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Hepatitis C virus-induced hepatocarcinogenesis

Open AccessPublished:May 25, 2009DOI:https://doi.org/10.1016/j.jhep.2009.05.008
      Although there is strong evidence that hepatitis C virus (HCV) is one of the leading causes of hepatocellular carcinoma (HCC), there is still much to understand regarding the mechanism of HCV-induced transformation. While liver fibrosis resulting from long-lasting chronic inflammation and liver regeneration resulting from immune-mediated cell death are likely factors that contribute to the development of HCC, the direct role of HCV proteins remains to be determined. In vitro studies have shown that HCV expression may interfere with cellular functions that are important for cell differentiation and cell growth. However, most studies were performed in artificial models which can only give clues for potential mechanisms that need to be confirmed in more relevant models. Furthermore, the difficulty to identify HCV proteins and infected liver cells in patients, contributes to the complexity of our current understanding. For these reasons, there is currently very little experimental evidence for a direct oncogenic role of HCV. Further studies are warranted to clarify these issues.

      Abbreviations:

      HCV (hepatitis C virus), HCC (hepatocellular carcinoma), NCR (non-coding region), NS (non-structural), vLDL (very low density lipoproteins), B-NHL (B-cell non-Hodgkin lymphoma), MAPK (mitogen-activated protein kinase), NAFLD (non-alcoholic fatty liver disease), NASH (non-alcoholic steatohepatitis), IR (insulin resistance), IRS (insulin resistance substrate), ER (endoplasmic reticulum), ROS (reactive oxygen species)

      Keywords

      1. Introduction

      Chronic hepatitis C virus (HCV) infection is characterized by inflammatory lesions in the liver, often accompanied by intrahepatic lipid accumulation (steatosis) and progressive fibrosis of variable degrees, and long-term progression to cirrhosis and hepatocellular carcinoma (HCC) [
      • Moradpour D.
      • Blum H.E.
      Pathogenesis of hepatocellular carcinoma.
      ,
      • Zoulim F.
      • Chevallier M.
      • Maynard M.
      • Trepo C.
      Clinical consequences of hepatitis C virus infection.
      ]. HCC incidence has increased sharply over recent decades and has been attributed to chronic HCV infection. Chronic HCV infection, therefore, is a major risk factor for HCC development. Indeed, each year, 4-5% of patients with chronic hepatitis C develop HCC. Serological markers of HCV infection in patients with HCC range from 27% up to 80%, and HCV infection increases the risk for HCC development by an estimated 17-fold compared to healthy individuals [
      • Donato F.
      • Tagger A.
      • Gelatti U.
      • Parrinello G.
      • Boffetta P.
      • Albertini A.
      • et al.
      Alcohol and hepatocellular carcinoma: the effect of lifetime intake and hepatitis virus infections in men and women.
      ,
      • Imazeki F.
      • Yokosuka O.
      • Fukai K.
      • Saisho H.
      Favorable prognosis of chronic hepatitis C after interferon therapy by long-term cohort study.
      ,
      • El-Serag H.B.
      Hepatocellular carcinoma: an epidemiologic view.
      ,
      • Sun C.A.
      • Wu D.M.
      • Lin C.C.
      • Lu S.N.
      • You S.L.
      • Wang L.Y.
      • et al.
      Incidence and cofactors of hepatitis C virus-related hepatocellular carcinoma: a prospective study of 12,008 men in Taiwan.
      ] (Table 1). Host, environmental and viral factors appear to play an important role in determining progression of chronic hepatitis C to liver cirrhosis and HCC, a process that frequently takes several decades (Fig. 1). The molecular mechanisms underlying HCC development remain ill-defined. So far, it has not been possible to correlate specific changes in gene expression patterns with HCC development. HCV does not integrate into its host genome and has a predominantly cytoplasmic life cycle [
      • Moradpour D.
      • Penin F.
      • Rice C.M.
      Replication of hepatitis C virus.
      ]. Hepatocarcinogenesis, therefore, must involve several indirect mechanisms including the interplay between chronic inflammation, steatosis, fibrosis and oxidative stress and their pathological consequences. In addition, several HCV proteins have been shown to have direct oncogenic effects and to upregulate mitogenic processes. Increased cell proliferation in a setting of oxidative stress leads to accumulation of DNA damage and is thought to compromise gene and chromosome stability and to form the genomic basis for the malignant transformation of the hepatocyte. Here, we review the epidemiology of HCV-induced HCC and the potential underlying molecular mechanisms.
      Table 1Association between HCV infection and human malignancies.
      MalignancyFold risk increaseReference
      Hepatocellular carcinoma3- to 17-fold
      • Donato F.
      • Tagger A.
      • Gelatti U.
      • Parrinello G.
      • Boffetta P.
      • Albertini A.
      • et al.
      Alcohol and hepatocellular carcinoma: the effect of lifetime intake and hepatitis virus infections in men and women.
      ,
      • Imazeki F.
      • Yokosuka O.
      • Fukai K.
      • Saisho H.
      Favorable prognosis of chronic hepatitis C after interferon therapy by long-term cohort study.
      ,
      • El-Serag H.B.
      Hepatocellular carcinoma: an epidemiologic view.
      ,
      • Sun C.A.
      • Wu D.M.
      • Lin C.C.
      • Lu S.N.
      • You S.L.
      • Wang L.Y.
      • et al.
      Incidence and cofactors of hepatitis C virus-related hepatocellular carcinoma: a prospective study of 12,008 men in Taiwan.
      B-cell non-Hodgkin lymphoma2- to 10-fold
      • Gisbert J.P.
      • Garcia-Buey L.
      • Pajares J.M.
      • Moreno-Otero R.
      Prevalence of hepatitis C virus infection in B-cell non-Hodgkin’s lymphoma: systematic review and meta-analysis.
      ,
      • Matsuo K.
      • Kusano A.
      • Sugumar A.
      • Nakamura S.
      • Tajima K.
      • Mueller N.E.
      Effect of hepatitis C virus infection on the risk of non-Hodgkin’s lymphoma: a meta-analysis of epidemiological studies.
      ,
      • Nieters A.
      • Kallinowski B.
      • Brennan P.
      • Ott M.
      • Maynadie M.
      • Benavente Y.
      • et al.
      Hepatitis C and risk of lymphoma: results of the European multicenter case-control study EPILYMPH.
      Intrahepatic cholangiocarcinoma2- to 6-fold
      • Kobayashi M.
      • Ikeda K.
      • Saitoh S.
      • Suzuki F.
      • Tsubota A.
      • Suzuki Y.
      • et al.
      Incidence of primary cholangiocellular carcinoma of the liver in japanese patients with hepatitis C virus-related cirrhosis.
      ,
      • Shaib Y.H.
      • El-Serag H.B.
      • Davila J.A.
      • Morgan R.
      • McGlynn K.A.
      Risk factors of intrahepatic cholangiocarcinoma in the United States: a case-control study.
      ,
      • Shaib Y.H.
      • El-Serag H.B.
      • Nooka A.K.
      • Thomas M.
      • Brown T.D.
      • Patt Y.Z.
      • et al.
      Risk factors for intrahepatic and extrahepatic cholangiocarcinoma: a hospital-based case-control study.

      2. HCV infection: the virus and the disease

      In the 1970s and 1980s, serological analyses developed for the detection of hepatitis A virus (HAV) and hepatitis B virus (HBV) infection, respectively, indicated that the majority of transfusion-transmitted hepatitis was not caused by either HAV or HBV and was therefore termed non-A, non-B hepatitis (NANBH). The etiological agent of NANBH was discovered in 1989 and was termed HCV. Based on its structural and functional organization HCV was classified into the family of the Flaviviridae, where it forms its own genus Hepacivirus’ [

      Lindenbach BD, Thiel HJ, Rice CM. Flaviviridae: the viruses and their replication. In: Knipe DM, Howley PM, editors. Fields virology, 5th ed. Philadelphia: Lippincott-Raven Publishers; 2007.

