Abstract 3561: Silencing of farnesoid X receptor in human colon cancer by epigenetic mechanisms is associated with cancer progression.

Background: Colon cancer is the third leading cause of cancer related deaths in the United States. Epidemiological studies suggest an increase in the intestinal bile-acid load resulting from the consumption of a high-fat diet is a significant risk factor for colon cancer. Bile acids are the endogenous ligands for the farnesoid X receptor (FXR), a ligand-activated transcription factor and member of the nuclear receptor superfamily, and high levels of bile acids can promote colon cancer development. FXR is essential for maintaining bile-acid homeostasis by regulating bile-acid synthesis and transport, preventing the accumulation of intestinal bile acid levels to cancer promoting levels. Previous studies have demonstrated that FXR knockout mice are more susceptible to the development of colon adenocarcinomas, indicating that FXR plays a suppressive role in colon tumor formation. This study investigates the role of FXR in the development of human colon cancer. Methods: Immunohistochemistry was used to label for FXR in normal human colon, colon polyps, and colon adenocarcinomas staged I-IV. SYBR green quantitative PCR and western blot analysis were used to measure expression of FXR and FXR target genes in normal human colon and colon cancers staged I-IV as well as colon cancer cell lines. Reverse phase protein array on colon cancer cell line lysates was used to correlate FXR expression with oncogenic signaling cascades. To test if FXR expression was suppressed by DNA methylation, colon cancer cell lines were treated with a DNA methyltransferase (DNMT) inhibitor and DNMT siRNA and FXR mRNA measured by real-time PCR. Immunoprecipitation with an antibody against 5-methylcytosine (MeDIP) analysis was done in human colon cancer cell lines to determine methylation of NR1H4 (gene encoding FXR) promoter. Results: IHC and qPCR analysis reveals that the expression and function of FXR is markedly reduced early in colon cancer progression, with suppression seen within precancerous lesions. Furthermore, FXR expression in colon cancer cell lines were negatively correlated to oncogenic PI3 kinase signaling cascades and associated with epithelial to mesenchymal transition (EMT). Results suggest DNA methylation as a mechanism of FXR silencing in colon cancer and confirms methylation of the FXR promoter. Conclusion: FXR deficiency in animals indicates FXR serves a tumor suppressive role. Our studies show that FXR is silenced in early in human colon cancer progression possibly by DNA methylation, which could be a cancer promoting event. The overall mechanism of FXR9s anti-tumorigenic activity is not fully established but may be due to FXR9s role in regulating EMT and bile acid homeostasis. Restoration and enhancement of FXR activity, by blocking DNA methylation or increasing baseline activity of FXR, represents a potential therapeutic option for the treatment of colon cancer. Citation Format: Ann M. Thomas, Le Zhan, Julie Izzo, Dipen Maru, Imad Shureiqi, Veera Baladandayuthapani, Han Liang, Grace L. Guo, Garth Powis. Silencing of farnesoid X receptor in human colon cancer by epigenetic mechanisms is associated with cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3561. doi:10.1158/1538-7445.AM2013-3561