Mutant p53: it’s not all one and the same

Mutation of the TP53 tumor suppressor gene is the most common genetic alteration in cancer, and almost 1000 alleles have been identified in human tumors. While virtually all TP53 mutations are thought to compromise wild type p53 activity, the prevalence and recurrence of missense TP53 alleles has motivated countless research studies aimed at understanding the function of the resulting mutant p53 protein. The data from these studies support three distinct, but perhaps not necessarily mutually exclusive, mechanisms for how different p53 mutants impact cancer: first, they lose the ability to execute wild type p53 functions to varying degrees; second, they act as a dominant negative (DN) inhibitor of wild type p53 tumor-suppressive programs; and third, they may gain oncogenic functions that go beyond mere p53 inactivation. Of these possibilities, the gain of function (GOF) hypothesis is the most controversial, in part due to the dizzying array of biological functions that have been attributed to different mutant p53 proteins. Herein we discuss the current state of understanding of TP53 allele variation in cancer and recent reports that both support and challenge the p53 GOF model. In these studies and others, researchers are turning to more systematic approaches to profile TP53 mutations, which may ultimately determine once and for all how different TP53 mutations act as cancer drivers and whether tumors harboring distinct mutations are phenotypically unique. From a clinical perspective, such information could lead to new therapeutic approaches targeting the effects of different TP53 alleles and/or better sub-stratification of patients harboring TP53 mutant cancers.Subject terms: Cancer genetics, Tumour-suppressor proteins     

Mutant p53 reactivation by small molecules makes its way to the clinic

The TP53 tumor suppressor gene is mutated in many human tumors, including common types of cancer such as colon and ovarian cancer. This illustrates the key role of p53 as trigger of cell cycle arrest or cell death upon oncogenic stress. Most TP53 mutations are missense mutations that result in single amino acid substitutions in p53 and expression of high levels of dysfunctional p53 protein. Restoration of wild type p53 function in such tumor cells will induce robust cell death and allow efficient eradication of the tumor. Therapeutic targeting of mutant p53 in tumors is a rapidly developing field at the forefront of translational cancer research. Various approaches have led to the identification of small molecules that can rescue mutant p53. These include compounds that target specific p53 mutations, including PK083 and PK5174 (Y220C mutant p53) and NSC319726 (R175H mutant p53), as well as PRIMA-1 and its analog APR-246 that affect a wider range of mutant p53 proteins. APR-246 has been tested in a Phase I/II clinical trial with promising results.     

Mutant p53R175H upregulates Twist1 expression and promotes epithelial–mesenchymal transition in immortalized prostate cells

A mutation within one allele of the p53 tumor suppressor gene can inactivate the remaining wild-type allele in a dominant-negative manner and in some cases can exert an additional oncogenic activity, known as mutant p53 ‘gain of function'' (GOF). To study the role of p53 mutations in prostate cancer and to discriminate between the dominant-negative effect and the GOF activity of mutant p53, we measured, using microarrays, the expression profiles of three immortalized prostate epithelial cultures expressing wild-type, inactivated p53 or mutated p53. Analysis of these gene expression profiles showed that both inactivated p53 and p53^R175H mutant expression resulted in the upregulation of cell cycle progression genes. A second group, which was upregulated exclusively by mutant p53^R175H, was predominantly enriched in developmental genes. This group of genes included the Twist1, a regulator of metastasis and epithelial–mesenchymal transition (EMT). Twist1 levels were also elevated in metastatic prostate cancer-derived cell line DU145, in immortalized lung fibroblasts and in a subset of lung cancer samples, all in a mutant p53-dependent manner. p53^R175H mutant bearing immortalized epithelial cells showed typical features of EMT, such as higher expression of mesenchymal markers, lower expression of epithelial markers and enhanced invasive properties in vitro. The mechanism by which p53^R175H mutant induces Twist1 expression involves alleviation of the epigenetic repression. Our data suggest that Twist1 expression might be upregulated following p53 mutation in cancer cells.     

