Causes of Liver Cancer in HCV:
Hepatitis C virus-induced hepatocarcinogenesis (HCC))
Birke Bartosch123, Robert Thimme4, Hubert Blum4, Fabien Zoulim123Corresponding Author Informationemail address
Articles in Press
Jnl of Hepatology
published online 25 May 2009.
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 infected 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.
"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"
"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"...."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"
"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) , , duration of infection , male sex (2- to 3-fold increased risk) , severity of liver disease at presentation, co-morbidities such as porphyria cutanea tarda , heavy alcohol intake , , , , diabetes mellitus , , steatosis , , obesity ,  and coinfections, especially with HBV , . 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"
"all HCV genotypes have been shown to interfere with glucose homeostasis, often at early stages in HCV infection"....."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"
"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."
"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"
"a complex interplay between steatosis, ER/oxidative stress and IR (insulin resistance), 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."
"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."..."HCV is thought to induce steatosis by interfering with lipid secretion and degradation and by increasing lipid synthesis"
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 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.
Associate Editor: K. Koike
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) , . 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 , , ,  (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 . 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.
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 H epacivirus' . The HCV genome is a single-stranded, positive sense RNA of approx. 9600 nt in length  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 proteins (NSI), designated NS2 to NS5B in the 3'-region.
HCV is considered hepatotropic, and only man and chimpanzees are susceptible to HCV infection and disease , . 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 , , . Circulating HCV particles have a diameter of 35-50nm and are frequently associated with either immune globulins or very low density lipoproteins (vLDL) . Indeed, the vLDL biosynthesis machinery plays a pivotal role in the life cycle of HCV , , .
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 . 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 .
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 , . 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 .
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 , ,  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 .
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 ,  (Table 1). HCV infection is estimated to increase the risk for HCC development up to 17-fold , . Host, environmental and viral factors appear to play an important role in determining progression of chronic hepatitis C to liver cirrhosis and HCC . Some , , , , but not all clinical studies , , , , 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 .
Apart from HCC, HCV is also a well-established risk factor of lymphoproliferative syndromes such as type II mixed cryoglobulinemia  and malignant lymphoma. Indeed, HCV infection increases the risk of B-cell non-Hodgkin lymphoma (B-NHL) 2- to 10-fold ,  (Table 1). This association is particularly striking in southern Europe but much less in northern Europe and north America , , 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  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 . HCV infection has also been linked to the development of intrahepatic cholangiocarcinoma (ICC) , , . 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 , . 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) , , duration of infection , male sex (2- to 3-fold increased risk) , severity of liver disease at presentation, co-morbidities such as porphyria cutanea tarda , heavy alcohol intake , , , , diabetes mellitus , , steatosis , , obesity ,  and coinfections, especially with HBV , . 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 , . 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 . Further, an association between the HLA DQ02 allele and HCC development has been reported .
4. HCV-induced hepatocarcinogenesis
The mechanisms underlying the progression of HCV infection to HCC, which usually takes many years or decades, remain ill-defined , . 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 , .
HCV is the only RNA virus with a predominantly cytoplasmic life cycle , . 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 ,  (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 , . Markers of intracellular oxidative stress have also been found to be increased in patients with chronic HCV infection ,  as well as HCV core transgenic mice , . 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 , . 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 , , .
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 . 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 , as well as some HCV non-structural proteins, have been shown to interfere with very low density lipoprotein (vLDL) secretion , . HCV infection also upregulates lipid synthesis , inhibits fatty acid oxidation ,  and increases release of fatty acids from adipocytes . 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 . With respect to IR, all HCV genotypes have been shown to interfere with glucose homeostasis, often at early stages in HCV infection . The mechanism underlying IR and its severity seem 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 . 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 .
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-ß, 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-ß induces EMT not only in HSCs but possibly also in hepatocytes . TGF-ß 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-ß signaling via SMAD interacts with other signaling pathways to mediate pro-oncogenic EMT. JNK activation by the pro-inflammatory cytokine interleukin (IL)-1ß can shift TGF-ß signaling from tumor suppression to oncogenesis with increased fibrogenesis, cell motility and transactivation of cell cycle regulatory genes , . 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 , , , .
The HCV core protein is a highly conserved, basic protein that multimerizes, probably in conjunction with microtubules  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 , , . Core has also been shown to localize to the outer membranes of mitochondria  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 , to bind the tumor suppressor proteins p53 , , p73  and pRb . Core also inhibits the expression of the cyclin-dependent kinase (CDK) inhibitor p21/Waf . 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 , , protects cells from serum starvation and growth arrest and drives cells into proliferation. NF-κB transcription has been shown to be activated , ,  and repressed  by HCV core. HCV core has also been shown to activate the Wnt/b-catenin pathway, which controls cell proliferation, DNA synthesis and cell-cycle progression . Furthermore, HCV core variants have been shown to interact with SMAD3 and to inhibit the TGF-ß pathway . TGF-ß signaling not only controls cell proliferation, differentiation and apoptosis but also stimulates liver regeneration and fibrogenesis through its actions on the extracellular matrix (see above). TGF-ß levels are frequently increased in patients with chronic HCV infection and correlate with the degree of fibrosis , . Finally, HCV core protein associates with cellular membranes ,  and lipid vesicles , binds to apolipoprotein II ,  and reduces microsomal triglyceride transfer protein activity , 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 ,  and HCC , .
Overexpression of E2 inhibits eIF2α phosphorylation by the dsRNA-activated protein kinase (PKR) or the ER-stress signaling kinase PERK , . 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 , , , , . NS2 has been shown to inhibit the cellular proapoptotic molecule CIDE-B  and to downregulate transcription .
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 , , . Indeed, RIG-I inactivation has been shown to render Huh-7 cells permissive to HCV replication , .
NS5A has been shown to interact with the geranylgeranylated cellular protein FBL2 , an F-box motif containing protein that is probably involved in targeting cellular proteins of yet unknown identity for ubiquitylation and degradation. A number of studies suggest that NS5A is also involved in IFN resistance  and one possible mechanism may be its ability to induce expression of the type I interferon anatagonist IL-8 . 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 , , . The accumulation of mutations in this region is thought to correlate with treatment efficacy , . 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 including p21 , ,  and of pRB . Other NS5A interaction partners include apoliporotein 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 ß3, a protein involved in nuclear trafficking, Cdk1/2, cyclin-dependent and Fyn, Hck, Lck, and Lyn, Src-family kinases , , , , . 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 ß-catenin and stimulation of ß-catenin-dependent 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 .
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 ß-catenin mutations that dysregulate the Wnt pathway . 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-ß-signaling , . 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 . 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 . Moreover, gankyrin, an inhibitor of p53 and Rb check point function is overexpressed in the vast majority of HCCs . Expression of insulin-like growth factor IGF-2 is frequent and thought to be an early event in hepatocarcinogenesis, present in more than 60% of dysplastic nodules and HCC . IGF-2 receptor impairs cell proliferation by promoting degradation of the IGF-2 mitogen and by activation of TGF-ß signaling .
ß-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 . ß-Catenin activation is mainly induced by ß-catenin gene mutations and/or Wnt signaling pathway alterations . Wnt/frizzled/ß-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/ß-catenin pathway is involved in cell growth and proliferation as well as developmental control and cell adhesion. Cellular levels of ß-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-3b (GSK-3b). A recent report indicates that Fzd-7, which stabilizes and activates ß-catenin , 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 . Hypomethylation of the IGF-2 locus in hepatitis C cirrhosis has been shown to predict HCC development . 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 . Finally, increased hTERT DNA levels have been found to predict HCC development .