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Molecular Basis of Hepatitis C Virus–Associated Hepatocarcinogenesis: Lessons From Animal Model Studies

2005; Elsevier BV; Volume: 3; Linguagem: Inglês

10.1016/s1542-3565(05)00700-7

1542-7714

Kazuhiko Koike,

Cancer, Lipids, and Metabolism

Despite numerous lines of epidemiologic evidence connecting HCV infection and the development of hepatocellular carcinoma (HCC), it remains controversial whether HCV itself plays a direct role or an indirect role in the pathogenesis of HCC. Through the use of transgenic mice, it has become evident that the core protein of HCV has oncogenic potential. HCV is directly involved in hepatocarcinogenesis, albeit other factors such as inflammation and environmental factors might also play a role. The direct involvement of HCV in hepatocarcinogenesis would be achieved via 2 pathways. In one pathway, the core protein acts on the function of mitochondria, leading to the overproduction of oxidative stress, which yields genetic aberrations in cell growth–related genes. The other pathway involves the modulation of cellular gene expressions and intracellular signal transductions, such as mitogen-activated protein kinase pathway, which results in the activation of transcription factors and cell cycle machineries. The combination of these alterations would be hypothesized to provoke the development of HCC in HCV infection. This would be a mechanism for HCC development in HCV infection that is distinct from those for other cancers. The presence of the HCV core protein, to which an oncogenic potential is ascribed, might allow some of the multiple steps to be bypassed in hepatocarcinogenesis. Therefore, unlike in other cancers, HCV infection can elicit HCC in the absence of a complete set of genetic aberrations. Such a scenario, "non-Vogelstein type" carcinogenesis, may explain the unusually high incidence and multicentric nature of HCC development in HCV infection. Despite numerous lines of epidemiologic evidence connecting HCV infection and the development of hepatocellular carcinoma (HCC), it remains controversial whether HCV itself plays a direct role or an indirect role in the pathogenesis of HCC. Through the use of transgenic mice, it has become evident that the core protein of HCV has oncogenic potential. HCV is directly involved in hepatocarcinogenesis, albeit other factors such as inflammation and environmental factors might also play a role. The direct involvement of HCV in hepatocarcinogenesis would be achieved via 2 pathways. In one pathway, the core protein acts on the function of mitochondria, leading to the overproduction of oxidative stress, which yields genetic aberrations in cell growth–related genes. The other pathway involves the modulation of cellular gene expressions and intracellular signal transductions, such as mitogen-activated protein kinase pathway, which results in the activation of transcription factors and cell cycle machineries. The combination of these alterations would be hypothesized to provoke the development of HCC in HCV infection. This would be a mechanism for HCC development in HCV infection that is distinct from those for other cancers. The presence of the HCV core protein, to which an oncogenic potential is ascribed, might allow some of the multiple steps to be bypassed in hepatocarcinogenesis. Therefore, unlike in other cancers, HCV infection can elicit HCC in the absence of a complete set of genetic aberrations. Such a scenario, "non-Vogelstein type" carcinogenesis, may explain the unusually high incidence and multicentric nature of HCC development in HCV infection. Worldwide HCV chronically infects hundreds of millions of people and induces a spectrum of chronic liver diseases.1Saito I. Miyamura T. Ohbayashi A. et al.Hepatitis C virus infection is associated with the development of hepatocellular carcinoma.Proc Natl Acad Sci U S A. 1990; 87: 6547-6549Crossref PubMed Scopus (1084) Google Scholar Hence, it impacts the society in a