Virginie Marcel

Biological functions of p53 isoforms through evolution: lessons from animal and cellular models

The TP53 tumour-suppressor gene is expressed as several protein isoforms generated by different mechanisms, including use of alternative promoters, splicing sites and translational initiation sites, that are conserved through evolution and within the TP53 homologues, TP63 and TP73. Although first described in the eighties, the importance of p53 isoforms in regulating the suppressive functions of p53 has only become evident in the last 10 years, by analogy with observations that p63 and p73 isoforms appeared indispensable to fully understand the biological functions of TP63 and TP73. This review summarizes recent advances in the field of ‘p53 isoforms’, including new data on p63 and p73 isoforms. Details of the alternative mechanisms that produce p53 isoforms and cis- and trans-regulators identified are provided. The main focus is on their biological functions (apoptosis, cell cycle, aging and so on) in cellular and animal models, including mouse, zebrafish and Drosophila. Finally, the deregulation of p53 isoform expression in human cancers is reviewed. Based on these latest results, several developments are expected in the future: the identification of drugs modulating p53 isoform expression; the generation of animal models and the evaluation of the use of p53 isoform as biomarkers in human cancers.

G-quadruplex structures in TP53 intron 3: role in alternative splicing and in production of p53 mRNA isoforms

The tumor suppressor gene TP53, encoding p53, is expressed as several transcripts. The fully spliced p53 (FSp53) transcript encodes the canonical p53 protein. The alternatively spliced p53I2 transcript retains intron 2 and encodes Δ40p53 (or ΔNp53), an isoform lacking first 39 N-terminal residues corresponding to the main transactivation domain. We demonstrate the formation of G-quadruplex structures (G4) in a GC-rich region of intron 3 that modulates the splicing of intron 2. First, we show the formation of G4 in synthetic RNAs encompassing intron 3 sequences by ultraviolet melting, thermal difference spectra and circular dichroism spectroscopy. These observations are confirmed by detection of G4-induced reverse transcriptase elongation stops in synthetic RNA of intron 3. In this region, p53 pre-messenger RNA (mRNA) contains a succession of short exons (exons 2 and 3) and introns (introns 2 and 4) covering a total of 333 bp. Site-directed mutagenesis of G-tracts putatively involved in G4 formation decreased by ~30% the excision of intron 2 in a green fluorescent protein-reporter splicing assay. Moreover, treatment of lymphoblastoid cells with 360A, a synthetic ligand that binds to single-strand G4 structures, increases the formation of FSp53 mRNA and decreases p53I2 mRNA expression. These results indicate that G4 structures in intron 3 regulate the splicing of intron 2, leading to differential expression of transcripts encoding distinct p53 isoforms.

P53 Acts as a Safeguard of Translational Control by Regulating Fibrillarin and rRNA Methylation in Cancer

Summary Ribosomes are specialized entities that participate in regulation of gene expression through their rRNAs carrying ribozyme activity. Ribosome biogenesis is overactivated in p53-inactivated cancer cells, although involvement of p53 on ribosome quality is unknown. Here, we show that p53 represses expression of the rRNA methyl-transferase fibrillarin (FBL) by binding directly to FBL. High levels of FBL are accompanied by modifications of the rRNA methylation pattern, impairment of translational fidelity, and an increase of internal ribosome entry site (IRES)-dependent translation initiation of key cancer genes. FBL overexpression contributes to tumorigenesis and is associated with poor survival in patients with breast cancer. Thus, p53 acts as a safeguard of protein synthesis by regulating FBL and the subsequent quality and intrinsic activity of ribosomes.

Detection of R337H, a germline TP53 mutation predisposing to multiple cancers, in asymptomatic women participating in a breast cancer screening program in Southern Brazil.

