Gain-of-function mutant p53-R280K mediates survival of breast cancer cells

Missense mutations in TP53 resulting in the expression of p53-R175H, p53-R273H, or p53-R280K are frequently detected in human breast cancer. Currently, the role of mutant p53-R280K in breast cancer is relatively unknown, and therefore, the present study analyzed the function of mutant p53-R280K in breast cancer cell growth. To this end, we used small interfering RNA to study the role of mutant p53-R280K in MDA-MB-231 cells, which endogenously express the mutant protein. We found that curcumin induced apoptosis in MDA-MB-231 cells and downregulated mutant p53-R280K. We also observed that knockdown of mutant p53 by small interfering RNA induced apoptosis in MDA-MB-231 cells. Curcumin-induced apoptosis was further enhanced by the overexpression of wild-type p53, but was decreased by mutant p53-R280K overexpression. Our findings indicate that mutant p53-R280K has an important role in mediating the survival of triple-negative breast cancer MDA-MB-231 cells. Furthermore, this study suggests mutant p53-R280K could be used as a therapeutic target for breast cancer cells harboring this TP53 missense mutation.     

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.     

Only Missense Mutations Affecting the DNA Binding Domain of P53 Influence Outcomes in Patients with Breast Carcinoma

The presence of a TP53 gene mutation can influence tumour response to some treatments, especially in breast cancer. In this study, we analysed p53 mRNA expression, LOH at 17p13 and TP53 mutations from exons 2 to 11 in 206 patients with breast carcinoma and correlated the results with disease-free and overall survival. The observed mutations were classified according to their type and location in the three protein domains (transactivation domain, DNA binding domain, oligomerization domain) and correlated with disease-free and overall survival. In our population, neither p53 mRNA expression nor LOH correlated with outcome. Concerning TP53 mutations, 27% of tumours were mutated (53/197) and the presence of a mutation in the TP53 gene was associated with worse overall survival (p = 0.0026) but not with disease-free survival (p = 0.0697), with median survival of 80 months and 78 months, respectively. When alterations were segregated into mutation categories and locations, and related to survival, tumours harbouring mutations other than missense mutations in the DNA binding domain of P53 had the same survival profiles as wild-type tumours. Concerning missense mutations in the DNA binding domain, median disease-free and overall survival was 23 months and 35 months, respectively (p = 0.0021 and p<0.0001, respectively), compared with 78 and 80 months in mutated tumours overall. This work shows that disease-free and overall survival in patients with a frameshift mutation of TP53 or missense mutation in the oligomerization domain are the same as those in wild-type TP53 patients.     

Splicing mutations in TP53 in human squamous cell carcinoma lines influence immunohistochemical detection.

The mutational status of the tumor suppressor gene TP53 is often examined by immunohistochemistry. We compared the incidence of TP53 mutations in 12 permanent squamous cell carcinoma lines of the head and neck with the immunohistochemical staining obtained with two different antibodies. The mutational status of the TP53 gene was assessed by sequencing the complete coding frame of the TP53 mRNA. All 12 tumor cell lines had TP53 mutations. Six of them showed missense mutations and five had premature stop codons caused either by splicing mutations or nonsense mutations or by exon skipping. One tumor cell line was heterozygous, with a truncating splicing mutation and an additional missense mutation located on different alleles. In one case, an in-frame insertion of 23 extra codons was found. All missense mutations were positive in immunhistochemistry and Western blotting. The truncated p53 was not immunohistochemically detected in three cases with the DO-7 antibody and in five cases with the G59-12 antibody, giving false-negative results in 25% or 40%, respectively, of all tumor cell lines examined. We conclude that splicing mutations are common in squamous cell carcinoma lines and that the incidence of p53 inactiviation by erroneous splicing is higher than yet reported. Sequencing of only the exons of TP53 may miss intronic mutations leading to missplicing and may therefore systematically underestimate the TP53 mutation frequency.     

