Chronic viral hepatitis and its association with liver cancer

Thomas Tu 1 , Sandra Bühler 1 ,  and Ralf Bartenschlager 1 , 2
  • 1 Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany
  • 2 Division of Virus-Associated Carcinogenesis, Germany Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany
Thomas Tu
  • Corresponding author
  • Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany
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, Sandra Bühler
  • Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany
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and Ralf Bartenschlager
  • Corresponding author
  • Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany
  • Division of Virus-Associated Carcinogenesis, Germany Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany
  • Email
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Abstract

Chronic infection with hepatitis viruses represents the major causative factor for end-stage liver diseases, including liver cirrhosis and primary liver cancer (hepatocellular carcinoma, HCC). In this review, we highlight the current understanding of the molecular mechanisms that drive the hepatocarcinogenesis associated with chronic hepatitis virus infections. While chronic inflammation (associated with a persistent, but impaired anti-viral immune response) plays a major role in HCC initiation and progression, hepatitis viruses can also directly drive liver cancer. The mechanisms by which hepatitis viruses induce HCC include: hepatitis B virus DNA integration into the host cell genome; metabolic reprogramming by virus infection; induction of the cellular stress response pathway by viral gene products; and interference with tumour suppressors. Finally, we summarise the limitations of hepatitis virus-associated HCC model systems and the development of new techniques to circumvent these shortcomings.

Introduction

Hepatitis viruses are amongst the most widespread pathogens of humans. These viruses are united only by their distinct hepatotropism. Apart from this, each virus belongs to separate families and differ profoundly in their biological features. Currently, five viruses are classified as hepatitis viruses: hepatitis A virus (HAV) and hepatitis E virus (HEV) cause almost exclusively acute self-limiting infections, while hepatitis B virus (HBV) and hepatitis C virus (HCV) frequently establishing persistent infections. The fifth virus, hepatitis D virus (HDV), is a satellite virus that depends on the HBV viral envelope proteins to produce new virions. Although acute infections with these viruses can be severe, and occasionally fulminant, chronic infection by HBV, HCV and HDV are responsible for the majority of the disease burden associated with hepatitis virus infections. According to the World Health Organisation (WHO), about 240 million people suffer from chronic HBV infection, ~15–20 million of them from HBV-HDV co-infection, and ~130 million people from chronic HCV infection. Chronically infected people have a high risk of serious liver disease, including liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Liver disease risk is enhanced by clinical co-factors, most notably a high body-mass index, alcohol consumption, or exposure to DNA-damaging agents (e.g. aflatoxins).

Current approaches to treat infection with these viruses remain suboptimal. Both HBV and HDV infections can be prevented by vaccination composed of recombinant hepatitis B surface antigen (HBsAg), which profoundly reduces the incidence of HCC (Kao, 2015). However, therapeutic options against established infections are limited; while anti-viral therapy against chronic HBV infection suppresses viral replication, the majority of patients relapse after cessation of treatment (Zoulim and Durantel, 2015). The situation for HCV is the inverse to that of HBV. While no prophylactic vaccine currently exists for HCV infection, interferon (IFN)-free anti-viral therapies lead to virus elimination in >95% of treated patients, witnessing a historical success (Pawlotsky et al., 2015). However, costs for these medications are extremely high and thus unaffordable for the majority of infected people, especially in countries with a high prevalence of HCV. Moreover, owing to the frequently asymptomatic course of chronic hepatitis virus infection, most infected people only enter the clinic after significant liver damage has occurred.

