title: Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression through Fxr 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 how APC inactivation influences the regulation of FXR expression in colonic mucosal cells. We hypothesized that APC 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 and Apc-deficient (Apc(Min) (/+)) male mice, respectively. The expression of Fxr, 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 of Fxr in bisulfonated genomic DNA. In vitro, we measured the impact of APC inactivation and deoxycholic acid (DCA) treatment on FXR expression in human colon cancer HCT-116 cells transfected with silencing RNA for APC and HT-29 cells carrying inactivated APC.
  
  
  RESULTS
  In Apc(Min) (/+) mice, constitutive CpG methylation of the Fxrα3/4 promoter was linked to reduced (60-90%) baseline Fxr, Ibabp, and Shp and increased Cox-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 induced FXR expression and lowered CpG methylation of FXR.
  
  
  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.
authors.affiliation: Department of Nutritional Sciences and University of Arizona Cancer Center, University of Arizona, Tucson, AZ; and.
authors.forename: Ornella I
authors.initials: OI
authors.lastname: Selmin
authors.affiliation: University of Arizona Cancer Center, University of Arizona, Tucson, AZ; and.
authors.forename: Changming
authors.initials: C
authors.lastname: Fang
authors.affiliation: Department of Nutritional Sciences and.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Lyon
authors.affiliation: University of Arizona Cancer Center, University of Arizona, Tucson, AZ; and.
authors.forename: Tom C
authors.initials: TC
authors.lastname: Doetschman
authors.affiliation: University of Arizona Cancer Center, University of Arizona, Tucson, AZ; and.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Thompson
authors.affiliation: University of Arizona Cancer Center, University of Arizona, Tucson, AZ; and.
authors.forename: Jesse D
authors.initials: JD
authors.lastname: Martinez
authors.affiliation: Sanford/Burnham Medical Research Institute, Cancer Center Division, La Jolla, CA.
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Smith
authors.affiliation: University of Arizona Cancer Center, University of Arizona, Tucson, AZ; and.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Lance
authors.affiliation: Department of Nutritional Sciences and University of Arizona Cancer Center, University of Arizona, Tucson, AZ; and donato@u.arizona.edu.
authors.forename: Donato F
authors.initials: DF
authors.lastname: Romagnolo
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics; D011125:Adenomatous Polyposis Coli / Q000235:genetics*; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D003106:Colon / Q000378:metabolism; D003110:Colonic Neoplasms / Q000235:genetics*; D051546:Cyclooxygenase 2 / Q000378:metabolism; D019175:DNA Methylation; D015870:Gene Expression; D020868:Gene Silencing; D017491:Genes, APC; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D008810:Mice, Inbred C57BL; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics* / Q000378:metabolism; D000099150:Receptor, Farnesoid X-Activated
chemical_list: D001647:Bile Acids and Salts; D051546:Cyclooxygenase 2; D016271:Proto-Oncogene Proteins c-myc; D018160:Receptors, Cytoplasmic and Nuclear; D000099150:Receptor, Farnesoid X-Activated; C489427:MYC protein, human; C099713:nuclear receptor subfamily 0, group B, member 2
keywords: CpG methylation; adenomatous polyposis coli; bile acid metabolism; colon cancer; deoxycholic acid; epigenetics; farnesoid X receptor; inflammation