre-clinical trials. Our attempt here is to identify additional targets of p53 mutants for rescue drugs and provide insight into the molecular level interactions of the drug with the mutant target. In this study PhiKan083 also known as PK083 is investigated, screened and validated on 28 different mutants of p53 using FlexX. Interaction of PhiKan08 with Y220C is found to be largely hydrophobic and a crucial single H-bond interaction with Asp228 in addition to few electrostatic interactions. Our study identified Y220S mutant as an additional target for PK083 as it shows a similar interaction pattern. Besides this, Docking and MD simulation studies, showed that Y220S binds to PK083 at higher efficiency as a result of improved steric and hydrophobic environment in the binding cavity in comparison with known mutant target, Y220C. Further, we point out that structure guided optimization of PhiKan08 can lead to an improved drug that can interact favourably with yet another mutant, Y220 N. In addition, this study revealed that Y220H and other mutants including native p53 does not provide any favourable interaction with PhiKan08 which is in accord with the experimental findings. These findings can facilitate the selection of patients for clinical studies and cancer survival analysis.

Biochemical study of survivin -31G/C promoter polymorphism in breast Cancer

Breast cancer is the most common cancer and also the leading cause of cancer mortality in women worldwide. Survivin is a member of the inhibitor of apoptosis (IAP) family, which has been identified recently. Unlike other IAP proteins, survivin is generally not found in normal adult tissues but notably expressed in the most common human cancers including stomach, colorectal, lung and breast. In cancer cells, overexpression of survivin at both protein and mRNA levels is linked to genetic variant -31G/C in the survivin promoter. P53-Abs were discovered 20 years ago during the course of tumor-associated antigens screening. The discovery of p53 mutation and accumulation of p53 in human tumors shed new light on the p53 humoral response. The aim of work: investigate -31 G/C single nucleotide polymorphism (SNP)of survivin promoter in breast cancer patients.Measurement of the level of p53 antibodies in them and detecting the association between it and the polymorphism also. Patients and methods: 100 subjects were enrolled in this study. All were women in the age between 40 and 65 (60 breast cancer cases and 40 control divided into two groups according to age: Group 1 ≤ 50 years (27.3 ± 4.87) and Group 2 > 50(24.83 ± 6.12). Results: Our study shows a significant difference in the prevalence of the survivin promoter polymorphism (–31G > C) between the case and control groups, P-value (P = 0.005*). Notably, the combined prevalence of the GC and CC genotypes (GC + CC), reflecting the prevalence of the C allele, was significantly greater in the breast cancer group than in the control group (P= 0.002*) with rates of 29% and 6%, respectively. These results imply that the C allele at position –31 in the promoter region of the survivin gene increases an individual’s susceptibility to breast cancer. Also, the risk of developing cancer was 4.05 times higher in patients with the GC or CC genotype (GC + CC) than in patients with the GG genotype, and this difference was statistically significant (95% CI: (1.48 – 11.09). Besides, in the breast cancer group, the GG genotype was present in 35 patients, whereas the GC + CC genotype was present in 25 patients. As regards the level of p53 antibodies, there was a significant difference (p=0.025) between cases (11.67±11.96) and controls (4.65 ± 0.48). But, no significant difference in the different genotypes of breast cancer patients(p=0.83). Conclusion Survivin promoter -31 G/C polymorphism is associated with the risk of developing breast cancer. C allele at position –31G/C in the promoter region of the survivin gene increases an individual’s susceptibility to breast cancer. As regards the level of p53 antibodies, there was a significant difference between cases and controls. But, no significant difference in the different genotypes of breast cancer patients.The presence of p53 Abs could be a useful marker to complement routine prognostic factors in breast cancer patients.

Tumor suppressor p53 and its gain-of-function mutants in cancer

Tumor suppressor p53 plays a pivotal role in tumor suppression. p53 is the most frequently mutated gene in cancer. As a transcription factor, p53 mainly exerts its role in tumor suppression through transcriptional regulation of its downstream target genes. Thus, p53 and its target genes form a complex p53 signaling pathway to regulate a wide variety of biological processes to prevent tumorigenesis. Recent studies have revealed that in addition to apoptosis, cell cycle arrest and senescence, p53's functions in the regulation of energy metabolism and anti-oxidant defense contribute significantly to its role in tumor suppression. Studies further show that many tumor-associated mutant p53 proteins not only lose tumor suppressive functions of wild-type p53, but also gain new oncogenic activities that are independent of wild-type p53, including promoting tumor cell proliferation, survival, metabolic changes, angiogenesis, and metastasis, which are defined as mutant p53 gain-of-function. The frequent loss of wild-type p53 function and the gain-of-function of mutant p53 in human tumors make p53 an extremely attractive target for cancer therapy. Different strategies and many small-molecule drugs are being developed for the p53-based tumor therapy. Here, we review the mechanisms of p53 in tumor suppression and gain-of-function mutant p53 in tumor development, as well as the recent advances in the development of the p53-based tumor therapy.

