Role of the bile acid receptor TGR5 (GPBAR1) in cholangiocarcinoma (CCA)

K Deutschmann; M Reich; A Lang; R Piekorz; D Häussinger; V Keitel

doi:10.1055/s-0037-1612786

Zeitschrift für Gastroenterologie, Table of Contents

Z Gastroenterol 2018; 56(01): E2-E89
DOI: 10.1055/s-0037-1612786

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Georg Thieme Verlag KG Stuttgart · New York

Role of the bile acid receptor TGR5 (GPBAR1) in cholangiocarcinoma (CCA)

Authors

K Deutschmann

¹University Hospital Düsseldorf, Medical Faculty of the Heinrich Heine University Düsseldorf, Department of Gastroenterology, Hepatology and Infectious Diseases, Düsseldorf
M Reich

¹University Hospital Düsseldorf, Medical Faculty of the Heinrich Heine University Düsseldorf, Department of Gastroenterology, Hepatology and Infectious Diseases, Düsseldorf
A Lang

²University Hospital Düsseldorf, Medical Faculty of the Heinrich Heine University Düsseldorf, Institute for Biochemistry and Molecular Biology II, Düsseldorf
R Piekorz

²University Hospital Düsseldorf, Medical Faculty of the Heinrich Heine University Düsseldorf, Institute for Biochemistry and Molecular Biology II, Düsseldorf
D Häussinger

¹University Hospital Düsseldorf, Medical Faculty of the Heinrich Heine University Düsseldorf, Department of Gastroenterology, Hepatology and Infectious Diseases, Düsseldorf
V Keitel

¹University Hospital Düsseldorf, Medical Faculty of the Heinrich Heine University Düsseldorf, Department of Gastroenterology, Hepatology and Infectious Diseases, Düsseldorf

Abstract

Full Text

Background:

The membrane bound G-protein coupled bile acid receptor TGR5 (GPBAR1) is expressed in epithelial cells of the liver and was found to be overexpressed in human cholangiocarcinoma (CCA) and cell lines generated from CCAs. It was shown that especially secondary bile acids (BAs) play a role in the development of different gastrointestinal malignant tumors, including malignancies of the liver. TGR5 activation triggers secretion as well as proliferative and anti-apoptotic effects in normal cholangiocytes and CCA cell lines. Whether TGR5 may also promote invasiveness and metastasis development of CCA is unclear.

Methods:

TGR5 receptor knockout was achieved in the CCA cell line TFK-1 via Crispr-Cas9 technique. A pool of four sgRNAs, designed to specifically mutate the TGR5 gene contiguous at TMD3 (transmembrane domain 3) were transfected into TFK-1 cells by nucleofection. Empty vector transfection served as control. Puromycin was used for selection of transfected and clonal cells, which were analyzed for their TGR5 genotype, including mRNA and protein expression using nested PCR and Sanger sequencing with two paired primer sets as well as western blot analysis and immunofluorescence staining. Receptor stimulations with the BA TLCA and a TGR5 agonist were performed to determine the migratory and invasive properties using transwell migration and CytoSelect cell invasion assays.

Results/Conclusion:

Using PCR, we identified a Cas9 generated TGR5-deficient clone that showed no TGR5 WT mRNA. Sanger sequencing revealed a 57bp deletion within the TMD3 of the TGR5 gene in this clone. Although TGR5 protein expression and plasma membrane localization is unaffected, ligand binding and downstream signalling seems to be disturbed, underscoring the importance of TMD3 for TGR5 receptor activity. For functional analysis, BA and TGR5 agonist stimulation of TFK-1 parental cells resulted in significantly enhanced migration compared to DMSO control treatment. However, using the TGR5 deficient clone cell migration in response to BA or TGR5 agonist was completely abrogated, indicating that TGR5 is essential for BA mediated migration of CCA cells. This further underscores the hypothesis that activation of TGR5 by BAs can promote CCA progression not only through increased cell proliferation but also through promotion of migration and thus tumor spread.