Advertisement

Beyond viral dependence: The pathological consequences of HCV-induced EGF signaling

  • Armando Andres Roca Suarez
    Affiliations
    Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France

    Université de Strasbourg, Strasbourg, France
    Search for articles by this author
  • Thomas F. Baumert
    Correspondence
    Corresponding author. Address: Inserm, U1110, 3 Rue Koeberlé, 67000 Strasbourg, France. Tel.: +33 3 68 85 37 03; fax: +33 3 68 85 37 05.
    Affiliations
    Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France

    Université de Strasbourg, Strasbourg, France

    Pôle Hépato-digestif, Institut Hospitalo-universitaire, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
    Search for articles by this author
  • Joachim Lupberger
    Affiliations
    Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France

    Université de Strasbourg, Strasbourg, France
    Search for articles by this author

      Keywords

      Linked Article

      • HCV modifies EGF signalling and upregulates production of CXCR2 ligands: Role in inflammation and antiviral immune response
        Journal of HepatologyVol. 69Issue 3
        • Preview
          As a leading cause of chronic liver disease worldwide, in more than 70% of infected individuals HCV establishes a persistent infection characterised by continuous replication and high serum titres. The high propensity for persistence and the insidious course of disease is suggestive of powerful mechanisms allowing HCV to subvert host antiviral immunity, to modify the inflammatory response, and to utilise host cell infrastructure without affecting cell viability. This enables the virus to avert development of overt disease over decades, despite ongoing viral replication.
        • Full-Text
        • PDF
      Chronic hepatitis C virus (HCV) infection affects approximately 71 million individuals worldwide,
      Global prevalence and genotype distribution of hepatitis C virus infection in 2015: a modelling study.
      being a major etiological factor for the development of liver cirrhosis and hepatocellular carcinoma (HCC). Acute HCV infection often progresses to chronicity and is characterized by a non-resolving liver inflammation leading to a broad range of alterations in the tissue microenvironment. About ninety percent of HCC cases arise in the context of chronic liver inflammation, highlighting the central role of this persistent immune response in disease pathogenesis.
      • Llovet J.M.
      • Zucman-Rossi J.
      • Pikarsky E.
      • Sangro B.
      • Schwartz M.
      • Sherman M.
      • et al.
      Hepatocellular carcinoma.
      Despite efficient antiviral therapy by direct acting antivirals (DAA), the risk of HCC development cannot be fully eliminated in patients with advanced liver disease.
      • Reig M.
      • Marino Z.
      • Perello C.
      • Inarrairaegui M.
      • Ribeiro A.
      • Lens S.
      • et al.
      Unexpected high rate of early tumor recurrence in patients with HCV-related HCC undergoing interferon-free therapy.
      In this regard, accumulating evidence suggests a potentially persisting proto-oncogenic environment created by virus-induced changes in cell signaling.
      • Hoshida Y.
      • Fuchs B.C.
      • Bardeesy N.
      • Baumert T.F.
      • Chung R.T.
      Pathogenesis and prevention of hepatitis C virus-induced hepatocellular carcinoma.
      • Van Renne N.
      • Roca Suarez A.A.
      • Duong F.H.
      • Gondeau C.
      • Calabrese D.
      • Fontaine N.
      • et al.
      MiR-135a-5p-mediated downregulation of protein tyrosine phosphatase receptor delta is a candidate driver of HCV-associated hepatocarcinogenesis.
      • Mailly L.
      • Xiao F.
      • Lupberger J.
      • Wilson G.K.
      • Aubert P.
      • Duong F.H.T.
      • et al.
      Clearance of persistent hepatitis C virus infection in humanized mice using a claudin-1-targeting monoclonal antibody.
      • Lupberger J.
      • Zeisel M.B.
      • Xiao F.
      • Thumann C.
      • Fofana I.
      • Zona L.
      • et al.
      EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.
      Therefore, even in the DAA era, the understanding of virus-host interactions during chronic HCV-associated inflammation is key to identify and treat patients at high risk of developing HCC.
      In this context, a recent article in Journal of Hepatology by Johannes G. Bode’s laboratory at the Heinrich-Heine University in Germany provides a novel mechanism by which HCV infection contributes to this pathologic inflammatory response.
      • Groepper C.
      • Rufinatscha K.
      • Schröder N.
      • Stindt S.
      • Ehlting C.
      • Albrecht U.
      • et al.
      HCV modifies EGF signaling and upregulates production of CXCR2 ligands: role in inflammation and antiviral immune response.
