Abstract 2178: ARID1B, a member of the human SWI/SNF chromatin remodeling complex, is a novel tumor suppressor gene in pancreatic cancer

Abstract

Abstract Lack of early diagnosis and efficient therapeutic options are the two major obstacles hindering improvement in pancreatic cancer (PaCa) survival rate. Traditionally, researchers have focused their attention on identification and characterization of oncogenes, given the comparative ease of therapeutic targeting. Recent studies have however suggested that up to 90% of cancer ‘driver’ genes are tumor suppressors. In our previous work, we identified a recurrent deletion located at 6q25.3 in pancreatic cancer cell lines and xenografts that included only one gene viz. ARID1B, a component of the human SWI/SNF chromatin remodeling complex. We utilized a two-pronged strategy for this study; ectopic expression in MiaPaCa2 PaCa cell line harboring an ARID1B homozygous deletion and analysis of promoter hypermethylation induced transcriptional silencing in cell lines and tumor tissues. The ARID1B cDNA was cloned into the pcDNA3.1 expression system in order to generate permanent transfectants in MiaPaCa2; vector transfectants were generated as a control. Two independent gene and vector clones were evaluated for several tumor-related characteristics. There was no significant difference in growth rates between the gene and vector clones as determined through MTT assay, crystal violet staining assay and FACS-based cell cycle analysis nor was a perceptible difference discernable in percentage apoptotic cells based on Caspase 3 and Propidium Iodide/Hoechst staining assays. However, the ARID1B clones generated significantly reduced number of colonies in liquid colony formation as well as soft agar assays indicating reduced ability to grow in the presence of contact inhibition and in the absence of anchorage, respectively. In addition, the ARID1B clones exhibited reduced motility in wound healing assays when compared to vector clones. We independently analyzed ten PaCa cell lines that exhibited a single copy loss and reduced expression of ARID1B. Azacytidine and Trichostatin A treatment resulted in a significant increase in ARID1B transcript levels indicating the possibility of methylation-induced transcriptional silencing. Bisulphite sequencing revealed extensive hypermethylation in the ARID1B promoter in cell lines exhibiting elevated transcript levels upon Azacytidine/TSA treatment but not in cell lines that exhibited no change in transcript levels. Immunohistochemistry and quantitative reverse transcription PCR-based analysis of pancreatic tumor samples revealed reduced ARID1B expression in tumour as compared to normal samples. Methylation analysis of the ARID1B promoter using DNA isolated from pancreatic cancer tissue is currently underway. Our results therefore suggest a tumor suppressor role for ARID1B in pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2178. doi:1538-7445.AM2012-2178