Best Practice & Research Clinical Gastroenterology 28 (2014) 937e947
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Best Practice & Research Clinical Gastroenterology
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Pathogenesis of hepatocellular carcinoma according to aetiology Jean-Charles Nault, MD, Hepatologist a, b, c, d, * Inserm, UMR-1162, G enomique fonctionnelle des Tumeurs solides, IUH, Paris, F-75010, France Universit e Paris Descartes, Labex Immuno-Oncology, Sorbonne Paris Cit e, Facult e de M edecine, Paris, France c ^pital Jean Verdier, AP-HP, Bondy, France Service d'H epatologie, Ho d Universit e Paris 13, Bobigny, France a
b
a b s t r a c t Keywords: Hepatocellular carcinoma Aetiology Hepatitis B Hepatitis C Genetic
Hepatocellular carcinoma is related to various etiologies including hepatitis B, hepatitis C, high alcohol intake, aflatoxin B1 and metabolic syndrome. Most of the time HCC developed on cirrhosis. Consequently, the mechanisms of carcinogenesis of these different risk factors are difficult to separate from the events leading to cirrhosis. In contrast, aflatoxin B1 and hepatitis B have a clear direct oncogenic role through point mutations in the TP53 tumour suppressor gene and insertional mutagenesis respectively. Finally, next-generation sequencing and transcriptome analysis will refine our knowledge of the relationship between aetiology and the genetic events that draw the mutational landscape of hepatocellular carcinoma. © 2014 Elsevier Ltd. All rights reserved.
Introduction Hepatocellular carcinoma (HCC) is a strongly heterogeneous disease both from a molecular and clinical point of view. It mirrors the different etiologies of HCC worldwide, HBV in eastern countries, and alcohol, Non-alcoholic steatohepatitis (NASH) and hepatitis C in western countries [1]. In addition,
Paris Descartes, 27 rue Juliette Dodu, Paris, 75010, France. Tel.: þ33 1 53 72 51 94; fax: þ33 1 * INSERM UMR-1162, Universite 53 72 51 92. E-mail address: [email protected].
http://dx.doi.org/10.1016/j.bpg.2014.08.006 1521-6918/© 2014 Elsevier Ltd. All rights reserved.
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the severity of the underlying liver disease may vary from normal liver to cirrhosis, although HCC develops on cirrhosis in 90% of patients in western countries [2]. This leads to differences in clinical care of HCC patients treated in western and eastern countries. These differences have been described in Asian and western guidelines and are partly explained by the genetic diversity of HCC [3e5]. HCC are heterogeneous at both the tumoral and non-tumoral level, and a major challenge lies in defining the relationship between aetiology, HCC development and molecular features [6,7]. HCC, like other cancers, is a disease of the genome and is defined by malignant hepatocytes accumulating somatic genetic alterations that combine mutations in both driver and passenger genes [8,9]. The recent technological breakthrough of next-generation sequencing has led to characterization of a whole genome and a whole exome of tumours within a few hours and at lower cost [10]. This again highlights the wide genetic diversity and the influence of aetiology, especially in HCC due to chronic HBV infection [11]. The aim of the present review was to describe the link between aetiology and the pathogenesis of HCC. Pathogenesis of HCC according to aetiology Hepatitis B Hepatitis B virus is a partially double-stranded circular DNA virus belonging to the hepadnavirus family [12]. Pre-S/S ORF encodes three surface proteins, pre-C/C ORF encodes a terminal protein and viral polymerase has a reverse transcriptase and a DNA polymerase function [11]. The X gene encodes the HBx protein that is mandatory for viral replication. Chronic infection by hepatitis B leads to HCC development, with an added risk that is 25- to 37-fold that of non-infected patients [13,14]. It is the leading aetiology in Asian and African countries. HCC related to HBV infection may develop on both cirrhotic liver and normal liver, whereas most HCC related to high alcohol intake or chronic HCV infection develop on cirrhosis [1]. Along this line, one of the main mechanisms of carcinogenesis due to HBV infection is through chronic inflammation, liver fibrosis and thus development of cirrhosis [11]. In this setting, chronic inflammation, oxidative stress and replicative senescence of hepatocytes due to telomere shortening induce malignant transformation of cirrhotic nodules, as in other cirrhotic backgrounds (Fig. 1) [7]. However, other
Fig. 1. The multistep process of liver carcinogenesis and the role of etiologies.