      Aiming to identify chemokines regulated by HCV, the authors performed a functional screen using an HCV subgenomic replicon system and identified an HCV-induced upregulation of C-X-C motif chemokine receptor 2 (CXCR2) ligands (CXCLs) 1, 2, 3 and 8. Consistently, similar results were obtained upon HCV infection using the cell culture-derived strain Jc1. Having previously shown that HCV infection enhances epidermal growth factor (EGF) signaling, the authors next explored the possible involvement of this pathway on CXCR2 ligand expression. EGF receptor (EGFR) perturbation studies combining RNAi knockdown of EGF and the use of MAPK inhibitors, confirmed an HCV-induced upregulation of CXCL8 via EGFR and the MAP kinase kinase, MEK1 (MAP2K1). Additionally, knockdown of the p65 subunit of the NF-κB complex was sufficient to abrogate basal and EGF-induced CXCL8 expression in replicon-expressing cells, while in HCV-infected cells this mainly affected basal CXCL8 levels. This suggests that the observed enhancement of chemokine expression during HCV infection not only depends on the EGFR pathway but also on the activation of additional transcription factors such as NF-κB. The in vivo relevance of the data is emphasized by an association of HCV viral load with CXCL8 serum levels in chronically infected patients. Similarly, serum levels of EGF and CXCL8 tend to positively correlate, although this did not reach statistical significance in their study cohort.
      In a previous study, the authors demonstrated that HCV enhances EGFR signaling via NS3/4A-mediated proteolytic cleavage of T-cell protein tyrosine phosphatase (TC-PTP [PTPN2]), one of the major negative regulators of EGFR tyrosine-kinase activity.
      • Brenndorfer E.D.
      • Karthe J.
      • Frelin L.
      • Cebula P.
      • Erhardt A.
      • Schulte am Esch J.
      • et al.
      Nonstructural 3/4A protease of hepatitis C virus activates epithelial growth factor-induced signal transduction by cleavage of the T-cell protein tyrosine phosphatase.
      Indeed, here they demonstrate that NS3/4A expression alone enhances EGF-inducible CXCL8 expression, an effect that can be mimicked by knocking down TC-PTP. As the major role of chemokines is the recruitment of immune cells to the site of inflammation, the authors next evaluated if in the context of HCV replication EGF-induced release of chemokines influences leukocyte migration. Remarkably, the authors demonstrate that media from EGF-treated cell lines expressing the HCV subgenomic replicon enhances the migration of neutrophils, an effect that was not observed with EGF-conditioned media alone. This suggests that HCV infection modulates chemoattraction of immune cells to the liver via EGF-regulated chemokine secretion.
      The findings of Christina Groepper and co-workers are not just relevant for our understanding of HCV-EGFR interaction but most importantly provide insight into the pathologic consequences of derailed EGF signaling for liver inflammation and HCC development (Fig. 1). EGFR is a host factor for HCV by facilitating the assembly of the host entry complex, viral glycoprotein-dependent membrane fusion and cell-to-cell transmission of the virus.
      • Lupberger J.
      • Zeisel M.B.
      • Xiao F.
      • Thumann C.
      • Fofana I.
      • Zona L.
      • et al.
      EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.
      HCV requires EGFR signaling to maintain its life cycle but also induces these signals itself during binding to the receptor complex.
      • Mailly L.
      • Xiao F.
      • Lupberger J.
      • Wilson G.K.
      • Aubert P.
      • Duong F.H.T.
      • et al.
      Clearance of persistent hepatitis C virus infection in humanized mice using a claudin-1-targeting monoclonal antibody.
      • Diao J.
      • Pantua H.
      • Ngu H.
      • Komuves L.
      • Diehl L.
      • Schaefer G.
      • et al.
      Hepatitis C virus induces epidermal growth factor receptor activation via CD81 binding for viral internalization and entry.
      Moreover, during HCV infection the non-structural protein NS5A prolongs EGFR signaling by perturbing its internalization and subsequent degradation.
      • Plissonnier M.L.
      • Lahlali T.
      • Michelet M.
      • Lebosse F.
      • Cottarel J.
      • Beer M.
      • et al.
      Epidermal growth factor receptor-dependent mutual amplification between netrin-1 and the hepatitis C virus.
      • Mankouri J.
      • Griffin S.
      • Harris M.
      The hepatitis C virus non-structural protein NS5A alters the trafficking profile of the epidermal growth factor receptor.
      This leads to a persistent EGFR activation during chronic HCV infection that potentially contributes to an impaired antiviral response by modulating interferon alpha signaling via STAT3.
      • Lupberger J.
      • Duong F.H.
      • Fofana I.
      • Zona L.
      • Xiao F.
      • Thumann C.
      • et al.
      Epidermal growth factor receptor signaling impairs the antiviral activity of interferon-alpha.
      Figure thumbnail gr1
      Fig. 1Refined model of HCV-EGFR modulation and its impact on liver disease development. HCV binding to the HCV entry receptor complex (i.e. CD81, CLDN1) at the cell surface induces EGFR phosphorylation and downstream signaling. EGFR activity is prolonged by the NS5A-mediated perturbation of EGFR internalization and degradation. As a consequence, prolonged EGFR activity is associated with an increased hepatocyte proliferation, HSC activation, fibrogenesis and a dampened antiviral response via modulation of STAT3. Groepper et al., demonstrated that HCV replication enhances the expression of CXCR2 ligands (e.g. CXCL8) by an EGF-dependent mechanism and activation of the NF-κB signaling pathway. This is further favored via the proteolytic cleavage of TC-PTP by NS3/4A, resulting in increased EGFR activation. Upon EGF stimulation, the production of CXCL8 during HCV replication promotes the recruitment of neutrophils.