      ]. The HCV genome is a single-stranded, positive sense RNA of approx. 9600 nt in length [
      • Lindenbach B.D.
      • Rice C.M.
      Molecular biology of flaviviruses.
      ] with genetic heterogeneity, resulting in its classification into six different genotypes. The HCV genome contains short non-coding regions (NCR) at each end. The coding sequence is translated into a polyprotein that is processed by viral and cellular proteases. The 5′-region of the genome encodes the structural proteins, including the nucleocapsid protein (core) and two envelope glycoproteins (E1 and E2) that form the viral particle, followed by a number of non-structural (NS) proteins, designated NS2 to NS5B in the 3′-region.
      HCV is considered hepatotropic, and only man and chimpanzees are susceptible to HCV infection and disease [
      • Bartosch B.
      • Cosset F.L.
      Cell entry of hepatitis C virus.
      ,
      • Lau D.T.
      • Fish P.M.
      • Sinha M.
      • Owen D.M.
      • Lemon S.M.
      • Gale M.
      • et al.
      Interferon regulatory factor-3 activation, hepatic interferon-stimulated gene expression, and immune cell infiltration in hepatitis C virus patients.
      ]. While HCV RNA has been unequivocally detected in hepatocytes in liver biopsies from chronically infected patients and chimpanzees, the HCV genome seems to replicate also in cells of lymphoid origin and dendritic cells [
      • Shimizu Y.K.
      • Igarashi H.
      • Kiyohara T.
      • Shapiro M.
      • Wong D.C.
      • Purcell R.H.
      • et al.
      Infection of a chimpanzee with hepatitis C virus grown in cell culture.
      ,
      • Sung V.M.
      • Shimodaira S.
      • Doughty A.L.
      • Picchio G.R.
      • Can H.
      • Yen T.S.
      • et al.
      Establishment of B-cell lymphoma cell lines persistently infected with hepatitis C virus in vivo and in vitro: the apoptotic effects of virus infection.
      ,
      • Pachiadakis I.
      • Pollara G.
      • Chain B.M.
      • Naoumov N.V.
      Is hepatitis C virus infection of dendritic cells a mechanism facilitating viral persistence?.
      ]. Circulating HCV particles have a diameter of 35–50 nm and are frequently associated with either immune globulins or very low density lipoproteins (vLDL) [
      • Andre P.
      • Perlemuter G.
      • Budkowska A.
      • Brechot C.
      • Lotteau V.
      Hepatitis C virus particles and lipoprotein metabolism.
      ]. Indeed, the vLDL biosynthesis machinery plays a pivotal role in the life cycle of HCV [
      • Andre P.
      • Perlemuter G.
      • Budkowska A.
      • Brechot C.
      • Lotteau V.
      Hepatitis C virus particles and lipoprotein metabolism.
      ,
      • Miyanari Y.
      • Atsuzawa K.
      • Usuda N.
      • Watashi K.
      • Hishiki T.
      • Zayas M.
      • et al.
      The lipid droplet is an important organelle for hepatitis C virus production.
      ,
      • Nielsen S.U.
      • Bassendine M.F.
      • Burt A.D.
      • Martin C.
      • Pumeechockchai W.
      • Toms G.L.
      Association between hepatitis C virus and very-low-density lipoprotein (VLDL)/LDL analyzed in iodixanol density gradients.
      ].
      Risk factors for HCV transmission include transfusion of blood and blood products, transplantation of solid organs from infected donors, injecting drug use, unsafe therapeutic injections and occupational exposure to blood [
      • Alter M.J.
      Epidemiology of hepatitis C virus infection.
      ]. The rate of transmission after a needle-stick injury from HCV positive blood ranges from 0 to 10% in most studies. The rate of perinatal HCV transmission is 4–7% and occurs only when HCV RNA is detectable in maternal serum at delivery. Importantly, coinfection with HIV increases the rate of perinatal transmission 4- to 5-fold [
      • Alter M.J.
      Epidemiology of hepatitis C virus infection.
      ].
      Persistence of HCV infection occurs in the majority of HCV-infected individuals. Indeed, acute hepatitis C resolves spontaneously only in about 10–40% of cases [
      • Afdhal N.H.
      The natural history of hepatitis C.
      ,
      • Poynard T.
      • Yuen M.F.
      • Ratziu V.
      • Lai C.L.
      Viral hepatitis C.
      ]. Chronic hepatitis C is characterized by the persistence of elevated aminotransferase levels and HCV RNA in serum, but is otherwise generally asymptomatic. The rate of progression to severe liver disease is highly variable. Factors that promote clinical progression include alcohol intake, coinfection with HIV and/or HBV, male sex and older age at infection [
      • Zoulim F.
      • Chevallier M.
      • Maynard M.
      • Trepo C.
      Clinical consequences of hepatitis C virus infection.
      ].
      The estimated prevalence of HCV infection worldwide is 2.2% and active or passive vaccination is not available to date. Antiviral combination therapy with a pegylated interferon and ribavirin is usually administered only to patients with more advanced and progressive disease [
      • Afdhal N.H.
      The natural history of hepatitis C.
      ,
      • Poynard T.
      • Yuen M.F.
      • Ratziu V.
      • Lai C.L.
      Viral hepatitis C.
      ,
      • Deutsch M.
      • Hadziyannis S.J.
      Old and emerging therapies in chronic hepatitis C: an update.
      ] due to cost, side effects and limited efficacy, especially in individuals infected with HCV genotype 1. Therefore, several novel antiviral agents are currently being evaluated including NS3-4A protease inhibitors, RNA dependent RNA polymerase inhibitors and different immune therapies [
      • Manns M.P.
      • Foster G.R.
      • Rockstroh J.K.
      • Zeuzem S.
      • Zoulim F.
      • Houghton M.
      The way forward in HCV treatment – finding the right path.
      ].

