Abstract 2883: Impact of novel CDKN2A/p16INK4a 5’UTR variants predisposing to melanoma on p16 translational regulation
Authors: Bisio Alessandra , Latorre Elisa , Andreotti Virginia , Bressac-de Paillerets Brigitte , Harland Mark , Cabaret Odile , Newton-Bishop Julia , Pastorino Lorenza , Bruno William , Bertorelli Roberto , De Sanctis Veronica , Menin Chiara , Fronza Gilberto , Queirolo Paola , Bianchi Scarrà Giovanna , Spitale Robert C. , Provenzani Alessandro , Inga Alberto , Ghiorzo Paola 
Published: July 2016

CDKN2A/p16INK4a is an important tumor-suppressor gene whose dysregulation is associated with melanoma. We have recently demonstrated that the p16INK4a expression can be modulated by IRES-dependent mRNA translation and this regulation might have an important role during tumorigenesis. We also identified YBX1 as RNA binding protein targeting and stimulating p16 mRNA translation efficiency. The identified IRES function was particularly active in hypoxia and inhibition of mTOR. This post-transcriptional regulatory mechanism would potentially be a target for mutational inactivation during melanomagenesis. Indeed, we previously showed that p16INK4a 5’UTR variants found in melanoma patients with a family predisposition can have a negative effect on p16 translation. Single Nucleotide Variants (SNVs) have been investigated during routine testing of CDKN2A/p16INK4a in Italian, English and French melanoma patients followed by GenoMEL, the International Melanoma Genetics Consortium and a total of 17 germline variants were identified in a cohort of nearly 6000 patients. These p16INK4a 5’UTR 17 variants were studied with multiple approaches, that included mono- and bi-cistronic reporter assays, western blot of endogenous protein, and quantification of allelic representation after polysomal profiling to investigate their impact on p16INK4a mRNA translation regulation. We devised a classification score based on the concordance between functional assays and the extent of dysfunction displayed by each variant compared to the wild type. Variants are classified as neutral (score 0) when no difference was observed in at least 3 assays. This applied to: c.-14C&gt;T, c.-20A&gt;G, c.-25C&gt;T+c.-180G&gt;A, c.-30G&gt;A, c.-40C&gt;T, c.-45G&gt;A, c.-59C&gt;G, c.-87T&gt;A, c.-252A&gt;T. Variants were considered as potential mutations when a defect was measured in either one or two assays (score 1-2; c.-42T&gt;A and c.-67G&gt;C variants). Finally, we classified variants as causal mutations when three or more assays showed impairment (score &gt;3). This applied to: c.-27del23, c.-56G&gt;T, c.-93-91delAGG, as well as to c.-21C&gt;T and c.-34G&gt;T, which were already considered as a causal mutations. We have also determined the structure of the wild type p16INK4a 5’UTR by Selective Hydroxyl Acylation or SHAPE assay. The variant c.-42T&gt;A encompassing the predicted YBX1 binding site was also studied and shown to induce a local change in conformation. The structure of all p16INK4a 5’UTR variants examined so far is being investigated as well as their impact on the interaction of RNA binding proteins such as YBX1, SRSF1 and RBM4. Our data indicate that the sequencing of the entire p16INK4a 5’UTR should be included in routine screening of melanoma families as nearly half of SNVs tested so far displayed a negative impact on the p16 mRNA translation efficiency.

Citation Format: Alessandra Bisio, Elisa Latorre, Virginia Andreotti, Brigitte Bressac-de Paillerets, Mark Harland, Odile Cabaret, Julia Newton-Bishop, Lorenza Pastorino, William Bruno, Roberto Bertorelli, Veronica De Sanctis, Chiara Menin, Gilberto Fronza, Paola Queirolo, Giovanna Bianchi Scarrà, Robert C. Spitale, Alessandro Provenzani, Alberto Inga, Paola Ghiorzo. Impact of novel CDKN2A/p16INK4a 5’UTR variants predisposing to melanoma on p16 translational regulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2883.