Signal transduction cascades and hepatitis B and C related hepatocellular carcinoma
Authors: Mark Branda, Jack R. Wands
Journal: Hepatology (2006)

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide.1, 2 The incidence ranges from <10 cases per 100,000 population in North America and western Europe to 50-150 cases per 100,000 population in parts of Africa and Asia where HCC is responsible for a large proportion of cancer deaths. A rise in the incidence and mortality from HCC has recently been observed in most industrialized countries.3 This likely reflects the increased prevalence of hepatitis C virus (HCV) infection.4, 5 In addition, with the obesity epidemic and increased incidence of Type II diabetes and insulin resistance, the emergence of non-alcoholic steatohepatitis (NASH) has begun to assume a larger role in the pathogenesis due to the development of cirrhosis, a risk factor for HCC. Among health care professionals, there is widespread concern regarding the increase in HCC and the lack of optimal screening techniques that lead to delay in diagnosis and treatment. It is hoped that timely intervention may improve the prognosis of this almost uniformly fatal disease. The major etiologies of HCC are now well-defined and include chronic viral hepatitis B, C and D, toxins and drugs (e.g., alcohol, aflatoxins, anabolic steroids), and metabolic liver diseases (e.g., hereditary hemochromatosis, α1-antitrypsin deficiency). On a global scale, chronic hepatitis B (HBV) and C virus infection account for well over 90% of HCCs.6 Indeed, the risk of the development of HCC is about 25-35 times higher among patients with chronic HBV infection compared to uninfected persons; coinfection with HBV and HCV increases this risk by 130-fold.7 Another major clinical risk factor for HCC development is liver cirrhosis. In fact, 70% to 90% of HCCs develop in a liver with cirrhosis.6 The HCC risk in patients with liver cirrhosis depends upon the activity, duration and etiology of the underlying liver disease. It is particularly high in cirrhosis resulting from chronic viral hepatitis and hemochromatosis, followed in descending order by alcoholic cirrhosis, autoimmune hepatitis, and primary biliary cirrhosis. The risk of HCC is low in Wilson's disease. Coexistence of etiologies, such as HBV and HCV coinfection, HBV and aflatoxin B1,3 or HCV and alcohol, increases the relative risk of developing HCC.8 The molecular factors and interactions involved in hepatocarcinogenesis are still poorly understood. This is particularly true with respect to genomic mutations, as it has been difficult to identify common genetic changes in more than 20% to 30% of tumors. In fact, it is becoming clear that HCCs are genetically heterogenous tumors. Regardless of the etiology of liver disease, malignant transformation of hepatocytes may result from a sequence of increased liver cell turnover induced by chronic liver injury and regeneration in the context of inflammation and oxidative DNA damage. This raises the question as to what the processes are that regulate hepatocyte proliferation, migration, and survival. Advances in our understanding of the molecular pathogenesis of HCC has led to the identification of signal transduction pathways that are activated during hepatic transformation.6 Two signal transduction cascades that appear to be very important are insulin/ IGF-1/IRS-1/MAPK and Wnt/ Frizzled/β-catenin pathways as shown in Fig. 1. These pathways are activated early in over 90% of HCC tumors. This review will focus on the characteristics and biological consequences of enhanced activity of these pathways. We will present emerging evidence that HBV and HCV structural and non-structural proteins interact with and promote these cascades, ultimately leading to increased cell growth. Major signaling pathways activated during hepatocarcinogenesis with points of interactions between cellular and hepatitis B and C viral proteins. These interactions lead to cellular behavioral and functional changes important in malignant transformation. Arrows indicate stimulatory pathways and targets of viral proteins, perpendicular lines indicate inhibitory pathways, P indicates phosphorylation. HBx, hepatitis B X protein; HCV core, hepatitis C core protein; HCV NS5a, hepatitis C nonstructural protein 5a; IRS-1, insulin receptor substrate-1; PI3K, phosphatidylinositol-3 kinase; FZD, frizzled receptor; GSK-3β, glycogen synthase kinase-3β; APC, adenomatous polyposis coli; ERK, extracellular signal-regulated kinase; JNK, jun N-terminal kinase; AP-1, activator protein-1. In an attempt to identify genes associated with hepatocyte transformation, several libraries of monoclonal antibodies to a human HCC cell line (FOCUS) were generated to screen for antigens distinguishing HCCs from normal hepatocytes.9, 10 Those studies led