Abstract 179: Identification of novel targets for aberrant methylation in pancreatic cancer by a newly developed method “methyl-CpG targeted transcriptional activation (MeTA)”

Abstract

Abstract Identification and characterization of genes selectively hypermethylated in cancer may improve our understanding of the role of epigenetic alterations in tumorigenesis. In addition, genes frequently methylated in a tumor-specific manner could be used as tumor markers. DNA methylation inhibitors such as 5-aza-cytidine or 5-aza-2’-deoxycytidine have been widely used to search epigenetically silenced genes. However, these inhibitors frequently upregulate genes whose promoters remain unmethylated. We tried to improve the specificity and sensitivity in detecting methylation-mediated silenced genes in cancer and successfully developed a new method termed “methyl-CpG targeted transcriptional activation (MeTA)” by using a transcriptional activating fragment with a methyl-CpG binding domain (MBD). Because MBD proteins in fact mediate transcriptional repression of tumor suppressor genes associated with promoter hypermethylation in cancer, MeTA is thought to be one of the ideal methods to search such genes. In the preliminary experiments using human embryonic kidney cell line 293T, we found that the combination of MeTA with gene expression microarray is useful; genes containing CpG islands can be efficiently identified (93.5% by MeTA vs. 66.8% by 5-aza-cytidine) even those that are difficult to find by the conventional DNA methylation inhibitors. We applied this method to twelve typical pancreatic cancer cell lines with a normal pancreatic ductal epithelial cell line HPDE as the control, and have searched for genes commonly upregulated after MeTA. Thirty-one genes were upregulated by two-folds or more in six pancreatic cancer cell lines or more. Among these, 26 genes have not been reported previously using the conventional DNA methylation inhibitors. Methylation statuses of three genes, NEFL, NEFM, and IRX4 were further examined by methylation-specific PCR and found that these three genes were aberrantly methylated in all of the analyzed tumors, 11 resected primary pancreatic cancer specimens and 22 pancreatic cancer cell lines. Our results suggest that “MeTA” is a highly efficient method to identify methylation-mediated transcriptionally silenced genes in human pancreatic cancer and that this method can be applied to other types of human cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 179.