      3. Association between HCV infection and development of HCC or other malignancies

      Chronic HCV infection is a major risk factor for HCC development and serological markers of HCV infection are found in up to 80% of patients with HCC in some areas of the world [
      • Bosch F.X.
      • Ribes J.
      • Cleries R.
      • Diaz M.
      Epidemiology of hepatocellular carcinoma.
      ,
      • Kiyosawa K.
      • Umemura T.
      • Ichijo T.
      • Matsumoto A.
      • Yoshizawa K.
      • Gad A.
      • et al.
      Hepatocellular carcinoma: recent trends in Japan.
      ] (Table 1). HCV infection is estimated to increase the risk for HCC development up to 17-fold [
      • Donato F.
      • Tagger A.
      • Gelatti U.
      • Parrinello G.
      • Boffetta P.
      • Albertini A.
      • et al.
      Alcohol and hepatocellular carcinoma: the effect of lifetime intake and hepatitis virus infections in men and women.
      ,
      • Imazeki F.
      • Yokosuka O.
      • Fukai K.
      • Saisho H.
      Favorable prognosis of chronic hepatitis C after interferon therapy by long-term cohort study.
      ]. Host, environmental and viral factors appear to play an important role in determining progression of chronic hepatitis C to liver cirrhosis and HCC [
      • Zoulim F.
      • Chevallier M.
      • Maynard M.
      • Trepo C.
      Clinical consequences of hepatitis C virus infection.
      ]. Some [
      • Silini E.
      • Bottelli R.
      • Asti M.
      • Bruno S.
      • Candusso M.E.
      • Brambilla S.
      • et al.
      Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a case-control study.
      ,
      • Bruno S.
      • Silini E.
      • Crosignani A.
      • Borzio F.
      • Leandro G.
      • Bono F.
      • et al.
      Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a prospective study.
      ,
      • Bruno S.
      • Crosignani A.
      • Maisonneuve P.
      • Rossi S.
      • Silini E.
      • Mondelli M.U.
      Hepatitis C virus genotype 1b as a major risk factor associated with hepatocellular carcinoma in patients with cirrhosis: a seventeen-year prospective cohort study.
      ,
      • Hatzakis A.
      • Katsoulidou A.
      • Kaklamani E.
      • Touloumi G.
      • Koumantaki Y.
      • Tassopoulos N.C.
      • et al.
      Hepatitis C virus 1b is the dominant genotype in HCV-related carcinogenesis: a case-control study.
      ], but not all clinical studies [
      • Fattovich G.
      • Stroffolini T.
      • Zagni I.
      • Donato F.
      Hepatocellular carcinoma in cirrhosis: incidence and risk factors.
      ,
      • Niederau C.
      • Lange S.
      • Heintges T.
      • Erhardt A.
      • Buschkamp M.
      • Hurter D.
      • et al.
      Prognosis of chronic hepatitis C: results of a large, prospective cohort study.
      ,
      • Fattovich G.
      • Ribero M.L.
      • Pantalena M.
      • Diodati G.
      • Almasio P.
      • Nevens F.
      • et al.
      Hepatitis C virus genotypes: distribution and clinical significance in patients with cirrhosis type C seen at tertiary referral centres in Europe.
      ,
      • Serfaty L.
      • Aumaitre H.
      • Chazouilleres O.
      • Bonnand A.M.
      • Rosmorduc O.
      • Poupon R.E.
      • et al.
      Determinants of outcome of compensated hepatitis C virus-related cirrhosis.
      ], suggest that the risk of HCC development is associated with certain HCV genotypes, particularly genotype 1b. Apart from chronic HCV infection other risk factors for HCC development are among others HBV infection, obesity in men, diabetes mellitus, heavy alcohol use and hereditary hemochromatosis. Successful clearance of chronic HCV infection has been shown to reduce the overall liver-related mortality and HCC incidence, providing further evidence for a causal role of HCV in this cancer [
      • Kasahara A.
      • Hayashi N.
      • Mochizuki K.
      • Takayanagi M.
      • Yoshioka K.
      • Kakumu S.
      • et al.
      Risk factors for hepatocellular carcinoma and its incidence after interferon treatment in patients with chronic hepatitis C. Osaka Liver Disease Study Group.
      ].
      Apart from HCC, HCV is also a well-established risk factor of lymphoproliferative syndromes such as type II mixed cryoglobulinemia [
      • Kayali Z.
      • Buckwold V.E.
      • Zimmerman B.
      • Schmidt W.N.
      Hepatitis C, cryoglobulinemia, and cirrhosis: a meta-analysis.
      ] and malignant lymphoma. Indeed, HCV infection increases the risk of B-cell non-Hodgkin lymphoma (B-NHL) 2- to 10-fold [
      • Gisbert J.P.
      • Garcia-Buey L.
      • Pajares J.M.
      • Moreno-Otero R.
      Prevalence of hepatitis C virus infection in B-cell non-Hodgkin’s lymphoma: systematic review and meta-analysis.
      ,
      • Matsuo K.
      • Kusano A.
      • Sugumar A.
      • Nakamura S.
      • Tajima K.
      • Mueller N.E.
      Effect of hepatitis C virus infection on the risk of non-Hodgkin’s lymphoma: a meta-analysis of epidemiological studies.
      ] (Table 1). This association is particularly striking in southern Europe but much less in northern Europe and north America [
      • Gisbert J.P.
      • Garcia-Buey L.
      • Pajares J.M.
      • Moreno-Otero R.
      Prevalence of hepatitis C virus infection in B-cell non-Hodgkin’s lymphoma: systematic review and meta-analysis.
      ,
      • Matsuo K.
      • Kusano A.
      • Sugumar A.
      • Nakamura S.
      • Tajima K.
      • Mueller N.E.
      Effect of hepatitis C virus infection on the risk of non-Hodgkin’s lymphoma: a meta-analysis of epidemiological studies.
      ], suggesting that differences in HCV prevalence in these geographic regions, in control populations and in methods of HCV detection may account for these findings. The mechanisms underlying HCV-related lymphoma development, including the contributing host and viral factors [
      • Nieters A.
      • Kallinowski B.
      • Brennan P.
      • Ott M.
      • Maynadie M.
      • Benavente Y.
      • et al.
      Hepatitis C and risk of lymphoma: results of the European multicenter case-control study EPILYMPH.
      ] remain to be identified. Interestingly, clinical data show a regression of lymphoma after successful treatment of HCV infection supporting the concept of HCV infection as a cause of lymphoma development in humans [
      • Hermine O.
      • Lefrere F.
      • Bronowicki J.P.
      • Mariette X.
      • Jondeau K.
      • Eclache-Saudreau V.
      • et al.
      Regression of splenic lymphoma with villous lymphocytes after treatment of hepatitis C virus infection.
      ]. HCV infection has also been linked to the development of intrahepatic cholangiocarcinoma (ICC) [
      • Kobayashi M.
      • Ikeda K.
      • Saitoh S.
      • Suzuki F.
      • Tsubota A.
      • Suzuki Y.
      • et al.
      Incidence of primary cholangiocellular carcinoma of the liver in japanese patients with hepatitis C virus-related cirrhosis.
      ,
      • Shaib Y.H.
      • El-Serag H.B.
      • Davila J.A.
      • Morgan R.
      • McGlynn K.A.
      Risk factors of intrahepatic cholangiocarcinoma in the United States: a case-control study.
      ,
      • Shaib Y.H.
      • El-Serag H.B.
      • Nooka A.K.
      • Thomas M.
      • Brown T.D.
      • Patt Y.Z.
      • et al.
      Risk factors for intrahepatic and extrahepatic cholangiocarcinoma: a hospital-based case-control study.
      ]. It remains unclear, however, whether this association is independent from the underlying liver disease/cirrhosis.
      Prospective and retrospective cohort studies of patients with HCV infection have shown the role of the duration of chronic hepatitis in HCC development and the link between HCC development and liver cirrhosis. These studies demonstrated the sequential occurrence of advanced liver fibrosis and the development of HCC. The incidence of HCC development was estimated to be between 3 and 5%/year in patients with liver cirrhosis [
      • Tong M.J.
      • el-Farra N.S.
      • Reikes A.R.
      • Co R.L.
      Clinical outcomes after transfusion-associated hepatitis C.
      ,
      • Tsukuma H.
      • Hiyama T.
      • Tanaka S.
      • Nakao M.
      • Yabuuchi T.
      • Kitamura T.
      • et al.
      Risk factors for hepatocellular carcinoma among patients with chronic liver disease.
      ]. In HCV-infected patients, host and environmental factors appear to be more important than viral factors in determining progression of the liver disease to cirrhosis and HCC. These factors include: older age at diagnosis (>55 years: 2- to 4-fold increased risk) [
      • Pradat P.
      • Tillmann H.L.
      • Sauleda S.
      • Braconier J.H.
      • Saracco G.
      • Thursz M.
      • et al.
      Long-term follow-up of the hepatitis C HENCORE cohort: response to therapy and occurrence of liver-related complications.
      ,
      • Pradat P.
      • Voirin N.
      • Tillmann H.L.
      • Chevallier M.
      • Trepo C.
      Progression to cirrhosis in hepatitis C patients: an age-dependent process.
      ], duration of infection [
      • Niederau C.
      • Lange S.
      • Heintges T.
      • Erhardt A.
      • Buschkamp M.
      • Hurter D.
      • et al.
      Prognosis of chronic hepatitis C: results of a large, prospective cohort study.
      ], male sex (2- to 3-fold increased risk) [
      • Khan M.H.
      • Farrell G.C.
      • Byth K.
      • Lin R.
      • Weltman M.
      • George J.
      • et al.
      ], severity of liver disease at presentation, co-morbidities such as porphyria cutanea tarda [
      • Fracanzani A.L.
      • Taioli E.
      • Sampietro M.
      • Fatta E.
      • Bertelli C.
      • Fiorelli G.
      • et al.
      Liver cancer risk is increased in patients with porphyria cutanea tarda in comparison to matched control patients with chronic liver disease.
      ], heavy alcohol intake [
      • Donato F.
      • Tagger A.
      • Gelatti U.
      • Parrinello G.
      • Boffetta P.
      • Albertini A.
      • et al.
      Alcohol and hepatocellular carcinoma: the effect of lifetime intake and hepatitis virus infections in men and women.
      ,
      • Morgan T.R.
      • Mandayam S.
      • Jamal M.M.
      Alcohol and hepatocellular carcinoma.
      ,
      • Ikeda K.
      • Saitoh S.
      • Koida I.
      • Arase Y.
      • Tsubota A.
      • Chayama K.
      • et al.
      A multivariate analysis of risk factors for hepatocellular carcinogenesis: a prospective observation of 795 patients with viral and alcoholic cirrhosis.
      ,
      • Aizawa Y.
      • Shibamoto Y.
      • Takagi I.
      • Zeniya M.
      • Toda G.
      Analysis of factors affecting the appearance of hepatocellular carcinoma in patients with chronic hepatitis C. A long term follow-up study after histologic diagnosis.
      ], diabetes mellitus [
      • El-Serag H.B.
      • Tran T.
      • Everhart J.E.
      Diabetes increases the risk of chronic liver disease and hepatocellular carcinoma.
      ,
      • Chen C.L.
      • Yang H.I.
      • Yang W.S.
      • Liu C.J.
      • Chen P.J.
      • You S.L.
      • et al.
      Metabolic factors and risk of hepatocellular carcinoma by chronic hepatitis B/C infection: a follow-up study in Taiwan.
      ], steatosis [
      • Kumar D.
      • Farrell G.C.
      • Kench J.
      • George J.
      Hepatic steatosis and the risk of hepatocellular carcinoma in chronic hepatitis C.
      ,
      • Ohata K.
      • Hamasaki K.
      • Toriyama K.
      • Matsumoto K.
      • Saeki A.
      • Yanagi K.
      • et al.
      Hepatic steatosis is a risk factor for hepatocellular carcinoma in patients with chronic hepatitis C virus infection.
      ], obesity [
      • Chen C.L.
      • Yang H.I.
      • Yang W.S.
      • Liu C.J.
      • Chen P.J.
      • You S.L.
      • et al.
      Metabolic factors and risk of hepatocellular carcinoma by chronic hepatitis B/C infection: a follow-up study in Taiwan.
      ,
      • Calle E.E.
      • Rodriguez C.
      • Walker-Thurmond K.
      • Thun M.J.
      Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults.
      ] and coinfections, especially with HBV [
      • Bruno S.
      • Silini E.
      • Crosignani A.
      • Borzio F.
      • Leandro G.
      • Bono F.
      • et al.
      Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a prospective study.
      ,
      • Degos F.
      • Christidis C.
      • Ganne-Carrie N.
      • Farmachidi J.P.
      • Degott C.
      • Guettier C.
      • et al.
      Hepatitis C virus related cirrhosis: time to occurrence of hepatocellular carcinoma and death.
      ]. Slightly elevated serum bilirubin levels, decreased platelet counts and skin manifestations of liver disease, such as vascular spiders and/or palmar erythema correlate with the HCC risk [
      • Bruno S.
      • Silini E.
      • Crosignani A.
      • Borzio F.
      • Leandro G.
      • Bono F.
      • et al.
      Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a prospective study.
      ,
      • Degos F.
      • Christidis C.
      • Ganne-Carrie N.
      • Farmachidi J.P.
      • Degott C.
      • Guettier C.
      • et al.
      Hepatitis C virus related cirrhosis: time to occurrence of hepatocellular carcinoma and death.
      ]. Specific HLA class II alleles have also been associated with progression of chronic hepatitis C to decompensated cirrhosis or HCC. In this context, studies documented an association between DRB1∗1301/2 alleles and an asymptomatic HCV infection [
      • Pradat P.
      • Tillmann H.L.
      • Sauleda S.
      • Braconier J.H.
      • Saracco G.
      • Thursz M.
      • et al.
      Long-term follow-up of the hepatitis C HENCORE cohort: response to therapy and occurrence of liver-related complications.
      ]. Further, an association between the HLA DQ02 allele and HCC development has been reported [
      • Pradat P.
      • Tillmann H.L.
      • Sauleda S.
      • Braconier J.H.
      • Saracco G.
      • Thursz M.
      • et al.
      Long-term follow-up of the hepatitis C HENCORE cohort: response to therapy and occurrence of liver-related complications.
      ].

