Role of the bile acid receptor TGR5 (GPBAR1) in cholangiocarcinoma (CCA) progression and tumor spread

Background The membrane bound G-protein coupled bile acid receptor TGR5 is expressed in liver epithelial cells. Its activation 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. Furthermore, TGR5 and the bile acid receptor S1PR2 show partly overlapping signalling functions in biliary epithelial cells. To date, it is unclear whether there is a potential crosstalk of both receptors.

Methods In the human CCA cell line TFK-1, the CRISPR/Cas9 mediated TGR5 knockout was achieved via nucleofection using sgRNAs that specifically mutate the transmembrane domain 3 (TMD3). Puromycin selected clonal cells were analyzed by Sanger sequencing, quantitative Realtime PCR, immunofluorescence staining and additionally by homology modelling. Furthermore, receptor stimulations of Cas9 mediated TGR5 knockout and control cells with BAs and a TGR5 agonist were performed to measure the proliferative response using BrdU-incorporation assay, 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 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. Moreover, 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. S1PR2 can induce proliferation and invasion in CCA cells as well. Elevated S1PR2 expression levels in TGR5 truncated cells imply a compensational crosstalk of both bile acid receptors TGR5 and S1PR2, nevertheless S1PR2 elevation cannot fully restore the TGR5 effects.

Publikationsverlauf

Artikel online veröffentlicht:
04. Januar 2021