FoxO3a Nuclear Localization and Its Association with β-Catenin and Smads in IFN-α-Treated Hepatocellular Carcinoma Cell Lines

Interferon-alpha 2b (IFN-alpha 2b) reduces proliferation and increases apoptosis in hepatocellular carcinoma cells by decreasing beta-catenin/TCF4/Smads interaction. Forkhead box O-class 3a (FoxO3a) participates in proliferation and apoptosis and interacts with beta-catenin and Smads. FoxO3a is inhibited by Akt, I kappa B kinase beta (IKK beta), and extracellular-signal-regulated kinase (Erk), which promote FoxO3a sequestration in the cytosol, and accumulates in the nucleus upon phosphorylation by c-Jun N-terminal kinase (JNK) and p38 mitogen-activated kinase (p38 MAPK). We analyzed FoxO3a subcellular localization, the participating kinases, FoxO3a/beta-catenin/Smads association, and FoxO3a target gene expression in IFN-alpha 2b-stimulated HepG2/C3A and Huh7 cells. Total FoxO3a and Akt-phosphorylated FoxO3a levels decreased in the cytosol, whereas total FoxO3a levels increased in the nucleus upon IFN-alpha 2b stimulus. IFN-alpha 2b reduced Akt, IKK beta, and Erk activation, and increased JNK and p38 MAPK activation. p38 MAPK inhibition blocked IFN-alpha 2b-induced FoxO3a nuclear localization. IFN-alpha 2b enhanced FoxO3a association with beta-catenin and Smad2/3/7. Two-step coimmunoprecipitation experiments suggest that these proteins coexist in the same complex. The expression of several FoxO3a target genes increased with IFN-alpha 2b. FoxO3a knockdown prevented the induction of these genes, suggesting that FoxO3a acts as mediator of IFN-alpha 2b action. Results suggest a beta-catenin/Smads switch from TCF4 to FoxO3a. Such events would contribute to the IFN-alpha 2b-mediated effects on cellular proliferation and apoptosis. These results demonstrate new mechanisms for IFN-alpha action, showing the importance of its application in antitumorigenic therapies.