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

Background:

The membrane bound G-protein coupled bile acid receptor TGR5 (GPBAR1) is expressed in epithelial cells of the liver. Activation of the TGR5 receptor through bile acids (BAs) triggers secretion, proliferation and anti-apoptotic effects in normal cholangiocytes and CCA cell lines. TGR5 was found to be overexpressed in human cholangiocarcinoma tissue (CCA) and cell lines generated from CCAs. Especially secondary bile acids play a role in the development of different malignant tumors in the gastrointestinal tract, including liver. Whether TGR5 activation also promotes invasiveness and metastasis development of CCA is unclear and aim of this work.

Methods:

Crispr/Cas9 mediated TGR5 knockout was achieved in the human CCA cell line TFK-1 using sgRNAs that specifically mutate the transmembrane domain 3 (TMD3). Cells were transfected sgRNA containing Cas9 vector via nucleofection while empty vector served as control. Puromycin selected clonal cells were analyzed for their TGR5 genotype and phenotype by Sanger sequencing, immunofluorescence staining and analyzed by homology modelling. Receptor stimulations of Cas9 mediated TGR5 knockout and control cells with the BAs and a TGR5 agonist were performed to measure the proliferative response using BrdU-incorporation, as well as migratory and invasive properties using transwell cell migration and invasion assays.

Results/Conclusion:

Using Crispr/Cas9 technique, we generated a TGR5 deletion variant Δ89 – 110, lacking 57 bp within TMD3. While protein expression and plasma membrane localization of the TGR5 variant was unaffected, bile acid and agonist induced cell proliferation, migration and invasion was abolished in TGR5Δ89 – 110 expressing cells. Modelling of the truncated receptor revealed complete obstruction of the receptor binding pocket by the shortened TMD3, rendering receptor activation impossible. Together, these data indicate that TMD3 is essential for BA mediated TGR5 activation. Furthermore, TGR5 truncated CCA cells were resistant to BA induced cell proliferation, migration and invasion, underscoring the role for TGR5 in CCA progression and tumor spread.