Abstract 4028: Identification of novel binding targets of hSNF5 in malignant rhabdoid tumors
Authors: Davies Brian , Kuwahara Yasumichi , Charboneau Aubri , Weissman Bernard 
Published: June 2012

Abstract
Loss of SNF5, a core subunit of the SWI/SNF ATP-dependent chromatin-remodeling complex, has been linked to development of malignant rhabdoid tumors (MRT). MRTs, a deadly form of pediatric cancer, can occur in almost any soft tissue of the body, including the brain. We have previously shown that introduction of SNF5, a known tumor suppressor gene, into deficient cell lines results in a G1 growth arrest through p21(CIP1/WAF1) mRNA induction. However, additional gene expression changes that may contribute to MRT development or may be involved in SNF5 cell cycle arrest remain uncharacterized. To discover novel binding targets of SNF5, we used a Biosciences Human Cell Cycle RT2Profiler PCR expression array to identify genes whose expression significantly changed upon reexpression of SNF5-GFP in the MRT cell line A204.1. The genes identified include p16(INK4A), p21(CIP1/WAF1), CCNG2, CCNH, CDK8, and HERC5. p16(INK4A) and p21(CIP1/WAF1) have been previously validated as direct binding targets of SNF5. Cyclin G2 (CCNG2) is a noncanonical cyclin that appears to negatively control cell cycle progression. HERC5 is involved in interferon signaling and ISGylation of proteins. CDK8 is a member of the mediator complex that plays a role in transcription. Cyclin H (CCNH) has been linked to development of some neural cancers. We developed an adenoviral vector system containing a novel HA tagged SNF5 construct. We used RT-PCR to validate the gene expression changes seen in the array in A204.1 and other MRT cell lines using this novel construct. An empty adenoviral vector was used as a control. While all genes showed increases in A204.1 after SNF5 reexpression, CCNG2 and CCNH mRNA levels increased in only a few other MRT cell lines. CDK8 expression levels were unchanged or decreased in all other MRT cell lines examined. HERC5 mRNA expression was greatly increased in all MRT cell lines at significantly higher levels than any of the other genes identified in this study. Chromatin immunoprecipitation (ChIP) analysis of the HERC5 and CCNG2 promoter regions in A204.1 confirmed direct SNF5 binding. Maximal enrichment was observed upstream of the transcriptional start site of both promoters. RNA Polymerase II also increased across the promoter regions in a pattern similar to SNF5 binding. Western blotting was also performed, confirming the upregulation of HERC5. CCNG2 protein levels did not follow mRNA expression, possibly because of tight regulation due to involvement in the cell cycle. Overall, we have identified the HERC5 and CCNG2 promoters as direct binding targets of SNF5 that may play roles in the development of this cancer. Interestingly, previous studies have shown that interferon signaling is activated upon SNF5 introduction into MRTs, indicating the potential biological relevance of HERC5. Future studies are needed to fully characterize their relationship to MRT oncogenesis and the mechanism of SWI/SNF regulation at their proximal promoter region.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4028. doi:10.1158/1538-7445.AM2011-4028