Therefore, the burden of severe liver disease and HCC will continue to increase until at least 2020 (Morgan et al., 2013; Thomas, 2013; Harris et al., 2014). It is becoming increasingly clear that the risk of HCC development remains after virus elimination, especially for patients who had advanced liver cirrhosis at the time of treatment (van der Meer et al., 2012; Pollicino and Saitta, 2014; El-Serag et al., 2016). However, it is unknown at which stage during liver disease progression this ‘point of no return’ is. Interestingly, three recent studies suggest that the risk for HCC is even increased when HCV has been eliminated by direct acting antiviral (DAA) treatment (Conti et al., 2016; Kozbial et al., 2016; Reig et al., 2016). Although the interpretation of this observation still controversial (Toyoda et al., 2016; Pol et al., 2017), these results clearly show that viral-associated HCC remains an unresolved and significant public health issue. Importantly, the underlying molecular mechanisms that drive hepatitis virus-associated HCC are not completely understood, making risk-assessment, prevention, and treatment difficult. In this review, we summarise what is currently known about these pro-oncogenic mechanisms and highlight tools required for future studies in the field.

Drivers of HCC common across hepatitis viruses

Chronic infections with hepatitis viruses cause immune-mediated cell death and subsequent inflammation. Chronic inflammation is a common feature in the majority of HCC cases, regardless of aetiology. With any given chronic injury to the liver, there is a generalised response consisting of inflammation, regeneration and formation of fibrotic septa, all of which promote carcinogenesis. We highlight here four main responses and their role in HCC formation (Figure 1): (1) oxidative stress; (2) accumulation of genomic alterations/instability; (3) activation of regeneration pathways, hepatocyte turnover and subsequent clonal expansion; and (4) formation of a fibrotic/cirrhotic microenvironment. Many of these features act in concert and can generate feed-forward loops (Tu et al., 2014). For example, oxidative stress generates liver damage and stimulates fibrosis pathways. Fibrosis and cirrhosis, in turn, cause altered intrahepatic blood flow, which increases hypoxia and oxidative stress in hepatocytes.

Figure 1:
Figure 1:

The components of chronic inflammation that drive hepatocellular carcinoma initiation and progression.

Factors of chronic inflammation (blue boxes) not only contribute to HCC, but also can drive other factors to perpetuate chronic inflammation. Hepatitis viruses can feed into this cycle at different stages (red) to prolong inflammatory responses and initiate HCC.

Citation: Biological Chemistry 398, 8; 10.1515/hsz-2017-0118

Oxidative stress

Oxidative stress is defined by an imbalance between the production of oxidative free radicals and the anti-oxidative responses within the hepatocyte. Chronic inflammation leads to build-up of reactive oxygen species (ROS) via multiple mechanisms, including the activation of infiltrating immune cells and the induction of nitric oxide synthase expression by activated endothelial cells (Kawanishi and Hiraku, 2006; Farinati et al., 2010). Chronic exposure to excessive ROS can alter cellular metabolism by damaging proteins, lipids and nucleic acids, thus causing cellular dysfunction, including a perturbed energy metabolism reviewed recently (Nogueira and Hay, 2013; Choi et al., 2014; Higgs et al., 2014; Tariq et al., 2014). Furthermore, stress-induced MAP kinases such as JNK are upregulated by oxidative stress and contribute to the development of metabolic syndrome-related disorders such as NASH, which in turn promote HCC (Nakagawa and Maeda, 2012). Finally, the build-up of ROS directly contributes to increases in DNA adducts (particularly at G bases) in host cells, increasing the probability of G:C to T:A transversions (Yasui et al., 2014) and thus, oncogenic genetic lesions (Barash et al., 2010).

Accumulation of genomic alterations/instability

HCC is accompanied, as with many other cancers, with multiple genetic alterations. The most common pathways that are mutated in HCC include the Wnt/β-catenin-, Hedgehog-, p53-, and JAK/STAT-dependent pathways (Feo et al., 2009). HCC represents a somewhat heterogeneous cancer, with at least six different subtypes and mutational profiles being described (Boyault et al., 2007; Alexandrov et al., 2013; Fujimoto et al., 2016). Indeed, more than 11 different molecular pathways have been found to be altered in ≥5% of HCC (Schulze et al., 2015), suggesting that there are multiple routes to HCC initiation and progression. For the sake of this review, we will focus mainly on those of hepatitis virus-associated HCC.