number of domains including medical, sociologic, and economic. Hepatocellular carcinoma (HCC) has become the most frequent cause of death in individuals persistently infected with HCV. In particular, HCV has received increasing attention because of its wide and deep penetration in the community, coupled with a very high incidence of HCC. Once cirrhosis is established in Japanese patients infected with HCV, HCC develops at a yearly rate of 5%–7%.2Ikeda K. Saitoh S. Suzuki Y. et al.Disease progression and hepatocellular carcinogenesis in patients with chronic viral hepatitis a prospective observation of 2215 patients.J Hepatol. 1998; 28: 930-938Abstract Full Text PDF PubMed Scopus (403) Google Scholar Knowledge about the mechanism of HCC development in chronic HCV infection, therefore, is required for the prevention of HCC. How HCV induces HCC is not yet clear, despite the fact that more than 70% of patients with HCC in Japan are infected with HCV.1Saito I. Miyamura T. Ohbayashi A. et al.Hepatitis C virus infection is associated with the development of hepatocellular carcinoma.Proc Natl Acad Sci U S A. 1990; 87: 6547-6549Crossref PubMed Scopus (1084) Google Scholar, 3Kiyosawa K. Sodeyama T. Tanaka E. et al.Interrelationship of blood transfusion, non-A, non-B hepatitis and hepatocellular carcinoma analysis by detection of antibody to hepatitis C virus.Hepatology. 1990; 12: 671-675Crossref PubMed Scopus (1187) Google Scholar, 4Yotsuyanagi H. Shintani Y. Moriya K. et al.Virological analysis of non-B, non-C hepatocellular carcinoma in Japan frequent involvement of hepatitis B virus.J Infect Dis. 2000; 181: 1920-1928Crossref PubMed Scopus (93) Google Scholar HCV infection is also common in patients with HCC in other countries, albeit to a lesser extent. These epidemiologic facts are a stimulus to determine the role of HCV in hepatocarcinogenesis. Inflammation induced by HCV, manifesting itself in various forms of hepatitis, should be considered in a study of the carcinogenic capacity of hepatitis viruses. It has been proposed repeatedly that necrosis of hepatocytes as a result of chronic inflammation and ensuing regeneration enhances mutagenesis in host cells, which can culminate in HCC. This theory presupposes an indirect involvement of hepatitis viruses in HCC via hepatic inflammation. However, this leaves specialists in hepatology with a serious question: can inflammation per se result in the development of HCC in such a high incidence or multicentric pattern in HCV infection? The secondary role of HCV would have to be weighed against the extremely rare occurrence of HCC in patients with autoimmune hepatitis in whom severe inflammation in the liver persists indefinitely. This background and line of reasoning lead to the hypothesis that viral proteins might play a role in inducing HCC. This possibility has been evaluated by introducing genes of HCV into hepatocytes in culture with little success. One of the difficulties in using cultured cells is that the carcinogenic capacity of HCV, if any, appears to be weak and would thus take a long time to manifest itself. Actually, it takes 30–40 years for HCC to develop in individuals infected with HCV. Another constraint common to studies of carcinogenesis is the development of HCC by transformed cells that might have resulted from uncontrolled growth and escaped surveillance of the host. If this is the case, the analysis of transformed cells would not be sufficient for solving the mystery of carcinogenesis. On the basis of these viewpoints, we initiated a study of carcinogenesis in chronic viral hepatitis by transgenic mouse technology. As illustrated in Figure 1, transgenic mouse lines with parts of the HCV genome were engineered by introducing genes excised from the cDNA of the HCV genome of genotype 1b.5Moriya K. Yotsuyanagi H. Shintani Y. et al.Hepatitis C virus core protein induces hepatic steatosis in transgenic mice.J Gen Virol. 