Multiple mutations of the p53 gene in human mammary carcinoma

Alteration of the p53 tumor suppressor gene is the most common genetic abnormality in human cancer. In breast cancer, depending on the stage of disease and method of detection, mutation rates of 25-60% have been observed. Multiple mutations of p53 gene in the same tumor however, are rarely reported. In this study we explored the frequency of multiple mutations of p53 gene in mammary carcinoma in a cohort of south Florida patients. Three hundred eighty-four cases of primary breast cancer diagnosed between 1984 and 1986 at the University of Miami, Jackson Medical Center were subjects of this study. Sequence analysis of exons 5 through 8 of p53 was performed on cloned PCR-amplified DNA of formalin-fixed, paraffin-embedded tumors. Two hundred thirty-four of 384 breast cancers (61%) had p53 mutation. Of those, 36 tumors showed more than one mutation; 31 tumors had two mutations, three showed three, one tumor had five mutations, and one case carried six mutations. The majority of mutations were missense (43) followed by silent (35); and most occurred within a single exon. Our study suggests that multiple mutations of p53 suppressor gene in breast cancer are more common than currently believed.     

Evaluation of arrayed primer extension for TP53 mutation detection in breast and ovarian carcinomas

Mutations in the tumor suppressor gene TP53 are associated with a wide range of different cancers and may have prognostic and therapeutic implications. Methods for rapid and sensitive detection of mutations in this gene are therefore required. In order to make screening more effective, a commercially available TP53 genotyping microarray from Asper Biotech has been constructed by arrayed primer extension (APEX). The present study is the first report that blindly evaluates the efficiency of the second generation APEX TP53 genotype chip outside the Asper laboratory and compares it to temporal temperature gradient electrophoresis (TTGE) and sequencing of TP53 for mutation detection in ovarian and breast cancer samples. All nucleotides in the TP53 gene from exon 2-9 are included on the chip by synthesis and application of sequence-specific oligonucleotides. The chip was validated by screening 48 breast and 11 ovarian cancer cases, all of which had previously been analyzed by TTGE and sequencing. APEX scored 17 of 20 sequence variants, missing one deletion, one insertion, and a missense mutation. Resequencing efficiency using APEX was 92% for both DNA strands and 99.5% for sense and/or antisense strand. We conclude that the APEX TP53 microarray is a robust, rapid, and comprehensive screening tool for sequence alterations in tumors.     

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     

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。     

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.     

TP53: an oncogene in disguise

The standard classification used to define the various cancer genes confines tumor protein p53 (TP53) to the role of a tumor suppressor gene. However, it is now an indisputable fact that many p53 mutants act as oncogenic proteins. This statement is based on multiple arguments including the mutation signature of the TP53 gene in human cancer, the various gains-of-function (GOFs) of the different p53 mutants and the heterogeneous phenotypes developed by knock-in mouse strains modeling several human TP53 mutations. In this review, we will shatter the classical and traditional image of tumor protein p53 (TP53) as a tumor suppressor gene by emphasizing its multiple oncogenic properties that make it a potential therapeutic target that should not be underestimated. Analysis of the data generated by the various cancer genome projects highlights the high frequency of TP53 mutations and reveals that several p53 hotspot mutants are the most common oncoprotein variants expressed in several types of tumors. The use of Muller''s classical definition of mutations based on quantitative and qualitative consequences on the protein product, such as ‘amorph'', ‘hypomorph'', ‘hypermorph'' ‘neomorph'' or ‘antimorph'', allows a more meaningful assessment of the consequences of cancer gene modifications, their potential clinical significance, and clearly demonstrates that the TP53 gene is an atypical cancer gene.     

neu/ERBB2 cooperates with p53-172H during mammary tumorigenesis in transgenic mice.

