Attenuation of wnt/beta-catenin/TCF pathway by in vivo interferon (IFN-a2b) treatment in preneoplastic rat livers

PARODY, JUAN PABLO; ALVAREZ, MARÍA DE LUJÁN; QUIROGA, ARIEL DARÍO; CEBALLOS, MARÍA PAULA; FRANCÉS, DANIEL; PISANI, GERARDO BRUNO; PELLEGRINO, JOSÉ MANUEL; CARNOVALE, CRISTINA; CARRILLO, MARÍA CRISTINA

Milán, Italia

Congreso; International Liver Cancer Association Third Annual Conference; 2009

International Liver Cancer Association

Background: It has been described that the wnt/b-catenin/TCF pathway is activated inseveral types of cancers. In previous works using our rat liver preneoplastic model, wehave demonstrated that IFN-a2b reduces the number and volume of altered hepatic fociinducing apoptosis through a mechanism mediated by reactive oxygen species (ROS)and TGF-b1. Furthermore, it is known that b-catenin interacts with FOXO transcriptionfactors 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 ofhepatocarcinogenesis and the effects of IFN-a2b treatment on it. Methods: Wistar ratswere subjected to a two-phase model of hepatocarcinogenesis (IP). A group of IP ratswas 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 byimmunofluorescence and immunoblotting. Activation of the wnt/b-catenin/TCFpathway was evaluated by immunoblotting with anti p-b-catenin and bysemiquantitative RT-PCR on several TCF target genes. b-catenin mutation analysis wasperformed by direct sequencing. Protein expression level of Frizzled-7 was analyzed byimmunoblotting. Finally, to evaluate a possible IFN-a2b-induced switching from bcatenin/TCF to b-catenin/FOXO pathway, expression of FOXO target genes wasevaluated by semiquantitative RT-PCR. Results: b-catenin membrane delocalization inIP and, to a lesser extent, in IP+IFN livers was observed. C and C+IFN groups showedb-catenin plasma membrane localization. Total b-catenin protein levels was unchangedin liver lysates from all groups, however p-b-catenin levels was significantly decreasedonly in IP group compared with C. Using semiquantitative RT-PCR we found that asubset of b-catenin target genes such as Cyclin-D1, MMP7, Axin 2 and SP5 wereoverexpressed at mRNA level only in IP group compared to C. Direct sequencinganalysis of b-catenin transcript revealed the absence of mutations in all groups. Inaddition, a significant overexpression of Frizzled-7 at the mRNA and protein levels inIP 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 othergroups showed transcript levels similar to C group. Conclusion: We demonstrated thatwnt/b-catenin/TCF pathway is activated at an early stage of hepatocarcinogenesis in thepresence of wild-type b-catenin as a consequence of Frizzled-7 overexpression. Weconclude that the production of ROS and TGF-b1 induced by IFN-a2b treatmentdescribed in previous works, leads to inhibition of TCF transcriptional activity andenhances FOXO activation. As a consequence, apoptosis pathway is activated favoringthe elimination of preneoplastic cells.