Phenotype Specific Analyses Reveal Distinct Regulatory Mechanism for Chronically Activated p53

The downstream functions of the DNA binding tumor suppressor p53 vary depending on the cellular context, and persistent p53 activation has recently been implicated in tumor suppression and senescence. However, genome-wide information about p53-target gene regulation has been derived mostly from acute genotoxic conditions. Using ChIP-seq and expression data, we have found distinct p53 binding profiles between acutely activated (through DNA damage) and chronically activated (in senescent or pro-apoptotic conditions) p53. Compared to the classical ‘acute’ p53 binding profile, ‘chronic’ p53 peaks were closely associated with CpG-islands. Furthermore, the chronic CpG-island binding of p53 conferred distinct expression patterns between senescent and pro-apoptotic conditions. Using the p53 targets seen in the chronic conditions together with external high-throughput datasets, we have built p53 networks that revealed extensive self-regulatory ‘p53 hubs’ where p53 and many p53 targets can physically interact with each other. Integrating these results with public clinical datasets identified the cancer-associated lipogenic enzyme, SCD, which we found to be directly repressed by p53 through the CpG-island promoter, providing a mechanistic link between p53 and the ‘lipogenic phenotype’, a hallmark of cancer. Our data reveal distinct phenotype associations of chronic p53 targets that underlie specific gene regulatory mechanisms.     

p53 Mutations: Gains or losses?

Although the case for p53 as a tumor suppressor gene appears very strong, one should still keep an open eye for the possibility that mutations in p53 do not necessarily imply a mere loss of "suppressor" activity. It is still possible that the presence of a p53 mutation in a tumor contributes, in a dominant positive manner, to tumorigenesis. In other words, certain p53 mutants may well be oncogenic in their own right, and carry distinct activities that promote growth deregulation and malignant progression. Elucidating this issue also has practical implications, since the nature of the resident mutations may greatly dictate the consequences of attempts to reintroduce wild-type (wt) p53 into particular types of tumor cells. There are two major obstacles along the road to meaningful answers: the limitations of the experimental systems used for evaluating the biological activities of wt and mutant p53 and a fundamental lack of knowledge about the relevant biochemistry of the p53 protein. These two aspects constitute primary experimental challenges for investigators in the field.     

Genetic selection of intragenic suppressor mutations that reverse the effect of common p53 cancer mutations.

Several lines of evidence suggest that the presence of the wild-type tumor suppressor gene p53 in human cancers correlates well with successful anti-cancer therapy. Restoration of wild-type p53 function to cancer cells that have lost it might therefore improve treatment outcomes. Using a systematic yeast genetic approach, we selected second-site suppressor mutations that can overcome the deleterious effects of common p53 cancer mutations in human cells. We identified several suppressor mutations for the V143A, G245S and R249S cancer mutations. The beneficial effects of these suppressor mutations were demonstrated using mammalian reporter gene and apoptosis assays. Further experiments showed that these suppressor mutations could override additional p53 cancer mutations. The mechanisms of such suppressor mutations can be elucidated by structural studies, ultimately leading to a framework for the discovery of small molecules able to stabilize p53 mutants.     

Codon 249 of the human TP53 tumor suppressor gene is no hot spot for aflatoxin B1 in a heterologous background

Mutations in the TP53 tumor suppressor gene are the most common alteration in cancer, and human primary liver cancers related to previous dietary exposure to the mycotoxin aflatoxin B1 (AFB1) exhibit a specific hot spot mutation at TP53 codon 249. We have asked whether the 249 hot spot is related to a particular susceptibility to AFB1 of this TP53 region or whether it is related to a phenotype of the 249S p53 mutant protein. This was addressed by constructing a metabolically competent variant of Saccharomyces cerevisiae strain yIG397 expressing human cytochrome P450 1A2 and P450-reductase and isolating AFB1-induced mutants that failed to express the genomic ADE2 reporter gene. Molecular analysis revealed that only 8/40 mutants had a mutation in the TP53 target gene, whereas 32/40 mutants were due to a recombination event eliminating the ADE2 reporter gene. None of 19 mutations identified in the eight mutant TP53 plasmids altered codon 249, thus this codon was no hot spot if the TP53 gene was in the heterologous background yeast. The genotoxic action of AFB1 was completely different from that of the alkylating agent ethyl-methane-sulfonate, where 28/30 induced mutations were linked to the TP53 target gene.     