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carcinogenic mechanisms are specific to chronic HBV infection and could explain the occurrence of HCC against a non-cirrhotic background. First, HBV DNA could integrate the human genome and be clonally selected in tumours [15]. Interestingly, only a small part of the HBV genome integrates the human genome; consequently, integrated HBV is not used for viral replication [16]. Overall, at least 70e80% of HCC due to HBV infection show clonal integration of the viral genome [17,18]. HBV insertion may occur randomly throughout the human genome, but preferential sites near the fragile site, the repeat DNA sequence and the highly transcribed site have been suggested. Most of the time, the 30 end of HBx and the 50 end of the preCore/core genes are preferentially inserted [17]. One of the cardinal effects of viral integration is a cis effect wherein the virus is integrated [19]. Several recurrent sites of insertion in key genes in carcinogenesis have been described: TERT, MLL4, CCNE1, CCNA2 and RARB (retinoic acid receptor b) [16,17,20]. HBV integration sites are frequently promoter regions or intron regions and, less frequently, coding regions [17,19]. Frequently, HBV insertion in the human genome leads to increased expression of this gene, as has been demonstrated in TERT and CCNE1 [17]. In addition, several pieces of evidence suggest that HBV integration might induce, or at least be associated with, chromosomal instability [21,22]. Another mechanism of viral carcinogenesis through viral integration into the human genome is a trans mechanism, through production of the truncated protein like HBx or the pre S2/S protein, due to partial integration of the virus [11]. This is related to another mechanism of HBV carcinogenesis, namely, the direct oncogenic properties of the viral protein [23]. Production of the truncated HBx protein and the pre S2/S protein can modulate the signalling pathway and induce transactivation of several genes [11,23]. While the truncapted pre S2/S protein is believed to be an oncogenic protein, one of the most thoroughly studied and controversial oncogenic functions is related to the HBx regulatory protein. Physiologically, the HBx protein is involved in HBV replication and transcription and could stimulate the viral promoter and enhancer via it transactivation function [24]. This function has been suggested to be a key mechanism that promotes carcinogenesis in humans through activation of several signalling pathways, control of apoptosis and DNA repair [25,26]. Recent studies have shown an interaction with acetyltransferase CBP/p300 that leads to CREB-dependent transcription controlling liver metabolism and cell proliferation [27]. In addition, frequent truncated HBx proteins are produced, and this protein could increase metastasis or cell invasiveness in vitro models [28]. However, its oncogenic properties are subject to debate. Indeed, several experimental studies identified an oncogenic effect [29,30], whereas others showed no effect or even a paradoxical tumour suppressive effect of truncated HBx [31]. Mouse models have shown both an oncogenic function of truncated Hbx [32] and no effect upon carcinogenesis [33]. Moreover, HBx gene harboured a pattern of tumour suppressor gene in human hepatocellular carcinoma including stop mutations and frameshift deletion [34].In addition, several studies have pointed to a possible role for the HBx protein as a regulator of a mitotic spindle that could constitute a link between HBV infection and chromosomal instability [35,36]. Currently, most data suggest an oncogenic role for the HBx truncated protein, but mysteries remain. Hepatitis C Hepatitis C is a single-stranded RNA virus belonging to the flaviridae virus family [37]. Most of the cell cycle of the HCV virus occurs in the cytoplasm and, in contrast to HBV, HCV cannot integrate the host genome. Most HCC related to HCV occurs on cirrhosis; consequently, the pathogenesis of hepatocarcinogenesis is roughly the same as that of HCC developing on alcohol- or NASH-related cirrhosis [38]. HCV leads to chronic inflammation, immune-mediated hepatocyte death, tissue damage, fibrosis synthesis by hepatic stellate cell and replicative senescence due to telomere shortening [38,39]. Moreover, additional factors such as oxidative stress, steatosis and insulin resistance accelerate the evolution to cirrhosis and hepatocellular carcinoma [[40e42] Bartosch, 2009 #3021]. One of the key determinants of fibrosis progression and HCC occurrence against a hepatitis C background is insulin resistance and steatosis [43e45]. Most of the time, steatosis is due to concomitant diabetes and obesity, two well known risk factors in liver disease and HCC [46]. However, genotype 3 hepatitis C is directly associated per se with liver steatosis through modulation of lipid degradation, synthesis and secretion [47]. Taken together, the main mechanisms leading to HCC are indirectly caused by HCV. However, some authors suggested that HCV proteins have direct oncogenic properties [38]. They might affect
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signalling pathways like the Wnt/B-catenine, TGFB, NFKB or P53 pathway by modulating transcription or translation or by a post-translational mechanism [48,49]. In addition, several studies have shown that the HCV protein core or NS3, NS4B and NS5A could induce transformation of fibroblasts in vitro [50]. Interestingly, HCV core protein expression in a transgenic mouse model leads to HCC development [51]. However, most studies dealing with direct oncogenic properties of the HCV protein used inadequate models that precluded drawing firm conclusions [38]. Consequently, the oncogenic function of the HCV protein in human disease remains to be demonstrated. Alcoholic liver disease According to the World Health Organization, alcohol is a carcinogen that predisposes to several cancers, ranging from esophageal cancer to breast cancer, often in conjunction with other carcinogens like tobacco [52]. Clearly, alcohol consumption is associated with increased risk of HCC development [53]. However, most of the time, HCC develops on alcoholic cirrhosis [54]. The role of alcohol in HCC developing on non-cirrhotic liver is unknown, although a French study suggested a link with high alcohol consumption [55]. In addition, alcohol is a co-factor in liver disease and HCC in patients with chronic hepatitis B, chronic hepatitis C, haemochromatosis and NASH [53,54]. Paradoxically, cirrhotic patients who stop drinking have a higher risk of HCC development than patients with persistent consumption [56]. This might be explained by decreased death from liver failure and longer exposure to HCC risk in abstinent patients. Taken together, the mechanisms inducing liver injury and cirrhosis and the mechanisms of liver carcinogenesis in patients who abuse alcohol are difficult to separate [52]. Ethanol has no direct carcinogenic properties when given to mice and rats [57]. Under certain conditions, often in conjunction with chemical carcinogens, an increased incidence of tumours in rodents exposed to ethanol has been demonstrated [58]. Acetaldehyde is an ethanol metabolite that has carcinogenic properties through DNA binding [59]. Production of acetaldehyde is dependent on alcohol dehydrogenase, an enzyme with polymorphisms regulating its activity [52]. However, the role of these polymorphisms in liver carcinogenesis is still unclear. In addition, CYP2E1, a cytochrome induced by ethanol consumption, converts ethanol into acetaldehyde, but also into reactive oxygen species (like superoxide anion and hydrogen