      4. HCV-induced hepatocarcinogenesis

      The mechanisms underlying the progression of HCV infection to HCC, which usually takes many years or decades, remain ill-defined [
      • McGivern D.R.
      • Lemon S.M.
      Tumor suppressors, chromosomal instability, and hepatitis C virus-associated liver cancer.
      ,
      • Levrero M.
      Viral hepatitis and liver cancer: the case of hepatitis C.
      ]. Transcriptomics and proteomics have helped to identify many genetic and epigenetic alterations associated with HCC clusters. However, the changes of gene expression identified in tumor cells are very heterogeneous, raising the question whether yet unidentified, specific changes at early, preneoplastic stages trigger the transformation process and whether differentiated hepatocytes or stem cells are at the origin of HCC [
      • Sell S.
      • Leffert H.L.
      Liver cancer stem cells.
      ,
      • Mishra L.
      • Banker T.
      • Murray J.
      • Byers S.
      • Thenappan A.
      • He A.R.
      • et al.
      Liver stem cells and hepatocellular carcinoma.
      ].
      HCV is the only RNA virus with a predominantly cytoplasmic life cycle [
      • Moradpour D.
      • Penin F.
      • Rice C.M.
      Replication of hepatitis C virus.
      ,

      Lindenbach BD, Thiel HJ, Rice CM. Flaviviridae: the viruses and their replication. In: Knipe DM, Howley PM, editors. Fields virology, 5th ed. Philadelphia: Lippincott-Raven Publishers; 2007.

      ]. All potentially pro-oncogenic events are therefore likely to be restricted to the cytoplasm, suggesting indirect mechanisms of hepatocarcinogenesis. While HCV infection leads to chronic inflammation, steatosis, fibrosis and oxidative DNA damage, several HCV proteins have been shown to have direct oncogenic effects and to upregulate mitogenesis [
      • McGivern D.R.
      • Lemon S.M.
      Tumor suppressors, chromosomal instability, and hepatitis C virus-associated liver cancer.
      ,
      • Koike K.
      Pathogenesis of HCV-associated HCC: Dual-pass carcinogenesis through activation of oxidative stress and intracellular signaling.
      ] (Table 2). The accumulation of oxidative stress and DNA damage in a setting of restricted cell cycle checkpoint control and/or accelerated cell division, is thought to compromise gene and chromosome stability and to form the genomic basis for the malignant transformation (Fig. 1). Indeed, in chronic HCV infection, changes in mitogen-activated protein kinase (MAPK) signaling, that regulates both cell metabolism and growth, are frequently detected [
      • Tardif K.D.
      • Mori K.
      • Siddiqui A.
      Hepatitis C virus subgenomic replicons induce endoplasmic reticulum stress activating an intracellular signaling pathway.
      ,
      • Waris G.
      • Felmlee D.J.
      • Negro F.
      • Siddiqui A.
      Hepatitis C virus induces proteolytic cleavage of sterol regulatory element binding proteins and stimulates their phosphorylation via oxidative stress.
      ]. Markers of intracellular oxidative stress have also been found to be increased in patients with chronic HCV infection [
      • Sumida Y.
      • Nakashima T.
      • Yoh T.
      • Nakajima Y.
      • Ishikawa H.
      • Mitsuyoshi H.
      • et al.
      Serum thioredoxin levels as an indicator of oxidative stress in patients with hepatitis C virus infection.
      ,
      • Shimoda R.
      • Nagashima M.
      • Sakamoto M.
      • Yamaguchi N.
      • Hirohashi S.
      • Yokota J.
      • et al.
      Increased formation of oxidative DNA damage, 8-hydroxydeoxyguanosine, in human livers with chronic hepatitis.
      ] as well as HCV core transgenic mice [
      • Moriya K.
      • Fujie H.
      • Shintani Y.
      • Yotsuyanagi H.
      • Tsutsumi T.
      • Ishibashi K.
      • et al.
      The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice.
      ,
      • Moriya K.
      • Nakagawa K.
      • Santa T.
      • Shintani Y.
      • Fujie H.
      • Miyoshi H.
      • et al.
      Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis.
      ]. However, direct interactions of the various HCV proteins with host cell factors have also been shown to lead to changes in cellular signaling cascades involved in regulation of cell metabolism and division and seem to be sufficient to induce hepatocarcinogenesis [
      • Moriya K.
      • Fujie H.
      • Shintani Y.
      • Yotsuyanagi H.
      • Tsutsumi T.
      • Ishibashi K.
      • et al.
      The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice.
      ,
      • Lerat H.
      • Honda M.
      • Beard M.R.
      • Loesch K.
      • Sun J.
      • Yang Y.
      • et al.
      Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus.
      ]. Overall, it is thought that the synergism between chronic inflammation and direct virus–host cell interactions triggers the malignant transformation of hepatocytes. The requirement for such a synergism would also explain the slow ‘multi-step’ transformation process that underlies human HCC development. Indeed, a considerable time lag between HCV infection and the development of cirrhosis and HCC is common and also explains the heterogeneity of genetic and epigenetic alterations observed in different HCCs [
      • McGivern D.R.
      • Lemon S.M.
      Tumor suppressors, chromosomal instability, and hepatitis C virus-associated liver cancer.
      ,
      • Levrero M.
      Viral hepatitis and liver cancer: the case of hepatitis C.
      ,
      • Thorgeirsson S.S.
      • Grisham J.W.
      Molecular pathogenesis of human hepatocellular carcinoma.
      ].
      Table 2HCV proteins, their role in the viral life cycle and possible roles in hepatocyte transformation.
      HCV proteinsRole in viral life cyclePossible roles in hepatocyte transformation (examples)
      Core proteinNucleocapsid assemblyInsulin resistance/steatosis/oxidative stress
      Interference (direct or indirect) with p53, p73, pRb
      Interference with host cell signaling
      (NF-κB, Raf1/MAPK, Wnt/β-catenin pathway)
      Interference with TGF-β signaling
      Transcriptional activation of cellular genes
      Apoptosis
      E1/E2 glycoproteinVirus morphogenesisInterference with PKR activity
      Cell entry
      p7Virus assembly, export and infectivity
      NS2Polyprotein processing and viral assemblyApoptosis
      NS3 N-terminal domainSerine protease activityInterference with hepatocyte innate response
      Interference with NF-KB
      Interference with p53
      NS3 C-terminal domainHelicase activity
      HCV genome replication
      NS4ACo-factor of NS4B and NS5AER stress
      NS4BFormation of membranous
      web structuresER stress
      NS5APart of the replication complexInterference with protein ubiquitinylation
      Inhibition of PKR activity
      Oxidative stress
      Modulation of transcription of cellular genes and rRNA
      Activation of cell signaling pathways (STAT-3, NF-κB etc)
      Accumulation of β-catenin by indirect mechanism
      NS5BRNA dependent RNA polymerase
      In chronic HCV infection, pro-carcinogenic cofactors are steatosis, oxidative stress and insulin resistance (IR). Thus chronic hepatitis C shares many similarities with non-alcoholic fatty liver disease (NAFLD), which may lead to non-alcoholic steatohepatitis (NASH) and HCC. In NAFLD, chronic excess of nutrients causes endoplasmic reticulum (ER) stress and leads to increased steatosis and IR. These phenomena are intrinsically linked, amplify each other and induce inflammation, which in turn favors hepatocarcinogenesis. The accumulation of unnaturally high levels of fat in the liver has been known to trigger the recruitment of inflammatory cells and to increase the levels of pro-oxidants. Reactive oxygen species (ROS) act directly on essential biomolecules and induce hepatotoxicity. ROS also indirectly activate redox sensitive transcriptional cascades, which trigger the production of cytotoxic, proinflammatory and fibrogenic mediators. Steatosis and ER/oxidative stress, have both causal roles in the development of IR by molecular pathways that are only beginning to be elucidated [
      • Hotamisligil G.S.
      Inflammation and metabolic disorders.
      ]. In IR, insulin receptor substrate (IRS)-induced signaling is frequently downregulated or blocked by various mechanisms, but the effects on downstream signaling pathways remain largely unexplored. Apart from ER stress and fatty acid metabolites, IR can also be triggered by peptide mediators and cytokines secreted by adipose tissue and/or the liver. Finally, IR and oxidative stress aggravate steatosis by stimulating lipid synthesis, increasing the transfer of fatty acids from adipose tissue to the liver and inhibiting lipid secretion by interference with mitochondrial function. In conclusion, a complex interplay between steatosis, ER/oxidative stress and IR, whose underlying molecular mechanisms remain largely undefined, can lead to chronic liver inflammation, apoptosis and fibrogenesis that are central to the development of liver cirrhosis and HCC in patients with chronic hepatitis C.