1997; 78: 1527-1531Crossref PubMed Scopus (579) Google Scholar, 6Moriya K. Fujie H. Shintani Y. et al.Hepatitis C virus core protein induces hepatocellular carcinoma in transgenic mice.Nat Med. 1998; 4: 1065-1068Crossref PubMed Scopus (1107) Google Scholar The background of the mouse lines is a C57BL/6 strain, which is known for a rare spontaneous occurrence of HCC.7Smith G.S. Walford R.L. Mickey M.R. Lifespan and incidence of cancer and other diseases in selected long-lived inbred mice and their F 1 hybrids.J Natl Cancer Inst. 1973; 50: 1195-1213Crossref PubMed Scopus (135) Google Scholar Three different transgenic mouse lines are established, which carry the core gene, envelope genes, or nonstructural genes under the same transcriptional control element. Among these mouse lines, only the transgenic mice carrying the core gene develop HCC in 2 independent lineages (Figure 1).6Moriya K. Fujie H. Shintani Y. et al.Hepatitis C virus core protein induces hepatocellular carcinoma in transgenic mice.Nat Med. 1998; 4: 1065-1068Crossref PubMed Scopus (1107) Google Scholar The envelope gene transgenic mice do not develop HCC, despite high expression levels of both E1 and E2 proteins.8Koike K. Moriya K. Ishibashi K. et al.Expression of hepatitis C virus envelope proteins in transgenic mice.J Gen Virol. 1995; 76: 3031-3038Crossref PubMed Scopus (80) Google Scholar, 9Koike K. Moriya K. Yotsuyanagi H. et al.Sialadenitis resembling Sjögren's syndrome in mice transgenic for hepatitis C virus envelope genes.Proc Natl Acad Sci U S A. 1997; 94: 233-236Crossref PubMed Scopus (216) Google Scholar The transgenic mice carrying the entire nonstructural genes have not developed HCC. The transgenic mice carry the core gene and express the core protein of an expected size, approximately 21 kd, the level of which in the liver is similar to that in the liver of chronic hepatitis C patients. Early in life, these mice develop hepatic steatosis, which is one of the histologic characteristics of chronic hepatitis C, along with lymphoid follicle formation and bile duct damage.10Bach N. Thung S.N. Schaffner F. The histological features of chronic hepatitis C and autoimmune chronic hepatitis a comparative analysis.Hepatology. 1992; 15: 572-577Crossref PubMed Scopus (481) Google Scholar Thus, the core gene transgenic mouse model reproduces well this feature of chronic hepatitis C. Of note, evidence of significant inflammation is observed in the liver of this animal model. Late in life, these transgenic mice develop HCC. Most hepatic nodules exhibit a pathology characterized by "nodule in nodule," and HCC with a low degree of differentiation develops within an adenoma as well as within HCC with a higher degree of differentiation.6Moriya K. Fujie H. Shintani Y. et al.Hepatitis C virus core protein induces hepatocellular carcinoma in transgenic mice.Nat Med. 1998; 4: 1065-1068Crossref PubMed Scopus (1107) Google Scholar Although numerous lipid droplets are found in cells forming an adenoma, as in nontumorous cells, they are rarely observed in HCC cells. These histologic features closely resemble those observed in HCC developing in chronic hepatitis C patients, in which prominent lipid droplets are found in small, differentiated HCC and its precursors; poorly differentiated HCC without lipid droplets develops from within differentiated HCC.6Moriya K. Fujie H. Shintani Y. et al.Hepatitis C virus core protein induces hepatocellular carcinoma in transgenic mice.Nat Med. 1998; 4: 1065-1068Crossref PubMed Scopus (1107) Google Scholar Notably, the development of steatosis and HCC has been reproduced by other HCV transgenic mouse lines, which harbor the entire HCV genome or structural genes including the core gene.11Lerat H. Honda M. Beard M.R. et al.Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus.Gastroenterology. 