TP53 mutations in human meningiomas

Overexpression of p53 has been reported to play a role in the development of neoplasms of the central nervous system. Meningiomas are generally benign intracranial tumors originating from the meninges. Overexpression of the p53 protein in meningiomas and an association with histological type and recurrence has been reported. Mutation of the TP53 gene leads to a more stable p53 protein in quantities high enough for detection by immunohistochemistry. In the search for these mutations the core domain of the TP53 gene of meningiomas has been analyzed. Only a very low incidence of mutations was reported. The apparent discordance between overexpression of p53 protein and TP53 gene mutations may be explained by mutations located outside the core domain. This issue was addressed in the present study. All 11 exons of 17 meningiomas were analyzed for DNA alterations by PCR single-strand conformation polymorphism (PCR-SSCP) analysis with subsequent sequencing. PCR-SSCP analysis showed a various number of band shifts and nucleotide alterations, caused either by alterations in the flanking introns or common polymorphisms (codon 36 and 72). The allele frequencies of the polymorphisms found in this small population of tumors resemble the frequencies reported in the literature. In addition, three nucleotide changes located in introns 2, 3 and 7 were found in 11, 3 and 4, respectively, of 17 specimens. Based on this study and on reports by others we conclude that it is not very likely that TP53 mutations are involved in the etiology of meningiomas.     

TP53 and TP53-related genes associated with protection from apoptosis in the radioadaptive response

We investigated the effect of administering priming low-dose radiation prior to high-dose radiation on the level of apoptosis and on the expression of TP53 and TP53-related genes in mouse splenocytes. The percentage of apoptotic cells was significantly lower in TP53(+/+) mice receiving priming radiation 2 to 168 h before the high-dose irradiation, compared to TP53(+/+) mice exposed to 2 Gy alone. In contrast, TP53(+/-) mice exhibited a reduced level of apoptosis only when priming was performed for 2 or 4 h prior to the high-dose irradiation. In TP53(+/+) mice, primed mice had higher TP53 expression than mice exposed to 2 Gy. Phospho-TP53 (ser15/18) expression was the highest in mice exposed to 2 Gy and intermediate in primed mice. Expression of p21 (CDKN1A) was higher in primed mice compared with mice exposed to 2 Gy. MDM2 expression remained at a high level in all mice receiving 2 Gy. Elevated phospho-ATM expression was observed only in mice exposed to 2 Gy. We conclude that TP53 plays a critical role in the radioadaptive response and that TP53 and TP53-related genes might protect cells from apoptosis through activation of the intracellular repair system.     

TP53 and mutations in human cancer

TP53 is the most frequently mutated gene in human cancer, with a predominance of missense mutations scattered over 200 codons. In many cancers, specific mutation patterns can be identified, which are shaped by site-specific mutagenesis and by biological selection. In tobacco-related cancers (lung, head and neck), organ-specific patterns are observed, with many mutations compatible with the ones experimentally induced by tobacco carcinogens. In several other cancers, such as squamous cell carcinoma of the oesophagus or hepatocellular carcinoma (HCC), mutation patterns show geographic variations between regions of high and low incidence, suggesting a role for region-specific risk factors. HCC from high-incidence regions showing also a high prevalence of a specific Ser-249 TP53 mutation is one of the most striking examples of a mutagen fingerprint. All such assessments are useful to generate clues on the mutagenic mechanisms involved in human cancer. Moreover, it has been shown that DNA retrieved from plasma can be successfully used for detection of TP53 mutations, which gives hope for earlier more accurate detection of human cancers.     

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.     

Uncoupling elephant TP53 and cancer

Elephant testicles do not descend, with implications for sperm production being hot enough to compromise germline DNA replication/repair. Uniquely, elephants also possess 20 copies of a gene encoding for the p53 protein. Did elephants evolve multiplication of the TP53 gene complex to protect their germline rather than to fight cancer?     