peroxide) [60]. Chronic oxidative stress induced by alcohol intake and cytokine production in the context of chronic inflammation is a well known phenomenon leading to cirrhosis and HCC development [52,54]. Accumulation of reactive species of oxygen favours lipid peroxidation and creates DNA adducts [61]. Polymorphism of superoxide dismutase and MnSoD has been associated with HCC occurrence in alcohol-related cirrhosis, but not in HCV-related cirrhosis [62]. Moreover, iron overload induced by chronic alcohol intake participates in liver injury through oxidative stress. A prospective study has shown that iron overload is associated with high risk of HCC development in alcohol-related cirrhosis [63]. Finally, additional mechanisms of alcohol-mediated carcinogenesis exist, including downregulation of levels of retinoic acid [64] and modulation of methylation by downregulation of S-adenosyl L methionine (SAMe) [65]. Non-alcoholic fatty liver disease (NAFLD) A large body of evidence supports the role of diabetes and obesity in HCC pathogenesis. In a large epidemiologic study, patients with BMI >35 had an increased risk of cancer, especially hepatocellular carcinoma, with a hazards ratio (HR) of 4.52 [66]. In addition, diabetes is associated with cirrhosis and HCC occurrence, with a HR of 2.39 in a US cohort [67]. Interestingly, diabetes was also a risk factor for HCC occurrence in a prospective cohort of HCV-related and alcohol cirrhotic patients [68]. While most HCC related to NASH develops on cirrhosis, several groups have reported the occurrence of HCC in NASH without underlying cirrhosis [69,70]. This suggests that specific carcinogenic mechanisms related to NASH could lead to HCC development outside the classical background of cirrhosis [71]. However, these specific carcinogenic mechanisms are still unknown, and at present, the pathogenesis of NASH is difficult to distinguish from the carcinogenic mechanism of HCC development on NASH. Obesity is characterized by chronic low-grade inflammation that predisposes to liver injury and HCC occurrence [71,72]. An elegant study by the team of Mickael Karin showed that obesity enhanced HCC
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formation induced by chemical carcinogens [73]. This phenomena was cytokine-dependent, especially IL6 and TNF-alpha production. In addition, studies have pinpointed the role of microbiota and inflammation in tumour promotion [74,75]. These models highlighted the pivotal role of inflammation in HCC initiation due to obesity. This inflammatory state is a conjunction of the action of hepatocytes, Kupffer cells and adipocytes. Moreover, an abnormal balance between leptin and adipokine characterizes NAFLD and predisposes to HCC development. A decreased level of adiponectin, an antinflammatory, antiproliferative cytokine exclusively produced by adipocytes, and a parallel increase in leptin, a pro-inflammatory, angiogenic and profibrogenic cytokine predominantly produced by adipocytes, are cardinal features of NAFLD [72,76,77]. This underlines the important role of visceral adiposity in liver disease. However, their exact role in tumour initiation and progression remains to be refined. In a mouse model of NASH, hypoadiponectinemia was associated with increased tumour occurrence, thus supporting a role in carcinogenesis [76]. In addition, accumulation of lipid droplets in hepatocytes leads to lipotoxicity that contributes to NASH and consequently to HCC pathogenesis [78]. Moreover, this phenomenon increases insulin resistance, that leads to hyperinsulinemia and production of IGF1 by the liver. IGF1 induces phosphorylation of IRS1, leading to activation of the PI3K/AKT/MTOR and MAPK kinase pathways, and consequently promotes cell proliferation and inhibits apoptosis [79]. In parallel, decreased production of IGFBP1 and IGFBP2 by the liver increases IGF1 bioavailability [[80] Siddique, 2011 #3218]. Consequently, the IGF/IRS1 axis is a key signalling pathway involved in NAFLD pathogenesis and related carcinogenesis. In conclusion, NAFLD pathogenesis and tumorogenesis are related to various factors, including chronic inflammation, oxidative stress, insulin resistance, lipotoxicity and an abnormal relationship with visceral adiposity. However, the exact role and time frame of each of these factors in tumour initiation in NAFLD are still unclear. Rare diseases Rare diseases, including primary biliary cirrhosis, autoimmune hepatitis, haemochromatosis and Wilson's disease, can lead to HCC development. In most cases, HCC occurs on cirrhosis, and the exact carcinogenic mechanisms linked to specific etiologies have not yet been identified. In addition, few experimental or clinical studies have focused on carcinogenesis in these rare diseases. However, clinical evidence concerning haemochromatosis provides additional insight into the role of iron in liver carcinogenesis. Iron deposits in the liver favour hepatic injury, occurrence of cirrhosis and tumour initiation [81]. However, the identification of iron-free dysplastic nodules and HCC suggests a two-hit hypothesis, with iron overload leading to cirrhosis and tumour initiation and, in a second step, to tumour promotion, with proliferation of iron-free malignant hepatocytes to avoid the cytotoxic effect of iron [82]. Genetic alterations in HCC and the influence of aetiology Next-generation sequencing has revealed that HCC harbours a mean of 40e50 damaging somatic mutations in coding sequences, including mutations both in driver and in passenger genes [83e86]. This can be compared with other types of tumours, including paediatric cancers (a mean of three mutations in rhabdoid tumours), melanoma and lung cancer (a mean of 110 and 120 mutations, respectively) [8]. In various next-generation studies, no differences in the mean number of genetic alterations between etiologies have been identified, although the number of studies is limited [83e88]. Among such mutations, several driver genes are recurrently mutated in HCC. The oldest somatic genetic alterations identified in HCC are activating mutations of b-catenin (coded by CTNNB1) and inactivating mutations of TP53, in 20e40% and 20e50% of cases, respectively [89,90]. CTNNB1 mutations appear to be less frequent in HCC developing on HBV cirrhosis [83e85]. In contrast, there is a strong overrepresentation of TP53 mutations in HBV-related HCC [91]. This is the strongest example of a relationship between aetiology, exposure to carcinogens and specific mutations of the tumour genome [92]. Aflatoxin B1 is a mycotoxin that contaminates dietary products in some regions of Asia and Africa [93]. It is associated with HCC development in conjunction with HBV infection. HCC related