Spontaneous inactivating p53 mutations and the “selfish cell”

Odell et al. reported in Aging that murine embryo fibroblasts (MEFs) undergo p53 mutations and subsequent immortalizations in culture. This “release from cell cycle arrest” occurs in murine fibroblasts with a humanized or murine WT p53 [1]. The authors also noted that the cultured cells had frequently sustained a mutation matching a human tumor p53 mutation. Jang, et al. also reported spontaneous “immortalization and tumorigenic transformation” of human keratinocytes associated with p53 inactivating mutations [2]. Odell's remarkable findings are of great interest to us and support a recently proposed hypothesis for tumorigenesis. We and others have proposed that cells seek to survive, in a Darwinian sense [3, 4, 5]. Specifically we have presented evidence to suggest that while WT p53 might primarily function as the “guardian of the genome” (inducing cells to undergo apoptosis or cell cycle arrest under stress), the WT p53 gene sequence is evolutionarily maintained such that it is susceptible to inactivating mutations and consequent cell survival (with the untoward result of tumorigenesis). The facts that these mutations occur spontaneously and frequently are the exact mutations seen in human cancers lends key support to this notion. Taken together, spontaneous mutations allowing cell survival and tumorigenesis might be referred to as the “Selfish Cell Theory” in recognition of Dawkin's The Selfish Gene published in 1976.

PTEN Has Tumor-Promoting Properties in the Setting of Gain-of-Function p53 Mutations

We show, for the first time, that the tumor suppressor PTEN can have tumor-promoting properties. We show that PTEN acquires these unexpected properties by enhancing gain-of-function mutant p53 (mut-p53) protein levels. We find that PTEN restoration to cells harboring mut-p53 leads to induction of G(1)-S cell cycle progression and cell proliferation and to inhibition of cell death. Conversely, PTEN inhibition in cells expressing wild-type PTEN and mut-p53 leads to inhibition of cell proliferation and inhibition of in vivo tumor growth. We show the dependency of the tumor-promoting effects of PTEN on mut-p53 by showing that knockdown of mut-p53 expression inhibits or reverses the tumor-promoting effects of PTEN. Mechanistically, we show that PTEN expression enhances mut-p53 protein levels via inhibition of mut-p53 degradation by Mdm2 and possibly also via direct protein binding. These findings describe a novel function of PTEN and have important implications for experimental and therapeutic strategies that aim at manipulating PTEN or p53 in human tumors. They suggest that the mutational status of PTEN and p53 should be considered to achieve favorable therapeutic outcomes. The findings also provide an explanation for the low frequency of simultaneous mutations of PTEN and p53 in human cancer.

Human tumor p53 mutations are selected for in mouse embryonic fibroblasts harboring a humanized p53 gene.

To date, there has been no way to examine induced human p53 gene mutations in cell cultures exposed to mutagenic factors, other than by restriction site analysis. Here, we used embryonic cells from our Hupki (human p53 knock-in) mouse strain to generate human p53 DNA-binding domain (DBD) mutations experimentally. Twenty cultures of untreated primary mouse Hupki fibroblasts and 20 short-wavelength UV light (UVC)-treated cultures (20J/m(2)) were passaged >20 times. Established Hupki embryonic fibroblast cell lines (HUFs) were genotyped by dideoxy DNA sequencing of p53 exons 4-9. Seven of the HUFs harbored point mutations in the humanized p53 DBD. Of the 9 mutations (6 single- and 1 triple-site mutation), 2 were at the most frequently mutated codons in human cancers (c.248 and c.273). The Affymetrix p53 GeneChip assay also readily identified the 6 single-base substitutions. All mutations in HUFs from UV-treated cultures were at dipyrimidine sites, including 3 nontranscribed strand C -->T transitions. The mutant HUFs were deficient in p53 transactivation function, and missense mutants had high levels of nuclear p53 protein. In a second experiment, primary Hupki cells were exposed to the carcinogen aristolochic acid I (AAI). Five of 10 cultures that became established within 2 months harbored p53 DBD mutations. All were transversions, including 4 A --> T substitutions on the nontranscribed strand, a hallmark of DNA mutation by AAI. We conclude that establishment of Hupki mouse fibroblasts in culture readily selects for p53 DBD mutations found in human tumors, providing a basis for generating experimental mutation patterns in human p53.

