Attenuation of Wnt/beta-catenin/TCF pathway by in vivo interferon-alpha2b (IFN-alpha2b) treatment in preneoplastic rat livers

PARODY, JUAN PABLO; ALVAREZ, MARIA DE LUJAN; QUIROGA, ARIEL DARIO; CEBALLOS, MARIA PAULA; FRANCES, DANIEL ELEAZAR ANTONIO; PISANI, GERARDO BRUNO; PELLEGRINO, JOSE MANUEL; CARNOVALE, CRISTINA ESTER; CARRILLO, MARÍA CRISTINA

Milan

Congreso; International Liver Cancer Association (ILCA)’s Third Annual Conference; 2009

Background: It has been described that the wnt/b-catenin/TCF pathway is activated in several types of cancers. In previous works using our rat liver preneoplastic model, we have demonstrated that IFN-a2b reduces the number and volume of altered hepatic foci inducing apoptosis through a mechanism mediated by reactive oxygen species (ROS) and TGF-b1. Furthermore, it is known that b-catenin interacts with FOXO transcription factors in response to oxidative stress and mediates TGF-b1-induced apoptosis process. Objective: To analyze the status of the wnt/b-catenin/TCF pathway in an early stage of hepatocarcinogenesis and the effects of IFN-a2b treatment on it. Methods: Wistar rats were subjected to a two-phase model of hepatocarcinogenesis (IP). A group of IP rats was treated with IFN-a2b (IP+IFN). Control rats were treated only with IFN-a2b (C+IFN) or saline (C). Foci detection and b-catenin localization was assessed by immunofluorescence and immunoblotting. Activation of the wnt/b-catenin/TCF pathway was evaluated by immunoblotting with anti p-b-catenin and by semiquantitative RT-PCR on several TCF target genes. b-catenin mutation analysis was performed by direct sequencing. Protein expression level of Frizzled-7 was analyzed by immunoblotting. Finally, to evaluate a possible IFN-a2b-induced switching from b-catenin/TCF to b-catenin/FOXO pathway, expression of FOXO target genes was evaluated by semiquantitative RT-PCR. Results: b-catenin membrane delocalization in IP and, to a lesser extent, in IP+IFN livers was observed. C and C+IFN groups showed b-catenin plasma membrane localization. Total b-catenin protein levels was unchanged in liver lysates from all groups, however p-b-catenin levels was significantly decreased only in IP group compared with C. Using semiquantitative RT-PCR we found that a subset of b-catenin target genes such as Cyclin-D1, MMP7, Axin 2 and SP5 were overexpressed at mRNA level only in IP group compared to C. Direct sequencing analysis of b-catenin transcript revealed the absence of mutations in all groups. In addition, a significant overexpression of Frizzled-7 at the mRNA and protein levels in IP compared with C was shown; this increment was not so pronounced in IP+IFN. Finally, transcriptional activity of FOXO is enhanced in IP+IFN animals, while all other groups showed transcript levels similar to C group. Conclusion: We demonstrated that wnt/b-catenin/TCF pathway is activated at an early stage of hepatocarcinogenesis in the presence of wild-type b-catenin as a consequence of Frizzled-7 overexpression. We conclude that the production of ROS and TGF-b1 induced by IFN-a2b treatment described in previous works, leads to inhibition of TCF transcriptional activity and enhances FOXO activation. As a consequence, apoptosis pathway is activated favoring the elimination of preneoplastic cells.