Restoration of mutant TP53 to normal TP53 function by glycerol as a chemical chaperone

We have previously reported that heat stress induces expression of wild-type TP53 (formerly known as p53) activated factor 1 (CDKN1A, formerly known as WAF1) only when TP53 protein is wild-type using cells of a human glioblastoma cell line (A-172) and cells of its transformant (A-172/mp53/ 143) with a mutant TP53 (point mutation at codon 143 from Val to Ala) vector. Transfection of A-172 cells with the mutant TP53 vector abolished the heat-induced expression of CDKN1A, demonstrating the dominant negative nature of this TP53 mutant over the endogenous wild-type TP53. This kind of dominant negative TP53 mutant occurs frequently in various types of cancer. Overcoming this dominance or restoring the normal functions to these TP53 mutants is a new strategy for TP53-targeted cancer therapies. We examined whether glycerol can act as a chemical chaperone to correct the mutant TP53 conformation. No CDKN1A expression was induced after heating or treatment with glycerol at concentrations of 0.6 and 1.2 M in these transformants. In contrast, A-172/mp53/ 143 cells showed CDKN1A expression when they were heated in the presence of glycerol at 0.6 or 1.2 M, which was similar to the response of the parental and neo vector-transfected control cells. To test the generality of the effects of glycerol on mutant TP53, we used human osteosarcoma Saos-2 cells (lacking TP53) transfected with mutant TP53 and cells of two other human glioblastoma cell lines carrying mutant TP53. These cells showed similar CDKN1A expression when heated in the presence of glycerol at 0.6 or 1.2 M. These results suggest that glycerol is effective in restoring several TP53 mutants to normal TP53 function, leading to normal CDKN1A expression after heat stress. This observation provides a novel tool for correction of mutant TP53 conformation and may be applicable for TP53-targeted cancer therapy.     

TP53 and lacZ mutagenesis induced by 3-nitrobenzanthrone in Xpa-deficient human TP53 knock-in mouse embryo fibroblasts

3-Nitrobenzanthrone (3-NBA) is a highly mutagenic compound and possible human carcinogen found in diesel exhaust. 3-NBA forms bulky DNA adducts following metabolic activation and induces predominantly G:C > T:A transversions in a variety of experimental systems. Here we investigated the influence of nucleotide excision repair (NER) on 3-NBA-induced mutagenesis of the human tumour suppressor gene TP53 and the reporter gene lacZ. To this end we utilised Xpa -knockout (Xpa-Null) human TP53 knock-in (Hupki) embryo fibroblasts (HUFs). As Xpa is essential for NER of bulky DNA adducts, we hypothesized that DNA adducts induced by 3-NBA would persist in the genomes of Xpa-Null cells and lead to an increased frequency of mutation. The HUF immortalisation assay was used to select for cells harbouring TP53 mutations following mutagen exposure. We found that Xpa-Null Hupki mice and HUFs were more sensitive to 3-NBA treatment than their wild-type (Xpa-WT) counterparts. However, following 3-NBA treatment and immortalisation, a similar frequency of TP53-mutant clones arose from Xpa-WT and Xpa-Null HUF cultures. In cells from both Xpa genotypes G:C > T:A transversion was the predominant TP53 mutation type and mutations exhibited bias towards the non-transcribed strand. Thirty-two percent of 3-NBA-induced TP53 mutations occurred at CpG sites, all of which are hotspots for mutation in smokers’ lung cancer (codons 157, 158, 175, 245, 248, 273, 282). We also examined 3-NBA-induced mutagenesis of an integrated lacZ reporter gene in HUFs, where we again observed a similar mutant frequency in Xpa-WT and Xpa-Null cells. Our findings suggest that 3-NBA-DNA adducts may evade removal by global genomic NER; the persistence of 3-NBA adducts in DNA may be an important factor in its mutagenicity.     

TP53 codon 72 polymorphism in pigmentary phenotypes

The p53 protein exerts different cellular functions, and recent findings have demonstrated its influence on the cascade of skin pigmentation during UV exposure. Among TP53 gene polymorphisms, the most studied is the G to C transversion in exon 4 at codon 72, which results in three distinct genotypes, Arg/Arg, Pro/Pro and Arg/Pro, each one encoding different p53 isoforms. Therefore, this study aimed to determine the relationship between TP53 codon 72 polymorphism and skin protection against sunburn. Genomic DNA was extracted from peripheral blood samples and genotyping was performed by PCR and confirmed by restriction enzyme digestion. The genotype frequency was 50% for Arg/Arg and 14.6% for Pro/Pro genotype. The frequency of heterozygous subjects was 35.4%. In our population, p53 genotypes were in Hardy-Weinberg (HW) equilibrium (X2 HM less than 3.84), showing a predominance of arginine allele (total Arg allele frequency of 68%). No significant association between p53 genotype and skin colour, hair or eye colour and susceptibility to sun exposure was found. However, further analysis demonstrated a significant association between the genotype Pro/Pro and blue/green eyes among participants who presented redness (P=0.016). Our findings indicate susceptibility to sun exposure when this phenotype (eye colour) occurs simultaneously with Pro/Pro genotype.     