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to aflatoxin B1 exposure harbours a highly specific mutation in codon 249 (R249S) of TP53 [90]. Consequently, the R249S mutation of TP53 is a surrogate marker of aflatoxin B1 exposure in patients with HCC (Fig. 1). More recently, a whole genome study identified, in some HCC, a specific mutational signature (G to A substitution) related to chronic intake of aristolochic acid, a plant also associated with urothelial cancer harbouring the same mutational signature [94]. Taken together, there exists a molecular imprint of exposure to carcinogens that could help to identify a new mechanism of carcinogenesis and new carcinogens. Whole exome sequencing of HCC has also identified a genotoxic signature overrepresented in HCC developing on normal liver [83]. At this time, the link between a carcinogen and this genotoxic signature on normal liver remains unknown. In the future, study of mutational signatures using next-generation sequencing could help to identify new carcinogens and assess the level of exposure to known carcinogens [95]. Differences in recently published next-generation studies could also be explained by the different etiologies of HCC analysed by whole exome or whole genome sequencing. While telomere shortening due to the absence of telomerase activity favours liver injury and tumour initiation through hepatocyte replicative senescence, in contrast, telomerase reactivation is required for tumour promotion so as to allow uncontrolled cancer cell proliferation [96]. TERT promoter mutations are the most frequent genetic alterations in HCC, with an overall frequency of 59% [97]. This leads to an increase in TERT promoter activity and explains a substantial part of telomerase overexpression in HCC. Strikingly, TERT promoter mutations are less frequent in HBV-related tumours [97,98]. This could be explained by the presence of HBV insertion in the TERT promoter, a well known mechanism leading to telomerase reactivation. New, recurrent genetic alterations have been identified using whole exome and whole genome sequencing [83e86]. ARID1A and ARID2 are recurrently mutated in 15% and 10% of cases, respectively [83]. ARID1A and ARID2 belong to the SWI/SNF complex, implicated in chromatin remodelling and transcription control. These mutations lead to inactivation of ARID1A and ARID2, a classical feature of the tumour suppressor gene. In one series, the ARID1A mutation was significantly associated with high alcohol intake, although this association is not supported by a physiopathological explanation [83]. Recurrent mutations in KEAP1 and NFE2L2 lead to the constitutive activation of the stress oxidative pathway [83,88]. Moreover, the ras/raf/map kinase pathway is a key signalling pathway, with rare activating mutations in PIK3CA (1%), RAS (1%) and, more frequently, mutations in RPS6KA3 (8%). Amplification of FGF19 has been described in up to 15% of HCC [99]. One study reported the association of 6p21.1 amplification with HCC developing on NASH. CULLIN7 has been underlined as a potential gene of interest in this region of the genome [100]. Molecular classification of HCC according to aetiology Several molecular classifications have thus far been reported in HCC [101]. The two main goals of these molecular classifications are to describe genetic heterogeneity and to identify prognostic biomarkers relevant to clinical care. We previously described a molecular classification based on transcriptome analysis in six subgroups (G1eG6) [102]. This molecular classification was closely related to genetic defects, signalling pathways and clinical features. HCC classified into G4eG6 subgroups have chromosomal stability; in contrast, HCC classified into the G1eG3 subgroups have chromosomal instability. HCC of the G5eG6 subgroups were strongly associated with activating mutations of CTNNB1 mutations. HCC classified into G3 subgroups have poor prognosis and harbour disregulation in the cell cycle gene. HCC of the G1eG2 subgroups are strongly related to chronic HBV infection, and the G1 subgroup to high levels of AFP. Taken together, these molecular classifications confirmed that HBV infection is an aetiology that harbours a strong specific molecular signature. Other molecular classifications have underlined the relationship between HBV and specific genetic defects. Work by the team of Snorri Thorgeisson has shown that HBV-related HCC are enriched in TP53 mutations related to a stem cell phenotype [91,103]. TP53-mutated tumours in this series of Asian patients with chronic HBV infection also carry a poor prognosis [91]. We recently confirmed the prognostic role of TP53 in HBVrelated HCC. Strikingly, in a series of HCC non-related to HBV infection, TP53 mutations had no significant impact on mortality [34]. This is an example of the relationship between aetiology, genetic defects and the impact on prognosis assessment. It underlined that genetic defects could have different
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clinical significance depending on the underlying aetiology. Another feature of HBV-related HCC is the higher proportion of stem cell phenotypes in Asian series of patients compared to western patients [91,101,104]. One explanation might be that of differing carcinogenesis related to HBV infection that promotes aggressive tumours of the stem cell phenotype, mutated for TP53. However, common molecular features linked to cancer aggressiveness among HCC of different etiologies have also been identified. A five-gene score derived from tumour analysis was able to predict tumour recurrence and survival after liver resection in 748 HCC of various etiologies, including high alcohol intake, HCV and HBV infection [105]. While molecular features may differ according to aetiology, nonetheless, common molecular mechanisms exist in liver carcinogenesis. Conclusion The various etiologies associated with liver disease and promoting of HCC complexify our understanding of the pathogenesis of HCC. This wide diversity reflects diverse exposure to risk factors, from viral infection to environmental factors like aflatoxin B1 and alcohol [106]. In addition, a conjunction of risk factors is frequent in clinical practice, and the specific impact of each factor in hepatocarcinogenesis is difficult to evaluate. The most clear-cut direct carcinogenic role is that of chronic hepatitis B infection, that can lead to HCC outside of the background of cirrhosis [11]. It includes well known viral integration into the tumour genome, but also the potential oncogenic role of viral protein. In contrast, the direct carcinogenic role of other risk factors like chronic hepatitis C, alcohol and metabolic syndrome is difficult to separate from mechanisms of liver injury leading to cirrhosis formation (Fig. 1). Overall, the conjunction of chronic inflammation, oxidative stress and telomere shortening favours liver injury and tumour initiation in various liver diseases [107]. The different steps of carcinogenesis against a cirrhotic background and the potential relationship with etiologies are still unclear and need further investigation. Finally, a small fraction of HCC develop on normal liver. Although some of these HCC could be due to malignant transformation of hepatocellular adenoma through accumulation of CTNNB1 and thus TERT promoter mutations, most cases are not related to any risk factors [108e110]. In the future, epidemiological and molecular studies should help to identify potential carcinogens and risk factors associated with HCC occurrence in normal liver, that is, a pure model of liver carcinogenesis without a cirrhotic background.
Practice points (1) Hepatitis B virus has a direct oncogenic effect due to viral insertional mutagenesis and the role of HBx protein. (2) Aflatoxin B1 is a direct carcinogen and induces specific point mutations in TP53 (R249S). (3) Some studies have reported the occurrence of HCC due to metabolic syndrome on noncirrhotic liver; however a specific mechanism has not been described so far. (4) Oxidative stress, chronic inflammation, telomere shortening and insulin resistance are common mechanisms that lead to chronic liver injury and HCC occurrence on cirrhosis.
Research agenda (1) The association between the different etiologies and the somatic genetic alterations of hepatocellular carcinoma should be refined. (2) The role of metabolic syndrome on liver carcinogenesis, specially outside the cirrhotic background, should be clarified. (3) The risk factors leading to HCC occurrence on normal liver needs to be identified.