to the observation that HCCs overexpress insulin receptor substrate-1 (IRS-1).11, 12 Insulin receptor substrate-1 is a ≈185 kDa docking protein that transmits growth factor-stimulated mitogenic and metabolic signals by interacting with downstream src-homology 2 domain (SH2)-containing molecules through specific tyrosyl phosphorylated motifs located within the C-terminus.13, 14 For example, the 897YVNI motif of IRS-1 binds to the growth factor receptor-bound protein 2 (Grb2) adapter molecule, the 1180YIDL motif binds to Syp protein tyrosine phosphatase, and 613YMPM and 942YMKM motifs bind to the p85 subunit of phosphatidylinositol-3 kinase (PI3K).13, 15-19 The Grb2 binds to PY-IRS-119, 20 leading to sequential activation of p21ras, mitogen-activated protein kinase kinase (MAPKK), and MAP kinase (MAPK)20-23 (see Fig. 1). When activated, extracellular signal-regulated kinase (ERK), a MAPK, contributes to growth factor-stimulated mitogenesis and gene expression.22, 24 The binding of PY-IRS-1 to p85 stimulates glucose uptake,25 inhibits apoptosis by activating Akt/protein kinase B,26-28 and inhibits glycogen synthase kinase-3β (GSK-3β).29-31 The net result of these interactions is to send survival signals to hepatocytes (Fig. 1). Several lines of evidence illustrate the importance of the insulin/IGF-1/IRS-1/MAPK cascade in regulating cell growth and survival. For example, it has been demonstrated to play a role in regulating liver regeneration following two-thirds hepatectomy and embryonic development of the liver.32, 33 Aberrant signaling through this pathway has been implicated in situations of unrestrained growth as well. For example, constitutive activation of the insulin/IGF-I/IRS-1/MAPK cascade due to enhanced IRS-1 expression has been identified in the majority of HCC. Indeed, IRS-1 overexpression is associated with activation of the ERK/MAPK cascade and results in increased HCC tumor size.34-36 Overexpression of IRS-1 and/or activation of one or more of the components of this signaling pathway occurs in over 90% of tumors.6 Furthermore, inhibition of the insulin/IGF-I/IRS-1 mediated signaling by a dominant-negative IRS-1 mutant protein has been shown to reverse the malignant phenotype of human HCC cells.37 Experimental overexpression of IRS-1 causes transformation of NIH-3T3 cells manifested by increased proliferation, anchorage-independent growth, tumor formation in nude mice, and resistance to apoptosis.34 In addition, IRS-1 overexpression in transgenic mouse livers results in increased cell proliferation, constitutive activation of ERK in hepatocytes, increased cell proliferation, and a 25% to 30% larger liver than those of non-transgenic littermates.36, 38 Therefore, IRS-1 mediated signaling regulates hepatocyte growth, and constitutive activation of IRS-1 pathways contributes to the transformed phenotype. HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; NASH, nonalcoholic steatohepatitis; HBx, hepatitis B X protein; HCV core, hepatitis C core protein; HCV NS5a, hepatitis C nonstructural protein 5a; IRS-1, insulin receptor substrate-1; PI3K, phosphatidylinositol-3 kinase; FZD, frizzled receptor; GSK-3β, glycogen synthase kinase-3β; APC, adenomatous polyposis coli; LEF/TCF, lymphoid enhancing factor/T-cell factor; ERK, extracellular signal-regulated kinase; JNK, jun N-terminal kinase; AP-1, activator protein-1; LRP, low density lipoprotein receptor; MAPKKK, mitogen-activated protein kinase kinase kinase; MAPKK, mitogen-activated protein kinase kinase; MAPK, mitogen-activated protein kinase; Grb2, growth factor receptor-bound protein 2; SH2, src-homology 2; NF-κB, nuclear factor-kappa B. There is substantial evidence that HCC has aberrant β-catenin signaling on the basis of its nuclear and/or cellular accumulation, which is the hallmark of canonical Wnt/Frizzled (FZD) signaling activation. The Wnt family of proteins consist of 350 - 380 amino acid molecules that serve as the ligands for FZD receptors. There are 19 known Wnt ligands in humans. These proteins are highly conserved throughout evolution and play major roles in the generation of cell polarity, embryonic patterning, and cell fate determination during embryogenesis. Wnt/β-catenin signaling appears to be important in hepatocyte proliferation during early embryonic liver development39, 40 as well as liver regeneration after partial hepatectomy in animal models41, 42 Wnt ligands bind to FZD receptors, which are seven transmembrane proteins (Fig. 1). There are ten known FZD receptors in humans and all contain a highly conserved cystein-rich domain which constitutes the ligand-binding region for Wnt proteins. The C-terminal region is essential for receptor signaling.43, 44 It is likely that most Wnt ligands will bind to several FZD receptors. These specific interactions depend, in part, on the