Knock-in mice with a chimeric human/murine p53 gene develop normally and show wild-type p53 responses to DNA damaging agents: a new biomedical research tool.

The high prevalence and great diversity of p53 tumor suppressor gene mutations in human tumors call for development of therapeutic molecules that rescue function of aberrant p53 protein. P53 mutations also offer new approaches to the study of the origins of mutations in human cancer. An experimental mouse model with a genetically modified but normal functioning p53 gene harboring the human rather than the murine core domain, would be of considerable benefit to research on both cancer therapeutics and etiology; however, it is uncertain whether such mice would permit biological functions of p53 to be retained. Using a Cre/lox P gene-targeting approach, we have constructed a human p53 knock-in (hupki) mouse strain in which exons 4-9 of the endogenous mouse p53 allele were replaced with the homologous, normal human p53 gene sequence. The chimeric p53 allele (p53(KI)) is properly spliced, transcribed in various tissues at levels equivalent to wild-type mice, and yields cDNA with the anticipated sequence, that is, with a core domain matching that of humans. The hupki p53 protein binds to p53 consensus sequences in gel mobility shift assays and accumulates in the nucleus of hupki fibroblasts in response to UV irradiation, as is characteristic of wild-type p53. Induction of various p53-regulated genes in spleen of gamma-irradiated homozygous hupki mice (p53(KI/KI)), and the kinetics of p53-dependent apoptosis in thymocytes are similar to results with wild-type (p53(+/+)) mice, further indicating normal p53 pathway function in the hupki strain. The mice are phenotypically normal and do not develop spontaneous tumors at an early age, in contrast to knock-out (p53(-/-)) strains with a defective p53 gene. The chimeric (p53(KI)) allele thus appears to provide a biological equivalent to the endogenous murine (p53(+)) gene. This strain is a unique tool for examining in vivo spontaneous and induced mutations in human p53 gene sequences for comparison with published human tumor p53 mutation spectra. In addition, the hupki strain paves the way for mouse models in pre-clinical testing of pharmaceuticals designed to modulate DNA-binding activity of human p53.

UV-induced mutagenesis of humanp53: Analysis using a double-selection method in yeast

Comparison of the mutation patterns of p53 in human tumors with those of selectable genes in model systems is a powerful approach to identify potential etiological factors for specific tumor types. Recently, we validated use of a yeast assay to permit direct determination of the mutation spectrum induced in human p53 by carcinogens that would reduce uncertainties inherent in comparing spectra induced in different target genes. Here, we describe modifications in the assay designed to facilitate screening for mutants and to permit intracellular exposure of the gene instead of in vitro treatment. This was accomplished by introducing growth-based selection for transactivation-deficient p53 mutants into yeast already possessing red/white colony color selection. This improved model system was able to detect cells harboring p53 mutations among cells with wild-type p53 at a frequency of 10(-4) or less. Additionally, UV light was used to verify that the majority of mutagenized cells with the appropriate phenotype on selective medium contained mutations in p53, not elsewhere in the genome. Sequence analysis of UV-induced mutations revealed that the nature of the mutations was similar to those obtained in previous studies of this mutagen. This system will prove useful in the determination of the ability of environmental agents to mutate the human p53 gene, and thus may contribute to hazard identification.

UV-induced mutagenesis of human p53: Analysis using a double-selection method in yeast

Comparison of the mutation patterns of p53 in human tumors with those of selectable genes in model systems is a powerful approach to identify potential etiological factors for specific tumor types. Recently, we validated use of a yeast assay to permit direct determination of the mutation spectrum induced in human p53 by carcinogens that would reduce uncertainties inherent in comparing spectra induced in different target genes. Here, we describe modifications in the assay designed to facilitate screening for mutants and to permit intracellular exposure of the gene instead of in vitro treatment. This was accomplished by introducing growth-based selection for transactivation-deficient p53 mutants into yeast already possessing red/white colony color selection. This improved model system was able to detect cells harboring p53 mutations among cells with wild-type p53 at a frequency of 10−4 or less. Additionally, UV light was used to verify that the majority of mutagenized cells with the appropriate phenotype on selective medium contained mutations in p53, not elsewhere in the genome. Sequence analysis of UV-induced mutations revealed that the nature of the mutations was similar to those obtained in previous studies of this mutagen. This system will prove useful in the determination of the ability of environmental agents to mutate the human p53 gene, and thus may contribute to hazard identification. Environ. Mol. Mutagen. 35:31–38, 2000 © 2000 Wiley-Liss, Inc.