2002; 122: 352-365Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar These outcomes indicate that the core protein of HCV has an oncogenic potential when expressed in vivo. It is difficult to determine the mechanism of carcinogenesis, even for our simple model in which only the core protein is expressed in otherwise normal liver tissues. There is a notable feature in the localization of the core protein in hepatocytes; whereas the core protein predominantly exists in the cytoplasm associated with lipid droplets, it is also present in the mitochondria and nuclei.6Moriya K. Fujie H. Shintani Y. et al.Hepatitis C virus core protein induces hepatocellular carcinoma in transgenic mice.Nat Med. 1998; 4: 1065-1068Crossref PubMed Scopus (1107) Google Scholar, 12Moriya K. Fujie H. Yotsuyanagi H. et al.Subcellular localization of hepatitis C virus structural proteins expressed in transgenic liver.Jpn J Med Sci Biol. 1997; 50: 169-177Crossref PubMed Scopus (26) Google Scholar On the basis of this finding, the pathways related to these 2 organelles, the mitochondria and nuclei, were meticulously analyzed. One activity of the core protein is an increased production of oxidative stress in the liver. We would like to draw particular attention to the fact that the production of oxidative stress is increased in our transgenic mouse model in the absence of inflammation in the liver, ie, hepatitis. This reflects a state of an overproduction of reactive oxygen species in the liver or predisposition to it, which is staged by the HCV core protein without any intervening inflammation.13Moriya K. Nakagawa K. Santa T. et al.Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocellular carcinogenesis.Cancer Res. 2001; 61: 4365-4370PubMed Google Scholar, 14Moriya K. Todoroki T. Tsutsumi T. et al.Increase in the concentration of carbon 18 monounsaturated fatty acids in the liver with hepatitis C analysis in transgenic mice and humans.Biophys Biochem Res Commun. 2001; 281: 1207-1212Crossref PubMed Scopus (63) Google Scholar The overproduction of oxidative stress results in the generation of deletions in the mitochondrial DNA, an indicator of genetic damage. Thus, the core protein induces excessive oxidative stress in the absence of inflammation and might, at least in part, contribute to hepatocarcinogenesis in HCV infection. If inflammation is induced in the liver with the HCV core protein, the production of oxidative stress is escalated to an extent that cannot be further scavenged by a physiologic antagonistic system. This indicates that the inflammation in chronic HCV infection would have a characteristic different in its quality from those of other types of hepatitis, such as autoimmune hepatitis. The basis for the overproduction of oxidative stress might be ascribed to the mitochondrial dysfunction.13Moriya K. Nakagawa K. Santa T. et al.Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocellular carcinogenesis.Cancer Res. 2001; 61: 4365-4370PubMed Google Scholar, 15Okuda M. Li K. Beard M.R. et al.Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein.Gastroenterology. 2002; 122: 366-375Abstract Full Text Full Text PDF PubMed Scopus (807) Google Scholar The function of the electron transfer system of the mitochondrion is suggested in association with the presence of the HCV core protein.16Moriya K. Tajima A. Tsutsumi T. et al.Hepatitis C virus core protein insults mitochondrial function through reducing the ETS complex 1 activity, p73. 2003Google Scholar Hepatic steatosis in hepatitis C might work as fuel for oxidative stress overproduction.14Moriya K. Todoroki T. Tsutsumi T. et al.Increase in the concentration of carbon 18 monounsaturated fatty acids in the liver with hepatitis C analysis in transgenic mice and humans.Biophys Biochem Res Commun. 2001; 281: 1207-1212Crossref PubMed Scopus (63) Google Scholar, 17Shintani Y. Fujie H. Miyoshi H. et al.Hepatitis C virus and diabetes direct involvement of the virus in the development of insulin resistance.Gastroenterology. 2004; 126: 840-848Abstract Full Text Full Text PDF PubMed Scopus (667) Google Scholar, 18Koike K. Moriya K. Metabolic aspects of hepatitis C steatohepatitis distinct from NASH.J Gastroenterol. 