Germline TP53 mutations predispose to a rare familial cancer syndrome, the Li-Fraumeni Syndrome (LFS), characterized by the early onset of multiple cancers including childhood adrenocortical carcinomas, sarcomas and brain tumors, and breast and colon cancer in young adults. An identical germline mutation at codon 337 in TP53 (R337H) has been shown to be causally related to an increased risk of multiple cancers in unrelated subjects with familial cancer risk in Southern Brazil. Here we have assessed the prevalence of R337H in 750 healthy women participating in a community-based breast cancer screening program in the area of Porto Alegre. The mutant was detected in two participants (0.3%) who were fourth-degree relatives and reported a familial history of cancer at multiple sites that did not match classical criteria for LFS and its variants. Testing in additional family members detected the mutation in three subjects, one of whom developed breast cancer at the age of 36. These findings indicate that R337H may be a low penetrance mutant which predisposes to multiple cancers and occurs in the population of Southern Brazil at a frequency 10-20 times higher than other TP53 mutants commonly associated with LFS.

TP53 PIN3 and MDM2 SNP309 polymorphisms as genetic modifiers in the Li–Fraumeni syndrome: impact on age at first diagnosis

Background: Li–Fraumeni and Li–Fraumeni-like syndromes (LFS/LFL), characterised by the development of multiple early onset cancers with heterogeneous tumour patterns, are associated with germline TP53 mutations. Polymorphisms in the TP53 pathway (TP53 PEX4 at codon 72, rs1042522; MDM2 SNP309, rs2279744) have modifier effects on germline TP53 mutations that may account for the individual and familial diversity of tumour patterns. Methods and results: Four polymorphisms were analysed in a series of 135 Brazilian LFS/LFL cancer patients (32 TP53 mutation carriers and 103 wild-type subjects). We report for the first time that another polymorphism in the TP53 gene, TP53 PIN3 (rs17878362), has a strong modifier effect on germline TP53 mutations. This polymorphism, which consists of a 16 bp duplication in intron 3 (A1, non-duplicated allele; A2, duplicated allele), is associated with a difference of 19.0 years in the mean age at the first diagnosis in TP53 mutation carriers (n = 25, A1A1: 28.0 years; n = 7, A1A2: 47.0 years; p = 0.01). In addition, cancer occurrence before the age of 35 years is exclusively observed in A1A1 homozygotes. In this series, the effect of TP53 PEX4 and MDM2 SNP309 on age at diagnosis was similar to the one reported in other series and was smaller than the one of TP53 PIN3 (TP53 PIN3: difference of 19.0 years; TP53 PEX4: 8.3 years; MDM2 SNP309: 12.5 years). Conclusion: These results suggest that TP53 PIN3 is another polymorphism in the TP53 pathway that may have a modifier effect on germline TP53 mutations and may contribute to the phenotypic diversity of germline TP53 mutations associated with LFS/LFL patients.

Δ160p53 is a novel N-terminal p53 isoform encoded by Δ133p53 transcript

p53 gene expresses several protein isoforms modulating p53‐mediated responses through regulation of gene expression. Here, we identify a novel p53 isoform, Δ160p53, lacking the first 159 residues. By knockdown experiments and site‐directed mutagenesis, we show that Δ160p53 is encoded by Δ133p53 transcript using ATG160 as translational initiation site. This hypothesis is supported by endogenous expression of Δ160p53 in U2OS, T47D and K562 cells, the latter ones carrying a premature stop codon that impairs p53 and Δ133p53 protein expression but not the one of Δ160p53. Overall, these results show that the Δ133p53 transcript generates two different p53 isoforms, Δ133p53 and Δ160p53.

Host microRNA molecular signatures associated with human H1N1 and H3N2 influenza A viruses reveal an unanticipated antiviral activity for miR-146a.

