Abstract 4033: A20 gene promoter methylation confers resistance to colorectal carcinoma cell lines

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 and most neoplasms show hypermethylation of one or more genes. A20, also known as TNF alpha induced protein3 (TNFAIP3), is a tumor suppressor gene and a well-known negative regulator of the NF-kB pathway. In colorectal carcinoma (CRC), aberrant NF-kB regulation has been associated with poor prognosis and resistance to therapy. Previously, we elucidated the biological role of A20, mechanism of its inactivation and its prognostic role in colorectal carcinoma. Loss of A20 protein expression by immunohistochemistry and A20 deletions by FISH were studied in a microarray cohort of CRC cases and cell lines for mutations in A20 gene and A20 promoter methylation were also screened. We further elucidated the mechanism of A20 inactivation in colorectal carcinoma cell lines in this extended study. Methylation status of A20 promoter in CRC cell lines correlated with the degree of A20 protein expression: unmethylated cell line SW948 showed the highest expression of A20 protein whereas LOVO, which was methylated, showed almost complete loss of A20 expression. CRC cell lines with high levels of A20 expression (SW948 and COLO320), intermediate levels (HCT-15 and DLD-1) and very low levels (LOVO-1) were immunoblotted for detection of A20 protein expression. Treatment of methylated colorectal cell lines with the methyltransferase inhibitor, 5-aza-2′deoxy-cytidine resulted in partial demethylation and re-expression of A20 gene, suggesting the role of methylation in silencing the gene. In addition, demethylation of A20 gene by 5-aza-2′deoxy-cytidine also decreased cell viability by 15-20X in CRC cell lines, suggesting that A20 expression may play an important role in CRC tumorigenesis. In conclusion, A20 methylation may be a promising molecular marker to predict patient outcome in colorectal cancer, and may be used as therapeutic demethylating target. 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 4033. doi:1538-7445.AM2012-4033