      Their finding that HCV replication promotes EGF expression is highly relevant in the study of HCV-induced chronic liver disease, as the EGF pathway is a key driver associated with progression towards cirrhosis
      • Fuchs B.C.
      • Hoshida Y.
      • Fujii T.
      • Wei L.
      • Yamada S.
      • Lauwers G.Y.
      • et al.
      Epidermal growth factor receptor inhibition attenuates liver fibrosis and development of hepatocellular carcinoma.
      and HCC development.
      • Hoshida Y.
      • Villanueva A.
      • Kobayashi M.
      • Peix J.
      • Chiang D.Y.
      • Camargo A.
      • et al.
      Gene expression in fixed tissues and outcome in hepatocellular carcinoma.
      Equally interesting is the observation that HCV-induced EGF expression is a regulator of CXCR2 ligands. For example, HCV infection has previously been described to promote CXCL8 expression, which inhibits interferon antiviral activity and facilitates viral infection.
      • Polyak S.J.
      • Khabar K.S.
      • Paschal D.M.
      • Ezelle H.J.
      • Duverlie G.
      • Barber G.N.
      • et al.
      Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response.
      Hepatic CXCL8 is detected at low maintenance levels during acute HCV infection, although marked increases in serum and hepatic levels have been observed in HCV-infected patients with progressive inflammation and cirrhosis.
      • Neuman M.G.
      • Benhamou J.P.
      • Marcellin P.
      • Valla D.
      • Malkiewicz I.M.
      • Katz G.G.
      • et al.
      Cytokine–chemokine and apoptotic signatures in patients with hepatitis C.
      Indeed, CXCL8, which is associated with poor outcome in patients with HCC, has been suggested as an HCC biomarker.
      • Ren Y.
      • Poon R.T.
      • Tsui H.T.
      • Chen W.H.
      • Li Z.
      • Lau C.
      • et al.
      Interleukin-8 serum levels in patients with hepatocellular carcinoma: correlations with clinicopathological features and prognosis.
      Here, Groepper and co-workers validated a mechanistic concept between EGFR signaling and CXCL8 during HCV infection, that has been previously proposed for hepatomas.
      • Huang P.
      • Xu X.
      • Wang L.
      • Zhu B.
      • Wang X.
      • Xia J.
      The role of EGF-EGFR signalling pathway in hepatocellular carcinoma inflammatory microenvironment.
      Moreover, they provide a previously undescribed mechanism linking EGFR signaling to chemoattraction of immune cells. In macrophages EGFR knockout attenuates HCC development in mice.
      • Lanaya H.
      • Natarajan A.
      • Komposch K.
      • Li L.
      • Amberg N.
      • Chen L.
      • et al.
      EGFR has a tumour-promoting role in liver macrophages during hepatocellular carcinoma formation.
      EGF-mediated recruitment of neutrophils during HCV infection is potentially relevant for liver pathobiology, since it has detrimental effects on the host by contributing to the necro-inflammatory process.
      • Wisniewska-Ligier M.
      • Wozniakowska-Gesicka T.
      • Glowacka E.
      • Lewkowicz P.
      • Banasik M.
      • Tchorzewski H.
      Involvement of innate immunity in the pathogenesis of chronic hepatitis C in children.
      Although further studies in larger patient cohorts are needed to consolidate the model proposed by Groepper and co-workers, the impact of their findings for liver disease and its association to EGF signaling is evident.
      • Nakagawa S.
      • Wei L.
      • Song W.M.
      • Higashi T.
      • Ghoshal S.
      • Kim R.S.
      • et al.
      Molecular liver cancer prevention in cirrhosis by organ transcriptome analysis and lysophosphatidic acid pathway inhibition.
      In future studies, it would be very interesting and potentially relevant to follow-up HCV-induced EGF expression pattern in liver tissue and blood samples before and after sustained viral response and to compare them to liver fibrosis scores. Furthermore, does HCV genotype influence EGF and chemokine expression profiles since genotype 3 is associated with more severe liver disease manifestations? Taken together, this paper represents a further corroboration for the clinical potential of HCC chemo-preventive strategies based on regulators of signal transduction. Indeed, EGFR which is phosphorylated in hepatic stellate cells has been successfully targeted by the clinical EGFR inhibitor erlotinib in animal models, demonstrating proof of concept that EGF-based therapies attenuates chemically induced liver fibrosis and HCC nodules.
      • Fuchs B.C.
      • Hoshida Y.
      • Fujii T.
      • Wei L.
      • Yamada S.
      • Lauwers G.Y.
      • et al.
      Epidermal growth factor receptor inhibition attenuates liver fibrosis and development of hepatocellular carcinoma.
      Therefore, EGFR or MAPK modulators could be part of a personalized immuno-therapeutic strategy modulating chemokine profiles and inflammatory responses associated with liver disease progression.