relative affinities of a particular FZD receptor for the Wnt ligands.45, 46 The canonical pathway is activated only when Wnt ligands are associated with both FZD and its co-receptor members of the single transmembrane low density lipoprotein receptor related family (LRP5 or LRP6).47, 48 The β-catenin protein is the key signaling molecule in the canonical Wnt/FZD pathway (Fig. 1). Under resting conditions, most of the cellular β-catenin is bound to type 1 cadherin which forms a tight junction between cells.49 In the absence of Wnt/FZD signaling, the low level cytoplasmic β-catenin is bound to its destruction complex that consists of casein kinase 1, adenomatous polyposis coli (APC), axin, and GSK-3β. In this context, binding of Wnt to the FZD receptor leads to phosphorylation of downstream molecules, which results in dissociation of the destruction complex. When dissociation of the destruction complex occurs, β-catenin escapes degradation and accumulates in the cytoplasm where it may then translocate to the nucleus. In the nucleus, β-catenin acts as an essential component for transcription factor binding of the lymphoid enhancing factor/T-cell factor (LEF/TCF) group. Binding of β-catenin to LEF/TCF proteins leads to transcription of a number of Wnt-responsive genes that regulate cell proliferation and migration such as c-myc, cyclin D-1, and members of the WISP family.50 In addition, mice demonstrating activated β-catenin signaling upregulate epidermal growth factor receptor (EGFR)51 and genes involved in glutamine metabolism, including glutamine synthetase (GS), ornithine aminotransferase (OAT), and glutamate transporter GLT-1.52 GS upregulation was noted in all tested human HCC specimens harboring activating mutations of β-catenin, as well as about half the tumors without these mutations.52 These findings suggest that such genes are also targets of Wnt/β-catenin signaling and may be involved in the oncogenic process in HCC. There is accumulating evidence that alterations of the canonical Wnt/FZD signaling pathway is a common early event in the molecular pathogenesis of HCC. Nuclear and/or cellular accumulation of β-catenin is generally accepted as the hallmark of activated canonical Wnt/FZD signaling, and this phenomenon can be demonstrated in tumor tissues by immunohistochemical staining. Indeed, in 33% to 67% of HCCs, nuclear and/or cellular accumulation of β-catenin has been described and is associated with the clinical and pathological features of the disease.53-55 Furthermore, these studies suggest that tumors with β-catenin accumulation are associated with a dismal prognosis due to a poorly differentiated morphology,56 high proliferative activity,57 and vascular invasion.55-57 It is well recognized that β-catenin mutations in human HCC ranges from 8 to 34%55, 58-63 and is the most common genetic abnormality involving the Wnt/ FZD signaling pathway. Huang et al. found β-catenin mutations in 41% of HCCs associated with HCV infection.64 In contrast, mutations in the AXIN/conductin gene are relatively rare in HCC (5%-10% reported),61, 65 while APC mutations have not been described.64, 66, 67 It is interesting to note, however, that a significant proportion of HCCs with β-catenin protein accumulation do not demonstrate mutations of pathway components such as β-catenin or AXIN.10 These observations suggest that other upstream elements may be involved in the upregulation of the canonical Wnt/FZD signaling cascade during hepatocarcinogenesis. Overexpression of the FZD receptors in HCC appears to be important in the pathogenesis of these tumors. For example, FZD-7 was markedly upregulated both in transgenic mouse models of HCC and in human tumors. Recently a study evaluated four different HCC transgenic mouse models produced by overexpression of c-myc or SV40-TAG alone, or coexpression of IRS-1/c-myc and HBx/c-myc. The FZD-7 gene was the only FZD mRNA species that was upregulated in all four models of hepatocarcinogenesis as measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). This upregulation was confirmed by Western blot analysis.68 In another study, 90% of human HCC tumors over-expressed FZD-7 when compared to adjacent non-tumorous tissue. Moreover, approximately 80% of non-tumorous surrounding liver tissue demonstrated low level FZD-7 overexpression compared to normal liver.69 These findings strongly support the hypothesis that activation of this pathway is an early event during the pathogenesis of HCC. Interestingly, in the presence of high level FZD-7 expression, HCC tissues containing only the wild-type β-catenin gene demonstrate nuclear accumulation of β-catenin. These findings suggest that upregulation of FZD-7 alone was sufficient to activate the canonical Wnt/FZD-7 signaling pathway; mutations in the β-catenin gene or other components of its