HBV- and HCV-associated HCC usually presents with mutations and altered transcription in various molecular pathways, causing dysregulation in mitotic cell cycle-, AKT activation-, AXIN1-, developmental-, and DNA imprinting-pathways (Boyault et al., 2007). HBV-associated HCC in particular tends to be associated with more DNA rearrangements (Guichard et al., 2012; Jiang et al., 2012b) and copy number variations (Sung et al., 2012; Jiang et al., 2012b) compared to HCCs of other aetiologies. HCV-associated HCC on the other hand appears to have similar mutational profiles to other inflammation-associated HCCs (Zucman-Rossi et al., 2015), suggesting that different pathways lead to HCC in the two different aetiologies.

A known early molecular change in HCC are mutations in the promoter of the human telomerase reverse transcriptase (hTERT) gene. Increased hTERT transcription as a result of these mutations become cumulatively more common as cells progress from early-stage preneoplastic lesions to HCC (Nault et al., 2013). Cell clones with these newly acquired hTERT mutations can then overcome senescence, undergo unlimited proliferation and thereby gather more pro-oncogenic mutations.

Hepatocyte turnover, clonal expansion and liver regeneration

For mild to moderate levels of cell death (<30% of the liver), hepatocytes killed by immune-mediated cell death are replaced by compensatory mitosis of surrounding mature hepatocytes (Malato et al., 2011). Chronic inflammation ensures constant hepatocyte turnover, which can drive HCC initiation and progression through clonal expansion.

Constant liver turnover amplifies the number of cells with premalignant mutations by allowing replicative space for clonal expansion to occur. Hepatocytes with a growth or survival advantage (e.g. premalignant changes) preferentially replace lost cells, thereby increasing the number of cells with such mutations. Indeed, clonal expansion of cellular sub-populations is a known risk factor for many cancers, including HCC (Cameron, 1989; Bralet et al., 1996; Chen et al., 2005; Marongiu et al., 2008; Merlo et al., 2010) in both rat models (Cameron, 1989; Bralet et al., 1996) and human genetic diseases that predispose for HCC (Marongiu et al., 2008). Further to this point, larger clone sizes are detected in patients with HBV-associated HCC compared to HBV carriers (Tu et al., 2015b; Mason et al., 2016).

Fibrosis and cirrhosis

Chronic liver inflammation stimulates the development of progressive fibrosis and cirrhosis (Giannelli et al., 2003). The activation of stellate cells by hepatocyte cell death and cytokines released by infiltrating immune response causes intrahepatic fibrous septa to form in order to preserve liver structure and function. With chronic inflammation, these septa build up and blood flow is disrupted in liver nodules, impairing nutrition and oxygen distribution to hepatocytes, leading to a subsequent cascade of cellular responses that contribute to HCC (Tu et al., 2014). Cirrhosis is a strong driver of HCC and is present in 80–90% of patients with HCC of all aetiologies (Fattovich et al., 2004).

The cirrhotic liver microenvironment can drive oncogenic pathways via multiple mechanisms. Firstly, the altered blood flow associated with cirrhosis impairs the immune response and induces a more tolerogenic phenotype in infiltrating and liver-resident immune cells (Murdoch and Lewis, 2005). This not only inhibits clearance of chronic hepatitis virus infections thereby prolonging intrahepatic inflammation, but also potentially allows pre-neoplastic hepatocytes to escape immune surveillance (Wherry, 2011). Furthermore, cirrhosis is associated with altered hepatic stromal cells that provide pro-oncogenic signals to surrounding hepatocytes, including TGF-β (van Zijl et al., 2009).

Cirrhosis also feeds into the HCC-driving mechanisms described earlier. Areas within the liver surrounded by cirrhotic tissue can undergo constant turnover due to hypoxic conditions. These areas are termed regenerative nodules and are associated with the expansion of premalignant hepatocytes (Su and Bannasch, 2003; Gong et al., 2010). Also, the hypoxic response in hepatocytes subjected to altered blood flow in the cirrhotic liver induces the production of ROS and feeds into oxidative stress pathways.