A degradative detour for mutant TP53

Accumulation of mutant TP53 proteins in cancer cells has been recognized as an important factor that promotes cancer progression and metastasis. Thus, strategies that promote the degradation of mutant TP53 might be beneficial for the treatment of cancers. In a recent issue of Genes & Development, we demonstrated that blocking macroautophagy under nutritional stress condition leads to the degradation of mutant TP53 through activating the chaperone-mediated autophagy (CMA) pathway in nonproliferating cancer cells. We propose CMA as a new degradative mechanism for mutant TP53 and the possibility of activating CMA as a new treatment for cancers with mutant TP53.     

Differential response to radiation of TP53-inactivated cells by overexpression of dominant-negative mutant TP53 or HPVE6

The inactivation of TP53 by transfection of a dominant- negative mutated TP53 (MP53.13 cells) was compared with inactivation of TP53 by transfection with the HPV E6 gene (RC10.1 cells) with respect to PLD repair, G(1)-phase arrest, and induction of color junctions. Functional G(1) arrest was demonstrated in parental (RKO) cells with wild-type TP53, while in RC10.1 cells the G(1) arrest was eliminated. In MP53.13 cells an intermediate G(1) arrest was found. Functionality of endogenous TP53 was confirmed in RKO and MP53.13 cells by accumulation of TP53 protein and its downstream target CDKN1A (p21). Radiation survival of MP53.13 cells was higher than that of RKO cells, and PLD repair was found in RKO cells and MP53.13 cells but not in RC10.1 cells. Both with and without irradiation, the number of color junctions was 50 to 80% higher in MP53.13 cells than in RKO and RC10.1 cells. In the MP53.13 cells, the genetic instability appears to lead to more aberrations and to radioresistance. In spite of the presence of an excess of mutated TP53, wild- type TP53 functions appear to be affected only partly or not at all.     

Centrosome amplification as a possible mechanism for numerical chromosome aberrations in cerebral primitive neuroectodermal tumors with TP53 mutations

Although alterations in chromosome number have frequently been detected in human tumor cells and associated with tumor initiation and progression, the causal mechanisms are still not understood. One protein known to be involved in maintaining genetic stability is tumor suppressor p53. In mice, p53 has been implicated in the maintenance of diploidy (Cross et al., 1995) and the regulation of centrosome duplication (Fukasawa et al., 1996). Here we report on cerebral primitive neuroectodermal tumors that lacked the wild-type p53 gene (TP53) and showed multiple numerical chromosome aberrations, as detected by comparative genomic hybridization. In these tumors, the centrosome number was significantly higher than in a control tumor without a detected TP53 mutation and with few chromosomal imbalances. These findings indicate that abnormal centrosome amplification can occur in human tumors lacking wild-type TP53 and may be a mechanism by which numerical chromosome aberrations are generated.     

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.     

Targeting vasculature in urologic tumors: mechanistic and therapeutic significance

Recent advances toward understanding the molecular mechanisms regulating cancer initiation and progression provide new insights into the therapeutic value of targeting tumor vascularity by interfering with angiogenic signaling pathways. The functional contribution of key angiogenic factors toward increased vascularity characterizing metastatic tumors and their therapeutic exploitation is considered in three major urologic malignancies, renal, bladder, and prostate cancer. With the realization that the success of the therapeutic efficacy of the various anti-angiogenic approaches for the treatment of urologic tumors has yet to be proven clinically, the challenge remains to select critical angiogenesis pathways that can be targeted for an individual tumor. Here we discuss the major mechanisms that support formation of vasculature in renal, bladder, and prostate tumors and the current results of targeting of specific molecules/regulators for therapeutic intervention against metastastic disease.     

