Smad3 linker phosphorylation drives carcinogenesis in cholangiocarcinoma (CCA)

Background TGF-β signal transduction initiates with receptor-mediated C-terminal phosphorylation of Smad2/3 with cytostatic outcome towards epithelial cell types. Regulatory phosphorylation steps in the Smad linker (L) domain create phosphorylated forms with mitogenic functions by debranching signalling and transcriptional activity or initiating proteasomal degradation. It is known that Smad3 linker phosphorylation modulates TGF-β signalling towards cancer progression. The contribution of variant Smad3 phosphorylation patterns in CCA has not been investigated.

Methods We examined healthy and tumorigenic liver tissues from CCA patients for their Smad phosphorylation status at C-tail- and linker residues using immunoblotting and IHC and will correlate it with clinical data. We treated cultured CCA cells with TGF-β, with/without inhibitors for TGF-βRI (LY2157299) and GSK-3β (SB216763) and investigated the impact on tumour features such as proliferation, invasion and chemoresistance by measuring cell growth and the number of invasive cells over 72 hours with/without drug treatment.

Results Tumorigenic areas from intrahepatic CCA patients show decreased pSmad3C and increased pSmad3L (S213 and S204) levels in cancer cells and stromal hepatocytes (or NPC), whereas non-tumorous liver tissue shows strong pSmad3C and weak pSmad3L staining. To correlate these results with clinical data, more patient samples are investigated. CCA cell lines display a counter regulation of C-terminal versus linker phosphorylation. Intrinsic pSmad3L (S204 and S213) was blunted by a GSK-3β Inhibitor, and led to increased phosphorylation of Smad3C, which was further enhanced by TGF-b. When instead inhibiting ALK5, TGF-β treatment increased levels of pSmad3L. Downregulated pSmad3C increased cell proliferation and c-Myc expression, and decreased p21 expression at mRNA level. Depletion of pSmad3C increased chemoresistance against different drugs used in CCA patients and invasive activity, which was associated with enhanced expression of MMP-9 and reduced expression of PAI-1.

Conclusion Our data suggest that Smad3L (S213 and S204) phosphorylation is predominant in CCA, and interferes with cytostatic pSmad3C (S423/425) facilitating a malignant switch in TGF-β signalling and cellular response.

Publication History

Article published online:
07 September 2021