TP53 Mutations in Human Cancers: Origins, Consequences, and Clinical Use

Somatic mutations in the TP53 gene are one of the most frequent alterations in human cancers, and germline mutations are the underlying cause of Li-Fraumeni syndrome, which predisposes to a wide spectrum of early-onset cancers. Most mutations are single-base substitutions distributed throughout the coding sequence. Their diverse types and positions may inform on the nature of mutagenic mechanisms involved in cancer etiology. TP53 mutations are also potential prognostic and predictive markers, as well as targets for pharmacological intervention. All mutations found in human cancers are compiled in the IARC TP53 Database (http://www-p53.iarc.fr/). A human TP53 knockin mouse model (Hupki mouse) provides an experimental model to study mutagenesis in the context of a human TP53 sequence. Here, we summarize current knowledge on TP53 gene variations observed in human cancers and populations, and current clinical applications derived from this knowledge.Genetic variations in the tumor suppressor gene TP53 (OMIM #191117) contribute to human cancers in different ways. First, somatic mutations are frequent in most cancers (Hollstein et al. 1991). The antiproliferative role of p53 protein in response to various stresses and during physiological processes such as senescence makes it a primary target for inactivation in cancer (Levine 1997). The main modes of TP53 inactivation are single-base substitution and loss of alleles, with inactivation by viral or cellular proteins playing a major role in specific cancers (Tommasino et al. 2003). Second, inheritance of a TP53 mutation causes predisposition to early-onset cancers including breast carcinomas, sarcomas, brain tumors, and adrenal cortical carcinomas, defining the Li-Fraumeni (LFS) and Li-Fraumeni-like (LFL) syndromes (Li et al. 1988; Olivier et al. 2003). Third, TP53 is highly polymorphic in coding and noncoding regions and some of these polymorphisms have been shown to increase cancer susceptibility and to modify cancer phenotypes in TP53 mutation carriers (Whibley et al. 2009).Whereas tumor suppressors are commonly inactivated by frameshift or nonsense mutations, most TP53 mutations are missense and cause single amino-acid changes at many different positions. Mutations are thus diverse in their type, sequence context, position, and structural impact, making it possible to identify mutation patterns in relation with cancer type and etiology. The occurrence of special mutation patterns may inform on the nature of the mutagens that have caused them, making TP53 an interesting gene to analyze in the realm of molecular epidemiology.Data on mutation prevalence in human cancer can be conveniently accessed through the IARC TP53 database (http://www-p53.iarc.fr/), a resource that compiles all TP53 gene variations reported in human cancers with annotations on tumor phenotype, patient characteristics, and structural and functional impact of mutations (Petitjean et al. 2007b). Recently, it has become possible to confront these observations with experimental data generated in a novel mouse model, the HupKi mouse, that contains a human TP53 sequence at the mouse TP53 locus and recapitulates the effects of environmental mutagens in a human sequence context (Luo et al. 2001). In this article, we review the current knowledge on the origin, causes, and consequences of TP53 variations and mutations in cancer and we discuss their significance as biomarkers in epidemiology and in the clinics.     

Mutant <Emphasis Type="Italic">TP53</Emphasis> G245C and R273H promote cellular malignancy in esophageal squamous cell carcinoma

Background

TP53 gene mutations occur in more than 50% of human cancers and the vast majority of these mutations in human cancers are missense mutations, which broadly occur in DNA binding domain (DBD) (Amino acids 102–292) and mainly reside in six “hotspot” residues. TP53 G245C and R273H point mutations are two of the most frequent mutations in tumors and have been verified in several different cancers. In the previous study of the whole genome sequencing (WGS), we found some mutations of TP53 DBD in esophageal squamous cell carcinoma (ESCC) clinical samples. We focused on two high-frequent mutations TP53 p.G245C and TP53 p.R273H and investigated their oncogenic roles in ESCC cell lines, p53-defective cell lines H1299 and HCT116 p53?/?.