destruction complex are not necessary as has been shown in squamous cell esophageal tumors.70 The molecular mechanisms by which FZD-7 is upregulated during hepatocarcinogenesis have not been fully elucidated. Recently, the peptidyl-proplyl-isomerase, PIN1, has been shown to activate β-catenin in HCC. PIN1 binds β-catenin in proximity to the APC binding site, thereby interfering with the interaction between these two molecules. This results in increased nuclear translocation and transcriptional activity of β-catenin.71 Pang et al. found PIN1 overexpression (at both the transcriptional and translational levels) in human HCC tumors compared to peritumoral tissue in over half of the specimens tested.72 All tumors overexpressing PIN1 also demonstrated β-catenin accumulation by immunohistochemistry. Furthermore, transfecting the human liver cell line, MIHA, with PIN1 resulted in increased β-catenin protein levels. Finally, no mutations in β-catenin were observed in tumors overexpressing PIN1, while tumors with β-catenin mutations did not have increased PIN1 levels.72 Thus, the role of Wnt signaling has been of great interest in understanding hepatic oncogenesis.73 It is important to note, however, that β-catenin signaling in HCC may occur though non-canonical pathways. For example, in vitro studies using human HCC cells have found that EGF stimulation results in β-catenin nuclear translocation through a mechanism involving tyrosine phosphorylation.74, 75 Likewise, tumor growth factor-β1 (TGF-β1) induces β-catenin translocation and epithelial-to-mesenchymal transformation in HCC76, 77 through a mechanism which appears to involve GSK-3β phosphorylation/inactivation via PI3K/AKT.78 It is likely that several signaling cascades are activated during malignant transformation and hepatic oncogenesis. Various pathways may be influenced by signals common to multiple cascades. Cross-talk between the IRS-1 and Wnt/FZD cascades occurs through GSK-3β as shown in Fig. 1. Activation of this pathway was demonstrated in dysplastic liver from both murine models and human HCC, suggesting it is an early event in hepatic oncogenesis.68, 69 In summary, we may surmise that aberrant levels of β-catenin accumulation in the cytoplasm or the nucleus leads to inappropriate transcription of various target genes involved in cell proliferation and migration. The abnormal β-catenin accumulation may be due to mutations of β-catenin, APC, and axin genes, but these are relatively rare in HCC. This suggests that regulation of the Wnt pathway can be affected by overexpression of other upstream components such as Wnt ligands and FZD receptors without concomitant genetic alterations. Future research will be required to identify which specific Wnt ligands and FZD receptors are responsible for activation of the canonical β-catenin pathway in HCC. In addition, given the general importance of this pathway in hepatic oncogenesis it will be essential to assess new TCF/β-catenin responsive target genes. Because of the interactions of this signaling cascade with GSK-3β, there is an identifiable entry point allowing cross-talk with other signaling systems such as the insulin/IGF-1/IRS-1 cascade. Therefore, it seems likely that precise definition of the canonical Wnt/FZD/β-catenin pathway and its role in hepatocarcinogenesis may reveal new molecular targets for potential therapy of HCC. The observation that HCC develops with significantly increased frequency in patients with chronic viral hepatitis and cirrhosis is well documented, however the molecular mechanisms responsible for this association have not been fully elucidated. While HCC usually occurs in the setting of cirrhosis, it is recognized that it may develop in patients with chronic HBV infection in the absence of advanced fibrosis. It was initially theorized that oncogenensis resulted from chronic inflammation with repeated hepatocyte necrosis and regeneration. The question arises, however, why the risk of HCC is so much higher in chronic viral hepatitis when compared with other hepatic inflammatory disorders such as chronic alcoholism or autoimmune hepatitis. This disparity suggests that HBV and HCV may play a direct role in malignant transformation via interactions between viral and hepatocellular components.79 Several examples of interactions between hepatitis B and C viral proteins and cell signaling pathways are presented below and are depicted in Fig. 1. Through these interactions the liver may be placed at great risk for the development of HCC. The hepatitis B virus open reading frame X gene encodes HBx, which is a 154 amino-acid protein whose function in HBV infection has not been definitively described. Its expression appears to be required for woodchuck hepatitis B virus infection80, 81 and HBV replication in vitro.82 Though it does not bind directly to