Are Hedgehog and Wnt/β-catenin pathways involved in hepatitis C virus-mediated EMT?
Authors: Beatrice Conti, Antonella Minutolo, Mario Arciello, Clara Balsano
Journal: Journal of Hepatology (2012)

Hepatitis C viral protein NS5A induces EMT and participates in oncogenic transformation of primary hepatocyte precursorsJournal of HepatologyVol. 57Issue 5PreviewApicobasal polarity, which is essential for epithelial structure and function, is targeted by several tumour-related pathogens and is generally perturbed in the course of carcinogenesis. Hepatitis C virus (HCV) infection is associated with a strong risk of hepatocellular carcinoma, typically preceded by dysplastic alterations of cell morphology. We investigated the molecular mechanisms and the functional consequences of HCV-driven perturbations of epithelial polarity. Full-Text PDF Reply to: “Are Hedgehog and Wnt/β-catenin pathways involved in hepatitis C virus-mediated EMT?”: Persistent activation of Hedgehog or Wnt/β-catenin pathways is not involved in EMT mediated by HCV NS5A proteinJournal of HepatologyVol. 58Issue 3PreviewWe thank Conti et al. for the interesting discussion of our recent paper. We agree that it is of major interest to delineate the molecular mechanisms leading from HCV protein NS5A to Twist2 induction, which we have shown to trigger EMT in hepatocytes. Several plausible effectors of NS5A could be involved in this process, including the Wnt/β-catenin and the Hedgehog (HH) pathways. Full-Text PDF Open Access We read with great interest the article by Akkari et al. [[1]Akkari L. Grégoire D. Floc’h N. Moreau M. Hernandez C. Simonin Y. et al.Hepatitis C viral protein NS5A induces EMT and participates in oncogenic transformation of primary hepatocyte precursors.J Hepatol. 2012; 57: 1021-1028Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar], who evaluated the EMT induction and correlated oncogenic transformation induced by HCV protein NS5A in primary hepatocyte precursors. Epithelial–mesenchymal transition (EMT) is a biological process occurring during tissue development and regeneration, moreover, it is known to be involved in tumor progression and metastasis. The EMT is a phenomenon, which well represents the cellular plasticity, i.e., the ability of polarized epithelial cells, which through multiple biochemical changes acquire a mesenchymal cell phenotype. These cells exhibit loss of cell adhesion, enhanced migratory capacity, invasiveness, elevated resistance to apoptosis and a great ability to produce extracellular matrix (ECM) components. Several molecular processes are involved in EMT onset and progression. Among them we have to mention: activation of transcription factors (e.g., Snail, Twist, etc.), expression of specific cell-surface proteins, remodelling of cytoskeletal components, production of ECM-degrading enzymes and changes in the expression of specific microRNAs (e.g., microRNA 200 family). Most of them are commonly used as biomarkers to highlight a cellular EMT [2Kalluri R. Weinberg R.A. The basics of epithelial–mesenchymal transition.J Clin Invest. 2009; 119: 1420-1428Crossref PubMed Scopus (7430) Google Scholar, 3Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial–mesenchymal transitions in development and disease.Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (7659) Google Scholar]. Akkari et al. have evaluated how a viral protein can alter the typical epithelial architecture of hepatic cells. In particular, they demonstrated that the ectopic expression of hepatitis C virus (HCV) non-structural protein 5a (NS5A) is able to induce EMT in bipotential mouse embryonic liver (BMEL) cells which acquire mesenchymal phenotype, increased motility and invasiveness, and are featured by the downregulation of epithelial markers (e.g., E-Cadherin) and the upregulation of mesenchymal markers (like Vimentin and Twist2). They also confirmed their results in an in vivo xenograft mouse model. To unravel the molecular mechanisms involved in EMT occurrence, they focused their experimental investigation on transforming growth factor (TGF)-β, a main cytokine involved in EMT induction. They have shown that NS5A is not responsible for TGF-β pathway activation but it acts in a synergistic manner with the cytokine in inducing EMT. Furthermore, they also screened, in their in vitro model, the modulation of expression of EMT biomarkers, such as Snail, Slug, Zeb and Twist 1/2. They demonstrated that only Twist2 expression was specifically increased by NS5A protein, and that, accordingly, through short hairpin RNA against Twist2, they were able to counteract EMT occurrence. Interestingly, the TGF-β treatment of cells lacking Twist2 is able to promote EMT in any case, suggesting that NS5A and TGF-β trigger EMT by distinct pathways and thus identifying Twist2 as an NS5A EMT specific effector. Unfortunately, as the authors stated in the discussion session, currently the mechanisms underlying Twist2 involvement in NS5A-dependent and TGF-β-independent EMT are not yet known. Regarding the HCV context, it is well known that NS5A activities are strictly connected to many cellular proteins and have an impact on several signaling pathways like, for instance, the hedgehog (HH) pathway [[4]Choi S.S. Bradrick S. Qiang G. Mostafavi A. Chaturvedi G. Weinman S.A. et al.Up-regulation of Hedgehog pathway is associated with cellular permissiveness for hepatitis C virus replication.Hepatology. 