Connection between Wnt/beta-catenin and TGF-beta/Smads pathways in Hepatocellular Carcinoma (HCC): Interferon-alfa2b (IFN) and Transforming Growth Factor-beta1 (TGF) treatment effects on HCC cell lines

CEBALLOS, MARIA PAULA; PARODY, JUAN PABLO; ALVAREZ, MARÍA DE LUJÁN; INGARAMO, PAOLA; CARNOVALE, CRISTINA ESTER; CARRILLO, MARÍA CRISTINA

Barcelona

Congreso; 47th Annual Meeting of the European Association for the Study of the Liver; 2012

European Association for the Study of the Liver (EASL)

Background and aims: Wnt/b-catenin signaling is frequently dysregulated in HCC. Activated b-catenin accumulates in the cytosol and nucleus associating with TCF/LEF factors like TCF4 and promoting proliferation. IFN has recently been recognized to harbor therapeutic potential in HCC prevention and treatment. TGF is a mediator of apoptosis and exerts its effects via Smads proteins. One form of interaction between Wnt/b-catenin and TGF/Smads pathways is the association of Smads with b-catenin/TCF4. We have previously found that IFN and TGF decrease proliferation and increase apoptosis in HepG2 and Huh7. We decided to analyze IFN and TGF effects on Wnt/b-catenin pathway, Smads proteins and b-catenin/TCF4/Smads interaction in these HCC cell lines. We also evaluated the effect of an increased b-catenin protein level, by using Wnt3a, on the content of Smads proteins. Methods: We treated HepG2 and Huh7 with or without IFN and/or TGF during 48hs and evaluated by western blot the contents of Frizzled 7 (a receptor of Wnt/b-catenin pathway) in total cell lysates, and b-catenin and Smads levels in cytosolic and nuclear extracts. Also, we analyzed the cytokines effect on b-catenin/TCF4/Smads complex by co-immunoprecipitation. Finally, we treated HCC cell lines with Wnt3a during 48hs and then evaluated b-catenin and Smads levels in cytosolic and nuclear extracts by western blot. Results: IFN and/or TGF attenuated Wnt/b-catenin signal by significantly decreasing b-catenin and Frizzled7 receptor proteins and b-catenin/TCF4 interaction. The truncated b-catenin form present in HepG2 also significantly diminished after treatments. Both cytokines significantly declined Smads proteins levels and their association with TCF4. Wnt3a treatment, which elevates b-catenin protein levels, also generated a significant increment of Smads proteins when comparing with untreated cells. Conclusions: IFN and TGF proved to be effective as negative modulators of Wnt/b-catenin pathway in HCC cell lines holding both wild-type and truncated b-catenin. The inhibition of b-catenin/TCF4/Smads complexes formation may have a critical role in slowing down oncogenesis, since both cytokines diminish proliferation and increase apoptosis. In this way, IFN and TGF could be useful as potential treatments in patients with HCC. Moreover, Wnt3a experiments demonstrate that Wnt/b-catenin signaling status is able to modulate Smads levels.