Abstract
Abstract
Gene silencing associated with aberrant methylation of promoter region CpG islands is an acquired epigenetic alteration. It serves as an alternative to genetic defects in the inactivation of tumor suppressor and other genes in human cancers. Several genes have been shown to be epigenetically inactivated in a wide range of tumors. TMS1 is a tumor suppressor gene which encodes for a CARD (caspase recruitment domain) containing regulatory protein and has been shown to be hyper-methylated in various cancers. Therefore, we studied the methylation status of TMS1 using a panel of colorectal cell lines and 15 CRC cases. Methylation status of the promoter region of TMS1 gene was determined using methylation specific PCR in colorectal cancer cell lines. Seven out of 13 colorectal cell lines showed to be methylated for TMS1 gene (54%). Initial analyses with 15 clinical colorectal cases demonstrated 53% methylation. Real time PCR and western bolt analysis was employed to assess the expression levels. 5-AZA-2′-deoxycytidine was used to demethylate colorectal cancer cell lines. Four out of 7 methylated colorectal cell lines were completely methylated and 3 out of 7 showed partial methylation for TMS1 gene resulting in loss of TMS1 expression. Treating methylated colorectal cell lines with the methyltransferase inhibitor, 5-aza-2’ deoxycytidine alone resulted in partial demethylation and re-expression of TMS1 gene, suggesting the role of methylation in silencing the gene. TMS1 gene has shown tumor suppressive effects after demethylation in thyroid cancer cell. Additionally the oncogenic role of TMS1 has been demonstrated in different cancers. We will further evaluate the role of TMS1 gene after demethylation for apoptosis. After demethylation of TMS1 gene with 5-aza-2’ deoxycytidine we will treat our colorectal cancer cell lines with increasing doses of TRAIL and will evaluate its response by Annexin/PI dual staining and western blot analysis.
Our results demonstrated that TMSI gene is methylated in colorectal cancer cells and repression of methylation by 5-Aza-2′-deoxycytidine restored the expression of TMS1 gene. In conclusion, methylation of TMS1 may prove to be a useful prognostic marker and/or predictor of patient survival in colorectal and can be utilized for targeted therapy.
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 4813. doi:10.1158/1538-7445.AM2011-4813