2011; 54: 1580-1590Crossref PubMed Scopus (34) Google Scholar]. This correlation is really interesting, because the HH pathway has emerged as an important mediator of liver cancer pathogenesis [[5]Maitah M.Y. Ali S. Ahmad A. Gadgeel S. Sarkar F.H. Up-regulation of sonic Hedgehog contributes to TGF-β1-induced epithelial to mesenchymal transition in NSCLC cells.PLoS One. 2011; 6: e16068Crossref PubMed Scopus (126) Google Scholar]. Recently, this pathway was involved in the reprogramming of cancer cells via EMT. In fact, the upregulation of HH was associated with increased cell motility, invasion and tumor cell aggressiveness [[5]Maitah M.Y. Ali S. Ahmad A. Gadgeel S. Sarkar F.H. Up-regulation of sonic Hedgehog contributes to TGF-β1-induced epithelial to mesenchymal transition in NSCLC cells.PLoS One. 2011; 6: e16068Crossref PubMed Scopus (126) Google Scholar]. It is interesting to underline that different liver cell lines, such as HuH 7.5 and LH86, which are featured by high expression levels of HH pathway genes (e.g., sonic hedgehog (SHH), glioblastoma protein 1 (Gli1), and patched (PTC)), are the most diffuse cells supporting a high HCV replication rate. It was also demonstrated that the upregulation of mesenchymal markers and the downregulation of epithelial markers were deeply connected to HH signaling [[4]Choi S.S. Bradrick S. Qiang G. Mostafavi A. Chaturvedi G. Weinman S.A. et al.Up-regulation of Hedgehog pathway is associated with cellular permissiveness for hepatitis C virus replication.Hepatology. 2011; 54: 1580-1590Crossref PubMed Scopus (34) Google Scholar]. Besides the intracellular pathways governed by HH, Wnt/β-catenin pathway is known to have a role in EMT induction [[3]Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial–mesenchymal transitions in development and disease.Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (7659) Google Scholar]. This pathway could be strikingly relevant, because it is also involved in HCV-related neoplastic transformation and EMT onset [6Liu J. Ding X. Tang J. Cao Y. Hu P. Zhou F. et al.Enhancement of canonical Wnt/β-catenin signaling activity by HCV core protein promotes cell growth of hepatocellular carcinoma cells.PLoS One. 2011; 6: e27496Crossref PubMed Scopus (91) Google Scholar, 7Bose S.K. Meyer K. Di Bisceglie A.M. Ray R.B. Ray R. Hepatitis C virus induces epithelial mesenchymal transition in primary human hepatocytes.J Virol. 2012; 86: 13621-13628Crossref PubMed Scopus (58) Google Scholar]. The authors suggest that the mechanism underlying EMT caused by HCV is really more intricate and may involve a lot of signaling pathways, like HH and Wnt/β-catenin, which are already known to be involved in organogenesis and cell transformation. Our preliminary data obtained through the use of an in vitro model of HCV infection, such as HuH 7.5 cells infected with the full length chimeric HCV strain J6/JFH1, at 48 h after infection indicate a significant upregulation of the EMT marker gene, Twist1 (fold change: 16.55 ± 0.20) and several molecules involved in Wnt/β-catenin pathway such as: Wnt5a (fold change: 5.32 ± 0.32), Wnt5b (fold change: 3.60 ± 0.11), and LRP5 (fold change: 5.12 ± 0.15). Taken into account the high expression levels of HH pathway components in our cell model, our results suggest that during HCV infection with a complete viral strain, other genes involved in the EMT induction, i.e., Twist1, may be upregulated. In conclusion, a deep investigation of the intricate molecular pathways modulated by HCV should be considered in order to improve the existent know-how and to identify new potential therapeutic targets useful to develop innovative and effective targeted therapies against the HCV-related liver diseases. This work was supported by grants from MIUR-Programma di ricerca di Rilevante Interesse Nazionale (PRIN) 2009 (prot. 2009YNERCE) titled “Intestinal microflora, steatosis and chronic liver injury” and Progetti di Ateneo (ex 60%), University of L’Aquila, and from the Andrea Cesalpino Foundation. M.A. has been supported by fellowships from the Andrea Cesalpino Foundation. The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.