In the context of viral hepatitis-associated HCC, both HBV and HCV-specific pro-oncogenic mechanisms ultimately feed into these generalised inflammation-associated pathways. However, it is clear that chronic inflammation is not the only factor that drives HCC formation. Cirrhosis is not necessarily present in HCC patients with chronic HCV and (in particular) HBV infections. For example, cirrhosis is absent in up to 35% of patients with HBV-associated HCC (Kew, 1989). This suggests that the viruses can directly contribute to hepatocarcinogenesis, the mechanisms of which will be the topic of the rest of this review (Tables 1 and 2 ).

Table 1:

Overview of HBV factors and their role in the viral replication cycle as well as pathogenicity.

Viral factorRole in replication cyclePossible role in pathogenicity
HBsAgEnvelope proteinER stress (Pre S mutants)
HBxTranscriptional transactivatorTransactivation of HCC-associated genes

Cis-activation of HCC-associated genes (integrated form)
HBeAgImmune modulationNone reported
HBcAgNucleocapsidNone reported
PolViral polymeraseNone reported
Splice proteinUnknownActivation of stress response
Table 2:

Overview of HCV factors and their role in the viral replication cycle as well as pathogenicity.

Viral factorRole in replication cyclePossible role in pathogenicity
RNA genomeTemplate for polyprotein synthesis and RNA replication; component of virus particlesSequestration of miR-122
CoreNucleocapsidInsulin resistance/steatosis/oxidative stress

Interference with tumour suppressors (p53, p73, pRb)

Interference with cellular signalling pathways (e.g. NF-κB; TGF-β)

Transcriptional activation of cellular genes
NS2Protease/assemblyModulation of apoptosis
NS3Protease/ helicaseInterference with innate immune response by proteolytic cleavage of MAVS

Interference with NF-κB

Interference with tumour suppressor p53
NS4BComponent of viral replicaseInduction of ER stress
NS5ARNA replication; virus assemblyInterference with protein ubiquitination

Inhibition of PKR activity

Induction of oxidative stress

Modulation of transcription of cellular genes

Accumulation of β-catenin by indirect mechanisms

Activation of signalling pathways (e.g. STAT-3, NF-κB)

Activation of phosphatidyl inositol-4-kinase
NS5BRNA-dependent RNA polymeraseSequestration of tumour suppressor pRb

Hepatitis B virus

Virus structure and replication

HBV is the prototypic member of the Hepadnaviridae family, composed of blood-borne, enveloped viruses that contain ~3.2 kb relaxed circular dsDNA genomes encapsidated within virally-encoded capsids. The HBV genome has four overlapping reading frames which code for seven proteins. These include: HBV surface antigen (HBsAg), of which there are three forms (large, medium and small); the HBV core antigen (HBcAg), which encodes the capsid protein; HBV e antigen (HBeAg), an immunomodulator and secreted form of HBcAg; the HBV polymerase (pol), essential for HBV replication; and the transcriptional transactivator HBV x protein (HBx).

HBV replication (Figure 2) occurs solely within hepatocytes, the main cell type of the liver. Following initial attachment via low-specificity interactions between HBsAg and heparin sulfate proteoglycans on the hepatocyte surface (Schulze et al., 2007), receptor-mediated entry via the cellular receptor sodium-taurocholate cotransporting polypeptide (NTCP, a hepatocyte-specific bile acid transporter) allows HBV to pass into the cytoplasm (Yan et al., 2012; Ni et al., 2014). The nucleocapsid containing the relaxed circular DNA (rcDNA) genome is then transported to the nucleus. There the rcDNA genome is converted (using host cell DNA repair proteins) into covalently closed circular DNA (cccDNA), the stable episomal transcriptional template for HBV mRNAs. Pregenomic (pg)RNA is also transcribed from cccDNA and packaged along with the viral polymerase into capsids composed of HBcAg dimers. Reverse transcription of the pgRNA occurs within the nucleocapsid resulting in rcDNA genomes in ~90% of nucleocapsids. The rcDNA-containing nucleocapsids either cycle back to the nucleus to increase the intranuclear cccDNA pool or or acquire the envelope by budding into the endoplasmic reticulum (ER). A fraction of virions is released in an immature non-infectious state, but gains infectivity via a maturation folding to the large viral envelope glycoprotein (Seitz et al., 2016). In ~10% of nucleocapsids, double stranded linear (dsl)DNA is formed as a product of reverse transcription (Figure 2). Like rcDNA containing nucleocapsids, these can cycle to the nucleus to form cccDNA or be released as virions (Yang and Summers, 1998). An additional possibility for intra-nuclear dslDNA genomes is its integration into the host cell genome by non-homologous end joining (Bill and Summers, 2004). Depending on the insertion site, this integration can contribute to HCC development (see below).