Results

MTS and colony formation assays showed that mutant TP53 G245C and R273H increased cell vitality and proliferation. Flow cytometry results revealed inhibition of ultraviolet radiation (UV)- and ionizing radiation (IR)- induced apoptosis and disruption of TP53-mediated cell cycle arrest after UV, IR and Nocodazole treatment. Transwell assays indicated that mutant TP53 G245C and R273H enhanced cell migration and invasion abilities. Moreover, western blot revealed that they were able to suppress the expression of TP53 downstream genes in the process of apoptosis and cell cycle arrest induced by UV, which suggests that these two mutations can influence apoptosis and growth arrest might be due, at least in part, to down-regulate the expression of P21, GADD45α and PARP.

Conclusions

These results indicate that mutant TP53 G245C and R273H can lead to more aggressive phenotypes and enhance cancer cell malignancy, which further uncover TP53 function in carcinogenesis and might be useful in clinical diagnosis and therapy of TP53 mutant cancers.

     

p53 mutational spectra and the role of methylated CpG sequences

The occurrence of tumor-specific mutational spectra in the p53 mutation database provides indirect evidence that implicates certain exogenous and possibly endogenous mutagenic events in human carcinogenesis. In some cases, the distribution of DNA damage along the p53 gene caused by environmental carcinogens can be correlated with the mutational spectra, i.e. hotspots and types of mutations of certain cancers, most notably for nonmelanoma skin cancers and lung cancers in smokers. This concept has been validated by experiments with sunlight and cigarette smoke components representing the polycyclic aromatic hydrocarbon class of carcinogens. A disproportionally high number of mutations in p53 (and other genes) are found at methylated CpG dinucleotides. These sequences are particularly prone to mutagenesis involving endogenous events as well as modification by exogenous carcinogens.     

Gaining insights into relevance across cancers based on mutation features of TP53 gene

The tumor suppressor gene TP53, one of the most frequently mutated genes, is recognized as the guardian of genome and can provide a significant barrier to neoplastic transformation and tumor progression. Traditional theory believes that TP53 mutations are equal among cancer types. However, to date, no study has explored the TP53 mutation profile from a holistic and systematic standpoint to discovery its relevance and feature with cancers. Mutation signature, an unbiased approach to identify the mutational processes, can be a potent indicator for exploring mutation-driven tumor occurrence and progression. In this research, several features such as hotspots, mutability and mutation signature of somatic TP53 mutations derived from 18 types of cancer tissues from cBioPortal were analyzed and manifested the organizational preference among cancers. Mutation signatures found in almost all cancer types were Signature 6 related to mismatch repair deficiency, and Signature 1 that reflects the natural decomposition of 5-methylcytosine into thymine associated with aging. Meanwhile, several signatures of TP53 mutations displayed tissue-selective. Mutations enriched in bladder, skin, lung cancer were associated with signatures of APOBEC activity (Signature 2 and 13), alkylating agents (Signature 11), and tobacco smoke (Signature 4), respectively. Moreover, Signature 4 and 29 associated with tobacco smoking or chewing found in lung, sarcoma, esophageal, and head and neck cancer may be related to their smoking history. In addition, several digestive cancers, including colorectal, stomach, pancreatic and esophageal cancers, showed the high correlation in context and mutation signature profiles. Our study suggests that the tissue-selective activity of mutational processes would reflect the tissue-specific enrichment of TP53 mutations and provides a new perspective to understand the relevance of diverse diseases based on the spectrum of TP53 mutations.     