TP53 on trial for cancer gene therapy

Adenovirus-mediated p53 gene transfer in patients with advanced recurrent head and neck squamous cell carcinomaClayman G.L. et al. (1998)J. Clin. Oncol. 16, 2221–2232Gene therapy for non-small cell lung cancer: a preliminary report of a phase I trial of adenoviral p53 gene replacementRoth, J.A. et al. (1998)Semin. Oncol. 25, 33–37     

TP53 in gastric cancer: mutations in the l3 loop and LSH motif DNA-binding domains of TP53 predict poor outcome

The aim of this study was to clarify whether specific p53 mutations may have biological relevance in terms of disease relapse or death in gastric carcinomas (GC). Resected specimens from a consecutive series of 62 patients with GC undergoing potentially curative surgery were prospectively studied. The mutational status of exons 5-8 of the p53 gene was investigated in 62 cases using the PCR-SSCP and sequencing. Presence of microsatellite instability (MSI) was evaluated in 56 cases by analyzing loci highly sensitive of MSI. Twenty mutations of p53 were detected in 17 of the 62 cases analyzed (27%). Ten mutations (50%) occurred in highly conserved domains. According to the p53 specific functional domains: 4/20 mutations (20%) were in the L3 loop and 3/20 (15%) in LSH motif. Eight of the 56 GC resulted MSI-H, 5 (9%) MSI-L, and 43 (77%) MSI stable (MSS). None of the 8 (14%) MSI-H GC showed p53 mutations. p53 mutations were associated with intestinal histotype. Moreover, specific mutations in functional domain (L3 and LSH), together with advanced TNM stage, node involvement, depth of invasion, diffuse histotype, proved to be significantly related to quicker relapse and to shorter overall survival. Specific mutations in p53 functional domains, rather than any mutations in this gene, may be biologically more significant in terms of patients outcome, indicating that these mutations might have biological relevance to identify subgroups of patients at higher risk of relapse or death who might benefit from a more aggressive therapeutic approach.     