Figure 2:
Figure 2:

Replication cycles of HBV, HCV and HDV, and virus-induced effects likely contributing to HCC formation.

The HBV replication cycle (green) is schematically shown on the left. After receptor-mediated entry (via sodium-taurocholate cotransporting polypeptide, NTCP), the HBV nucleocapsid is transferred from the cytoplasm to the nucleus, where its relaxed circular (rc)DNA genome is converted into covalent closed circular (ccc)DNA. HBV RNA transcripts (vRNAs) are transcribed from the cccDNA and translated at the ER. Pregenomic (pg)RNA is also transcribed and then encapsidated by HBV core protein. Reverse transcription occurs within the viral nucleocapsid, producing either rcDNA (in ~90% of nucleocapsids) or double-stranded linear (dsl)DNA. Both rcDNA-and dslDNA-containing-mature nucleocapsids can be converted into additional cccDNA copies or be secreted as virions. Intra-nuclear dslDNA can integrate into the host-cell genome via non-homologous end joining. The HDV replication cycle (blue) is schematically shown on the top. HDV, like HBV, enters the cell via NTCP-mediated entry into the cytoplasm, where the single-stranded, negative-sense genome complexed with HDV antigen (HDAg, blue circles) enters the nucleus. The intranuclear genomic HDV RNA then undergoes rolling circle amplification through a positive-strand antigenomic RNA intermediate. HDV mRNA is transcribed from the amplified genomic RNA and used for the synthesis of HDAg, which is complexed with genomic HDV RNA. These ribonuclear protein complexes are enveloped at the ER with membranes including the HBV surface proteins and are then secreted as new HDV virions. The HCV replication cycle (purple) is schematically shown on the right. Upon HCV entry, the viral RNA is translated at the rough ER, giving rise to the polyprotein that is cleaved into 10 different proteins that all remain membrane bound. Viral RNA is amplified at membranes in close proximity of lipid droplets where infectious virus particles are assembled. These are released via the secretory pathway. The replication steps at which each virus can directly contribute to HCC initiation and progression are highlighted with red squares with the associated driving factor shown in the key legend (upper right).

Citation: Biological Chemistry 398, 8; 10.1515/hsz-2017-0118

Natural history of chronic HBV infection

The natural history of chronic HBV infection is categorised into five phases (EASL, 2012) (Figure 3). Chronic infection begins in the immune tolerance phase, characterised by high HBV serum titres and poorly-activated HBV-specific CD8+ T cells (Wang et al., 2010a; Kennedy et al., 2012). When an anti-viral immune response becomes increasing stimulated (through mechanisms not fully understood), patients enter the immune reactive HBeAg-positive phase, when fluctuations of immune-mediated liver damage and HBV titres occur (Wang et al., 2010a). As a result, many (but not all) HBV-infected hepatocytes are eliminated, viral titres are lowered and patients seroconvert to an anti-HBeAg state, which characterises the inactive HBV carrier phase. In the HBeAg-negative chronic hepatitis phase, low level viral replication can produce mutants that escape the immune response, generating flares of viral titres that are met by reactive flares of inflammation. This can drive further liver disease progression and is associated with increased risk of HCC. Occasionally (at a rate of ~1% of patients a year) patients can progress to the HBsAg-negative phase, in which HBV titres are at undetectable levels and antibodies to the HBV surface antigen are detected. The HBsAg-negative phase is classed as a cured state where liver disease progression ceases and reversal of fibrosis state can occur.