突变体p53研究进展

抑癌基因突变是癌症发生过程中一个极为关键的事件。p53作为体内最重要的抑癌基因之一, 在人类癌症中发生突变的频率高达50%。同时, p53突变也是人类遗传病Li-Fraumeni综合征的主要病因。p53最常见的突变形式是错义突变, 所形成的突变体p53不但失去了野生型p53的抑癌功能, 而且还获得了一系列类似于癌基因的功能, 促进了肿瘤的进程。文章拟对突变体p53的结构功能改变, 获得癌基因活性的分子机制, 以及近年来对封闭突变体p53活性所进行的探索等研究方向所取得的进展做一综述。     

TP53 mutations in circulating tumor DNA in advanced epidermal growth factor receptor-mutant lung adenocarcinoma patients treated with gefitinib

Tumor protein p53 (TP53) is a tumor suppressor gene and TP53 mutations are associated with poor prognosis in non-small cell lung cancer. However, the in-depth classification of TP53 and its relationship with treatment response and prognosis in epidermal growth factor receptor (EGFR)-mutant tumors treated with EGFR tyrosine kinase inhibitors are unclear. Circulating tumor DNA was prospectively collected at baseline in advanced treatment-naïve EGFR-mutant lung adenocarcinoma patients treated with gefitinib in an open-label, single-arm, prospective, multicenter, phase 2 clinical trial (BENEFIT trial) and analyzed using next-generation sequencing. Survival was estimated using the Kaplan–Meier method. Of the 180 enrolled patients, 115 (63.9%) harbored TP53 mutations. The median progression-free survival (PFS) and overall survival (OS) of patients with TP53-wild type tumors were significantly longer than those of patients with TP53-mutant tumors. Mutations in exons 5–8 accounted for 80.9% of TP53 mutations. Mutations in TP53 exons 6 and 7 were significantly associated with inferior PFS and OS compared to wild-type TP53. TP53 mutation also influenced the prognosis of patients with different EGFR mutations. Patients with TP53 and EGFR exon 19 mutations had significantly longer PFS and OS than patients with TP53 and EGFR L858R mutations, and both groups had worse survival than patients with only EGFR mutations. Patients with TP53 mutations, especially in exons 6 and 7, had a lower response rate and shorter PFS and OS when treated with gefitinib. Moreover, TP53 exon 5 mutation divided TP53 mutations in disruptive and non-disruptive types.     

Gain-of-function hot spot mutant p53R248Q regulation of integrin/FAK/ERK signaling in esophageal squamous cell carcinoma

PurposeTP53, encoding the protein p53, is among the most frequently mutated genes in all cancers. A high frequency of 60 – 90% mutations is seen in esophageal squamous cell carcinoma (ESCC) patients. Certain p53 mutants show gain-of-function (GoF) oncogenic features unrelated to its wild type functions.MethodsThis study functionally characterized a panel of p53 mutants in individual ESCC cell lines and assayed for GoF oncogenic properties.ResultsThe ESCC cell line with endogenous p53^R248Q expression showed suppressed tumor growth in an immunocompromised mouse model and suppressed colony growth in in vitro three-dimensional culture, when depleted of the endogenous p53 protein expression. This suppression is accompanied by suppressed cell cycle progression, along with reduced integrin expression and decreased focal adhesion kinase and extracellular-regulated protein kinase signaling and can be compensated by expression of a constitutively active mitogen-activated protein. P53^R248Q enhances cell proliferation upon glutamine deprivation, as compared to other non-GoF mutants.ConclusionsIn summary, study of the functional contributions of endogenous p53 mutants identified a novel GoF mechanism through which a specific p53 mutant exerts oncogenic features and contributes to ESCC tumorigenesis.     

