Abstract 401: Functional characterization of the 14q24 renal cancer susceptibility locus implicates SWI/SNF complex member DPF3 via inhibition of apoptosis
Authors: Colli Leandro Machado , Jessop Lea , Myers Timothy A. , Machiela Mitchell J. , Choi Jiyeon , Purdue Mark , Brown Kevin , Chanock Stephen J. 
Published: August 2018

To identify promising regulatory regions under the peaks of Renal Cell Carcinoma (RCC) GWAS signals, we deployed the Massively Parallel Reporter Assay (MPRA), a powerful tool to dissect putative regulatory regions in parallel. Using MPRA, we identified rs4903064 within the 14q24 RCC risk region as a potential transcriptional enhancer with allele-preferential activity. By MPRA, the risk allele C leads to higher expression, an effect confirmed by luciferase assay in three RCC cell lines. Using electromobility shift assays, we observed preferential protein binding to the T allele of rs4903064, one of the strongest signals in the RCC GWAS at 14q24. Motif analysis shows that this SNP resides in a binding site for the transcriptional repressors IRX2 and IRX5 and the C allele is predicted to disrupt the binding site. These results suggest that the higher expression seen in MPRA and luciferase assays when the C allele is present is likely due to reduced binding of transcriptional repressor proteins. Consistent with this, an eQTL analysis of RCC TCGA data showed that C allele is associated with higher expression of the double PHD Fingers 3 gene (DPF3) (p=5.5e-8). DPF3, a plausible candidate gene influenced by the RCC susceptibility allele is a member of the nucleosome remodeling SWI/SNF complex, components of which are frequently mutated in multiple cancers. PBRM1, a member of SWI/SNF complex, is the second most common mutated gene in RCC (40%), demonstrating the importance of SWI/SNF complex for RCC oncogenesis. We generated three kidney cells lines (two RCC- ACHN and UOK-121 and one normal kidney cell line- HK2) with DOX-inducible expression of both DPF3 isoforms (a or b), separately. ATAC-seq analysis of HK2 cells overexpressing DPF3a or DPF3b showed reduced chromatin accessibility compared to controls, suggesting that DPF3 overexpression is disrupting SWI/SNF activity. RNA-seq analysis of DPF3a or DPF3b over expressing cell lines showed deregulation of apoptotic genes, especially overexpression of CEMIP. Cell lines over expressing DPF3a or DPF3b had a higher growth rate, suggesting an oncogenic effect of both DPF3 isoforms. Knockdown of CEMIP by si-RNA blocked the effect of DPF3a and DPF3b on growth rate, suggesting that part of the DPF3 effect on cell growth can be attributed to overexpression of the apoptosis inhibitor CEMIP. We treated cells overexpression of either DPF3a or DPF3b with cisplatin or camptothecin to induce apoptosis and observed cleavage of Caspase 3 and PARP by Western Blot, both markers of apoptosis. Using AnexinV and 7-AAD, flow cytometery analysis confirmed that DPF3a or DPF3b overexpression reduces apoptosis in RCC cell lines. In summary, our results suggest that the RCC risk C-rs4903064 at the 14q24 locus increases expression of DPF3, which disrupts the normal function of the SWI/SNF complex leading to dysregulation of CEMIP and a reduction of apoptosis.

Citation Format: Leandro Machado Colli, Lea Jessop, Timothy A. Myers, Mitchell J. Machiela, Jiyeon Choi, Mark Purdue, Kevin Brown, Stephen J. Chanock. Functional characterization of the 14q24 renal cancer susceptibility locus implicates SWI/SNF complex member DPF3 via inhibition of apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 401.