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Conflicts of interest None. Acknowledgements This work was supported by HECAM (BPI), EBCI, INCa (WntHCC) project. J-C.N. was supported by a fellowship from INCa. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]
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Contents lists available at ScienceDirect
Best Practice & Research Clinical Gastroenterology
14
Pathogenesis of hepatocellular carcinoma according to aetiology Jean-Charles Nault, MD, Hepatologist a, b, c, d, * Inserm, UMR-1162, G enomique fonctionnelle des Tumeurs solides, IUH, Paris, F-75010, France Universit e Paris Descartes, Labex Immuno-Oncology, Sorbonne Paris Cit e, Facult e de M edecine, Paris, France c ^pital Jean Verdier, AP-HP, Bondy, France Service d'H epatologie, Ho d Universit e Paris 13, Bobigny, France a
b
a b s t r a c t Keywords: Hepatocellular carcinoma Aetiology Hepatitis B Hepatitis C Genetic
Hepatocellular carcinoma is related to various etiologies including hepatitis B, hepatitis C, high alcohol intake, aflatoxin B1 and metabolic syndrome. Most of the time HCC developed on cirrhosis. Consequently, the mechanisms of carcinogenesis of these different risk factors are difficult to separate from the events leading to cirrhosis. In contrast, aflatoxin B1 and hepatitis B have a clear direct oncogenic role through point mutations in the TP53 tumour suppressor gene and insertional mutagenesis respectively. Finally, next-generation sequencing and transcriptome analysis will refine our knowledge of the relationship between aetiology and the genetic events that draw the mutational landscape of hepatocellular carcinoma. © 2014 Elsevier Ltd. All rights reserved.
Introduction Hepatocellular carcinoma (HCC) is a strongly heterogeneous disease both from a molecular and clinical point of view. It mirrors the different etiologies of HCC worldwide, HBV in eastern countries, and alcohol, Non-alcoholic steatohepatitis (NASH) and hepatitis C in western countries [1]. In addition,
Paris Descartes, 27 rue Juliette Dodu, Paris, 75010, France. Tel.: þ33 1 53 72 51 94; fax: þ33 1 * INSERM UMR-1162, Universite 53 72 51 92. E-mail address: [email protected].
http://dx.doi.org/10.1016/j.bpg.2014.08.006 1521-6918/© 2014 Elsevier Ltd. All rights reserved.
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the severity of the underlying liver disease may vary from normal liver to cirrhosis, although HCC develops on cirrhosis in 90% of patients in western countries [2]. This leads to differences in clinical care of HCC patients treated in western and eastern countries. These differences have been described in Asian and western guidelines and are partly explained by the genetic diversity of HCC [3e5]. HCC are heterogeneous at both the tumoral and non-tumoral level, and a major challenge lies in defining the relationship between aetiology, HCC development and molecular features [6,7]. HCC, like other cancers, is a disease of the genome and is defined by malignant hepatocytes accumulating somatic genetic alterations that combine mutations in both driver and passenger genes [8,9]. The recent technological breakthrough of next-generation sequencing has led to characterization of a whole genome and a whole exome of tumours within a few hours and at lower cost [10]. This again highlights the wide genetic diversity and the influence of aetiology, especially in HCC due to chronic HBV infection [11]. The aim of the present review was to describe the link between aetiology and the pathogenesis of HCC. Pathogenesis of HCC according to aetiology Hepatitis B Hepatitis B virus is a partially double-stranded circular DNA virus belonging to the hepadnavirus family [12]. Pre-S/S ORF encodes three surface proteins, pre-C/C ORF encodes a terminal protein and viral polymerase has a reverse transcriptase and a DNA polymerase function [11]. The X gene encodes the HBx protein that is mandatory for viral replication. Chronic infection by hepatitis B leads to HCC development, with an added risk that is 25- to 37-fold that of non-infected patients [13,14]. It is the leading aetiology in Asian and African countries. HCC related to HBV infection may develop on both cirrhotic liver and normal liver, whereas most HCC related to high alcohol intake or chronic HCV infection develop on cirrhosis [1]. Along this line, one of the main mechanisms of carcinogenesis due to HBV infection is through chronic inflammation, liver fibrosis and thus development of cirrhosis [11]. In this setting, chronic inflammation, oxidative stress and replicative senescence of hepatocytes due to telomere shortening induce malignant transformation of cirrhotic nodules, as in other cirrhotic backgrounds (Fig. 1) [7]. However, other
Fig. 1. The multistep process of liver carcinogenesis and the role of etiologies.