brca2 and tp53 Collaborate in Tumorigenesis in Zebrafish

Germline mutations in the tumor suppressor genes BRCA2 and TP53 significantly influence human cancer risk, and cancers from humans who inherit one mutant allele for BRCA2 or TP53 often display loss of the wildtype allele. In addition, BRCA2-associated cancers often exhibit mutations in TP53. To determine the relationship between germline heterozygous mutation (haploinsufficiency) and somatic loss of heterozygosity (LOH) for BRCA2 and TP53 in carcinogenesis, we analyzed zebrafish with heritable mutations in these two genes. Tumor-bearing zebrafish were examined by histology, and normal and neoplastic tissues were collected by laser-capture microdissection for LOH analyses. Zebrafish on a heterozygous tp53^M214K background had a high incidence of malignant tumors. The brca2^Q658X mutation status determined both the incidence of LOH and the malignant tumor phenotype. LOH for tp53 occurred in the majority of malignant tumors from brca2 wildtype and heterozygous mutant zebrafish, and most of these were malignant peripheral nerve sheath tumors. Malignant tumors in zebrafish with heterozygous mutations in both brca2 and tp53 frequently displayed LOH for both genes. In contrast, LOH for tp53 was uncommon in malignant tumors from brca2 homozygotes, and these tumors were primarily undifferentiated sarcomas. Thus, carcinogenesis in zebrafish with combined mutations in tp53 and brca2 typically requires biallelic mutation or loss of at least one of these genes, and the specific combination of inherited mutations influences the development of LOH and the tumor phenotype. These results provide insight into cancer development associated with heritable BRCA2 and TP53 mutations.     

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     

p53 and disease: when the guardian angel fails

The p53 tumor suppressor gene (TP53) is mutated more often in human cancers than any other gene yet reported. Of importance, it is mutated frequently in the common human malignancies of the breast and colorectum and also, but less frequently, in other significant human cancers such as glioblastomas. There is also one inherited cancer predisposing syndrome called Li-Fraumeni that is caused by TP53 mutations. In this review, we discuss the significance of p53 mutations in some of the above tumors with a view to outlining how p53 contributes to malignant progression. We also discuss the usefulness of TP53 status as a prognostic marker and its role as a predictor of response to therapy. Finally, we outline some evidence that abnormalities in p53 function contribute to the etiology of other non-neoplastic diseases.     

Co-Evolution of Somatic Variation in Primary and Metastatic Colorectal Cancer May Expand Biopsy Indications in the Molecular Era

IntroductionMetastasis is thought to be a clonal event whereby a single cell initiates the development of a new tumor at a distant site. However the degree to which primary and metastatic tumors differ on a molecular level remains unclear. To further evaluate these concepts, we used next generation sequencing (NGS) to assess the molecular composition of paired primary and metastatic colorectal cancer tissue specimens.Methods468 colorectal tumor samples from a large personalized medicine initiative were assessed by targeted gene sequencing of 1,321 individual genes. Eighteen patients produced genomic profiles for 17 paired primary:metastatic (and 2 metastatic:metastatic) specimens.ResultsAn average of 33.3 mutations/tumor were concordant (shared) between matched samples, including common well-known genes (APC, KRAS, TP53). An average of 2.3 mutations/tumor were discordant (unshared) among paired sites. KRAS mutational status was always concordant. The overall concordance rate for mutations was 93.5%; however, nearly all (18/19 (94.7%)) paired tumors showed at least one mutational discordance. Mutations were seen in: TTN, the largest gene (5 discordant pairs), ADAMTS20, APC, MACF1, RASA1, TP53, and WNT2 (2 discordant pairs), SMAD2, SMAD3, SMAD4, FBXW7, and 66 others (1 discordant pair).ConclusionsWhereas primary and metastatic tumors displayed little variance overall, co-evolution produced incremental mutations in both. These results suggest that while biopsy of the primary tumor alone is likely sufficient in the chemotherapy-naïve patient, additional biopsies of primary or metastatic disease may be necessary to precisely tailor therapy following chemotherapy resistance or insensitivity in order to adequately account for tumor evolution.     