2005; 40: 329-336Crossref PubMed Scopus (61) Google Scholar Other possible pathways would be alteration in the expression of cellular genes, interacting with cellular proteins, and modulation of intracellular signaling pathways (Table 1). For example, tumor necrosis factor–α and interleukin-1β have been found to be transcriptionally activated.19Tsutsumi T. Suzuki T. Moriya K. et al.Intrahepatic cytokine expression and AP-1 activation in mice transgenic for hepatitis C virus core protein.Virology. 2002; 304: 415-424Crossref PubMed Scopus (82) Google Scholar The core protein has also been found to interact with some cellular proteins, such as retinoid X receptor–α, that play pivotal roles in cell proliferation and metabolism.20Tsutsumi T. Suzuki T. Shimoike T. et al.Interaction of hepatitis C virus core protein with retinoid X receptor-a modulates its transcriptional activity.Hepatology. 2002; 35: 937-946Crossref PubMed Scopus (152) Google Scholar The mitogen-activated protein kinase (MAPK) cascade is also activated in the liver of the core gene transgenic mouse model. The MAPK pathway, which consists of 3 routes, c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase, is involved in numerous cellular events including cell proliferation. In the liver of the core gene transgenic mouse model before HCC development, only the JNK route is activated. In the downstream of the JNK activation, transcription factor AP-1 activation is markedly enhanced.19Tsutsumi T. Suzuki T. Moriya K. et al.Intrahepatic cytokine expression and AP-1 activation in mice transgenic for hepatitis C virus core protein.Virology. 2002; 304: 415-424Crossref PubMed Scopus (82) Google Scholar, 21Tsutsumi T. Suzuki T. Moriya K. et al.Hepatitis C virus core protein activates ERK and p38 MAPK in cooperation with ethanol in transgenic mice.Hepatology. 2003; 38: 820-828PubMed Google Scholar Far downstream, both the mRNA and protein levels of cyclin D1 and CDK4 are increased. Thus, the HCV core protein modulates the intracellular signaling pathways and gives an advantage for cell proliferation to hepatocytes.Table 1Biomolecular Alterations With the Core Protein Expression Observed in the Transgenic Mouse Model1. Induction of cytokines including tumor necrosis factor–α and interleukin-1β19Tsutsumi T. Suzuki T. Moriya K. et al.Intrahepatic cytokine expression and AP-1 activation in mice transgenic for hepatitis C virus core protein.Virology. 2002; 304: 415-424Crossref PubMed Scopus (82) Google Scholar2. Activation of MAPK pathway and enhancement of AP-1 activation19Tsutsumi T. Suzuki T. Moriya K. et al.Intrahepatic cytokine expression and AP-1 activation in mice transgenic for hepatitis C virus core protein.Virology. 2002; 304: 415-424Crossref PubMed Scopus (82) Google Scholar, 21Tsutsumi T. Suzuki T. Moriya K. et al.Hepatitis C virus core protein activates ERK and p38 MAPK in cooperation with ethanol in transgenic mice.Hepatology. 2003; 38: 820-828PubMed Google Scholar3. Overproduction of oxidative stress or reactive oxygen species in the absence of inflammation13Moriya K. Nakagawa K. Santa T. et al.Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocellular carcinogenesis.Cancer Res. 2001; 61: 4365-4370PubMed Google Scholar4. Synergy of HCV core and alcohol in inducing oxidative stress and activating MAPK13Moriya K. Nakagawa K. Santa T. et al.Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocellular carcinogenesis.Cancer Res. 2001; 61: 4365-4370PubMed Google Scholar, 21Tsutsumi T. Suzuki T. Moriya K. et al.Hepatitis C virus core protein activates ERK and p38 MAPK in cooperation with ethanol in transgenic mice.Hepatology. 2003; 38: 820-828PubMed Google Scholar5. Interaction of HCV core and retinoid X receptor–α and peroxisome proliferator activated receptor–α20Tsutsumi T. Suzuki T. Shimoike T. et al.Interaction of hepatitis C virus core protein with retinoid X receptor-a modulates its transcriptional activity.Hepatology. 