Smad3 linker phosphorylation is predominant in cholangiocarcinoma
13. August 2018 (online)
Transforming growth factor (TGF)-β has been implicated in development and progression of cholangiocellular carcinoma (CCC). The canonical TGF-β pathway involves TGF-β receptor-mediated C-terminal phosphorylation of Smad3. On the other hand, non Smad TGF-β signaling as well as TGF-β independent signaling may activate several serine/threonine kinases to phosphorylate Smad3 in its linker region, which impacts on canonical Smad signaling, target gene expression signature and cellular fate. We identified presence of significant Smad3 linker region phosphorylation in cholangiocarcinoma tissues and now try to delineate the function.
Material and methods:
We performed immunoblotting using specific antibodies against the different Smad3 phosphosites (p-Smad3Cser423/425, p-Smad3LThr179, p-Smad3LSer204, p-Smad3LSer208, p-Smad3LSer213) with and without TGF-β stimulation in CCC cells. To determine upstream kinases necessary for the respective phosphorylation events, CCC cells were (pre)-treated with inhibitors: JNK, ERK, p38, GSK3β, CDK and TGF-βRI/ALK5 Inhibitors for 30 min before being treated with TGF-β1 for 1h (or not).
It is observed in 5 tested CCC patients that phospho-Smad3LSer204 and phospho-Smad3LSer213 positive areas exclude positivity for phospho-Smad3Cser423/425 staining, indicating a mechanism of counter-regulation between Smad3 C-terminal and linker site patterns. In the screen with kinase inhibitors performed in CCSW1 cells, we found that GSK3 and CDK are involved in phosphorylation of Smad3LSer204 and Smad3LSer213.
Conclusions acurrent steps:
Our preliminary findings suggest that pSmad3Lser204 and pSmad3Lser213 are predominant in cholangiocarcinoma cells and may play a tumor promoter role by antagonizing cytostatic pSmad3Cser423/425 and there with tumor suppressor function. To deeply investigate the direct influence of Smad3 linker phosphorylation in cholangiocarcinoma, we have established and currently use cholangiocarcinoma patient-derived organoids for further testing and will delineate downstream effects in vitro with phosphosite mutated Smad3 expression plasmids.