      Financial support

      TFB acknowledges support by the European Union Horizon 2020 research and innovation program (ERC AdG HEPCIR – No. 667273; H2020 HEP-CAR – No. 667273; ERC POC PRELICAN – No. 755460), the NCI of the National Institutes of Health (1R21CA209940–01A1), the French National Research Agency (LABEX ANR-10-LABX-0028 HEPSYS) and the Office of the Assistant Secretary of Defense for Health Affairs (No. W81XWH-16–1-0363). JL and AARS acknowledge the French Agence Nationale de Recherche sur le Sida et les Hépatites Virales (ANRS) (ECTZ4236; ECTZ4446).

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Authors’ contributions

      All authors conceived, wrote and reviewed the manuscript.

      Acknowledgements

      All authors thank Atish Mukherji (Inserm U1110, Strasbourg, France) for critical reading of the manuscript.

      Supplementary data

      References

      Author names in bold designate shared co-first authorship

      1. Global prevalence and genotype distribution of hepatitis C virus infection in 2015: a modelling study.
        Lancet Gastroenterol Hepatol. 2015; 2017: 161-176
        • Llovet J.M.
        • Zucman-Rossi J.
        • Pikarsky E.
        • Sangro B.
        • Schwartz M.
        • Sherman M.
        • et al.
        Hepatocellular carcinoma.
        Nat Rev Dis Primers. 2016; 2: 16018
        • Reig M.
        • Marino Z.
        • Perello C.
        • Inarrairaegui M.
        • Ribeiro A.
        • Lens S.
        • et al.
        Unexpected high rate of early tumor recurrence in patients with HCV-related HCC undergoing interferon-free therapy.
        J Hepatol. 2016; 65: 719-726
        • Hoshida Y.
        • Fuchs B.C.
        • Bardeesy N.
        • Baumert T.F.
        • Chung R.T.
        Pathogenesis and prevention of hepatitis C virus-induced hepatocellular carcinoma.
        J Hepatol. 2014; 61: S79-S90
        • Van Renne N.
        • Roca Suarez A.A.
        • Duong F.H.
        • Gondeau C.
        • Calabrese D.
        • Fontaine N.
        • et al.
        MiR-135a-5p-mediated downregulation of protein tyrosine phosphatase receptor delta is a candidate driver of HCV-associated hepatocarcinogenesis.
        Gut. 2018; 67: 953-962
        • Mailly L.
        • Xiao F.
        • Lupberger J.
        • Wilson G.K.
        • Aubert P.
        • Duong F.H.T.
        • et al.
        Clearance of persistent hepatitis C virus infection in humanized mice using a claudin-1-targeting monoclonal antibody.
        Nat Biotechnol. 2015; 33: 549-554
        • Lupberger J.
        • Zeisel M.B.
        • Xiao F.
        • Thumann C.
        • Fofana I.
        • Zona L.
        • et al.
        EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.
        Nat Med. 2011; 17: 589-595
        • Groepper C.
        • Rufinatscha K.
        • Schröder N.
        • Stindt S.
        • Ehlting C.
        • Albrecht U.
        • et al.
        HCV modifies EGF signaling and upregulates production of CXCR2 ligands: role in inflammation and antiviral immune response.
        J Hepatol. 2018; 69: 594-602
        • Brenndorfer E.D.
        • Karthe J.
        • Frelin L.
        • Cebula P.
        • Erhardt A.
        • Schulte am Esch J.
        • et al.
        Nonstructural 3/4A protease of hepatitis C virus activates epithelial growth factor-induced signal transduction by cleavage of the T-cell protein tyrosine phosphatase.
        Hepatology. 2009; 49: 1810-1820
        • Diao J.
        • Pantua H.
        • Ngu H.
        • Komuves L.
        • Diehl L.