      5. HCV-induced changes in the hepatic glucose and lipid metabolism

      Similar to NAFLD, ER/oxidative stress, steatosis and IR are involved in the pathogenesis of chronic HCV infection, either as metabolic predisposition or directly induced by HCV (Fig. 1 and Table 2). An increased prevalence of steatosis and IR has been observed in patients with HCV infection and has prognostic implications, as it is associated with faster progression to cirrhosis and HCC as well as with a poorer response to treatment. In patients infected with HCV genotypes 1 and 2, steatosis often develops in the context of a pre-existing diabetes, IR or increased body mass index. By comparison, in patients infected with HCV genotype 3, steatosis is directly induced by HCV, because it correlates with the viral load and reverses with response to antiviral treatment [
      • Negro F.
      Mechanisms and significance of liver steatosis in hepatitis C virus infection.
      ]. HCV is thought to induce steatosis by interfering with lipid secretion and degradation and by increasing lipid synthesis. The HCV core protein, which localizes to the surface of lipid droplets and mediates viral assembly in close association with the cellular fatty acid metabolism [
      • Miyanari Y.
      • Atsuzawa K.
      • Usuda N.
      • Watashi K.
      • Hishiki T.
      • Zayas M.
      • et al.
      The lipid droplet is an important organelle for hepatitis C virus production.
      ], as well as some HCV non-structural proteins, have been shown to interfere with vLDL secretion [
      • Wetterau J.R.
      • Lin M.C.
      • Jamil H.
      Microsomal triglyceride transfer protein.
      ,
      • Domitrovich A.M.
      • Felmlee D.J.
      • Siddiqui A.
      Hepatitis C virus nonstructural proteins inhibit apolipoprotein B100 secretion.
      ]. HCV infection also upregulates lipid synthesis [
      • Waris G.
      • Felmlee D.J.
      • Negro F.
      • Siddiqui A.
      Hepatitis C virus induces proteolytic cleavage of sterol regulatory element binding proteins and stimulates their phosphorylation via oxidative stress.
      ], inhibits fatty acid oxidation [
      • Cheng Y.
      • Dharancy S.
      • Malapel M.
      • Desreumaux P.
      Hepatitis C virus infection down-regulates the expression of peroxisome proliferator-activated receptor alpha and carnitine palmitoyl acyl-CoA transferase 1A.
      ,
      • Dharancy S.
      • Malapel M.
      • Perlemuter G.
      • Roskams T.
      • Cheng Y.
      • Dubuquoy L.
      • et al.
      Impaired expression of the peroxisome proliferator-activated receptor alpha during hepatitis C virus infection.
      ] and increases release of fatty acids from adipocytes [
      • Negro F.
      Mechanisms and significance of liver steatosis in hepatitis C virus infection.
      ]. Overall the effects of HCV proteins on lipid synthesis, secretion and oxidation seem to be most pronounced in HCV genotype 3 infection, but also occur in patients infected with other genotypes. Besides changes in the lipid metabolism, HCV core and several non-structural proteins, induce systemic oxidative stress and related signaling by various mechanisms [
      • Tardif K.D.
      • Waris G.
      • Siddiqui A.
      Hepatitis C virus, ER stress, and oxidative stress.
      ]. With respect to IR, all HCV genotypes have been shown to interfere with glucose homeostasis, often at early stages in HCV infection [
      • Negro F.
      Mechanisms and significance of liver steatosis in hepatitis C virus infection.
      ]. The mechanism underlying IR and its severity seems again to be genotype dependent. HCV has been shown to interfere with insulin signaling by proteasomal degradation of IRS-1 and -2 either via SOCS proteins or the PI3K/Akt/mTOR pathway, as well as by IRS-1 inactivation via transforming growth factor (TGF)-α and PI3K/Akt [
      • Aytug S.
      • Reich D.
      • Sapiro L.E.
      • Bernstein D.
      • Begum N.
      Impaired IRS-1/PI3-kinase signaling in patients with HCV: a mechanism for increased prevalence of type 2 diabetes.
      ]. Thus, the initial or early stages of HCV infection are strongly associated with IR. In contrast, at late stages of disease and in particular in tumorigenesis, transformed cells have been shown to require more glucose and to upregulate insulin sensitivity and glucose uptake [
      • Grobholz R.
      • Hacker H.J.
      • Thorens B.
      • Bannasch P.
      Reduction in the expression of glucose transporter protein GLUT 2 in preneoplastic and neoplastic hepatic lesions and reexpression of GLUT 1 in late stages of hepatocarcinogenesis.
      ].

      6. From liver inflammation to liver cancer

      The interdependence between steatosis, IR and oxidative stress is important for disease progression in NAFLD as well as in hepatitis C and induces tissue damage and inflammation with activation of hepatic stellate cells (HSCs). Activated HSCs become responsive to both proliferative and fibrogenic cytokines and undergo epithelial to mesenchymal trans-differentiation (EMT) into contractile myo-fibroblast-like cells, that synthesize extracellular matrix (ECM) components, which accumulate over time to form fibrous scars or fibrosis. Ultimately, regenerating hepatocytes become enclosed by scar tissue and form nodules that define cirrhosis. HSCs are activated by products and effectors of oxidative stress and growth factors, cytokines, adipokines and chemokines, secreted by hepatocytes, Kupffer and inflammatory cells that infiltrate the liver in response to infection. The cytokine TGF-β, a potent inhibitor of epithelial cell growth and tumor suppressor, is a key regulator of EMT and also has pro-oncogenic functions. Importantly, recent findings indicate that TGF-β induces EMT not only in HSCs but possibly also in hepatocytes [
      • Matsuzaki K.
      • Murata M.
      • Yoshida K.
      • Sekimoto G.
      • Uemura Y.
      • Sakaida N.
      • et al.
      Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor beta signaling, promoting cirrhosis and hepatocellular carcinoma.
      ]. TGF-β signaling is upregulated in fibrosis in HCV-infected patients and stimulates ECM deposition and accumulation. IR may link fibrosis and steatosis, since it stimulates HSCs to deposit ECM. Several signaling cascades are involved in fibrogenesis, including SMADs, PI3K-Akt and various MAPK pathways, such as p38 and JNK. While SMADs are indispensable for EMT, TGF-β signaling via SMAD interacts with other signaling pathways to mediate pro-oncogenic EMT. JNK activation by the pro-inflammatory cytokine interleukin (IL)-1β can shift TGF-β signaling from tumor suppression to oncogenesis with increased fibrogenesis, cell motility and transactivation of cell cycle regulatory genes [
      • Matsuzaki K.
      • Murata M.
      • Yoshida K.
      • Sekimoto G.
      • Uemura Y.
      • Sakaida N.
      • et al.
      Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor beta signaling, promoting cirrhosis and hepatocellular carcinoma.
      ,
      • Giannelli G.
      • Bergamini C.
      • Fransvea E.
      • Sgarra C.
      • Antonaci S.
      Laminin-5 with transforming growth factor-beta1 induces epithelial to mesenchymal transition in hepatocellular carcinoma.
      ]. Thus, in the context of chronic inflammation, the interplay between ER/oxidative stress, steatosis and IR induces a pro-oncogenic microenvironment that results in fibrogenesis and genomic instability. Even though HCV has been reported to have direct transforming properties, the liver microenvironment is thought to significantly modulate the transformation process because HCC develops in chronic HCV infection only over long periods of time.