DNA, HBx has been shown to modulate gene transcription by activating a wide variety of viral and cellular transcriptional elements (reviewed in 62, 63, 77). Among these are promoters, enhancers, transcription factors, and proto-oncogenes.80-82 The Hbx protein localizes to both the cytoplasm and the nucleus. It appears to alter cellular activity by stimulating cell signaling cascades in the former, and activating transcriptional elements in the latter.81, 83-85 In addition, HBx has been reported to influence cell cycle progression, apoptosis, and DNA repair through various mechanisms (reviewed in62, 77). There is a large body of evidence suggesting that HBx plays an important role in the development of HCC in the setting of chronic HBV infection. Studies have found that patients with HCC produce HBx-specific antibodies with much greater frequency than patients with chronic HBV infection alone, with or without cirrhosis (70% vs. 5-15% respectively)86-88 In addition, HBx RNA and protein can be demonstrated in HCC tissue samples.81, 86, 87, 89 Animal studies have provided evidence which implicates HBx in hepatocyte transformation and oncogenesis. Transgenic mice expressing HBx established by Koike et al. frequently develop liver neoplasms (including HCC), particularly those demonstrating high levels of X gene product.90, 91 In contrast to these findings, however, other studies of HBx transgenic mice did not demonstrate a tendency to develop HCC.80, 81 These conflicting findings may be due to the fact that some strains of mice did not produce detectable levels of HBx protein in the liver, and others expressed it only transiently.92 This suggests that chronic HBV infection with continuous HBx production may be required for oncogenesis to occur. Alternatively, a "second hit" hypothesis may apply. Chronic HBV infection with persistent inflammation may predispose to mutations which result in oncogenesis when they occur in the context of HBx expression.80 In support of this hypothesis, Slagle et al. reported that the addition of a hepatic chemical insult (diethylnitrosamine) increases the incidence and tumor burden of HBx transgenic mice.93 Similarly, crossing HBx transgenic mice with cancer-prone c-myc transgenic mice shortened the time to hepatic tumor development.94 Although HBx by itself may not result in oncogenesis, these studies suggest that it is an important factor in this process. Various cell signaling pathways and transcription factors have been implicated in HBx-induced oncogenesis.80, 81, 95 In this review, we will focus on the interactions of HBx with the Wnt/β-catenin and Ras/Raf/MAPK cell signaling pathways. There is some evidence to suggest that expression of HBx results in increased β-catenin activity. Ding et al. recently reported a significant correlation between HBx expression and β-catenin accumulation in HCC samples. In vitro studies with HBx-transfected hepatoma cell lines demonstrated increased β-catenin protein compared with controls, as well as increased nuclear translocation and transcriptional activation.96 This was due to decreased proteosomal degredation of β-catenin in HBx-transfected cells. In addition, HBx-transfected cells had higher protein levels of β-catenin's downstream targets, c-myc and cyclin D-1, when compaired with untransfected controls. Transfected cells also demonstrated higher proliferative activity, which was blocked by dominant negative TCF-4 and β-catenin siRNA. Finally, this study demonstrated that the increased β-catenin activity was the result of GSK-3β inactivation, suggesting a mechanism by which HBx influences this signaling pathway.96 Cha et al. studied the effect of transfecting hepatoma cell lines with combinations of Wnt-1, β-catenin, and TCF-4. They found that certain cell lines which were originally derived from HBV-infected livers (Hep3B, PLC/PRF/5) demonstrated increased nuclear staining of β-catenin and TCF-4-dependent transcription when co-transfected with Wnt-1 and β-catenin.97 The PLC/PRF/5 cell line has previously been shown to express low levels of HBx,98 while HepB3 cells do not express HBx.99 In addition, co-transfecting Huh-7 cells (derived from an HBV-negative liver) with HBx and Wnt-1 caused a dose-dependent increase in TCF-4-dependent transcriptional activity, and increased nuclear translocation of β-catenin.97 Introduction of a Src dominant-negative mutant blocked nuclear translocation of β-catenin in these same cells. Finally, Wnt-1/HBx co-transfected Huh-7 cells demonstated a marked reduction of GSK-3β activity, which was restored by adding the Src dominant-negative mutant construct.97 These studies suggest that HBx in conjunction with Wnt-1 signaling results in diminished GSK-3β activity, and β-catenin stabilization via decreased proteosomal degradation. This results in increased nuclear