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carcinogenic mechanisms are specific to chronic HBV infection and could explain the occurrence of HCC against a non-cirrhotic background. First, HBV DNA could integrate the human genome and be clonally selected in tumours [15]. Interestingly, only a small part of the HBV genome integrates the human genome; consequently, integrated HBV is not used for viral replication [16]. Overall, at least 70e80% of HCC due to HBV infection show clonal integration of the viral genome [17,18]. HBV insertion may occur randomly throughout the human genome, but preferential sites near the fragile site, the repeat DNA sequence and the highly transcribed site have been suggested. Most of the time, the 30 end of HBx and the 50 end of the preCore/core genes are preferentially inserted [17]. One of the cardinal effects of viral integration is a cis effect wherein the virus is integrated [19]. Several recurrent sites of insertion in key genes in carcinogenesis have been described: TERT, MLL4, CCNE1, CCNA2 and RARB (retinoic acid receptor b) [16,17,20]. HBV integration sites are frequently promoter regions or intron regions and, less frequently, coding regions [17,19]. Frequently, HBV insertion in the human genome leads to increased expression of this gene, as has been demonstrated in TERT and CCNE1 [17]. In addition, several pieces of evidence suggest that HBV integration might induce, or at least be associated with, chromosomal instability [21,22]. Another mechanism of viral carcinogenesis through viral integration into the human genome is a trans mechanism, through production of the truncated protein like HBx or the pre S2/S protein, due to partial integration of the virus [11]. This is related to another mechanism of HBV carcinogenesis, namely, the direct oncogenic properties of the viral protein [23]. Production of the truncated HBx protein and the pre S2/S protein can modulate the signalling pathway and induce transactivation of several genes [11,23]. While the truncapted pre S2/S protein is believed to be an oncogenic protein, one of the most thoroughly studied and controversial oncogenic functions is related to the HBx regulatory protein. Physiologically, the HBx protein is involved in HBV replication and transcription and could stimulate the viral promoter and enhancer via it transactivation function [24]. This function has been suggested to be a key mechanism that promotes carcinogenesis in humans through activation of several signalling pathways, control of apoptosis and DNA repair [25,26]. Recent studies have shown an interaction with acetyltransferase CBP/p300 that leads to CREB-dependent transcription controlling liver metabolism and cell proliferation [27]. In addition, frequent truncated HBx proteins are produced, and this protein could increase metastasis or cell invasiveness in vitro models [28]. However, its oncogenic properties are subject to debate. Indeed, several experimental studies identified an oncogenic effect [29,30], whereas others showed no effect or even a paradoxical tumour suppressive effect of truncated HBx [31]. Mouse models have shown both an oncogenic function of truncated Hbx [32] and no effect upon carcinogenesis [33]. Moreover, HBx gene harboured a pattern of tumour suppressor gene in human hepatocellular carcinoma including stop mutations and frameshift deletion [34].In addition, several studies have pointed to a possible role for the HBx protein as a regulator of a mitotic spindle that could constitute a link between HBV infection and chromosomal instability [35,36]. Currently, most data suggest an oncogenic role for the HBx truncated protein, but mysteries remain. Hepatitis C Hepatitis C is a single-stranded RNA virus belonging to the flaviridae virus family [37]. Most of the cell cycle of the HCV virus occurs in the cytoplasm and, in contrast to HBV, HCV cannot integrate the host genome. Most HCC related to HCV occurs on cirrhosis; consequently, the pathogenesis of hepatocarcinogenesis is roughly the same as that of HCC developing on alcohol- or NASH-related cirrhosis [38]. HCV leads to chronic inflammation, immune-mediated hepatocyte death, tissue damage, fibrosis synthesis by hepatic stellate cell and replicative senescence due to telomere shortening [38,39]. Moreover, additional factors such as oxidative stress, steatosis and insulin resistance accelerate the evolution to cirrhosis and hepatocellular carcinoma [[40e42] Bartosch, 2009 #3021]. One of the key determinants of fibrosis progression and HCC occurrence against a hepatitis C background is insulin resistance and steatosis [43e45]. Most of the time, steatosis is due to concomitant diabetes and obesity, two well known risk factors in liver disease and HCC [46]. However, genotype 3 hepatitis C is directly associated per se with liver steatosis through modulation of lipid degradation, synthesis and secretion [47]. Taken together, the main mechanisms leading to HCC are indirectly caused by HCV. However, some authors suggested that HCV proteins have direct oncogenic properties [38]. They might affect
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signalling pathways like the Wnt/B-catenine, TGFB, NFKB or P53 pathway by modulating transcription or translation or by a post-translational mechanism [48,49]. In addition, several studies have shown that the HCV protein core or NS3, NS4B and NS5A could induce transformation of fibroblasts in vitro [50]. Interestingly, HCV core protein expression in a transgenic mouse model leads to HCC development [51]. However, most studies dealing with direct oncogenic properties of the HCV protein used inadequate models that precluded drawing firm conclusions [38]. Consequently, the oncogenic function of the HCV protein in human disease remains to be demonstrated. Alcoholic liver disease According to the World Health Organization, alcohol is a carcinogen that predisposes to several cancers, ranging from esophageal cancer to breast cancer, often in conjunction with other carcinogens like tobacco [52]. Clearly, alcohol consumption is associated with increased risk of HCC development [53]. However, most of the time, HCC develops on alcoholic cirrhosis [54]. The role of alcohol in HCC developing on non-cirrhotic liver is unknown, although a French study suggested a link with high alcohol consumption [55]. In addition, alcohol is a co-factor in liver disease and HCC in patients with chronic hepatitis B, chronic hepatitis C, haemochromatosis and NASH [53,54]. Paradoxically, cirrhotic patients who stop drinking have a higher risk of HCC development than patients with persistent consumption [56]. This might be explained by decreased death from liver failure and longer exposure to HCC risk in abstinent patients. Taken together, the mechanisms inducing liver injury and cirrhosis and the mechanisms of liver carcinogenesis in patients who abuse alcohol are difficult to separate [52]. Ethanol has no direct carcinogenic properties when given to mice and rats [57]. Under certain conditions, often in conjunction with chemical carcinogens, an increased incidence of tumours in rodents exposed to ethanol has been demonstrated [58]. Acetaldehyde is an ethanol metabolite that has carcinogenic properties through DNA binding [59]. Production of acetaldehyde is dependent on alcohol dehydrogenase, an enzyme with polymorphisms regulating its activity [52]. However, the role of these polymorphisms in liver carcinogenesis is still unclear. In addition, CYP2E1, a cytochrome induced by ethanol consumption, converts ethanol into acetaldehyde, but also into reactive oxygen species (like superoxide anion and hydrogen peroxide) [60]. Chronic oxidative stress induced by alcohol intake and cytokine production in the context