cells Article Additional Inhibition of Wnt/β-Catenin Signaling by Metformin in DAA Treatments as a Novel Therapeutic Strategy for HCV-Infected Patients Dong Lin *, Venu Reddy, Hanadi Osman, Adriana Lopez , Ali Riza Koksal , Sadeq Mutlab Rhadhi, Srikanta Dash and Yucel Aydin *   Citation: Lin, D.; Reddy, V.; Osman, H.; Lopez, A.; Koksal, A.R.; Rhadhi, S.M.; Dash, S.; Aydin, Y. Additional Inhibition of Wnt/β-Catenin Signaling by Metformin in DAA Treatments as a Novel Therapeutic Strategy for HCV-Infected Patients. Cells 2021, 10, 790. https://doi.org/ 10.3390/cells10040790 Academic Editor: Philippe Gallay Received: 1 March 2021 Accepted: 27 March 2021 Published: 2 April 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Laboratory Medicine and Department of Pathology, Tulane University School of Medicine, New Orleans, LA 70112, USA; vreddy2@tulane.edu (V.R.); hosman1@tulane.edu (H.O.); alopez15@tulane.edu (A.L.); akoksal@tulane.edu (A.R.K.); srhadhi@tulane.edu (S.M.R.); sdash@tulane.edu (S.D.) * Correspondence: dlin6@tulane.edu (D.L.); yaydin@tulane.edu (Y.A.); Tel.: +1-504-988-2421 (D.L.); +1-443-579-6318 (Y.A.) Abstract: Chronic hepatitis C virus (HCV) infection causes hepatocellular carcinoma (HCC). Al- though HCV clearance has been improved by the advent of direct-acting antiviral agents (DAA), retrospective studies have shown that the risk of subsequent HCC, while considerably decreased compared with active HCV infection, persists after DAA regimens. However, either the mechanisms of how chronic HCV infection causes HCC or the factors responsible for HCC development after viral eradication in patients with DAA treatments remain elusive. We reported an in vitro model of chronic HCV infection and determined Wnt/β-catenin signaling activation due to the inhibition of GSK-3β activity via serine 9 phosphorylation (p-ser9-GSK-3β) leading to stable non-phosphorylated β-catenin. Immunohistochemical staining demonstrated the upregulation of both β-catenin and p-Ser9-GSK-3β in HCV-induced HCC tissues. Chronic HCV infection increased proliferation and colony-forming ability, but knockdown of β-catenin decreased proliferation and increased apoptosis. Unexpectedly, Wnt/β-catenin signaling remained activated in chronic HCV-infected cells after HCV eradication by DAA, but metformin reversed it through PKA/GSK-3β-mediated β-catenin degra- dation, inhibited colony-forming ability and proliferation, and increased apoptosis, suggesting that DAA therapy in combination with metformin may be a novel therapy to treat HCV-associated HCC where metformin suppresses Wnt/β-catenin signaling for HCV-infected patients. Keywords: chronic hepatitis C virus (HCV) infection; Wnt/β-catenin signaling; glycogen synthase kinase-3β (GSK-3β); direct-acting antivirus agents (DAA); metformin 1. Introduction Chronic hepatitis C virus (HCV) infection is a major risk factor for the development of hepatocellular carcinoma (HCC). Over the past decade, deaths caused by chronic HCV- induced HCC increased by 21.1% [1]. HCV is a positive-sense single-stranded RNA virus with 9.6 kb in length and is unable to integrate into the host genome [2,3]. The HCV genomes encodes a single polyprotein. The polyprotein is processed by cellular and viral proteases to generate 10 polypeptides including three structural proteins (core, E1, and E2), viroprotein p7, and six non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The Wnt/β-catenin signaling strongly contributes to tumorigenesis in a variety of tumor types [4–9]. β-catenin plays a critical role in the Wnt/β-catenin signaling path- way. Its cellular protein turnover is tightly regulated by ubiquitin-mediated degradation controlled by a phosphorylation-dependent ubiquitylation signal. The stabilization and accumulation of β-catenin is usually induced by aberrant Wnt/β-catenin pathway acti- vation, destruction complex components, or somatic gene mutations of β-catenin [10,11]. Under normal physiological conditions, β-catenin in the cytosol is phosphorylated by Cells 2021, 10, 790. https://doi.org/10.3390/cells10040790 https://www.mdpi.com/journal/cells