Abstract 3821: Elucidating the role of PBRM1 in SNF5 regulated gene expression in malignant rhadboid tumors.

Abstract

Abstract Malignant Rhabdoid Tumors (MRTs), a pediatric renal cancer, lack SNF5, a subunit of the SWI/SNF chromatin remodeling complex, a regulator of nucleosome positioning and gene expression. Recent studies showed that PBRM1, another subunit of SWI/SNF, was mutated in adult clear cell renal carcinoma (ccRCC). Intriguingly, both SNF5 and PBRM1 regulate the cell cycle through control of expression of p21WAF1/CIP1. We hypothesized that SWI/SNF complex mutations provide a link between adult and pediatric renal cancers. To test this notion, we re-expressed SNF5 in MRT cell lines and followed PBRM1 and p21 expression and cell growth. We also examined SWI/SNF complex composition and recruitment to the p21 promoter. Finally, we investigated PBRM1’s role in SNF5-induced G1 cell cycle arrest using PBRM1-deficient MRT cell lines. We utilized adenoviral vectors to express human SNF5 (hSNF5) in several different MRT cell lines. The expression of hSNF5 and other complex members was quantified through immunoblotting and QT-PCR. Immunoprecipitation (IP) was utilized to determine the composition of the SWI/SNF complex. Cell cycle analysis was conducted using flow cytometry with BrdU and PI staining. PBRM1 knockdown cell lines were generated using lentiviral-RNAis (Open Biosystems). Reexpression of SNF5 in MRT cell lines led to increased PBRM1 protein levels without concomitant increases in mRNA levels. Immunoprecipitation experiments showed that SNF5 re-expression caused PBRM1 levels in the complex to increase, as compared to the BRG1 ATPase levels that remained constant after SNF5 reexpression. These results were consistent across 4 MRT cell lines examined. Interestingly, levels of other complex proteins, such as BAF155, also increased in a similar fashion to PBRM1. After reexpression of SNF5, we also observed an increase of both SNF5 and PBRM1 at the p21 promoter, with a peak at the transcription start site. In contrast, other complex members showed only a modest increase across the entire promoter region with no apparent peak of binding. To further investigate PBRM1’s role in SNF5-induced G1 cell cycle arrest, we generated PBRM1-deficient MRT cell lines using RNAi technology. We are currently testing these knockdown cell lines for cell cycle arrest and p21 induction after hSNF5 reexpression. Our data show that SNF5 expression increases PBRM1 protein levels, presumably through stabilization within the SWI/SNF complex, suggesting a high degree of interplay between PBRM1 and SNF5 during chromatin remodeling activity. Therefore, identifying genes regulated by both proteins will prove critical in further understanding their contribution to MRT development. Ultimately, our studies will identify key signaling pathways that contribute to the initiation and/or progression of renal cancers including MRTs and ccRCCs. Citation Format: Darmood Wei, Bernard E. Weissman, Yasumichi Kuwahara. Elucidating the role of PBRM1 in SNF5 regulated gene expression in malignant rhadboid tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3821. doi:10.1158/1538-7445.AM2013-3821