Polymorphisms in p53 and the p53 pathway: roles in cancer susceptibility and response to treatment

The p53 tumour suppressor protein lies at the crossroads of multiple cellular response pathways that control the fate of the cell in response to endogenous or exogenous stresses and inactivation of the p53 tumour suppressor signalling pathway is seen in most human cancers. Such aberrant p53 activity may be caused by mutations in the TP53 gene sequence producing truncated or inactive mutant proteins, or by aberrant production of other proteins that regulate p53 activity, such as gene amplification and overexpression of MDM2 or viral proteins that inhibit or degrade p53. Recent studies have also suggested that inherited genetic polymorphisms in the p53 pathway influence tumour formation, progression and/or response to therapy. In some cases, these variants are clearly associated with clinico-pathological variables or prognosis of cancer, whereas in other cases the evidence is less conclusive. Here, we review the evidence that common polymorphisms in various aspects of p53 biology have important consequences for overall tumour susceptibility, clinico-pathology and prognosis. We also suggest reasons for some of the reported discrepancies in the effects of common polymorphisms on tumourigenesis, which relate to the complexity of effects on tumour formation in combination with other oncogenic changes and other polymorphisms. It is likely that future studies of combinations of polymorphisms in the p53 pathway will be useful for predicting tumour susceptibility in the human population and may serve as predictive biomarkers of tumour response to standard therapies.     

Co-localization of mutant p53 and amyloid-like protein aggregates in breast tumors

P53 is one of the most important tumor suppressor proteins in human cancers. Mutations in the TP53 gene are common features of malignant tumors and normally correlate to a more aggressive disease. In breast cancer, these gene alterations are present in approximately 20% of cases and are characteristically of missense type. In the present work we describe TP53 mutations in breast cancer biopsies and investigate whether wild and mutant p53 participate in protein aggregates formation in these breast cancer cases. We analyzed 88 biopsies from patients residing in the metropolitan area of Rio de Janeiro, and performed TP53 mutation screening using direct sequencing of exons 5-10. Seventeen mutations were detected, 12 of them were of missense type, 2 nonsenses, 2 deletions and 1 insertion. The presence of TP53 mutation was highly statistically associated to tumor aggressiveness of IDC cases, indicated here by Elston Grade III (p<0.0001). Paraffin embedded breast cancer tissues were analyzed for the presence of p53 aggregates through immunofluorescence co-localization assay, using anti-aggregate primary antibody A11, and anti-p53. Our results show that mutant p53 co-localizes with amyloid-like protein aggregates, depending on mutation type, suggesting that mutant p53 may form aggregates in breast cancer cells, in vivo.     

Inhibition of nonsense-mediated decay rescues p53β/γ isoform expression and activates the p53 pathway in MDM2-overexpressing and select p53-mutant cancers

Inactivation of p53 is present in almost every tumor, and hence, p53-reactivation strategies are an important aspect of cancer therapy. Common mechanisms for p53 loss in cancer include expression of p53-negative regulators such as MDM2, which mediate the degradation of wildtype p53 (p53α), and inactivating mutations in the TP53 gene. Currently, approaches to overcome p53 deficiency in these cancers are limited. Here, using non–small cell lung cancer and glioblastoma multiforme cell line models, we show that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising exons 1 to 9β or 9γ, respectively) and that lack the C-terminal MDM2-binding domain have markedly reduced susceptibility to MDM2-mediated degradation but are highly susceptible to nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53β and p53γ and restores activation of the p53 pathway. Consistent with p53 pathway activation, NMD inhibition induces tumor suppressive activities such as apoptosis, reduced cell viability, and enhanced tumor radiosensitivity, in a relatively p53-dependent manner. In addition, NMD inhibition also inhibits tumor growth in a MDM2-overexpressing xenograft tumor model. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in p53-deficient tumors bearing MDM2 overexpression or p53 mutations downstream of exon 9, subgroups that comprise approximately 6% of all cancers.     

