Reply to: “Are Hedgehog and Wnt/β-catenin pathways involved in hepatitis C virus-mediated EMT?”
Authors: Damien Grégoire, Leila Akkari, Christophe Carenco, Urszula Hibner
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 Are Hedgehog and Wnt/β-catenin pathways involved in hepatitis C virus-mediated EMT?Journal of HepatologyVol. 58Issue 3PreviewWe read with great interest the article by Akkari et al. [1], who evaluated the EMT induction and correlated oncogenic transformation induced by HCV protein NS5A in primary hepatocyte precursors. Full-Text PDF Open Access We 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.Interestingly, at least three different HCV proteins have been reported to trigger EMT in hepatocytes: some isotypes of core [[1]Battaglia S. Benzoubir N. Nobilet S. Charneau P. Samuel D. Zignego A.L. et al.Liver cancer-derived hepatitis C virus core proteins shift TGF-beta responses from tumor suppression to epithelial–mesenchymal transition.PLoS One. 2009; 4: e4355Crossref PubMed Scopus (103) Google Scholar], E1/E2 [[2]Wilson G.K. Brimacombe C.L. Rowe I.A. Reynolds G.M. Fletcher N.F. Stamataki Z. et al.A dual role for hypoxia inducible factor-1alpha in the hepatitis C virus lifecycle and hepatoma migration.J Hepatol. 2012; 56: 803-809Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar] and, as reported by us, NS5A [[3]Akkari L. Gregoire 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 (51) Google Scholar]. While the end results of these processes appear quite similar, the different viral proteins employ distinct mechanisms of EMT induction, with the notable involvement of TGF-β signalling for the core and envelope proteins but not for NS5A-mediated EMT. Moreover, they probably operate with different kinetics following their expression (discussed in [[3]Akkari L. Gregoire 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 (51) Google Scholar]). The very fact that HCV has evolved several mechanisms to trigger EMT strongly suggests that the process is important for viral physiology and/or spread. It also means that the mechanisms ascribed to NS5A-mediated induction of EMT are expected to be present in experimental models that include the whole complement of viral proteins, while the reverse is not necessarily true. Following this line of argument, we note that the increased level of expression of the components of the Hedgehog pathway and their impact on HCV replication were observed in the model based on JFH1 infection of HuH7.5 cells [[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]. We have obtained comparable results in the infection of human primary hepatocytes (PHH) (Fig. 1A), thus confirming the results of Choi et al. in this experimental setting. To our knowledge, there are no reports of effects of NS5A on HH expression or signalling. Our data suggest that increased synthesis of components of the Hedgehog pathway is not required for NS5A-mediated EMT, since expression of Sonic Hedgehog, Gli1 or Patched is not altered in cells that undergo this process under the influence of NS5A [[3]Akkari L. Gregoire 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 (51) Google Scholar], the BMEL-NS5A (Fig. 1B).The situation is a little different for the Wnt/β-catenin pathway. NS5A activates β-catenin signalling through interactions with the catalytic subunit of PI(3)K [[5]Street A. Macdonald A. McCormick C. Harris M. Hepatitis C virus NS5A-mediated activation of phosphoinositide 3-kinase results in stabilization of cellular beta-catenin and stimulation of beta-catenin-responsive transcription.J Virol. 2005; 79: 5006-5016Crossref PubMed Scopus (120) Google Scholar] or directly [[6]Milward A. Mankouri J. Harris M. Hepatitis C virus NS5A protein interacts with beta-catenin and stimulates its transcriptional activity in a phosphoinositide-3 kinase-dependent fashion.J Gen Virol. 2010; 91: 373-381Crossref PubMed Scopus (46) Google Scholar], leading in both cases to accumulation and nuclear translocation of β-catenin and transcription of its target genes, such as axin2, glutamine synthase or c-myc. This scheme has recently been elegantly confirmed in several cellular and in vivo contexts by Higgs and colleagues [[7]Higgs MR, Lerat H, Pawlotsky JM. Hepatitis C virus-induced activation of beta-catenin promotes c-Myc expression and a cascade of pro-carcinogenetic events. Oncogene 2012, http://dx.doi.org/10.1038/onc.2012.484. [Epub ahead of print].Google Scholar]. In addition to this non-transcriptional regulation of β-catenin signalling, HCV core protein has been reported to induce transcription of several Wnt ligands, their receptors (Frizzled) and co-receptors (LRP5) in hepatoma cells [8Fukutomi T. Zhou Y. Kawai S. Eguchi H. Wands J.R. Li J. Hepatitis C virus core protein stimulates hepatocyte growth: correlation with upregulation of wnt-1 expression.Hepatology. 2005; 41: 1096-1105Crossref PubMed Scopus (140) Google Scholar, 9Liu J. Wang Z. Tang J. Tang R. Shan X. Zhang W. et al.Hepatitis C virus core protein activates Wnt/beta-catenin signaling through multiple regulation of upstream molecules in the SMMC-7721 cell line.Arch Virol. 