translocation of β-catenin, and ultimately TCF-4-dependent transcription. Src kinase activity appears to mediate this signaling, which is not typically considered to play a role in the canonical Wnt signaling pathway. The biological consequences of activating this pathway are increased cell proliferation, migration, and survival (Fig. 1). Mitogen-activated protein kinases are a highly conserved family of enzymes which phosphorylate protein substrates, thereby influencing their activity. There are three well-characterized groups of MAPKs: (1) extracellular signal-regulated kinases (ERK), (2) c-jun NH2-terminal kinases (JNK), and (3) p38 enzymes.100 MAPK signal transduction influences many cellular activities, and regulates the functioning of various transcription factors.100 HBx has been linked to the activation of MAPK cell signaling pathways, which results in transcriptional factor activation. Several reports indicated that HBx induces activation of the transcription factor activator protein-1 (AP-1), a dimer consisting of Fos and Jun proteins; however, it does not accomplish this through a direct interaction.101-106 This led to the discovery that HBx increases the activation of Ras, a GTP-binding protein which lies upstream in the MAPK signaling cascade. As shown in Fig. 1, Ras activation by HBx resulted in augmented AP-1 DNA-binding, and was blocked by co-transfection with a Ras dominant negative mutant.107 Therefore, AP-1 activation by HBx is mediated through Ras-MAPK signaling. Furthermore, HBx also activates nuclear factor-κB (NF-κB) in a Ras-dependent manner.83, 108 This occurs by phosphorylation and degradation of IκBα, as well as reduction of p105 in the cytoplasm.108 Both IκBα and p105 inhibit nuclear translocation of NF-κB. Ras-MAPK signaling and NF-κB/AP-1-dependent transcription are only activated when HBx is located within the cytoplasm, not the nucleus.83 In the Ras-MAPK signal cascade, GTP-bound Ras (active state) next activates Raf, a MAPKKK.100 Raf goes on to phosphorylate MEK, a MAPKK, which then activates ERK, a MAPK.95, 100, 107, 109 If HBx stimulates this cascade, signal transduction would not occur without activation of Raf and ERK. Indeed, Cross et al. demonstrated that HBx-induced AP-1 promotor activity is blocked by co-transfection with a Raf dominant negative mutant.106 Natoli et al. confirmed that HBx induces c-jun, and that both Ras and Raf are required for this effect.110, 111 Schneider and Benn demonstrated Ras-dependent activation of AP-1 by HBx, and went on to show that MAPK (ERK) was also activated confirming its role in the pathway.107 They later demonstrate that the c-fos component of AP-1 was activated through Ras-Raf-ERK signaling, while the c-jun component was stimulated through the Ras-MEKK-JNK cascade.112 This would appear to be at odds with the findings of Natoli et al. as their study emphasized the requirement of Raf in c-jun activation.110 Likewise, an earlier study reported activation of c-jun by MAPK/ERK.113 A more recent study demonstrated that HBx induces AP-1 activation through MEK-ERK stimulation as well as JNK activity, and that blocking MEK reduced, but did not prevent, AP-1 activation.114 This implied that AP-1 activation was able to continue, at least temporarily, via an alternative signaling pathway such as Ras-MEKK-JNK. Similarly, another group reported increased phosphorylation of Raf, ERK, and JNK leading to increased activation of c-jun and AP-1; these events appear to be mediated by increased cytosolic calciums level resulting from HBx transfection.112 These data seem to corroborate the conclusions of Schneider and Benn that both Ras-MEKK-JNK and Ras-Raf-ERK pathways are involved in AP-1 activation.102 Importantly, these in vitro findings are supported by in vivo experiments using a mouse model. Nijhara et al. delivered the HBx gene into the hepatocytes of mice using a hepatocyte-specific virosome vector. HBx-expressing cells demonstrated a dose-dependent increase in ERK levels and activity, which could be blocked by a MEK inhibitor. Likewise, JNK and AP-1 were activated by HBx expression.115 Further experimentation by Klein and Schneider revealed that HBx activates the Ras-Raf-ERK pathway earlier in the cascade. Activation of Ras by HBx was shown to be mediated by cytoplasmic Src kinase, causing increased association of Shc-Grb2-Sos. The formation of Shc-Grb2-Sos complex activates Ras and leads to increased activity of ERK and AP-1. Inhibiting Src kinase by co-transfection with Csk abolished this effect, indicating its importance in this signaling cascade.116 Finally, HBx expression causes an increase in cytosolic calcium levels, which appears to mediate signaling through the Ras-Raf-ERK and Ras-MEKK-JNK cascades.112 Calcium has been shown to activate Pyk2, a cytoplasmic kinase, which then activates Src