While post-transcriptional regulation of gene expression by microRNAs (miRNAs) has been shown to be involved in influenza virus replication cycle, only a few studies have further investigated this aspect in a human cellular model infected with human influenza viruses. In this study, we performed miRNA global profiling in human lung epithelial cells (A549) infected by two different subtypes of human influenza A viruses (H1N1 and H3N2). We identified a common miRNA signature in response to infection by the two different strains, highlighting a pool of five miRNAs commonly deregulated, which are known to be involved in the innate immune response or apoptosis. Among the five miRNA hits, the only upregulated miRNA in response to influenza infection corresponded to miR-146a. Based on a previously published gene expression dataset, we extracted inversely correlated miR-146a target genes and determined their first-level interactants. This functional analysis revealed eight distinct biological processes strongly associated with these interactants: Toll-like receptor pathway, innate immune response, cytokine production and apoptosis. To better understand the biological significance of miR-146a upregulation, using a reporter assay and a specific anti-miR-146a inhibitor, we confirmed that infection increased the endogenous miR-146a promoter activity and that inhibition of miR-146a significantly increased viral propagation. Altogether, our results suggest a functional role of miR-146a in the outcome of influenza infection, at the crossroads of several biological processes.

Influenza A Viruses Control Expression of Proviral Human p53 Isoforms p53β and Δ133p53α

ABSTRACT Previous studies have described the role of p53 isoforms, including p53β and Δ133p53α, in the modulation of the activity of full-length p53, which regulates cell fate. In the context of influenza virus infection, an interplay between influenza viruses and p53 has been described, with p53 being involved in the antiviral response. However, the role of physiological p53 isoforms has never been explored in this context. Here, we demonstrate that p53 isoforms play a role in influenza A virus infection by using silencing and transient expression strategies in human lung epithelial cells. In addition, with the help of a panel of different influenza viruses from different subtypes, we also show that infection differentially regulates the expressions of p53β and Δ133p53α. Altogether, our results highlight the role of p53 isoforms in the viral cycle of influenza A viruses, with p53β and Δ133p53α acting as regulators of viral production in a p53-dependent manner.

Cellular transcriptional profiling in human lung epithelial cells infected by different subtypes of influenza A viruses reveals an overall down-regulation of the host p53 pathway

BackgroundInfluenza viruses can modulate and hijack several cellular signalling pathways to efficiently support their replication. We recently investigated and compared the cellular gene expression profiles of human lung A549 cells infected by five different subtypes of human and avian influenza viruses (Josset et al. Plos One 2010). Using these transcriptomic data, we have focused our analysis on the modulation of the p53 pathway in response to influenza infection.ResultsOur results were supported by both RT-qPCR and western blot analyses and reveal multiple alterations of the p53 pathway during infection. A down-regulation of mRNA expression was observed for the main regulators of p53 protein stability during infection by the complete set of viruses tested, and a significant decrease in p53 mRNA expression was also observed in H5N1 infected cells. In addition, several p53 target genes were also down-regulated by these influenza viruses and the expression of their product reduced.ConclusionsOur data reveal that influenza viruses cause an overall down-regulation of the host p53 pathway and highlight this pathway and p53 protein itself as important viral targets in the altering of apoptotic processes and in cell-cycle regulation.

The associations of sequence variants in DNA-repair and cell-cycle genes with cancer risk: genotype-phenotype correlations

DNA-repair systems maintain the integrity of the human genome, and cell-cycle checkpoints are a critical component of the cellular response to DNA damage. Thus the presence of sequence variants in genes involved in these pathways that modulate their activity might have an impact on cancer risk. Many molecular epidemiological studies have investigated the association between sequence variants, particularly SNPs (single nucleotide polymorphisms), and cancer risk. For instance, ATM (ataxia telangiectasia mutated) SNPs have been associated with increased risk of breast, prostate, leukaemia, colon and early-onset lung cancer, and the intron 3 16-bp repeat in TP53 (tumour protein 53) is associated with an increased risk of lung cancer. In contrast, the variant allele of the rare CHEK2 (checkpoint kinase 2 checkpoint homologue) missense variant (accession number rs17879961) was significantly associated with a lower incidence of lung and upper aerodigestive cancers. For some sequence variants, a strong gene-environment interaction has also been noted. For instance, a greater absolute risk reduction of lung and upper aerodigestive cancers in smokers than in non-smokers carrying the I157T CHEK2 variant has been observed, as has an interaction between TP53 intron 3 16-bp repeats and multiple X-ray exposures on lung cancer risk. The challenge now is to understand the molecular mechanisms underlying these associations.