New approaches to understanding p53 gene tumor mutation spectra

The first p53 gene mutation arising in a human tumor was described a decade ago by Baker et al. [S.J. Baker, E.R. Fearon, J.M. Nigro, S.R. Hamilton, A.C. Preisinger, J.M. Jessup, P. van Tuinen, D.H. Ledbetter, D.F. Barker, Y. Nakamura, R. White, B. Vogelstein, Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas, Science 244 (1989) 217–221]. There are now over 10,000 mutations extracted from the published literature in the IARC database of human p53 tumor mutations [P. Hainaut, T. Hernandez, A. Robinson, P. Rodriguez-Tome, T. Flores, M. Hollstein, C.C. Harris, R. Montesano, IARC database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualization tools, Nucleic Acids Res. 26 (1998) 205–213; Version R3, January 1999]. A large and diverse collection of tumor mutations in cancer patients provides important information on the nature of environmental factors or biological processes that are important causes of human gene mutation, since xenobiotic mutagens as well as endogenous mechanisms of genetic change produce characteristic types of patterns in target DNA [J.H. Miller, Mutational specificity in bacteria, Annu. Rev. Genet. 17 (1983) 215–238; T. Lindahl, Instability and decay of the primary structure of DNA, Nature 362 (1993) 709–715; S.P. Hussain, C.C. Harris, Molecular epidemiology of human cancer: contribution of mutation spectra studies of tumor suppressor genes, Cancer Res. 58 (1998) 4023–4037; P. Hainaut, M. Hollstein, p53 and human cancer: the first ten thousand mutations, Adv. Cancer Res. 2000]. P53 gene mutations in cancers can be compared to point mutation spectra at the HPRT locus of human lymphocytes from patients or healthy individuals with known exposure histories, and accumulated data indicate that mutation patterns at the two loci share certain general features. Hypotheses regarding specific cancer risk factors can be tested by comparing p53 tumor mutations typical of a defined patient group against mutations generated experimentally in rodents or in prokaryotic and eukaryotic cells in vitro. Refinements of this approach to hypothesis testing are being explored that employ human p53 sequences introduced artificially into experimental organisms used in laboratory mutagenesis assays. P53-specific laboratory models, combined with DNA microchips designed for high through-put mutation screening promise to unmask information currently hidden in the compilation of human tumor p53 mutations.

WILD-TYPE P53 IN CELLULAR-TRANSFORMATION - A REASSESSMENT

We found that a mouse p53 cDNA clone (pP53-5; Jenkins et al, Nature 312: 651-654, 1984), which previously was characterized as encoding mutant p53 protein, in fact represents wild-type mouse p53 cDNA, as we were able to demonstrate that the mutations described represented sequencing artefacts (compressions). Such sequencing artefacts were also observed with a p53 cDNA isolated from mouse T3T3 cells, encoding a mutant p53 with an Arg-Cys exchange at position 270, and could be resolved by sequencing of the opposite DNA strands. As pP53-5 had been successfully used in cellular immortalization and transformation assays (Jenkins et al, Nature 312: 651-654, 1984; Nature 317: 816-818, 1985), our results suggest that wild-type p53 under certain circumstances can induce transformation. This finding is relevant to the recent findings of overexpressed wild-type p53 in human tumors.

Different Tumor-Derived p53 Mutants Exhibit Distinct Biological Activities

In its wild-type form, the protein p53 can interfere with neoplastic processes. Tumor-derived cells often express mutant p53. Full-length mutant forms of p53 isolated so far from transformed mouse cells exhibit three common properties in vitro: loss of transformation-suppressing activity, gain of pronounced transforming potential, and ability to bind the heat shock protein cognate hsc70. A tumor-derived mouse p53 variant is now described, whose site of mutation corresponds to a hot spot for p53 in human tumors. While absolutely nonsuppressing, it is only weakly transforming and exhibits no detectable hsc70 binding. The data suggest that the ability of a p53 mutant to bind endogenous p53 is not the sole determinant of its oncogenic potential. The data also support the existence of gain-of-function p53 mutants.