Thirty percent of human breast cancers have amplification of ERBB2, often in conjunction with mutations in p53. The most common p53 mutation in human breast cancers is an Arg-to-His mutation at codon 175, an allele that functions in a dominant oncogenic manner in tumorigenesis assays and is thus distinct from loss of p53. Transgenic mice expressing mouse mammary tumor virus-driven neu transgene (MMTV-neu) develop clonal mammary tumors with a latency of 234 days, suggesting that other events are necessary for tumor development. We have examined the role of mutations in p53 in tumor development in these mice. We have found that 37% of tumors arising in these mice have a missense mutations in p53. We have directly tested for cooperativity between neu and mutant p53 in mammary tumorigenesis by creating bitransgenic mice carrying MMTV-neu and 172Arg-to-His p53 mutant (p53-172H). In these bitransgenic mice, tumor latency is shortened to 154 days, indicating strong cooperativity. None of the nontransgenic mice or the p53-172H transgenic mice developed tumors within this time period. Tumors arising in the p53-172H/neu bitransgenic mice were anaplastic and aneuploid and exhibited increased apoptosis, in distinction to tumors arising in p53-null mice, in which apoptosis is diminished. Further experiments address potential mechanisms of cooperativity between the two transgenes. In these bitransgenic mice, we have recapitulated two common genetic lesions that occur in human breast cancer and have shown that p53 mutation is an important cooperating event in neu-mediated oncogenesis.     

Activating Somatic FGFR2 Mutations in Breast Cancer

It is known that FGFR2 gene variations confer a risk for breast cancer. FGFR2 and FGF10, the main ligand of FGFR2, are both overexpressed in 5–10% of breast tumors. In our study, we sequenced the most important coding regions of FGFR2 in somatic tumor tissue of 140 sporadic breast cancer patients and performed MLPA analysis to detect copy number variations in FGFR2 and FGF10. We identified one somatic heterozygous missense mutation, p.K660N (c.1980G>C), within the tyrosine kinase domain of FGFR2 in tumor tissue of a sporadic breast cancer patient, which is likely mediated by the FGFR2-IIIb isoform. The presence of wild type and mutated alleles in equal quantities suggests that the mutation has driven clonal amplification of mutant cells. We have analyzed the tyrosine kinase activity of p.K660N and another recently described somatic breast cancer mutation in FGFR2, p.R203C, after expression in HEK293 cells and demonstrated that the intrinsic tyrosine kinase activity of both mutant proteins is strongly increased resulting in elevated phosphorylation and activity of downstream effectors. To our knowledge, this is the first report of functional analysis of somatic breast cancer mutations in FGFR2 providing evidence for the activating nature of FGFR2-mediated signalling in the pathogenesis of breast cancer.     

Missense mutations in cancer suppressor gene TP53 are colocalized with exonic splicing enhancers (ESEs)

Mutation databases can be viewed as footprints of functional organization of a gene and thus can be used to infer its functional organization. We studied the association of exonic splicing enhancers (ESEs) with missense mutations in the tumor suppressor gene TP53 using the International Agency for Research on Cancer (IARC) mutation database. The goals of the study were: (i) to verify the hypothesis that deleterious missense mutations are colocalized with ESEs; (ii) to identify potentially functional ESE sites in the open reading frame (ORF) of the TP53. If some sequence functions as a splicing enhancer, then nucleotide substitutions in the site will disturb splicing, abrogate p53 function, and cause an increased susceptibility to cancer. Therefore, among cancers showing p53 mutations, more missense mutations are expected within functional ESE sites as compared to non-functional ESE motifs. Using several statistical tests, we found that missense mutations in TP53 are strongly colocalized with ESEs, and that only a small fraction of ESE sites contributes to the association. There are usually one or two ESEs per exon showing a statistically significant association with missense mutations--so-called significant ESE sites. In many respects significant ESE sites are different from those that do not show association with missense mutations. We found that positions of significant ESE sites are codon-dependent--significant ESEs preferentially start from the first position of a codon, whereas non-significant ESEs show no position dependence. Significant ESEs showed a more limited set of sequences compared to non-significant ESEs. These findings suggest that there is a limited number of missense mutations that influence ESE sites and our analysis provides further insight into the types of sites that harbor exonic enhancer elements.     