2002; 35: 937-946Crossref PubMed Scopus (152) Google Scholar6. Induction of insulin resistance17Shintani Y. Fujie H. Miyoshi H. et al.Hepatitis C virus and diabetes direct involvement of the virus in the development of insulin resistance.Gastroenterology. 2004; 126: 840-848Abstract Full Text Full Text PDF PubMed Scopus (667) Google Scholar7. Development of steatosis by inhibiting microsomal triglyceride transfer protein activity5Moriya K. Yotsuyanagi H. Shintani Y. et al.Hepatitis C virus core protein induces hepatic steatosis in transgenic mice.J Gen Virol. 1997; 78: 1527-1531Crossref PubMed Scopus (579) Google Scholar, 14Moriya K. Todoroki T. Tsutsumi T. et al.Increase in the concentration of carbon 18 monounsaturated fatty acids in the liver with hepatitis C analysis in transgenic mice and humans.Biophys Biochem Res Commun. 2001; 281: 1207-1212Crossref PubMed Scopus (63) Google Scholar, 24Perlemuter G. Sabile A. Letteron P. 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.FASEB J. 2002; 16: 185-194Crossref PubMed Scopus (517) Google Scholar8. Interaction of HCV core and proteasome activator PA28γ25Moriishi K. Okabayashi T. Nakai K. et al.Proteasome activator PA28g-dependent nuclear retention and degradation of hepatitis C virus core protein.J Virol. 2003; 77: 10237-10249Crossref PubMed Scopus (140) Google Scholar9. Inhibition of suppressor of cytokine signaling–126Miyoshi H. Fujie H. Shintani Y. et al.Hepatitis C virus core protein exerts an inhibitory effect on suppressor of cytokine signaling (SOCS)-1 gene expression.J Hepatol. 2005Google Scholar Open table in a new tab Such an effect of the core protein on the MAPK pathway, combined with that on oxidative stress, might explain the extremely high incidence of HCC development in chronic hepatitis C. The results of our studies on transgenic mice have indicated a carcinogenic potential of the HCV core protein in vivo; thus, HCV might be directly involved in hepatocarcinogenesis. In research studies of carcinogenesis, the theory of Kinzler and Vogelstein22Kinzler K.W. Vogelstein B. Lessons from hereditary colorectal cancer.Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4269) Google Scholar has gained wide popularity. They have proposed that the development of colorectal cancer is induced by the accumulation of a complete set of cellular gene mutations. They have deduced that mutations in the APC gene for inactivation, those in K-ras for activation, and those in the p53 gene for inactivation accumulate, which together lead toward the development of colorectal cancer. Their theory has been extended to the carcinogenesis of other cancers as well, so-called Vogelstein-type carcinogenesis (Figure 2). On the basis of the results we obtained for the induction of HCC by the HCV core protein, we would like to introduce a mechanism different from that of Kinzler and Vogelstein22Kinzler K.W. Vogelstein B. Lessons from hereditary colorectal cancer.Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4269) Google Scholar for the hepatocarcinogenesis in HCV infection. We do allow multistages in the induction of all cancers; it would be mandatory for hepatocarcinogenesis that many mutations accumulate in hepatocytes. Some of these steps, however, might be bypassed in the development of HCC in HCV infection to which the core protein would contribute (Figure 2). The overall effects achieved by the expression of the viral protein would be the induction of HCC, even in the absence of a complete set of genetic aberrations, required for carcinogenesis. By considering such a non-Vogelstein–type process for the induction of HCC, a plausible explanation might be given for many unusual events happening in HCV carriers.23Koike K. Tsutsumi T. Fujie H. et al.Role of hepatitis viruses in hepatocarcinogenesis.Oncology. 2002; 62: 29-37Crossref PubMed Scopus (116) Google Scholar Our theory might explain why HCC develops in persistent HCV infection at such a high incidence. Our theory might also account for the nonmetastatic and multicentric de novo occurrence characteristics of HCC, which would be the result of persistent HCV infection.