UV and melanoma: the TP53 link

Ultraviolet radiation (UVR) is a major risk factor for melanoma development, but it has been unclear exactly how UVR leads to melanomagenesis. In a recent publication in Nature, Viros et al. identify TP53/Trp53 as a UVR-target gene in melanoma and show that UVR-induced TP53/Trp53 mutations accelerate BRAF(V600E)-driven melanomagenesis.Melanoma is the deadliest skin cancer, and its incidence has relentlessly increased over recent decades. According to the American Cancer Society''s estimates for melanoma in the United States for 2014, about 76 100 new melanomas will be diagnosed and about 9 710 people are expected to die from melanoma. It is well known that UVR is the major environmental factor contributing to melanomagenesis¹. This suggests that there is at least a component of melanoma risk which may be preventable through UVR protective strategies — an issue of immense public health importance due to the availability of sunblocks and sun-safe behaviors. Importantly, while many studies have been conducted to elucidate the link between UVR and melanoma, the precise molecular mechanism(s) by which UVR triggers melanoma formation have remained incompletely understood.Recently, a powerful UVR-induced skin inflammatory response has been shown to provoke metastasis of melanoma² and the presence of UV signature mutations has also been reported throughout the melanoma exome, including recurrent melanoma genes such as RAC1, PPP6C, and STK19^3,4. Previous mouse models for UVR-induced melanoma revealed that UVR-induced inflammation promoted melanomagenesis in neonatal mice^5,6,7. These studies underlined UVR''s significant contribution to melanoma formation. In a recent study published in Nature, Viros et al.⁸ address the role of UVR in previously established BRAF(V600E)-expressing melanocytes in vivo, and demonstrate that significantly accelerated melanoma formation often associated with mutations in TP53/Trp53. To mimic both somatic mutation acquisition and mild sunburn in humans, BRAF(V600E) was expressed at physiological levels in adult mice which were subsequently exposed to repeated low doses of UVR. In addition, certain mice were partially covered with UVR-proof cloth or topically treated with SunSense Milk Sunscreen SPF50 (2.2 mg/cm²) 30 min before UVR exposure, to assess the impact of these protective strategies.UVR was seen to significantly accelerate melanoma formation in mice whose melanocytes express BRAF(V600E), but not in BRAF wild-type mice (which unlike BRAF(V600E)-expressing mice do not develop long latency melanomas independently of UVR). Application of UVR-proof cloth or sunscreen delayed the onset of UVR-driven melanoma and partially prevented acceleration of BRAF(V600E)-driven melanomagenesis by UVR, and sunscreen-protected UVR-exposed BRAF(V600E) mice developed a reduced number of melanomas compared with unprotected UVR-exposed BRAF(V600E) mice (Figure 1). More somatic single nucleotide variants and a significantly higher proportion of C-to-T transitions at the 3′ end of pyrimidine dimers were observed in UVR-exposed melanomas, providing direct evidence of UVR-induced DNA damage. In addition, Trp53 mutations (H39Y, S124F, R245C, R270C, C272G) were detected in UVR-exposed BRAF(V600E) mouse melanomas, indicating a direct role of UVR in the induction of Trp53 mutations in melanoma. The mutated corresponding residues (S127F/S124F, R248C/R245C, R273C/R270C, C275G/C272G) were also identified in TP53 mutations in human melanoma, suggesting that TP53 mutations are linked to evidence of UVR-induced DNA damage in human melanoma. These results are consistent with previous reports that p53 deletion accelerates BRAF(V600E)-driven melanomagenesis both in mice⁹ and in zebrafish¹⁰, but demonstrate the ability of UVR to inflict UV signature mutations within the gene as has been widely observed in non-melanoma skin cancers and also in human melanomas.Open in a separate windowFigure 1A diagram depicting feasible routes of BRAF(V600E)-driven melanomagenesis.This elegant study by Viros et al. clearly helps to establish key roles of UVR in melanomagenesis, and further validates the functional importance of TP53 as a UVR-targeted tumor suppressor gene in a fraction of melanomas. The study also raises several intriguing questions worthy of follow-up analysis. For example, through which mechanism(s) did sunscreen or sunshielding delay but not prevent UVR-induced melanoma? Induction of cutaneous inflammatory changes that are less anatomically restricted to UV irradiated fields, would seem to be an attractive mechanism. This may help to explain the known risk of melanoma in both sun-exposed and less-exposed skin of lightly pigmented people. It is also valuable to better understand the role of UVB vs UVA wavelengths in melanomagenesis. Mechanistically, these distinct regions of the UV spectrum inflict largely distinctive chemical alterations on the genome. Efforts to block UVA as well as UVB in commercial sunscreen products are currently being promoted by the US Food and Drug Administration, a welcome improvement to sun protection strategies. Still, the precise role(s) of UVA in melanomagenesis remain incompletely understood and may involve both cell-autonomous and non-cell-autonomous targets. In addition to the acceleration of BRAF(V600E)-driven melanoma formation by UVR, red pigment (pheomelanin) has also been observed to accelerate BRAF(V600E)-driven melanomagenesis even in the absence of UVR¹¹. Pheomelanin has been identified as an intrinsic risk factor for melanoma with the red pigment itself producing reactive oxygen species that cause DNA damage in the skin, and consequently promote melanomagenesis independently of UVR. UVR likely exacerbates red pigment-induced BRAF(V600E)-driven melanoma, and still remains as a major contributor to melanomagenesis. Therefore, along with UV shielding by sunscreens, further preventative strategies should be investigated to diminish UVR-independent melanoma risk mechanisms.Viros et al. provide intriguing answers to several controversial questions regarding melanomagenesis: Does UVR really trigger melanoma? And can sunscreen actually prevent melanoma? The studies by Viros et al. provide experimental evidence for acceleration of BRAF(V600E)-driven melanoma by UVR-induced TP53/Trp53 mutation and demonstrate that sunscreen delayed but did not completely block UVR-driven melanoma. The current study clearly shows that UVR boosts melanoma and sunscreens may provide partial UVR protection against melanoma — evidence which matches human epidemiologic data. Nevertheless, to protect the public from melanoma, Viros et al. advise that sunscreen should be utilized in combination with additional sun avoidance strategies. In addition, measures that may prevent UV-independent melanoma formation will require additional research and may also be needed in order to optimally battle the incidence of this life-threatening malignancy.