Different mutation profiles associated to P53 accumulation in colorectal cancer

The tumor suppressor TP53 gene is one of the most frequently mutated in different types of human cancer. Particularly in colorectal cancer (CRC), it is believed that TP53 mutations play a role in the adenoma–carcinoma transition of tumors during pathological process. In order to analyze TP53 expressed alleles in CRC, we examined TP53 mRNA in tumor samples from 101 patients with sporadic CRC. Samples were divided in two groups defined according to whether they exhibit positive or negative P53 protein expression as detected by immunohistochemistry (IHC). The presence of TP53 mutation was a common event in tumors with an overall frequency of 54.5%. By direct sequencing, we report 42 different TP53 sequence changes in 55 CRC patients, being two of them validated polymorphisms. TP53 mutations were more frequent in positive than in negative P53 detection group (p < 0.0001), being the precise figures 79.6% and 30.8%, respectively. In addition, the mutation profiles were also different between the two groups of samples; while most of the mutations detected in P53 positive group were missense (38 out of 39), changes in P53 negative detection group include 7 insertions/deletions, 6 missense, 2 nonsense and 1 silent mutation. As previously observed, most mutations were concentrated in regions encoding P53 DNA binding domain (DBD). Codons 175, 248 and 273 together account for 36.7% of point mutations, in agreement with previous observations provided that these codons are considered mutation hotspots. Interestingly, we detected two new deletions and two new insertions. In addition, in three samples we detected two deletions and one insertion that could be explained as putative splicing variants or splicing errors.     

Anti-P53 antibodies in Brazilian brain tumor patients

Gliomas of astrocytic origin are the most common primary brain tumors, accounting for over 40 to 50% of all central nervous system tumors. The TP53 tumor suppressor gene is the most frequently mutated gene found in human malignancies. A mutation of this gene can lead to an increased half-life of the resulting protein and loss of biological function. High levels of p53 have been detected in the serum of colon cancer patients, although p53 protein has not been detected in the serum of brain tumor patients. Besides circulating p53, several studies have detected antibodies against p53 in patients with lung and breast cancer, as well as those with other types of cancer. We studied p53 protein and anti-p53 antibodies in the plasma of Brazilian brain tumor patients. Plasma samples were drawn from 24 untreated brain tumor patients and from 15 healthy donors without clinical signs of cancer. Western blotting techniques were used to detect p53 protein and anti-p53 antibodies. We found anti-p53 antibodies in 5/24 brain tumor patients. Age appears to affect the immune response, as four of six tumor patients under 16 years old had detectable anti-p53 antibodies, while these were found in only 1 of 18 adults (over 16 years old). We found no p53 protein in any of the serum samples from the brain tumors. Possibly the presence of this protein is affected by tumor type or by the organs that are sampled.     

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.     

Mutational spectra of human cancer

The purpose of this review is to summarize the evidence that can be used to reconstruct the etiology of human cancers from mutations found in tumors. Mutational spectra of the tumor suppressor gene p53 (TP53) are tumor specific. In several cases, these mutational spectra can be linked to exogenous carcinogens, most notably for sunlight-associated skin cancers, tobacco-associated lung cancers, and aristolochic acid-related urothelial tumors. In the TP53 gene, methylated CpG dinucleotides are sequences selectively targeted by endogenous and exogenous mutagenic processes. Recent high-throughput sequencing efforts analyzing a large number of genes in cancer genomes have so far, for the most part, produced mutational spectra similar to those in TP53 but have unveiled a previously unrecognized common G to C transversion mutation signature at GpA dinucleotides in breast cancers and several other cancers. Unraveling the origin of these G to C mutations will be of importance for understanding cancer etiology.