TP53: a key gene in human cancer

TP53 is mutated in most types of human cancers and is one of the most popular genes in cancer research. The p53 protein is a sensor of multiple forms of genotoxic, oncogenic and non-genotoxic stress. It suppresses growth and controls survival of stressed cells, and as such, is the focal point of selection pressures in tissues exposed to carcinogens or to oncogenic changes. Thus, the clonal expansion of cells with mutations in TP53 may be seen as the result of a selection process intrinsic to the natural history of cancer. In this review, we discuss the nature of these various forms of selection pressure. We present a hypothesis to explain why TP53 is often mutated as either an early or a late event in cancer. Furthermore, we also summarise current knowledge on the molecular consequences of mutation for loss of wild-type protein function, dominant-negative activity, and a possible gain of oncogenic function.     

Predicting Positive p53 Cancer Rescue Regions Using Most Informative Positive (MIP) Active Learning

Many protein engineering problems involve finding mutations that produce proteins with a particular function. Computational active learning is an attractive approach to discover desired biological activities. Traditional active learning techniques have been optimized to iteratively improve classifier accuracy, not to quickly discover biologically significant results. We report here a novel active learning technique, Most Informative Positive (MIP), which is tailored to biological problems because it seeks novel and informative positive results. MIP active learning differs from traditional active learning methods in two ways: (1) it preferentially seeks Positive (functionally active) examples; and (2) it may be effectively extended to select gene regions suitable for high throughput combinatorial mutagenesis. We applied MIP to discover mutations in the tumor suppressor protein p53 that reactivate mutated p53 found in human cancers. This is an important biomedical goal because p53 mutants have been implicated in half of all human cancers, and restoring active p53 in tumors leads to tumor regression. MIP found Positive (cancer rescue) p53 mutants in silico using 33% fewer experiments than traditional non-MIP active learning, with only a minor decrease in classifier accuracy. Applying MIP to in vivo experimentation yielded immediate Positive results. Ten different p53 mutations found in human cancers were paired in silico with all possible single amino acid rescue mutations, from which MIP was used to select a Positive Region predicted to be enriched for p53 cancer rescue mutants. In vivo assays showed that the predicted Positive Region: (1) had significantly more (p<0.01) new strong cancer rescue mutants than control regions (Negative, and non-MIP active learning); (2) had slightly more new strong cancer rescue mutants than an Expert region selected for purely biological considerations; and (3) rescued for the first time the previously unrescuable p53 cancer mutant P152L.     

Gain of Function of p53 Cancer Mutants in Disrupting Critical DNA Damage Response Pathways

Loss of the tumor suppression activity of p53 is required for the progression of most human cancers. In this context, p53 gene is somatically mutated in about half of all human cancers; in the rest human cancers, p53 is mostly inactivated due to the disruption of pathways important for its activation. Most p53 cancer mutations are missense mutations within the core domain, leading to the expression of full-length mutant p53 protein. The expression of p53 mutants is usually correlated with the poor prognosis of the cancer patients. Accumulating evidence has indicated that p53 cancer mutants not only lose the tumor suppression activity of WT p53, but also gain novel oncogenic activities to promote tumorigenesis and drug resistance. Therefore, to improve current cancer therapy, it is critical to elucidate the gain-of-functions of p53 cancer mutants. By analyzing the humanized p53 mutant knock-in mouse models, we have identified a new gain of function of the common p53 cancer mutants in inducing genetic instability by disrupting ATM-mediated cellular responses to DNA double-stranded break (DSB) damage. Considering that some current cancer therapies such as radiotherapy kills the cancer cells by inducing DSBs in their genome DNA, our findings will have important implications on the treatment of human cancers that express common p53 mutants.