      7. Direct oncogenic effects of HCV

      Apart from complex interactions among themselves, HCV proteins interact with a number of host factors and signaling pathways and thus contribute to the progression from chronic hepatitis C to liver cirrhosis and HCC (Table 2). By modulating gene transcription and translation as well as post-translational events, the HCV proteins interfere with innate immunity to favor viral persistence and liver inflammation; they alter cell signaling, apoptosis, membrane physiology and protein trafficking, induce oxidative stress, genomic instability as well as malignant transformation. Among the HCV proteins core, NS3, NS4B and NS5A have all been shown to have transforming potential when transiently or stably expressed in cell culture, or in transgenic mice expressing the different viral proteins or the HCV polyprotein [
      • Sakamuro D.
      • Furukawa T.
      • Takegami T.
      Hepatitis C virus nonstructural protein NS3 transforms NIH 3T3 cells.
      ,
      • Ray R.B.
      • Lagging L.M.
      • Meyer K.
      • Ray R.
      Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype.
      ,
      • Gale Jr., M.
      • Kwieciszewski B.
      • Dossett M.
      • Nakao H.
      • Katze M.G.
      Antiapoptotic and oncogenic potentials of hepatitis C virus are linked to interferon resistance by viral repression of the PKR protein kinase.
      ,
      • Park J.S.
      • Yang J.M.
      • Min M.K.
      Hepatitis C virus nonstructural protein NS4B transforms NIH3T3 cells in cooperation with the Ha-ras oncogene.
      ].
      The HCV core protein is a highly conserved, basic protein that multimerizes, probably in conjunction with microtubules [

      Roohvand F, Maillard P, Lavergne JP, Boulant S, Walic M, Andreo U, et al. Initiation of hepatitis C virus infection requires the dynamic microtubule network: role of the viral nucleocapsid protein. J Biol Chem, 2009 [Epub ahead of print].

      ] to form the viral nucleocapsid and to package the viral RNA genome. It is localized at the cytoplasmic surface of the ER and on lipid droplets, and the latter observation is likely related to the induction of liver steatosis observed in HCV-infected patients as well as in transgenic mice overexpressing HCV core [
      • Moradpour D.
      • Englert C.
      • Wakita T.
      • Wands J.R.
      Characterization of cell lines allowing tightly regulated expression of hepatitis C virus core protein.
      ,
      • Rouille Y.
      • Helle F.
      • Delgrange D.
      • Roingeard P.
      • Voisset C.
      • Blanchard E.
      • et al.
      Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus.
      ,
      • Barba G.
      • Harper F.
      • Harada T.
      • Kohara M.
      • Goulinet S.
      • Matsuura Y.
      • et al.
      Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets.
      ]. Core has also been shown to localize to the outer membrane of mitochondria [
      • Schwer B.
      • Ren S.
      • Pietschmann T.
      • Kartenbeck J.
      • Kaehlcke K.
      • Bartenschlager R.
      • et al.
      Targeting of hepatitis C virus core protein to mitochondria through a novel C-terminal localization motif.
      ] and is involved in changes of apoptosis and lipid metabolism as well as in malignant transformation. Among many interactions with cellular factors, core has been shown to induce ROS production via interaction with heat shock protein Hsp60 [

      Kang SM, Kim SJ, Kim JH, Lee W, Kim GW, Lee KH, et al. Interaction of hepatitis C virus core protein with Hsp60 triggers the production of reactive oxygen species and enhances TNF-alpha-mediated apoptosis. Cancer Lett, 2009 [Epub ahead of print].

      ], and to bind the tumor suppressor proteins p53 [
      • Ray R.B.
      • Steele R.
      • Meyer K.
      • Ray R.
      Transcriptional repression of p53 promoter by hepatitis C virus core protein.
      ,
      • Lu W.
      • Lo S.Y.
      • Chen M.
      • Wu K.
      • Fung Y.K.
      • Ou J.H.
      Activation of p53 tumor suppressor by hepatitis C virus core protein.
      ], p73 [
      • Alisi A.
      • Giambartolomei S.
      • Cupelli F.
      • Merlo P.
      • Fontemaggi G.
      • Spaziani A.
      • et al.
      Physical and functional interaction between HCV core protein and the different p73 isoforms.
      ] and pRb [
      • Cho J.
      • Baek W.
      • Yang S.
      • Chang J.
      • Sung Y.C.
      • Suh M.
      HCV core protein modulates Rb pathway through pRb down-regulation and E2F-1 up-regulation.
      ]. Core also inhibits the expression of the cyclin-dependent kinase (CDK) inhibitor p21/Waf [
      • Wang Q.M.
      • Hockman M.A.
      • Staschke K.
      • Johnson R.B.
      • Case K.A.
      • Lu J.
      • et al.
      Oligomerization and cooperative RNA synthesis activity of hepatitis C virus RNA-dependent RNA polymerase.
      ]. p21 is a transcriptional target of p53 and blocks the cyclin/CDK complexes involved in cell-cycle control and tumor formation. Core induces activation of the Raf1/MAPK pathway [
      • Aoki H.
      • Hayashi J.
      • Moriyama M.
      • Arakawa Y.
      • Hino O.
      Hepatitis C virus core protein interacts with 14-3-3 protein and activates the kinase Raf-1.
      ,
      • Hayashi J.
      • Aoki H.
      • Kajino K.
      • Moriyama M.
      • Arakawa Y.
      • Hino O.
      Hepatitis C virus core protein activates the MAPK/ERK cascade synergistically with tumor promoter TPA, but not with epidermal growth factor or transforming growth factor alpha.
      ], protects cells from serum starvation and growth arrest and drives cells into proliferation. NF-κB transcription has been shown to be activated [
      • Ray R.B.
      • Steele R.
      • Basu A.
      • Meyer K.
      • Majumder M.
      • Ghosh A.K.
      • et al.
      Distinct functional role of hepatitis C virus core protein on NF-kappaB regulation is linked to genomic variation.
      ,
      • Soo H.M.
      • Garzino-Demo A.
      • Hong W.
      • Tan Y.H.
      • Tan Y.J.
      • Goh P.Y.
      • et al.
      Expression of a full-length hepatitis C virus cDNA up-regulates the expression of CC chemokines MCP-1 and RANTES.
      ,
      • Tai D.I.
      • Tsai S.L.
      • Chang Y.H.
      • Huang S.N.
      • Chen T.C.
      • Chang K.S.
      • et al.
      Constitutive activation of nuclear factor kappaB in hepatocellular carcinoma.
      ] and repressed [
      • Joo M.
      • Hahn Y.S.
      • Kwon M.
      • Sadikot R.T.
      • Blackwell T.S.
      • Christman J.W.
      Hepatitis C virus core protein suppresses NF-kappaB activation and cyclooxygenase-2 expression by direct interaction with IkappaB kinase beta.
      ] by HCV core. HCV core has also been shown to activate the Wnt/β-catenin pathway, which controls cell proliferation, DNA synthesis and cell-cycle progression [
      • Fukutomi 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.
      ]. Furthermore, HCV core variants have been shown to interact with SMAD3 and to inhibit the TGF-β pathway [
      • Pavio N.
      • Battaglia S.
      • Boucreux D.
      • Arnulf B.
      • Sobesky R.
      • Hermine O.
      • et al.
      Hepatitis C virus core variants isolated from liver tumor but not from adjacent non-tumor tissue interact with Smad3 and inhibit the TGF-beta pathway.
      ]. TGF-β signaling not only controls cell proliferation, differentiation and apoptosis but also stimulates liver regeneration and fibrogenesis through its actions on the ECM (see above). TGF-β levels are frequently increased in patients with chronic HCV infection and correlate with the degree of fibrosis [

      Nelson DR, Gonzalez-Peralta RP, Qian K, Xu Y, Marousis CG, Davis GL, et al. Transforming growth factor-beta 1 in chronic hepatitis C. J Viral Hepat 4:29–35.

