Z Gastroenterol 2012; 50 - K102
DOI: 10.1055/s-0032-1324037

Differenzial Smad expression contributes to severity of cholangiocarcinoma

S Munker 1, HL Weng 1, Q Li 1, Y Liu 1, C Meyer 1, S Dooley 1, J Li 2
  • 1II. Medizinische Klinik, Universitätsklinikum Mannheim, Mannheim, Germany
  • 2University Hospital, Tübingen, General, Visceral Surgery and Transplantation, Tübingen, Germany

Background and aims: Incidence of cholangiocarcinoma (CC) rises in Western countries. TGF-β plays a dual role in the progression of human cancer and has been implicated in CC. It has been shown that Smad2 and Smad3, two canonical downstream signaling proteins of TGF-β, exert different roles in cancer. In the present study, we investigated the role of Smad2 and Smad3 in CC.

Methods: Immunohistochemical staining for TGF-β1 and phospho-Smad2/3 was performed in 27 paraffin-embedded CC specimens paired with adjacent non-cancerous tissue. The correlation between TGF-β1/Smad immunohistochemical score and characteristics of CC was evaluated. Western blot was used for analysis of TGF-β1/Smad signalling and target genes in tissue samples of 7 CC patients. In vitro, TFK-1, a cholangiocellular carcinoma cell line, was used to assess the impact of TGF-β1 on cancer cell proliferation and epithelial to mesenchymal transition (EMT). Interfering with Smad2 and Smad3 by RNAi and adenoviral overexpression was used to estimate effects of the two Smad proteins in proliferation and EMT of TFK-1 cells.

Results: IHC revealed that decreased Smad2/3 activation in cancer cells correlates significantly with grading, metastasis and lymphnode metastasis of CC in 27 patients. In Western blot of 7 CC samples, we observed overexpression of Smad2 in 3 tumor samples. This significantly correlated with strong Smad2 activation and interestingly also with Smad3 activation. Tumor samples with undetectable Smad2 expression, demonstrated significantly decreased Smad2/3 activation. Smad3 activation was significantly associated with p21 expression. In vitro, Smad3 knockdown reversed TGF-β mediated cell cycle arrest, as measured by an increased incorporation of BrdU, elevated PCNA and reduced p21 levels. Adenoviral overexpression only of Smad3 but not Smad2 resulted in elevated p21 levels. Knockdown of Smad3 decreased TGF-β mediated stress fiber formation, loss of cell cell contacts and cellular spreading, suggesting a central role of Smad3 in EMT of this cancer.

Conclusion: Decreased Smad2/3 signaling is significantly associated with cancer properties indicating a poor prognosis, such as metastasis and invasiveness. We hypothesise that loss of Smad2 expression is a key event in this cancer.