Breast cancer risk variants at 6q25 display different phenotype associations and regulate ESR1, RMND1 and CCDC170.

We analyzed 3,872 common genetic variants across the ESR1 locus (encoding estrogen receptor α) in 118,816 subjects from three international consortia. We found evidence for at least five independent causal variants, each associated with different phenotype sets, including estrogen receptor (ER(+) or ER(-)) and human ERBB2 (HER2(+) or HER2(-)) tumor subtypes, mammographic density and tumor grade. The best candidate causal variants for ER(-) tumors lie in four separate enhancer elements, and their risk alleles reduce expression of ESR1, RMND1 and CCDC170, whereas the risk alleles of the strongest candidates for the remaining independent causal variant disrupt a silencer element and putatively increase ESR1 and RMND1 expression.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ng.352

Polymorphismes situés dans les régions de type G-quadruplexe du gène suppresseur de tumeur TP53 : Impact sur la susceptibilité au cancer et l’expression des isoformes en N-terminal de p53

The TP53 gene is a highly polymorphic gene with 85 polymorphisms described. Some of these have been associated with an increase of cancer susceptibility, for example rs10425222 that can modulate certain p53 activities. However for others such as rs17878362, the most studied intronic polymorphism, the association with cancer risk is more controversial. To investigate the influence of rs17878362 on cancer susceptibility, we analysed its role in sporadic and familial contexts. The results are paradoxical with an increase of sporadic cancer associated with the rs17878362 A2A2 genotype whereas the rs17878362 A2 allele is associated with a “protective” effect in the context of Li-Fraumeni patients carrying a TP53 germline mutation on an A1 haplotype. These observations suggest that specific TP53 haplotypes could modulate p53’s tumour suppression capacities. A possible hypothesis to explain this could be that somatic mutations are carried on different haplotypes of TP53 present at different allele frequencies in the population. In addition, TP53 is expressed as several protein isoforms, such as D40p53, which inhibits p53’s suppressive activity. D40p53 can be produced from an alternative spliced transcript that retains intron 2. We have shown that G-quadruplexes, tri-dimensional structures formed in G-rich sequences, are formed in intron 3 and regulate the retention of intron 2 and the formation of the p53I2 transcript. We also observed that rs1642785 (located in intron 2) could regulate p53I2’s stability. These results suggest that the TP53 polymorphisms located in a 412 bp region located between exon 2 and exon 4 regulate the expression of p53 isoforms in a temporal sequence of events by modulating the pre-mRNA formation (rs17878362), mRNA stability (rs1642785) and protein functions (rs1042522).p53 isoforms’ expression is thus finely regulated by mechanisms involving TP53 polymorphisms, which are also associated with altered cancer susceptibility.Le gène TP53 est extrêmement polymorphique avec 85 polymorphismes décrits. Certains de ces polymorphismes sont associés à une augmentation du risque de cancer, par exemple rs10425222 peut moduler les fonctions de p53. Cependant, pour d’autres, comme le rs17878362 qui est le polymorphisme intronique le plus étudié, leur association avec une augmentation du riques au cancer est controversée.Pour analyser l’association entre le polymorphisme rs17878362 et la susceptibilité au cancer, nous avons analysé son rôle dans des contextes de cancers sporadiques et familiaux. Les résultats obtenus pour le polymorphisme rs17878362 sont paradoxaux avec une augmentation des cancers sporadiques associée avec le génotype A2A2 alors que l’allèle A2 est associé avec un effet « protectif » chez les patients atteints du syndrome de Li-Fraumeni porteurs d’une mutation germinale de TP53 situé sur l’haplotype A1. Ces observations suggèrent que des haplotypes spécifiques de TP53 pourraient moduler les capacités suppressives de p53. Une hypothèse possible est que les différents haplotypes de TP53 présenteraienrt des mutations somatiques à des fréquences différentes dans la population.De plus, le gène TP53 exprime différentes isoformes, comme le D40p53, inhibant l’activité suppressive de p53. Le D40p53 peut être produite par le maintien de l’intron 2 par épissage alternatif. Nous avons montré que les G-quadruplexes, des structures tridimensionnelles formées dans des régions riches en G, sont formés dans l’intron 3 et régulent la rétention de l’intron 2 et la formation du transcrit p53I2. Nous avons aussi observé que le polymorphisme rs1652785 (localisé dans l’intron 2) semble réguler la stabilité du p53I2. Ces résultats suggèrent que les polymorphismes de TP53 localisés dans une région de 412 pb située entre l’exon 2 et l’exon 4 régulent l’expression des isoformes de p53 dans une séquence temporelle d’évènements en modulant la formation des pré-ARNm (rs17878362), la stabilité des ARNm (rs1642785) et les fonctions protéiques (rs10425222).L’expression des isoformes de p53 est donc finement régulée par des mécanismes impliquant les polymorphismes de TP53 qui sont aussi associés avec une altération dans la susceptibilité au cancer

PARP inhibition and the radiosensitizing effects of the PARP inhibitor ABT-888 in in vitro hepatocellular carcinoma models

International audienceBackground: Hepatocellular carcinoma is the third cause of cancer related death for which new treatment strategies are needed. Targeting DNA repair pathways to sensitize tumor cells to chemo-or radiotherapy is under investigation for the treatment of several cancers with poly(ADP-ribose) polymerase (PARP) inhibitors showing great potential. The aim of this preclinical study was to evaluate the expression of PARP and PARG genes in a panel of liver cancer cell lines and primary human hepatocytes, their DNA repair capacity and assess the impact on cell survival of PARP inhibitors alone and in combination with radiotherapy. Methods: Quantitative PCR was used to measure PARP-1, −2, −3 and PARG mRNA levels and western blotting for PARP-1 protein expression and ADP-ribose polymer formation after exposure of cells to doxorubicin, a topoisomerase II poison. DNA repair capacity was assessed using an in vitro DNA lesion excision/synthesis assay and the effects on cell killing of the PARP inhibitor ABT-888 alone and in combination with ionizing radiation using clonogenic survival. Results: Although a wide range in expression of the PARPs and PARG was found correlations between PARP-1 and PARP-2 mRNA levels and PARP-1 mRNA and protein levels were noted. However these expression profiles were not predictive of PARP activity in the different cell lines that also showed variability in excision/synthesis repair capacity. 4 of the 7 lines were sensitive to ABT-888 alone and the two lines tested showed enhanced radiosensitivity in the presence of ABT-888. Conclusions: PARP inhibitors combined with radiotherapy show potential as a therapeutic option for hepatocellular carcinoma

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 D40p53 (or DNp53), 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 Gtracts 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

Genome-wide association and transcriptome studies identify target genes and risk loci for breast cancer

Abstract Genome-wide association studies (GWAS) have identified more than 170 breast cancer susceptibility loci. Here we hypothesize that some risk-associated variants might act in non-breast tissues, specifically adipose tissue and immune cells from blood and spleen. Using expression quantitative trait loci (eQTL) reported in these tissues, we identify 26 previously unreported, likely target genes of overall breast cancer risk variants, and 17 for estrogen receptor (ER)-negative breast cancer, several with a known immune function. We determine the directional effect of gene expression on disease risk measured based on single and multiple eQTL. In addition, using a gene-based test of association that considers eQTL from multiple tissues, we identify seven (and four) regions with variants associated with overall (and ER-negative) breast cancer risk, which were not reported in previous GWAS. Further investigation of the function of the implicated genes in breast and immune cells may provide insights into the etiology of breast cancer