Bile acids increase levels of microRNAs 221 and 222, leading to degradation of CDX2 during esophageal carcinogenesis.
OBJECTIVE
Bile reflux contributes to development of Barrett's esophagus (BE) and could be involved in its progression to esophageal adenocarcinoma (EAC). We investigated whether bile acids affect levels or functions of microRNAs (MIRs) 221 and 222, which bind to the 3'-UTR of p27Kip1 messenger RNA to inhibit its translation. Reduced p27Kip1 increases degradation of the transcription factor CDX2; levels of CDX2 have been reported to decrease during progression of BE to EAC.


METHODS
We used quantitative reverse transcriptase polymerase chain reaction to compare levels of MIRs 221 and 222 and immunohistochemistry to compare levels of p27Kip1 and CDX2 proteins in areas of BE and EAC from each of 11 patients. We examined the effects of bile acid exposure on levels of MIRs 221 and 222 and CDX2 in EAC cells. We investigated the effects of inhibitors of MIRs 221 and 222 on growth of human EAC xenograft tumors in NOD/SCID/IL-2Rγ(null) mice.


RESULTS
Levels of MIRs 221 and 222 increased and levels of p27Kip1 and CDX2 decreased in areas of EAC vs BE. Levels of MIRs 221 and 222 increased, along with activity of nuclear bile acid receptor/farnesoid X receptor (FXR), when cultured cells were exposed to bile acids. Incubation of cells with bile acids increased degradation of CDX2; this process was reduced when cells were also incubated with proteasome inhibitors. Overexpression of MIRs 221 and 222 reduced levels of p27Kip1 and CDX2, and knockdown of these MIRs increased levels of these proteins in cultured cells. Inhibitors of MIRs 221 and 222 increased levels of p27Kip1 and CDX2 in EAC cells and reduced growth of xenograft tumors in NOD/SCID/IL-2Rγ(null) mice.


CONCLUSIONS
We observed increased levels of MIRs 221 and 222 in human EAC tissues, compared with areas of BE from the same patient. We found that exposure of esophageal cells to bile acids activates FXR and increases levels of MIRs 221 and 222, reducing levels of p27Kip1 and promoting degradation of CDX2 by the proteasome. Our work opened the perspective of therapeutically targeting this pathway either via FXR antagonists or inhibitors of MIRs as a treatment option for BE and EAC.
Division of Gastroenterology and Hepatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan; Department of Internal Medicine, Saiseikai Central Hospital, Tokyo, Japan.
Juntaro
J
Matsuzaki
Hidekazu
H
Suzuki
Hitoshi
H
Tsugawa
Mitsuhiro
M
Watanabe
Sharif
S
Hossain
Eri
E
Arai
Yoshimasa
Y
Saito
Shigeki
S
Sekine
Toshihiro
T
Akaike
Yae
Y
Kanai
Ken-ichi
K
Mukaisho
Johan
J
Auwerx
Toshifumi
T
Hibi
D000230:Adenocarcinoma / Q000378:metabolism* / Q000473:pathology; D000368:Aged; D000818:Animals; D001471:Barrett Esophagus / Q000378:metabolism / Q000473:pathology; D001647:Bile Acids and Salts / Q000494:pharmacology*; D000071616:CDX2 Transcription Factor; D063646:Carcinogenesis / Q000378:metabolism*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D018450:Disease Progression; D004938:Esophageal Neoplasms / Q000378:metabolism* / Q000473:pathology; D005260:Female; D018398:Homeodomain Proteins / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D018345:Mice, Knockout; D016513:Mice, SCID; D035683:MicroRNAs / Q000187:drug effects / Q000378:metabolism*; D008875:Middle Aged; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays; D000099150:Receptor, Farnesoid X-Activated
D001647:Bile Acids and Salts; D000071616:CDX2 Transcription Factor; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018398:Homeodomain Proteins; D035683:MicroRNAs; D046988:Proteasome Endopeptidase Complex; D018160:Receptors, Cytoplasmic and Nuclear; D000099150:Receptor, Farnesoid X-Activated; C491270:CDX2 protein, human; C525709:MIR221, human; C525710:MIR222, human
BE; Barrett's esophagus; CA; CDCA; CDX2; Cancer; EAC; FXR; MIR; Mouse Model; Post-Transcriptional Modification; Tumor Development; caudal-related homologue 2; chenodeoxycholic acid; cholic acid; esophageal adenocarcinoma; mRNA; messenger RNA; microRNA; nuclear bile acid receptor/farnesoid X receptor