Figure 3:
Figure 3:

The natural serological history of chronic HBV and HCV infections.

The clinical course of a chronic HBV infection is shown in the top panel. Relative levels of serum HBV DNA titres (green), serum alanine aminotransferase (ALT) levels and HCC risk (red) are shown for each of the 5 phases of chronic HBV infection (maroon, described in greater detail in the text). Patients progress from HBsAg- and HBeAg-seropositive (light green and light blue, respectively) to anti-HBs- and anti-HBe-seropositive (dark green and dark blue, respectively) at the beginning of the inactive HBV carrier and HBsAg-negative phases, respectively. During flares (dashed lines), serum ALT and HBV DNA titres fluctuate in an inverse relationship as immune-mediated cell death of HBV-producing hepatocytes occurs. The clinical course of a chronic HCV infection is shown in the bottom panel. During the acute phase, ALT levels might be elevated, but most infections are asymptomatic. In any case, persistent HCV infection increases the risk for HCC development.

Citation: Biological Chemistry 398, 8; 10.1515/hsz-2017-0118

The role of HBV infection in hepatocarcinogenesis

Throughout chronic HBV infection, HCC risk generally coincides with the inflammatory phases (Figure 3). Several key clinical characteristics that affect intrahepatic inflammation have been shown to be predictive factors of HBV-associated HCC, including: level of immune-mediated liver damage (measured by serum ALT levels); concomitant alcohol consumption or exposure to aflatoxin B1; and high viral titres after the immune reactive phase (>2000 HBV IU/mL) (Chen et al., 2006; Lai et al., 2007; Tsang et al., 2008; Yang et al., 2008a, 2010). Indeed the anti-HBV immune response plays a critical role in HCC in HBV in vivo models. This is exemplified by a study by Chisari and colleagues showing that transgenic mice constitutively expressing HBV in the liver do not spontaneously develop HCC, but instead require adoptively transferred anti-HBV cytotoxic T-lymphocytes for hepatocarcinogenesis (Nakamoto et al., 1998). Some virological aspects also appear to play a role in HCC risk. HBV genotype (HBV genotype C has a higher risk) and the presence of HBV mutations, in particular basal core promoter and PreS mutations, are predictive risk factors for HCC development.

Chronic infection with HBV has been reported to feed into HCC-associated pathways via several molecular mechanisms (Figures 1 and 2) including HBV DNA integration, induction of cellular stress, transactivation of HCC pathways, and supporting HDV replication.

HBV DNA integration

One of the unique ways that HBV is reported to drive HCC is through the integration of virus DNA into the host cell genome (the mechanisms and clinical implications of which are reviewed in Tu et al., 2017), leading to insertional mutagenesis, chromosomal instability, and cis-activation of tumour associated genes.

In studies of chronic human HBV infection, no cellular gene has been observed to be consistently disrupted by HBV DNA integration (Paterlini-Brechot et al., 2003; Brechot, 2004; Huang et al., 2012; Jiang et al., 2012a,b; Sung et al., 2012), contrasting with studies of woodchuck hepatitis virus (WHV)-infected woodchucks where the N-Myc gene is a common target for WHV DNA integration (Jacob et al., 2004). While HBV DNA integration into oncogenes [such as the mixed-lineage leukaemia 4 (MLL4) and hTERT genes] have been repeatedly observed in HBV-associated HCCs (Paterlini-Brechot et al., 2003; Brechot, 2004; Sung et al., 2012), these only occur in a minority HCCs and usually in later stages of tumour progression.