kinases.117 HBx-transfected hepatic cell lines demonstrate increased Pyk2 phosphorylation, with subsequent Src kinase activation and AP-1-dependent transcription.118 Pyk2 activation is blocked by disrupting mitochondrial calcium signaling.118 These data suggest that HBx may induce signaling through the Ras-Raf-ERK and Ras-MEKK-JNK cascades by increasing cytosolic calcium levels, possibly by acting on the mitochondria. Thus, there is ample evidence that HBx interacts with and activates the Ras-Raf-MAPK signaling cascade. During persistent HBV infection, such interactions lead to transcriptional factor activation and hepatocyte proliferation (Fig. 1). Hepatitis C core protein has a molecular weight of 23 kDa and consists of 191 amino acids located at the N-terminus of the HCV polyprotein.119, 120 This peptide, called p23, is cleaved from the polyprotein by cellular signal peptidase, then further processed to its 21 kDa mature form, p21, by a cellular signal peptide peptidase.119-121 The p21 core protein is located predominantly in the cytoplasm, but can also be found in the nucleus.121, 122 The function of core protein is to assemble and package the HCV RNA genome, and is presumed to constitute the viral capsid.119, 120 There is substantial evidence from both in vivo and in vitro models supporting the role of HCV core protein in hepatocyte transformation and tumorogenesis. Transgenic mice have been instrumental in furthering our understanding of the role of core protein in the development of HCC. Koike et al. developed and studied two separate transgenic C57BL/6N mouse lines carrying the HCV 1b core gene. These animals developed steatosis starting from the age of 2 months,123 and many developed liver neoplasms resembling HCC by age 16-19 months.124 In mice which developed neoplasms, immunohistology demonstrated relatively higher levels of core protein within hepatic tumors compared with surrounding tissue. Transgenic mice carrying the HCV envelope genes, E1 and E2, did not develop hepatic neoplasms,124, 125 nor did transgenic mice carrying the gene encoding all nonstructural elements.79 Similarly, Lerat et al. developed several transgenic C57BL/6 lines expressing full-length HCV polyprotein or structural proteins (core, E1, E2p7 only).126 Some animals developed hepatic neoplasms after the age of 13 months. Conversely, Kawamura et al. found that transgenic mice expressing hepatic HCV 1b core, E1, and E2 exhibited histologically "normal" livers at 6 months of age.127 These contradictory findings may be due to the young age at which the livers were analysed (up to 6 months), since neoplasms were not observed in mice younger than 13 or 16 months in the studies reported by Lerat et al. and Koike et al., respectively.124, 126 However, a different study by Pasquinelli et al. also observed "no significant liver injury" in transgenic mice expressing HCV core protein up to 18 months of age.128 Nuclear translocation of the core protein may be important in hepatocyte transformation. Nuclear localization of core protein was observed in the livers of the older, cancer-prone transgenic mice studied by Koike et al.,124 while this was a rare finding in the younger transgenic mice studied by Kawamura et al.127 The transgenic mice studied by Pasquinelli et al. exhibited core protein in the cytoplasm fraction only by Western blot analysis; none was observed in nuclear extracts.128 In fact, core protein was not detected by immunohistochemical staining of liver sections. Therefore, it is possible that the contradictory observations regarding tumorogenesis in HCV core transgenic mice reported in these studies are the result of differences in core protein expression and nuclear localization where it may function as a transcription factor. On balance, there is reasonable evidence from animal models to suggest that chronic expression of HCV core protein may result in malignant transformation of the hepatocyte. This appears to involve a direct oncogenic effect produced by the mere presence of HCV core protein, and may act independently or in concert with a mechanism involving chronic inflammation, necrosis, and regeneration/repair resulting in cumulative mutations. In vitro studies have corroborated evidence from transgenic mouse studies implicating HCV core protein in malignant transformation. In a study using primary rat embryo fibroblasts (REFs), Ray et al.129 demonstrated that co-expression of HCV 1a core protein products (16 kDa and 20 kDa) and oncogenes, particularly H-ras, resulted in immortalization, accelerated growth, anchorage-independent growth, and tumor formation in nude mice consistent with a transformed phenotype.119 Immunohistochemical staining revealed a predominantly nuclear localization of the core protein.129 Chang et al. also transfected primary REFs with H-ras and core