Distinct mutational backgrounds and clonal architectures implicated prognostic discrepancies in small-cell carcinomas of the esophagus and lung

Small-cell carcinoma of the esophagus (SCCE) is a rare and aggressive cancer. Although several consistent genomic changes were observed previously between SCCE and small-cell lung cancer (SCLC), detailed mutational landscapes revealing discrepancies in genetic underpinnings of tumorigenesis between these two cancers are scarce, and little attention has been paid to answer whether these genetic alterations were related to the prognosis. Herein by performing whole-exome sequencing of 48 SCCE and 64 SCLC tumor samples, respectively we have shown that the number of driver mutations in SCCE was significantly lower than in SCLC (p = 0.0042). In SCCE, 46% of recurrent driver mutations were clonal, which occurred at an early stage during tumorigenesis, while 16 driver mutations were found clonal in SCLC. NOTCH1/3, PIK3CA, and ATM were specifically clonal in SCCE, while TP53 was clonal in SCLC. The total number of clonal mutations differed between two cancers and presented lower in SCCE compared to SCLC (p = 0.0036). Moreover, overall survival (OS) was shorter in patients with higher numbers of clonal mutations for both cancers. In summary, SCCE showed distinct mutational background and clonal architecture compared with SCLC. Organ-specific clonal events revealed different molecular mechanisms underlying tumorigenesis, tumor development, patients’ prognosis, and possible variations in therapeutic outcomes to candidate treatments.Subject terms: Oesophageal cancer, Small-cell lung cancer     

Computational Screening and Molecular Dynamic Simulation of Breast Cancer Associated Deleterious Non-Synonymous Single Nucleotide Polymorphisms in TP53 Gene

Breast cancer is one of the most common cancers among the women around the world. Several genes are known to be responsible for conferring the susceptibility to breast cancer. Among them, TP53 is one of the major genetic risk factor which is known to be mutated in many of the breast tumor types. TP53 mutations in breast cancer are known to be related to a poor prognosis and chemo resistance. This renders them as a promising molecular target for the treatment of breast cancer. In this study, we present a computational based screening and molecular dynamic simulation of breast cancer associated deleterious non-synonymous single nucleotide polymorphisms in TP53. We have predicted three deleterious coding non-synonymous single nucleotide polymorphisms rs11540654 (R110P), rs17849781 (P278A) and rs28934874 (P151T) in TP53 with a phenotype in breast tumors using computational tools SIFT, Polyphen-2 and MutDB. We have performed molecular dynamics simulations to study the structural and dynamic effects of these TP53 mutations in comparison to the wild-type protein. Results from our simulations revealed a detailed consequence of the mutations on the p53 DNA-binding core domain that may provide insight for therapeutic approaches in breast cancer.     

Deep sequencing of multiple regions of glial tumors reveals spatial heterogeneity for mutations in clinically relevant genes

Background

The extent of intratumoral mutational heterogeneity remains unclear in gliomas, the most common primary brain tumors, especially with respect to point mutation. To address this, we applied single molecule molecular inversion probes targeting 33 cancer genes to assay both point mutations and gene amplifications within spatially distinct regions of 14 glial tumors.

Results

We find evidence of regional mutational heterogeneity in multiple tumors, including mutations in TP53 and RB1 in an anaplastic oligodendroglioma and amplifications in PDGFRA and KIT in two glioblastomas (GBMs). Immunohistochemistry confirms heterogeneity of TP53 mutation and PDGFRA amplification. In all, 3 out of 14 glial tumors surveyed have evidence for heterogeneity for clinically relevant mutations.

Conclusions

Our results underscore the need to sample multiple regions in GBM and other glial tumors when devising personalized treatments based on genomic information, and furthermore demonstrate the importance of measuring both point mutation and copy number alteration while investigating genetic heterogeneity within cancer samples.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0530-z) contains supplementary material, which is available to authorized users.     

