Inactivation of adenomatous polyposis coli reduces bile acid/farnesoid X receptor expression through FXR gene cpgmethylation in mouse colon tumors and human colon cancer cells

Ornella Selmin, Changming Fang, Adam M. Lyon, Thomas C Doetschman, Patricia A. Thompson, Jesse D Martinez, Jeffrey W. Smith, Michael P Lance, Donato Romagnolo

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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 (ApcMin/+) 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, wemeasured 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 ApcMin/+ 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.

Original languageEnglish (US)
Pages (from-to)236-242
Number of pages7
JournalJournal of Nutrition
Volume146
Issue number2
DOIs
StatePublished - 2016

Fingerprint

Adenomatous Polyposis Coli
Bile Acids and Salts
Colonic Neoplasms
Colon
Mucous Membrane
Methylation
Deoxycholic Acid
Genes
Neoplasms
HCT116 Cells
Cyclooxygenase 2
HT29 Cells
Cytoplasmic and Nuclear Receptors
RNA Interference
Names
Real-Time Polymerase Chain Reaction
Colorectal Neoplasms
Homeostasis
Inflammation
Gene Expression

Keywords

  • Adenomatous polyposis coli
  • Bile acid metabolism
  • Colon cancer
  • CpG methylation
  • Deoxycholic acid
  • Epigenetics
  • Farnesoid X receptor
  • Inflammation

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Nutrition and Dietetics

Cite this

@article{0a1f6147c9094e16a5d5cbe9193a2d3d,
title = "Inactivation of adenomatous polyposis coli reduces bile acid/farnesoid X receptor expression through FXR gene cpgmethylation 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 (ApcMin/+) 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, wemeasured 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 ApcMin/+ 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.",
keywords = "Adenomatous polyposis coli, Bile acid metabolism, Colon cancer, CpG methylation, Deoxycholic acid, Epigenetics, Farnesoid X receptor, Inflammation",
author = "Ornella Selmin and Changming Fang and Lyon, {Adam M.} and Doetschman, {Thomas C} and Thompson, {Patricia A.} and Martinez, {Jesse D} and Smith, {Jeffrey W.} and Lance, {Michael P} and Donato Romagnolo",
year = "2016",
doi = "10.3945/jn.115.216580",
language = "English (US)",
volume = "146",
pages = "236--242",
journal = "Journal of Nutrition",
issn = "0022-3166",
publisher = "American Society for Nutrition",
number = "2",

}

TY - JOUR

T1 - Inactivation of adenomatous polyposis coli reduces bile acid/farnesoid X receptor expression through FXR gene cpgmethylation in mouse colon tumors and human colon cancer cells

AU - Selmin, Ornella

AU - Fang, Changming

AU - Lyon, Adam M.

AU - Doetschman, Thomas C

AU - Thompson, Patricia A.

AU - Martinez, Jesse D

AU - Smith, Jeffrey W.

AU - Lance, Michael P

AU - Romagnolo, Donato

PY - 2016

Y1 - 2016

N2 - 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 (ApcMin/+) 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, wemeasured 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 ApcMin/+ 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.

AB - 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 (ApcMin/+) 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, wemeasured 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 ApcMin/+ 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.

KW - Adenomatous polyposis coli

KW - Bile acid metabolism

KW - Colon cancer

KW - CpG methylation

KW - Deoxycholic acid

KW - Epigenetics

KW - Farnesoid X receptor

KW - Inflammation

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U2 - 10.3945/jn.115.216580

DO - 10.3945/jn.115.216580

M3 - Article

C2 - 26609171

AN - SCOPUS:84959301201

VL - 146

SP - 236

EP - 242

JO - Journal of Nutrition

JF - Journal of Nutrition

SN - 0022-3166

IS - 2

ER -