        • Schaefer G.
        • et al.
        Hepatitis C virus induces epidermal growth factor receptor activation via CD81 binding for viral internalization and entry.
        J Virol. 2012; 86: 10935-10949
        • Plissonnier M.L.
        • Lahlali T.
        • Michelet M.
        • Lebosse F.
        • Cottarel J.
        • Beer M.
        • et al.
        Epidermal growth factor receptor-dependent mutual amplification between netrin-1 and the hepatitis C virus.
        PLoS Biol. 2016; 14e1002421
        • Mankouri J.
        • Griffin S.
        • Harris M.
        The hepatitis C virus non-structural protein NS5A alters the trafficking profile of the epidermal growth factor receptor.
        Traffic. 2008; 9: 1497-1509
        • Lupberger J.
        • Duong F.H.
        • Fofana I.
        • Zona L.
        • Xiao F.
        • Thumann C.
        • et al.
        Epidermal growth factor receptor signaling impairs the antiviral activity of interferon-alpha.
        Hepatology. 2013; 58: 1225-1235
        • Fuchs B.C.
        • Hoshida Y.
        • Fujii T.
        • Wei L.
        • Yamada S.
        • Lauwers G.Y.
        • et al.
        Epidermal growth factor receptor inhibition attenuates liver fibrosis and development of hepatocellular carcinoma.
        Hepatology. 2014; 59: 1577-1590
        • Hoshida Y.
        • Villanueva A.
        • Kobayashi M.
        • Peix J.
        • Chiang D.Y.
        • Camargo A.
        • et al.
        Gene expression in fixed tissues and outcome in hepatocellular carcinoma.
        N Engl J Med. 2008; 359: 1995-2004
        • Polyak S.J.
        • Khabar K.S.
        • Paschal D.M.
        • Ezelle H.J.
        • Duverlie G.
        • Barber G.N.
        • et al.
        Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response.
        J Virol. 2001; 75: 6095-6106
        • Neuman M.G.
        • Benhamou J.P.
        • Marcellin P.
        • Valla D.
        • Malkiewicz I.M.
        • Katz G.G.
        • et al.
        Cytokine–chemokine and apoptotic signatures in patients with hepatitis C.
        Transl Res. 2007; 149: 126-136
        • Ren Y.
        • Poon R.T.
        • Tsui H.T.
        • Chen W.H.
        • Li Z.
        • Lau C.
        • et al.
        Interleukin-8 serum levels in patients with hepatocellular carcinoma: correlations with clinicopathological features and prognosis.
        Clin Cancer Res. 2003; 9: 5996-6001
        • Huang P.
        • Xu X.
        • Wang L.
        • Zhu B.
        • Wang X.
        • Xia J.
        The role of EGF-EGFR signalling pathway in hepatocellular carcinoma inflammatory microenvironment.
        J Cell Mol Med. 2014; 18: 218-230
        • Lanaya H.
        • Natarajan A.
        • Komposch K.
        • Li L.
        • Amberg N.
        • Chen L.
        • et al.
        EGFR has a tumour-promoting role in liver macrophages during hepatocellular carcinoma formation.
        Nat Cell Biol. 2014; 16: 972-977
        • Wisniewska-Ligier M.
        • Wozniakowska-Gesicka T.
        • Glowacka E.
        • Lewkowicz P.
        • Banasik M.
        • Tchorzewski H.
        Involvement of innate immunity in the pathogenesis of chronic hepatitis C in children.
        Scand J Immunol. 2006; 64: 425-432
        • Nakagawa S.
        • Wei L.
        • Song W.M.
        • Higashi T.
        • Ghoshal S.
        • Kim R.S.
        • et al.
        Molecular liver cancer prevention in cirrhosis by organ transcriptome analysis and lysophosphatidic acid pathway inhibition.
        Cancer Cell. 2016; 30: 879-890