In HBV infection, integration of HBV dslDNA leading to destabilisation and mass rearrangement of the host-cell genome has been studied in detail (Bonilla Guerrero and Roberts, 2005; Brechot et al., 2010; Sung et al., 2012). In many virus infections, DNA integration into the host cell chromosome induces chromosomal instability (CIN) (Ohshima et al., 1998; Pett et al., 2004; Herath et al., 2006; Nishida and Goel, 2011). In these cases, CIN can be induced by the integration into cellular genome sequences called structural and matrix-attachment regions (S/MAR), which modulate the activity of gene enhancers, interact with transcription-associated proteins, and are also associated with origins of cellular replication (Boulikas, 1993; Bode et al., 1996). Virus DNA integration into S/MAR and the resulting CIN has been observed in HCC associated with chronic WHV infections (Bruni et al., 2003) and HBV-infected hepatoma cells (Shera et al., 2001). This enrichment of integration may be due to that base unpairing regions in the cellular genome, including S/MAR, are particularly susceptible to double stranded DNA breakage (Liu et al., 2003), the substrate for HBV integration (Bill and Summers, 2004). Thus, the association between HBV DNA integration and CIN has led to the speculation that CIN induces the initiation and progression of HCC (Levy et al., 2002; Brechot, 2004; Bonilla Guerrero and Roberts, 2005). Oxidative stress likely drives greater integration rates. Indeed, increased HBV integration has been observed in HBV-expressing HepG2 cells exposed to H2O2-induced oxidative stress (Dandri et al., 2002).

Chronic stress response

The contributions towards HCC initiation and progression by HBV gene products (particularly HBsAg, HBV splice variants, and HBx) have been widely reported (Table 1) (Lakhtakia et al., 2003; Pollicino et al., 2011; Li et al., 2012). These gene products have been suggested to induce chronic stress responses and direct transcriptional activation of HCC-associated genes.

Immune pressure during HBeAg seroconversion selects for escape HBV mutants, which become common in the latter phases of HBV infection. One such class of escape mutants are PreS mutants. Mutant Pre-S proteins have been described to accumulate in the ER, induce oxidative and ER stress responses, and increase the risk of HCC (Wang et al., 2003, 2006; Liu et al., 2009a). Furthermore, over-expression of HBsAg containing these Pre-S mutations reportedly induces precancerous liver lesions and HCC in animal models (Chisari et al., 1987; Pasquinelli et al., 1992) and stimulates clonal hepatocyte expansion (Fan et al., 2001; Su et al., 2008; Wang et al., 2003, 2012). Indeed, meta-analyses show that the presence of HBV PreS mutations (particularly A1762T and G1764A) predicts HCC risk with adjusted odds ratio of 6.18 (Liu et al., 2009b).

The presence of HBV pgRNA splice variants also predicts HCC development and disease progression of HBV infection (Soussan et al., 2003; Marschenz et al., 2006; Bayliss et al., 2013). Pregenomic HBV RNA contains several conserved splice donor and acceptor sites (Gunther et al., 1997), many of which have been confirmed by deep sequencing of both HBV-infected cells and patient sera (Chen et al., 2015; Betz-Stablein et al., 2016). While the HBx ORF is generally undisrupted, deletions in the pol and surface ORFs are present in most splice variants, with around half also containing deletions in the core ORF (Chen et al., 2015). One possible mechanism by which HBV splice variants might increase the risk of HCC may be related to the accumulation of HBc and HBx transcripts upon expression of splice variants, leading to intracellular stress (Rosmorduc et al., 1995). However, in general, these splice-associated prooncogenic mechanisms are still unclear.