2011; 156: 1013-1023Crossref PubMed Scopus (39) Google Scholar]. In contrast to these reports and the interesting data of Conti et al., we see no long-term alteration of wnt5 expression in the JFH1-infected PHH. Interestingly, expression of LRP5 is high in these cells and transcription of axin2, one of β-catenin target genes, is increased. These results suggest that the pathway is persistently activated up to at least three weeks following HCV infection of PHH (Fig. 1C). However, although we cannot exclude an early transient activation of Wnt/β-catenin signalling in BMEL expressing NS5A, we found no evidence either of increased expression of components of this pathway or, more significantly, of sustained transcriptional activation of axin2 in BMEL-NS5A cells undergoing EMT (Fig. 1D). Thus, our results suggest that neither Hedgehog nor β-catenin signalling is required for NS5A-mediated EMT. It remains an open question whether these pathways participate in EMT induction orchestrated by other HCV proteins.Conflict of interestThe 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. We 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. Interestingly, at least three different HCV proteins have been reported to trigger EMT in hepatocytes: some isotypes of core [[1]Battaglia S. Benzoubir N. Nobilet S. Charneau P. Samuel D. Zignego A.L. et al.Liver cancer-derived hepatitis C virus core proteins shift TGF-beta responses from tumor suppression to epithelial–mesenchymal transition.PLoS One. 2009; 4: e4355Crossref PubMed Scopus (103) Google Scholar], E1/E2 [[2]Wilson G.K. Brimacombe C.L. Rowe I.A. Reynolds G.M. Fletcher N.F. Stamataki Z. et al.A dual role for hypoxia inducible factor-1alpha in the hepatitis C virus lifecycle and hepatoma migration.J Hepatol. 2012; 56: 803-809Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar] and, as reported by us, NS5A [[3]Akkari L. Gregoire 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 (51) Google Scholar]. While the end results of these processes appear quite similar, the different viral proteins employ distinct mechanisms of EMT induction, with the notable involvement of TGF-β signalling for the core and envelope proteins but not for NS5A-mediated EMT. Moreover, they probably operate with different kinetics following their expression (discussed in [[3]Akkari L. Gregoire 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 (51) Google Scholar]). The very fact that HCV has evolved several mechanisms to trigger EMT strongly suggests that the process is important for viral physiology and/or spread. It also means that the mechanisms ascribed to NS5A-mediated induction of EMT are expected to be present in experimental models that include the whole complement of viral proteins, while the reverse is not necessarily true. Following this line of argument, we note that the increased level of expression of the components of the Hedgehog pathway and their impact on HCV replication were observed in the model based on JFH1 infection of HuH7.5 cells [[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]. We have obtained comparable results in the infection of human primary hepatocytes (PHH) (Fig. 1A), thus confirming the results of Choi et al. in this experimental setting. To our knowledge, there are no reports of effects of NS5A on HH expression or signalling. Our data suggest that increased synthesis of components of the Hedgehog pathway is not required for NS5A-mediated EMT, since expression of Sonic Hedgehog, Gli1 or Patched is not altered in cells that undergo this process under the influence of NS5A [[3]Akkari L. Gregoire 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 (51) Google Scholar], the BMEL-NS5A (Fig. 1B). The situation is a little different for the Wnt/β-catenin pathway. NS5A activates β-catenin signalling through interactions with the catalytic subunit of PI(3)K [[5]Street A. Macdonald A. McCormick C. Harris M. Hepatitis C virus NS5A-mediated activation of phosphoinositide 3-kinase results in stabilization of cellular beta-catenin and stimulation of beta-catenin-responsive transcription.J Virol. 2005; 79: 5006-5016Crossref PubMed Scopus (120) Google Scholar] or directly [[6]Milward A. Mankouri J. Harris M. Hepatitis C virus NS5A protein interacts with beta-catenin and stimulates its transcriptional activity in a phosphoinositide-3 kinase-dependent fashion.J Gen Virol. 2010; 91: 373-381Crossref PubMed Scopus (46) Google Scholar], leading in both cases to accumulation and nuclear translocation of β-catenin and transcription of its target genes, such as axin2, glutamine synthase or c-myc. This scheme has recently been elegantly confirmed in several cellular and in vivo contexts by Higgs and colleagues [[7]Higgs MR, Lerat H, Pawlotsky JM. Hepatitis C virus-induced activation of beta-catenin promotes c-Myc expression and a cascade of pro-carcinogenetic events. Oncogene 2012, http://dx.doi.org/10.1038/onc.2012.484. [Epub ahead of print].Google Scholar]. In addition to this non-transcriptional regulation of β-catenin signalling, HCV core protein has been reported to induce transcription of several Wnt ligands, their receptors (Frizzled) and co-receptors (LRP5) in hepatoma cells [8Fukutomi T. Zhou Y. Kawai S. Eguchi H. Wands J.R. Li J. Hepatitis C virus core protein stimulates hepatocyte growth: correlation with upregulation of wnt-1 expression.Hepatology. 2005; 41: 1096-1105Crossref PubMed Scopus (140) Google Scholar, 9Liu J. Wang Z. Tang J. Tang R. Shan X. Zhang W. et al.Hepatitis C virus core protein activates Wnt/beta-catenin signaling through multiple regulation of upstream molecules in the SMMC-7721 cell line.Arch Virol. 2011; 156: 1013-1023Crossref PubMed Scopus (39) Google Scholar]. In contrast to these reports and the interesting data of Conti et al., we see no long-term alteration of wnt5 expression in the JFH1-infected PHH. Interestingly, expression of LRP5 is high in these cells and transcription of axin2, one of β-catenin target genes, is increased. These results suggest that the pathway is persistently activated up to at least three weeks following HCV infection of PHH (Fig. 1C). However, although we cannot exclude an early transient activation of Wnt/β-catenin signalling in BMEL expressing NS5A, we found no evidence either of increased expression of components of this pathway or, more significantly, of sustained transcriptional activation of axin2 in BMEL-NS5A cells undergoing EMT (Fig. 1D). Thus, our results suggest that neither Hedgehog nor β-catenin signalling is required for NS5A-mediated EMT. It remains an open question whether these pathways participate in EMT induction orchestrated by other HCV proteins. Conflict of interestThe 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. 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.