      ,
      • Marcellin P.
      • Asselah T.
      • Boyer N.
      Fibrosis and disease progression in hepatitis C.
      ]. Finally, HCV core protein associates with cellular membranes [
      • Barba G.
      • Harper F.
      • Harada T.
      • Kohara M.
      • Goulinet S.
      • Matsuura Y.
      • et al.
      Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets.
      ,
      • Moriya K.
      • Fujie H.
      • Yotsuyanagi H.
      • Shintani Y.
      • Tsutsumi T.
      • Matsuura Y.
      • et al.
      Subcellular localization of hepatitis C virus structural proteins in the liver of transgenic mice.
      ] and lipid vesicles [
      • Moriya K.
      • Fujie H.
      • Yotsuyanagi H.
      • Shintani Y.
      • Tsutsumi T.
      • Matsuura Y.
      • et al.
      Subcellular localization of hepatitis C virus structural proteins in the liver of transgenic mice.
      ], binds to apolipoprotein II [
      • Sabile A.
      • Perlemuter G.
      • Bono F.
      • Kohara K.
      • Demaugre F.
      • Kohara M.
      • et al.
      Hepatitis C virus core protein binds to apolipoprotein AII and its secretion is modulated by fibrates.
      ,
      • Perlemuter G.
      • Sabile A.
      • Letteron P.
      • Vona G.
      • Topilco A.
      • Chretien Y.
      • et al.
      Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis.
      ] and reduces microsomal triglyceride transfer protein activity [
      • Perlemuter G.
      • Sabile A.
      • Letteron P.
      • Vona G.
      • Topilco A.
      • Chretien Y.
      • et al.
      Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis.
      ], resulting in impaired assembly and secretion of vLDL, steatosis and oxidative stress. These in vitro findings are likely to be relevant for HCV pathogenesis because transgenic mice expressing HCV core protein also develop steatosis [
      • Perlemuter G.
      • Sabile A.
      • Letteron P.
      • Vona G.
      • Topilco A.
      • Chretien Y.
      • et al.
      Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis.
      ,
      • Moriya K.
      • Yotsuyanagi H.
      • Shintani Y.
      • Fujie H.
      • Ishibashi K.
      • Matsuura Y.
      • et al.
      Hepatitis C virus core protein induces hepatic steatosis in transgenic mice.
      ] and HCC [
      • Moriya K.
      • Nakagawa K.
      • Santa T.
      • Shintani Y.
      • Fujie H.
      • Miyoshi H.
      • et al.
      Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis.
      ,
      • Lerat H.
      • Honda M.
      • Beard M.R.
      • Loesch K.
      • Sun J.
      • Yang Y.
      • et al.
      Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus.
      ].
      Overexpression of E2 inhibits eIF2α phosphorylation by the dsRNA-activated protein kinase (PKR) or the ER-stress signaling kinase PERK [
      • Pavio N.
      • Romano P.R.
      • Graczyk T.M.
      • Feinstone S.M.
      • Taylor D.R.
      Protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis C virus envelope protein E2 through the eukaryotic initiation factor 2alpha kinase PERK.
      ,
      • Taylor D.R.
      • Shi S.T.
      • Romano P.R.
      • Barber G.N.
      • Lai M.M.
      Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein.
      ]. Similarly, overexpression of NS4A, NS4B, or NS4A-4B has been reported to induce an ER stress-mediated unfolded protein response, to reduce ER-to-Golgi trafficking, to inhibit protein synthesis, and to cause cytopathic effects [
      • Florese R.H.
      • Nagano-Fujii M.
      • Iwanaga Y.
      • Hidajat R.
      • Hotta H.
      Inhibition of protein synthesis by the nonstructural proteins NS4A and NS4B of hepatitis C virus.
      ,
      • Hugle T.
      • Fehrmann F.
      • Bieck E.
      • Kohara M.
      • Krausslich H.G.
      • Rice C.M.
      • et al.
      The hepatitis C virus nonstructural protein 4B is an integral endoplasmic reticulum membrane protein.
      ,
      • Kato J.
      • Kato N.
      • Yoshida H.
      • Ono-Nita S.K.
      • Shiratori Y.
      • Omata M.
      Hepatitis C virus NS4A and NS4B proteins suppress translation in vivo.
      ,
      • Konan K.V.
      • Giddings Jr., T.H.
      • Ikeda M.
      • Li K.
      • Lemon S.M.
      • Kirkegaard K.
      Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and ultrastructure of host secretory apparatus.
      ,
      • Zheng Y.
      • Gao B.
      • Ye L.
      • Kong L.
      • Jing W.
      • Yang X.
      • et al.
      Hepatitis C virus non-structural protein NS4B can modulate an unfolded protein response.
      ]. NS2 has been shown to inhibit the cellular proapoptotic molecule CIDE-B [
      • Erdtmann L.
      • Franck N.
      • Lerat H.
      • Le Seyec J.
      • Gilot D.
      • Cannie I.
      • et al.
      The hepatitis C virus NS2 protein is an inhibitor of CIDE-B-induced apoptosis.
      ] and to downregulate transcription [
      • Dumoulin F.L.
      • von dem Bussche A.
      • Li J.
      • Khamzina L.
      • Wands J.R.
      • Sauerbruch T.
      • et al.
      Hepatitis C virus NS2 protein inhibits gene expression from different cellular and viral promoters in hepatic and nonhepatic cell lines.
      ].
      NS3-4A serine protease has been reported to interact with p53 to repress p21 function, to block activation of the transcription factors IRF-3 and NF-κB and to antagonize the innate antiviral defenses by interfering with RIG-1, MDA5 and TLR3 mediated signal transduction [
      • Johnson C.L.
      • Gale Jr., M.
      CARD games between virus and host get a new player.
      ,
      • Meylan E.
      • Curran J.
      • Hofmann K.
      • Moradpour D.
      • Binder M.
      • Bartenschlager R.
      • et al.
      Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.
      ,
      • Li X.D.
      • Sun L.
      • Seth R.B.
      • Pineda G.
      • Chen Z.J.
      Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity.
      ]. Indeed, RIG-I inactivation has been shown to render Huh-7 cells permissive to HCV replication [
      • Sumpter Jr., R.
      • Loo Y.M.
      • Foy E.
      • Li K.
      • Yoneyama M.
      • Fujita T.
      • et al.
      Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I.
      ,
      • Blight K.J.
      • McKeating J.A.
      • Rice C.M.
      Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication.
      ].
      NS5A has been shown to interact with the geranylgeranylated cellular protein FBL2 [
      • Wang C.
      • Gale Jr., M.
      • Keller B.C.
      • Huang H.
      • Brown M.S.
      • Goldstein J.L.
      • et al.
      Identification of FBL2 as a geranylgeranylated cellular protein required for hepatitis C virus RNA replication.
      ], an F-box motif containing protein that is probably involved in targeting cellular proteins of yet unknown identity for ubiquitinylation and degradation. A number of studies suggest that NS5A is also involved in IFN resistance [

      Lindenbach BD, Thiel HJ, Rice CM. Flaviviridae: the viruses and their replication. In: Knipe DM, Howley PM, editors. Fields virology, 5th ed. Philadelphia: Lippincott-Raven Publishers; 2007.

      ] and one possible mechanism may be its ability to induce expression of the type I interferon antagonist IL-8 [
      • 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.
      ]. In addition, NS5A has been described to contain an ‘interferon sensitivity determining region’ (ISDR), that has been described to mediate inhibition of PKR, an activator of the innate immunity [
      • Gale Jr., M.
      • Kwieciszewski B.
      • Dossett M.
      • Nakao H.
      • Katze M.G.
      Antiapoptotic and oncogenic potentials of hepatitis C virus are linked to interferon resistance by viral repression of the PKR protein kinase.
      ,
      • Gale Jr., M.J.
      • Korth M.J.
      • Tang N.M.
      • Tan S.L.
      • Hopkins D.A.
      • Dever T.E.
      • et al.
      Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein.
      ,
      • Gale Jr., M.
      • Blakely C.M.
      • Kwieciszewski B.
      • Tan S.L.
      • Dossett M.
      • Tang N.M.
      • et al.
      Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation.
      ]. The accumulation of mutations in this region is thought to correlate with treatment efficacy [
      • Enomoto N.
      • Sakuma I.
      • Asahina Y.
      • Kurosaki M.
      • Murakami T.
      • Yamamoto C.
      • et al.
      Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection.
      ,
      • Enomoto N.
      • Sakuma I.
      • Asahina Y.
      • Kurosaki M.
      • Murakami T.
      • Yamamoto C.
      • et al.
      Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region.
      ]. Importantly, overexpression of NS5A has been reported to induce a number of effects in cells, including oxidative stress, activation of signaling pathways such as STAT-3, PI3K, and NF-κB and altered transcriptional regulation of p21 [
      • Gong G.
      • Waris G.
      • Tanveer R.
      • Siddiqui A.
      Human hepatitis C virus NS5A protein alters intracellular calcium levels, induces oxidative stress, and activates STAT-3 and NF-kappa B.
      ,
      • He Y.
      • Nakao H.
      • Tan S.L.
      • Polyak S.J.
      • Neddermann P.
      • Vijaysri S.
      • et al.
      Subversion of cell signaling pathways by hepatitis C virus nonstructural 5A protein via interaction with Grb2 and P85 phosphatidylinositol 3-kinase.
      ,
      • Street A.
      • Macdonald A.
      • Crowder K.
      • Harris M.
      The Hepatitis C virus NS5A protein activates a phosphoinositide 3-kinase-dependent survival signaling cascade.
      ] and of pRB [
      • Munakata T.
      • Nakamura M.
      • Liang Y.
      • Li K.
      • Lemon S.M.
      Down-regulation of the retinoblastoma tumor suppressor by the hepatitis C virus NS5B RNA-dependent RNA polymerase.
      ]. Other NS5A interaction partners include apolipoprotein A1, the major protein found on HDLs, the tumor suppressor p53, Grb-2, an adaptor protein involved in mitogen signaling, SRCAP, an adenosine triphosphatase (ATPase) that activates cellular transcription, karyopherin β3, a protein involved in nuclear trafficking, cyclin-dependent kinases 1 and 2 and Src-family kinases Fyn, Hck, Lck, and Lyn [

      Lindenbach BD, Thiel HJ, Rice CM. Flaviviridae: the viruses and their replication. In: Knipe DM, Howley PM, editors. Fields virology, 5th ed. Philadelphia: Lippincott-Raven Publishers; 2007.