of chronic inflammation is a well known phenomenon leading to cirrhosis and HCC development [52,54]. Accumulation of reactive species of oxygen favours lipid peroxidation and creates DNA adducts [61]. Polymorphism of superoxide dismutase and MnSoD has been associated with HCC occurrence in alcohol-related cirrhosis, but not in HCV-related cirrhosis [62]. Moreover, iron overload induced by chronic alcohol intake participates in liver injury through oxidative stress. A prospective study has shown that iron overload is associated with high risk of HCC development in alcohol-related cirrhosis [63]. Finally, additional mechanisms of alcohol-mediated carcinogenesis exist, including downregulation of levels of retinoic acid [64] and modulation of methylation by downregulation of S-adenosyl L methionine (SAMe) [65]. Non-alcoholic fatty liver disease (NAFLD) A large body of evidence supports the role of diabetes and obesity in HCC pathogenesis. In a large epidemiologic study, patients with BMI >35 had an increased risk of cancer, especially hepatocellular carcinoma, with a hazards ratio (HR) of 4.52 [66]. In addition, diabetes is associated with cirrhosis and HCC occurrence, with a HR of 2.39 in a US cohort [67]. Interestingly, diabetes was also a risk factor for HCC occurrence in a prospective cohort of HCV-related and alcohol cirrhotic patients [68]. While most HCC related to NASH develops on cirrhosis, several groups have reported the occurrence of HCC in NASH without underlying cirrhosis [69,70]. This suggests that specific carcinogenic mechanisms related to NASH could lead to HCC development outside the classical background of cirrhosis [71]. However, these specific carcinogenic mechanisms are still unknown, and at present, the pathogenesis of NASH is difficult to distinguish from the carcinogenic mechanism of HCC development on NASH. Obesity is characterized by chronic low-grade inflammation that predisposes to liver injury and HCC occurrence [71,72]. An elegant study by the team of Mickael Karin showed that obesity enhanced HCC
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formation induced by chemical carcinogens [73]. This phenomena was cytokine-dependent, especially IL6 and TNF-alpha production. In addition, studies have pinpointed the role of microbiota and inflammation in tumour promotion [74,75]. These models highlighted the pivotal role of inflammation in HCC initiation due to obesity. This inflammatory state is a conjunction of the action of hepatocytes, Kupffer cells and adipocytes. Moreover, an abnormal balance between leptin and adipokine characterizes NAFLD and predisposes to HCC development. A decreased level of adiponectin, an antinflammatory, antiproliferative cytokine exclusively produced by adipocytes, and a parallel increase in leptin, a pro-inflammatory, angiogenic and profibrogenic cytokine predominantly produced by adipocytes, are cardinal features of NAFLD [72,76,77]. This underlines the important role of visceral adiposity in liver disease. However, their exact role in tumour initiation and progression remains to be refined. In a mouse model of NASH, hypoadiponectinemia was associated with increased tumour occurrence, thus supporting a role in carcinogenesis [76]. In addition, accumulation of lipid droplets in hepatocytes leads to lipotoxicity that contributes to NASH and consequently to HCC pathogenesis [78]. Moreover, this phenomenon increases insulin resistance, that leads to hyperinsulinemia and production of IGF1 by the liver. IGF1 induces phosphorylation of IRS1, leading to activation of the PI3K/AKT/MTOR and MAPK kinase pathways, and consequently promotes cell proliferation and inhibits apoptosis [79]. In parallel, decreased production of IGFBP1 and IGFBP2 by the liver increases IGF1 bioavailability [[80] Siddique, 2011 #3218]. Consequently, the IGF/IRS1 axis is a key signalling pathway involved in NAFLD pathogenesis and related carcinogenesis. In conclusion, NAFLD pathogenesis and tumorogenesis are related to various factors, including chronic inflammation, oxidative stress, insulin resistance, lipotoxicity and an abnormal relationship with visceral adiposity. However, the exact role and time frame of each of these factors in tumour initiation in NAFLD are still unclear. Rare diseases Rare diseases, including primary biliary cirrhosis, autoimmune hepatitis, haemochromatosis and Wilson's disease, can lead to HCC development. In most cases, HCC occurs on cirrhosis, and the exact carcinogenic mechanisms linked to specific etiologies have not yet been identified. In addition, few experimental or clinical studies have focused on carcinogenesis in these rare diseases. However, clinical evidence concerning haemochromatosis provides additional insight into the role of iron in liver carcinogenesis. Iron deposits in the liver favour hepatic injury, occurrence of cirrhosis and tumour initiation [81]. However, the identification of iron-free dysplastic nodules and HCC suggests a two-hit hypothesis, with iron overload leading to cirrhosis and tumour initiation and, in a second step, to tumour promotion, with proliferation of iron-free malignant hepatocytes to avoid the cytotoxic effect of iron [82]. Genetic alterations in HCC and the influence of aetiology Next-generation sequencing has revealed that HCC harbours a mean of 40e50 damaging somatic mutations in coding sequences, including mutations both in driver and in passenger genes [83e86]. This can be compared with other types of tumours, including paediatric cancers (a mean of three mutations in rhabdoid tumours), melanoma and lung cancer (a mean of 110 and 120 mutations, respectively) [8]. In various next-generation studies, no differences in the mean number of genetic alterations between etiologies have been identified, although the number of studies is limited [83e88]. Among such mutations, several driver genes are recurrently mutated in HCC. The oldest somatic genetic alterations identified in HCC are activating mutations of b-catenin (coded by CTNNB1) and inactivating mutations of TP53, in 20e40% and 20e50% of cases, respectively [89,90]. CTNNB1 mutations appear to be less frequent in HCC developing on HBV cirrhosis [83e85]. In contrast, there is a strong overrepresentation of TP53 mutations in HBV-related HCC [91]. This is the strongest example of a relationship between aetiology, exposure to carcinogens and specific mutations of the tumour genome [92]. Aflatoxin B1 is a mycotoxin that contaminates dietary products in some regions of Asia and Africa [93]. It is associated with HCC development in conjunction with HBV infection. HCC related