Base damage,local sequence context and TP53 mutation hotspots: a molecular dynamics study of benzo[a]pyrene induced DNA distortion and mutability

The mutational pattern for the TP53 tumour suppressor gene in lung tumours differs to other cancer types by having a higher frequency of G:C>T:A transversions. The aetiology of this differing mutation pattern is still unknown. Benzo[a]pyrene,diol epoxide (BPDE) is a potent cigarette smoke carcinogen that forms guanine adducts at TP53 CpG mutation hotspot sites including codons 157, 158, 245, 248 and 273. We performed molecular modelling of BPDE-adducted TP53 duplex sequences to determine the degree of local distortion caused by adducts which could influence the ability of nucleotide excision repair. We show that BPDE adducted codon 157 has greater structural distortion than other TP53 G:C>T:A hotspot sites and that sequence context more distal to adjacent bases must influence local distortion. Using TP53 trinucleotide mutation signatures for lung cancer in smokers and non-smokers we further show that codons 157 and 273 have the highest mutation probability in smokers. Combining this information with adduct structural data we predict that G:C>T:A mutations at codon 157 in lung tumours of smokers are predominantly caused by BPDE. Our results provide insight into how different DNA sequence contexts show variability in DNA distortion at mutagen adduct sites that could compromise DNA repair at well characterized cancer related mutation hotspots.     

Reporting Tumor Molecular Heterogeneity in Histopathological Diagnosis

Background

Detection of molecular tumor heterogeneity has become of paramount importance with the advent of targeted therapies. Analysis for detection should be comprehensive, timely and based on routinely available tumor samples.

Aim

To evaluate the diagnostic potential of targeted multigene next-generation sequencing (TM-NGS) in characterizing gastrointestinal cancer molecular heterogeneity.

Methods

35 gastrointestinal tract tumors, five of each intestinal type gastric carcinomas, pancreatic ductal adenocarcinomas, pancreatic intraductal papillary mucinous neoplasms, ampulla of Vater carcinomas, hepatocellular carcinomas, cholangiocarcinomas, pancreatic solid pseudopapillary tumors were assessed for mutations in 46 cancer-associated genes, using Ion Torrent semiconductor-based TM-NGS. One ampulla of Vater carcinoma cell line and one hepatic carcinosarcoma served to assess assay sensitivity. TP53, PIK3CA, KRAS, and BRAF mutations were validated by conventional Sanger sequencing.

Results

TM-NGS yielded overlapping results on matched fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues, with a mutation detection limit of 1% for fresh-frozen high molecular weight DNA and 2% for FFPE partially degraded DNA. At least one somatic mutation was observed in all tumors tested; multiple alterations were detected in 20/35 (57%) tumors. Seven cancers displayed significant differences in allelic frequencies for distinct mutations, indicating the presence of intratumor molecular heterogeneity; this was confirmed on selected samples by immunohistochemistry of p53 and Smad4, showing concordance with mutational analysis.

Conclusions

TM-NGS is able to detect and quantitate multiple gene alterations from limited amounts of DNA, moving one step closer to a next-generation histopathologic diagnosis that integrates morphologic, immunophenotypic, and multigene mutational analysis on routinely processed tissues, essential for personalized cancer therapy.     

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.     

Advances in carcinogenesis: a historical perspective from observational studies to tumor genome sequencing and TP53 mutation spectrum analysis

Tumor sequencing projects have been initiated over the last decade with the promising goal of identifying novel cancer genes and potential therapeutic targets. One of the unexpected findings of these projects was the discovery that cancer genomes contain thousands of passenger mutations that are irrelevant to tumor development and are coselected by a small number of driver mutations that constitute the true selection power in cancer progression. Although often discarded and considered to be irrelevant, the value of passenger mutations should not be underestimated, as they are the most important markers of the exposure to various carcinogens and are essential to assess the etiology of individual tumors. Over the last century, the history of cancer epidemiology evolved in different stages and concepts from occupational observational studies beginning in the 18th century, in vitro and in vivo experimental analyses and cancer gene analyses, such as Ha-ras or TP53. Mutation spectra of passenger mutations from various types of cancers not only confirm the findings of molecular epidemiology analysis, but also reveal novel profiles that will extend this knowledge to single tumors in all types of cancer.