      ,
      • Bartenschlager R.
      • Frese M.
      • Pietschmann T.
      Novel insights into hepatitis C virus replication and persistence.
      ,
      • Macdonald A.
      • Harris M.
      Hepatitis C virus NS5A: tales of a promiscuous protein.
      ,
      • Reyes G.R.
      The nonstructural NS5A protein of hepatitis C virus: an expanding, multifunctional role in enhancing hepatitis C virus pathogenesis.
      ,
      • Tellinghuisen T.L.
      • Rice C.M.
      Interaction between hepatitis C virus proteins and host cell factors.
      ]. It has also been reported that NS5A expression in the context of the HCV polyprotein results in the inhibition of the transcription factor Forkhead as well as in the phosphorylation and inactivation of the GSK-3, leading to accumulation of β-catenin and stimulation of β-catenin-dependent transcription [
      • 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.
      ]. Finally, NS5A dependent activation of upstream binding factor, a Pol I DNA binding transcription factor, which occurs as a result of up-regulation of both cyclin D1 and CDK4, leads to enhancement of rRNA transcription activation [
      • Raychaudhuri S.
      • Fontanes V.
      • Barat B.
      • Dasgupta A.
      Activation of ribosomal RNA transcription by hepatitis C virus involves upstream binding factor phosphorylation via induction of cyclin D1.
      ].

      8. Genetic and epigenetic changes in HCV-induced hepato-carcinogenesis

      HCCs are genetically very heterogeneous tumors. This is not unexpected, given the number of etiological factors implicated in its development, the complexity of hepatocyte physiology and the advanced stage at which HCCs are usually diagnosed. Genome-wide analysis of genetic alterations occurring in HCC revealed two major mechanisms of hepatocarcinogenesis. In the first, genetic alterations are acquired in the context of elevated oxidative stress caused by the vicious circle between chronic inflammation, IR and steatosis; in the second, transformation is induced by β-catenin mutations that dysregulate the Wnt pathway [
      • Laurent-Puig P.
      • Legoix P.
      • Bluteau O.
      • Belghiti J.
      • Franco D.
      • Binot F.
      • et al.
      Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis.
      ]. So far, it has not been possible to correlate chromosome instability with a consistent pattern of proto-oncogene activation in HCC, but several growth factor signaling pathways are frequently affected, including insulin-growth factor (IGF)-, hepatocyte growth factor-, Wnt-, TGF-α/EGF- and TGF-β-signaling [
      • Levrero M.
      Viral hepatitis and liver cancer: the case of hepatitis C.
      ,
      • Breuhahn K.
      • Longerich T.
      • Schirmacher P.
      Dysregulation of growth factor signaling in human hepatocellular carcinoma.
      ]. The interplay between these pathways and their respective roles and contributions to HCC development remain to be elucidated, however. One of the most commonly affected pathways involved in cell cycle check control is the p53 pathway, that limits cell survival and proliferation in response to telomere shortening and oncogene activation in order to ensure genome integrity. Loss of p53 by e.g. deletion, mutation, degradation or direct inhibition during progression of chronic hepatitis C to cirrhosis likely results in proliferation of hepatocytes with shortened telomeres or chromosomal damage and to predispose to hepatocarcinogenesis. Indeed, p21 expression, a downstream target of p53 that blocks cell entry into the S phase, is increased in cirrhosis when presumably significant numbers of hepatocytes with genome damage have accumulated, but is lost in premalignant liver lesions and HCC [
      • Nonomura A.
      • Ohta G.
      • Hayashi M.
      • Izumi R.
      • Watanabe K.
      • Takayanagi N.
      • et al.
      Immunohistochemical detection of ras oncogene p21 product in liver cirrhosis and hepatocellular carcinoma.
      ]. Besides p53, the retinoblastoma (Rb) pathway is another major checkpoint that limits cell proliferation in response to DNA damage, telomere shortening and oncogene activation. In human HCC, the Rb pathway is defective in more than 80% of cases [

      Ozturk M. Genetic aspects of hepatocellular carcinogenesis. Semin Liver Dis 19:235–42.

      ]. Moreover, gankyrin, an inhibitor of p53 and Rb check point function is overexpressed in the vast majority of HCCs [
      • Fu X.Y.
      • Wang H.Y.
      • Tan L.
      • Liu S.Q.
      • Cao H.F.
      • Wu M.C.
      Overexpression of p28/gankyrin in human hepatocellular carcinoma and its clinical significance.
      ]. Expression of IGF-2 is frequent and thought to be an early event in hepatocarcinogenesis, present in more than 60% of dysplastic nodules and HCC [
      • Cariani E.
      • Seurin D.
      • Lasserre C.
      • Franco D.
      • Binoux M.
      • Brechot C.
      Expression of insulin-like growth factor II (IGF-II) in human primary liver cancer: mRNA and protein analysis.
      ]. IGF-2 receptor impairs cell proliferation by promoting degradation of the IGF-2 mitogen and by activation of TGF-β signaling [
      • Villevalois-Cam L.
      • Rescan C.
      • Gilot D.
      • Ezan F.
      • Loyer P.
      • Desbuquois B.
      • et al.
      The hepatocyte is a direct target for transforming-growth factor beta activation via the insulin-like growth factor II/mannose 6-phosphate receptor.
      ].
      β-catenin pathway activation is very common in hepatocarcinogenesis and detectable in more than 50% of HCCs. It can directly induce hepatocyte transformation without the need for multiple genetic/epigenetic alterations [
      • Legoix P.
      • Bluteau O.
      • Bayer J.
      • Perret C.
      • Balabaud C.
      • Belghiti J.
      • et al.
      Beta-catenin mutations in hepatocellular carcinoma correlate with a low rate of loss of heterozygosity.
      ]. β-catenin activation is mainly induced by β-catenin gene mutations and/or Wnt signaling pathway alterations [

      Ozturk M. Genetic aspects of hepatocellular carcinogenesis. Semin Liver Dis 19:235–42.

      ]. Wnt/frizzled/β-catenin signaling is mediated by a complex interaction between a Wnt ligand (Wnt) and a Frizzled receptor (Fzd), mostly in cooperation with the low density lipoprotein receptor (LDLR)-related proteins LRP-5 or -6. Normally, the Wnt/β-catenin pathway is involved in cell growth and proliferation as well as developmental control and cell adhesion. Cellular levels of β-catenin are tightly regulated by proteasome-dependent degradation, which is in turn controlled by the activity of the APC and Axin1 proteins, and the glycogen synthase kinase (GSK)-3b. A recent report indicates that Fzd-7, which stabilizes and activates β-catenin [
      • Merle P.
      • de la Monte S.
      • Kim M.
      • Herrmann M.
      • Tanaka S.
      • Von Dem Bussche A.
      • et al.
      Functional consequences of frizzled-7 receptor overexpression in human hepatocellular carcinoma.
      ], is overexpressed in more than 90% of HCCs and in around 75% of the peritumorous/precancerous liver tissue.
      In addition to the dysregulation of the above pathways, HCV infection has been shown to induce or correlate with epigenetic changes that are likely to contribute to hepatocarcinogenesis. HCV-induced ROS have been shown to activate histone deacetylase in a fashion similar to hydrogen peroxide and to cause hypoacetylation of histones [
      • Miura K.
      • Taura K.
      • Kodama Y.
      • Schnabl B.
      • Brenner D.A.
      Hepatitis C virus-induced oxidative stress suppresses hepcidin expression through increased histone deacetylase activity.
      ]. Hypomethylation of the IGF-2 locus in hepatitis C cirrhosis has been shown to predict HCC development [
      • Couvert P.
      • Carrie A.
      • Paries J.
      • Vaysse J.
      • Miroglio A.
      • Kerjean A.
      • et al.
      Liver insulin-like growth factor 2 methylation in hepatitis C virus cirrhosis and further occurrence of hepatocellular carcinoma.
      ]. Another study reported the hypermethylation of p16, p15, p14, pRB and the PTEN promoters in patients with sustained viral response in comparison to non-responders [
      • Hayashi T.
      • Tamori A.
      • Nishikawa M.
      • Morikawa H.
      • Enomoto M.
      • Sakaguchi H.
      • et al.
      Differences in molecular alterations of hepatocellular carcinoma between patients with a sustained virological response and those with hepatitis C virus infection.
      ]. Finally, increased hTERT DNA levels have been found to predict HCC development [
      • Divella R.
      • Lacalamita R.
      • Tommasi S.
      • Coviello M.
      • Daniele A.
      • Garrisi V.M.
      • et al.
      PAI-1, t-PA and circulating hTERT DNA as related to virus infection in liver carcinogenesis.
      ].

      9. Conclusions

      Our current view is that the mechanism of HCV-induced HCC is multifactorial. However, because of the lack of adequate models, it has been difficult to demonstrate the specific roles of HCV proteins and the liver micro environment in the malignant transformation of hepatocytes. To identify and characterize these mechanisms, primary human hepatocyte cultures supporting chronic HCV infection would be most useful to examine the accumulation of transforming events, leading to the selection of transformed cells after several cell passages. An immunocompetent animal model, susceptible to chronic HCV infection, would be important to analyze not only the different viral proteins, but also the liver microenvironment involved in HCC development (including IR, steatosis, oxidative stress, cytokine expression in response to HCV expression, liver regeneration, fibrosis, etc.). The recent discovery of cellular co-receptors required for virus-entry and the better understanding to the molecular biology of HCV replication should open new avenues to address these important questions.

      Acknowledgment

      We thank Thierry Buronfosse for critical reading of the manuscript.

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