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to aflatoxin B1 exposure harbours a highly specific mutation in codon 249 (R249S) of TP53 [90]. Consequently, the R249S mutation of TP53 is a surrogate marker of aflatoxin B1 exposure in patients with HCC (Fig. 1). More recently, a whole genome study identified, in some HCC, a specific mutational signature (G to A substitution) related to chronic intake of aristolochic acid, a plant also associated with urothelial cancer harbouring the same mutational signature [94]. Taken together, there exists a molecular imprint of exposure to carcinogens that could help to identify a new mechanism of carcinogenesis and new carcinogens. Whole exome sequencing of HCC has also identified a genotoxic signature overrepresented in HCC developing on normal liver [83]. At this time, the link between a carcinogen and this genotoxic signature on normal liver remains unknown. In the future, study of mutational signatures using next-generation sequencing could help to identify new carcinogens and assess the level of exposure to known carcinogens [95]. Differences in recently published next-generation studies could also be explained by the different etiologies of HCC analysed by whole exome or whole genome sequencing. While telomere shortening due to the absence of telomerase activity favours liver injury and tumour initiation through hepatocyte replicative senescence, in contrast, telomerase reactivation is required for tumour promotion so as to allow uncontrolled cancer cell proliferation [96]. TERT promoter mutations are the most frequent genetic alterations in HCC, with an overall frequency of 59% [97]. This leads to an increase in TERT promoter activity and explains a substantial part of telomerase overexpression in HCC. Strikingly, TERT promoter mutations are less frequent in HBV-related tumours [97,98]. This could be explained by the presence of HBV insertion in the TERT promoter, a well known mechanism leading to telomerase reactivation. New, recurrent genetic alterations have been identified using whole exome and whole genome sequencing [83e86]. ARID1A and ARID2 are recurrently mutated in 15% and 10% of cases, respectively [83]. ARID1A and ARID2 belong to the SWI/SNF complex, implicated in chromatin remodelling and transcription control. These mutations lead to inactivation of ARID1A and ARID2, a classical feature of the tumour suppressor gene. In one series, the ARID1A mutation was significantly associated with high alcohol intake, although this association is not supported by a physiopathological explanation [83]. Recurrent mutations in KEAP1 and NFE2L2 lead to the constitutive activation of the stress oxidative pathway [83,88]. Moreover, the ras/raf/map kinase pathway is a key signalling pathway, with rare activating mutations in PIK3CA (1%), RAS (1%) and, more frequently, mutations in RPS6KA3 (8%). Amplification of FGF19 has been described in up to 15% of HCC [99]. One study reported the association of 6p21.1 amplification with HCC developing on NASH. CULLIN7 has been underlined as a potential gene of interest in this region of the genome [100]. Molecular classification of HCC according to aetiology Several molecular classifications have thus far been reported in HCC [101]. The two main goals of these molecular classifications are to describe genetic heterogeneity and to identify prognostic biomarkers relevant to clinical care. We previously described a molecular classification based on transcriptome analysis in six subgroups (G1eG6) [102]. This molecular classification was closely related to genetic defects, signalling pathways and clinical features. HCC classified into G4eG6 subgroups have chromosomal stability; in contrast, HCC classified into the G1eG3 subgroups have chromosomal instability. HCC of the G5eG6 subgroups were strongly associated with activating mutations of CTNNB1 mutations. HCC classified into G3 subgroups have poor prognosis and harbour disregulation in the cell cycle gene. HCC of the G1eG2 subgroups are strongly related to chronic HBV infection, and the G1 subgroup to high levels of AFP. Taken together, these molecular classifications confirmed that HBV infection is an aetiology that harbours a strong specific molecular signature. Other molecular classifications have underlined the relationship between HBV and specific genetic defects. Work by the team of Snorri Thorgeisson has shown that HBV-related HCC are enriched in TP53 mutations related to a stem cell phenotype [91,103]. TP53-mutated tumours in this series of Asian patients with chronic HBV infection also carry a poor prognosis [91]. We recently confirmed the prognostic role of TP53 in HBVrelated HCC. Strikingly, in a series of HCC non-related to HBV infection, TP53 mutations had no significant impact on mortality [34]. This is an example of the relationship between aetiology, genetic defects and the impact on prognosis assessment. It underlined that genetic defects could have different
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clinical significance depending on the underlying aetiology. Another feature of HBV-related HCC is the higher proportion of stem cell phenotypes in Asian series of patients compared to western patients [91,101,104]. One explanation might be that of differing carcinogenesis related to HBV infection that promotes aggressive tumours of the stem cell phenotype, mutated for TP53. However, common molecular features linked to cancer aggressiveness among HCC of different etiologies have also been identified. A five-gene score derived from tumour analysis was able to predict tumour recurrence and survival after liver resection in 748 HCC of various etiologies, including high alcohol intake, HCV and HBV infection [105]. While molecular features may differ according to aetiology, nonetheless, common molecular mechanisms exist in liver carcinogenesis. Conclusion The various etiologies associated with liver disease and promoting of HCC complexify our understanding of the pathogenesis of HCC. This wide diversity reflects diverse exposure to risk factors, from viral infection to environmental factors like aflatoxin B1 and alcohol [106]. In addition, a conjunction of risk factors is frequent in clinical practice, and the specific impact of each factor in hepatocarcinogenesis is difficult to evaluate. The most clear-cut direct carcinogenic role is that of chronic hepatitis B infection, that can lead to HCC outside of the background of cirrhosis [11]. It includes well known viral integration into the tumour genome, but also the potential oncogenic role of viral protein. In contrast, the direct carcinogenic role of other risk factors like chronic hepatitis C, alcohol and metabolic syndrome is difficult to separate from mechanisms of liver injury leading to cirrhosis formation (Fig. 1). Overall, the conjunction of chronic inflammation, oxidative stress and telomere shortening favours liver injury and tumour initiation in various liver diseases [107]. The different steps of carcinogenesis against a cirrhotic background and the potential relationship with etiologies are still unclear and need further investigation. Finally, a small fraction of HCC develop on normal liver. Although some of these HCC could be due to malignant transformation of hepatocellular adenoma through accumulation of CTNNB1 and thus TERT promoter mutations, most cases are not related to any risk factors [108e110]. In the future, epidemiological and molecular studies should help to identify potential carcinogens and risk factors associated with HCC occurrence in normal liver, that is, a pure model of liver carcinogenesis without a cirrhotic background.
Practice points (1) Hepatitis B virus has a direct oncogenic effect due to viral insertional mutagenesis and the role of HBx protein. (2) Aflatoxin B1 is a direct carcinogen and induces specific point mutations in TP53 (R249S). (3) Some studies have reported the occurrence of HCC due to metabolic syndrome on noncirrhotic liver; however a specific mechanism has not been described so far. (4) Oxidative stress, chronic inflammation, telomere shortening and insulin resistance are common mechanisms that lead to chronic liver injury and HCC occurrence on cirrhosis.
Research agenda (1) The association between the different etiologies and the somatic genetic alterations of hepatocellular carcinoma should be refined. (2) The role of metabolic syndrome on liver carcinogenesis, specially outside the cirrhotic background, should be clarified. (3) The risk factors leading to HCC occurrence on normal liver needs to be identified.
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Conflicts of interest None. Acknowledgements This work was supported by HECAM (BPI), EBCI, INCa (WntHCC) project. J-C.N. was supported by a fellowship from INCa. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]
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