Pegylated interferon alpha targets Wnt signaling by inducing nuclear export of β-catenin

Abstract

Background & aims: Pegylated-Interferon-α2a (peg-IFN), a first line therapy for Hepatitis C virus (HCV) patients, also impacts the recurrence of hepatocellular carcinoma (HCC). The activation of the Wnt pathway due to β-catenin gene mutations contributes to the development of a significant subset of HCC. Herein, we explored the effect of peg-IFN on Wnt/β-catenin signaling in vitro and in vivo.

Methods: Multiple human hepatoma cell lines were treated with Peg-IFN to assess its effect on the Wnt pathway and the mechanisms involved. Transgenic (TG) mice expressing stable β-catenin mutant in the liver were exposed to diethylnitrosamine (DEN) and treated with peg-IFN.

Results: In vitro, peg-IFN decreased the transcriptional activity of β-catenin/Tcf and did so independently of JAK/Stat signaling. Peg-IFN treatment led to increased mRNA and protein expression of RanBP3, a known β-catenin nuclear export factor, in all hepatoma cells. Co-precipitation studies showed an increased association between RanBP3 and β-catenin after peg-IFN treatment. The siRNA-mediated RanBP3 knockdown abrogated Peg-IFN-induced decrease in TOPFlash reporter activity. In vivo, Peg-IFN treatment led to increased nuclear RanBP3, decreased nuclear β-catenin and cyclin D1, and decreased cytoplasmic glutamine synthetase. Increased association of RanBP3 and β-catenin was also observed in vivo in response to Peg-IFN that led to decreased hepatocyte proliferation.

Conclusions: Peg-IFN inhibits β-catenin signaling through the up-regulation of RanBP3, which may be a contributory mechanism for the delayed HCC and improved survival in treated HCV patients. This observation might have chemo-preventive or chemo-therapeutic implications in tumor with aberrant Wnt pathway activation.

Figure 1. Peg-IFN decreases β-catenin activity in vitro and does so independent of JAK/STAT signaling

A. Relative luciferase activity following transfection with either TOPFlash or FOPFlash reporter plasmid and treatment with three or four doses of Peg-IFN in HepG2 cells. Experiments were done in triplicate and averaged. (*p<0.05) B. Relative luciferase activity following transfection with either TOPFlash or FOPFlash reporter plasmid and one treatment with Peg-IFN or with one treatment with lithium followed by one dose treatment of Peg-IFN in Hep3Bcells. Experiments were done in triplicate and averaged. (**p<0.005) C. Inhibition of JAK/Stat pathway with AG490 does not reverse the effect of Peg-IFN on Topflash indicating that this effect is independent of the JAK/Stat pathway.

Figure 2. Peg-IFN increases Dkk-1 and RanBP3 expression in multiple hepatoma cell lines, and increases β-catenin-RanBP3 association

A. Real-time PCR showing increase in expression of Dkk-1 and RanBP3 in Hep3B cells after Peg-IFN. Values normalized to control and presented as fold-change. B. Real-time PCR showing increase in expression of Dkk-1 and RanBP3 in HepG2 cells after Peg-IFN. Values normalized to control and presented as fold-change. C. Increased total RanBP3 protein is evident after dose 1 (D1) and D2 in Hep3B, D2 in HepG2 and D2 and D3 in Huh-7 cells by western blots. D. Immunoprecipitation studies demonstrate an increase in RanBP3-β-catenin association following Peg-IFN doses. Densitometric values representing an increase in β-catenin-RanBP3 association following Peg-IFN treatment relative to saline-treated controls are indicated.

Figure 3. Negative effect on Wnt pathway after Peg-IFN treatment is via upregulation of RanBP3

A. TOPFlash luciferase assay showing a decrease in reporter activity after overexpression of RanBP3 in HepG2 cells. B. Topflash luciferase reporter assay shows a significant decrease in β-catenin activity after Peg-IFN treatment in control siRNA transfected Hep3B cells (p<0.01). Transfection of RanBPB3 siRNA that leads to a dramatic suppression of RanBP3 protein expression after 48 hours (upper panel) led to a significant abrogation of Peg-IFN-induced reduction of TOPFlash reporter activity (p<0.05).

Figure 4. Peg-IFN decreases β-catenin activity in vivo

A. Immunoblotting shows decreased levels of nuclear β-catenin and cyclin D1 after 6 weekly treatments with 7000U of Peg-IFN. B. Immunoblotting shows decreased cytoplasmic levels of glutamine synthetase after 6 weekly treatments with 7000U of Peg-IFN. C. IHC for PCNA in liver of animals treated with saline or Peg-IFN. Arrows indicate representative PCNA positive hepatocytes. D. Significant difference in the numbers of PCNA positive hepatocytes per high power field (200x) in the liver of animals treated with saline or Peg-IFN at either dose (*p<0.05). E. An increase in total RanBP3 is observed in whole cell liver lysates from mice that underwent treatment with 6 weekly doses of Peg-IFN at 7000U. F. Coprecipitation studies identify a dramatic increase in RanBP3-β-catenin association in whole liver cell lysates of mice treated with 6 weekly Peg-IFN injections of 7000U each as compared to saline treated animals.