Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression throughFxr gene CpG Methylation in Mouse Colon Tumors and Human Colon Cancer Cells

Abstract

Background: The farnesoid X receptor (FXR) regulates bile acid (BA) metabolism and possesses tumor suppressor functions. FXR expression is reduced in colorectal tumors of subjects carrying inactivated adenomatous polyposis coli (APC). Identifying the mechanisms responsible for this reduction may offer new molecular targets for colon cancer prevention.Objective: We investigated howAPC inactivation influences the regulation ofFXR expression in colonic mucosal cells. We hypothesized thatAPC inactivation would epigenetically repress nuclear receptor subfamily 1, group H, member 4 (FXR gene name) expression through increased CpG methylation.Methods: Normal proximal colonic mucosa and normal-appearing adjacent colonic mucosa and colon tumors were collected from wild-type C57BL/6J andApc-deficient (ApcMin/+) male mice, respectively. The expression ofFxr, ileal bile acid-binding protein (Ibabp), small heterodimer partner (Shp), and cyclooxygenase-2 (Cox-2) were determined by real-time polymerase chain reaction. In both normal and adjacent colonic mucosa and colon tumors, we measured CpG methylation ofFxr in bisulfonated genomic DNA. In vitro, we measured the impact ofAPC inactivation and deoxycholic acid (DCA) treatment onFXR expression in human colon cancer HCT-116 cells transfected with silencing RNA forAPC and HT-29 cells carrying inactivatedAPC.Results: InApcMin/+ mice, constitutive CpG methylation of theFxrα3/4 promoter was linked to reduced (60–90%) baselineFxr,Ibabp, andShp and increasedCox-2 expression in apparently normal adjacent mucosa and colon tumors.Apc knockdown in HCT-116 cells increased cellular myelocytomatosis(c-MYC) and lowered (∼50%)FXR expression, which was further reduced (∼80%) by DCA. In human HCT-116 but not HT-29 colon cancer cells, DCA inducedFXR expression and lowered CpG methylation ofFXR.Conclusions: We conclude that the loss of APC function favors the silencing of FXR expression through CpG hypermethylation in mouse colonic mucosa and human colon cells, leading to reduced expression of downstream targets (SHP, IBABP) involved in BA homeostasis while increasing the expression of factors (COX-2, c-MYC) that contribute to inflammation and colon cancer.