protein from HCV genotypes 1a and 1b, however these cells did not exhibit the transformed phenotype.130 Their transfected cells produced core protein products of 19 kDa and 21 kDa, but not 16 kDa. When they co-transfected cells from an established REF cell line, however, they did observe the transformed phenotype. These cells produced only the 19 kDa product. They postulated that the gene product species, level of expression, and intracellular localization (e.g., nuclear vs cytoplasmic), as well as cellular factors, may determine its ability to transform cells. Similarly, Tsuchihara et al. co-transfected a murine fibroblast cell line with H-ras and HCV core and observed the transformed phenotype.131 These cells produced a 21 kDa core gene product. Finally, Ray et al. transfected primary human hepatocytes from different donors with HCV core and noted morphologic changes, immortalization, telomerase activity, and anchorage-independent growth indicating a transformed phenotype.132 The dominant core gene product in these cells was 22 kDa. Transfecting these same cells with the antisense core gene, thereby inhibiting core protein expression, induced apoptosis, decreased telomerase activity, and essentially reversed their immortalization.133 Collectively, these studies support the hypothesis that HCV core protein itself, or in conjunction with other factors, is involved in the oncogenic transformation process. The precise mechanisms by which this occurs have not been delineated, however there is some compelling evidence that the Wnt/FZD cell signaling pathway may be involved. Fukutomi et al. recently reported that HCV 1b core-transfected Huh-7 cells upregulated Wnt-1 and WISP-2 transcription.134 These cells demonstrated increased proliferation, DNA synthesis, and cell cycle progression. Proliferation was blocked by Wnt-1 siRNA. Supernatant taken from cultures of these core-transfected cells increased the proliferation of other cells, suggesting that a secreted factor such as Wnt-1 provides a mitogenic stimulus. Finally, transfecting Huh-7 cells with Wnt-1 also resulted in increased proliferation. From these experiments it is reasonable to suspect that overexpression of Wnt-1 (and possibly other Wnt ligands) with activation of the canonical Wnt signaling pathway is important in the malignant transformation of hepatocytes. Nonstructural protein 5a (NS5a) is a polypeptide formed by proteolytic cleavage of the viral polyprotein precursor. It has two major forms, p56 and p58, which differ by the degree of phosphorylation of the serine residues.135 However, other forms (p50, p53) have also been described in the Huh-7 hepatoma cell line.136 Though expression of this protein in transgenic mice has not demonstrated direct oncongenicity,137, 138 NS5a has been associated with cell transformation in vitro.139-141 Transfecting murine embryo fibroblasts (NIH 3T3) with NS5a results in a transformed phenotype, defined by increased proliferation, colony formation in soft-agar medium, and tumor formation in nude mice.139, 140 Proposed mechanisms for NS5a-mediated transformation include cell cycle deregulation, decreased apoptosis, and activation of transcription factors. NS5a has been shown to activate by directly interacting with the p85 subunit.142 This results in activation of downstream Akt, which in turn phosphorylates and inactivates Bad, a pro-apoptotic Bcl-2 homologue, thereby inhibiting apoptosis.142, 143 As shown in Fig. 1, another downstream target of Akt is GSK-3β. Introducing HCV polyprotein or NS5a genes into hepatoma cell lines results in increased GSK-3β phosphorylation, decreased β-catenin degradation, and subsequently increased β-catenin-dependent transcription; these effects are blocked by restoring Akt.144 Activation/dysregulation of the PI3K/Akt pathway has been associated with many different malignancies.145, 146 Hepatocellular carcinoma is a dreaded consequence of chronic viral hepatitis. In recent years we have witnessed an increased incidence of this disease. There is mounting evidence that hepatitis B and C viral proteins may contribute to oncogenesis through direct interactions with cellular components. Various viral proteins have been observed to interact with growth factor signaling cascades known to influence cell behaviors such as proliferation, migration, and apoptosis. Such interactions may ultimately result in a malignant transformed phenotype and tumorogenesis. This review has focused on a few pathways that appear to be important in both HBV- and HCV-related hepatocarcinogenesis. A better understanding of the viral pathogenesis of HCC may yield molecular targets useful for the diagnosis and treatment of this devastating disease. We wish to thank Rolf Carlson for illustrating Figure 1 and Donna Pratt for editorial assistance in producing this publication.