Alterations of HCC-associated pathways

HBx is a necessary factor for HBV replication (Tsuge et al., 2010) and releases HBV transcriptional repression by inducing ubiquitination of the Smc5/6 complex (Decorsiere et al., 2016). HBx has also been reported to induce the transcription of host HCC-associated proteins (Brechot et al., 2010). For example, expression of HBx upregulates hTERT, and the vasoinvasion-associated genes hypoxia inducible factor 1-α and histone deacetylase 1, and increases degradation of β-catenin in models of HCC both in vitro and in vivo (Lin et al., 2005; Qu et al., 2005; Zhang et al., 2005; Xie et al., 2008). Moreover, C-terminal truncated HBx (generated by integrated HBV DNA sequences) has also been described to drive a panoply of oncogenic phenotypes, including: stem-cell like properties (Ng et al., 2016); cell transformation and inhibition of apoptosis (Tu et al., 2001; Ma et al., 2008); and cell growth and invasion (Wang et al., 2010b; Sze et al., 2013). However, these results should be taken as provisional, as the HBx gene in many of these studies was expressed under the control of a highly-active promoters rather than its native promoter. Indeed, the results of HBx studies have been so variable (due to the dependency of the systems used) that stringent technical standards have now been strongly recommended by leaders in the field for future work (Slagle et al., 2015).

Hepatitis D virus (HDV) co-infection

HDV is a negative-sense RNA satellite virus of HBV that requires the HBsAg for formation of new virions (Figure 2). Coinfection of HBV with HDV has been shown to increase the risk of HCC by 2-fold and the risk of cirrhosis by 3-fold (Tamura et al., 1993; Fattovich et al., 2000, 2008), although epidemiological studies show high variation in these risks between different populations (Abbas et al., 2015). Coinfection of HBV patients with HDV is associated with increased hepatocyte necroinflammation (Verme et al., 1986). As expression of HDV antigens (as with HBV) is non-cytopathic (Verme et al., 1986; Guilhot et al., 1994), the increased liver damage associated with HDV infection is generally assumed to be due to an anti-viral immune-mediated response. Indeed, increased interferon production and downstream signalling has been detected in HDV-infected cells (Giersch et al., 2015; He et al., 2015b). This increased induction of immune-mediated inflammation is likely to be a mechanism by which HDV increases HCC risk. While other mechanisms have been proposed (Reviewed in Abbas et al., 2015), the full extent of HDV-associated pathogenesis remains unclear at the moment.

Hepatitis C virus

HCV replication cycle

HCV is an enveloped virus that is characterised by its tight association with lipids and components of the lipoprotein pathway, most notably apolipoprotein E (reviewed in Bartenschlager et al., 2011). These lipoviroparticles escape neutralising antibodies, at least in part, by lipid-based shielding (Bartosch et al., 2005). The HCV genome is a single stranded RNA of positive polarity and has a length of about 9600 nucleotides. Owing to error-prone replication, within a patient HCV exists as quasispecies and on a global scale seven different genotypes have been identified differing in their nucleotide sequences by more than 30% (Smith et al., 2014). HCV belongs to the family Flaviviridae and therefore is phylogenetically related to, e.g. Dengue virus or the Zika virus.

The HCV infection process is initiated by a complex interplay of the viral envelope glycoproteins E1 and E2 with multiple surface-resident host cell factors, resulting in uptake of HCV particles by clathrin-mediated endocytosis (Figure 2). The viral RNA genome is released into the cytosol and used for the synthesis of a polyprotein that is cleaved into 10 products executing various functions (reviewed in Paul et al., 2014). These include: the structural proteins, Core, E1 and E2 (major constituents of the virus particle); and the HCV replicase (composed of several non-structural proteins, some of which induce ER membrane rearrangements). In tight association with these remodelled membranes, the viral RNA genome is amplified via a negative-strand intermediate that serves as template for the production of multiple positive-strand RNA molecules. These are used as template for RNA translation, for negative-strand RNA synthesis or incorporated into infectious virus particles. Assembly is tightly linked to cytosolic lipid droplets, lipid signalling and components of the very-low-density lipoprotein (VLDL) pathway giving rise to new lipoviroparticles. Upon release from the cell they can infect new hepatocytes; alternatively HCV can spread via cell-cell transmission (Timpe et al., 2008), which appears to be the predominant route of transmission in vivo (reviewed in Zeisel et al., 2013).

Natural history of chronic HCV infection

Around 80% of HCV infections persist, indicating that HCV employs efficient strategies to avoid or counteract anti-viral immune responses (reviewed in Heim and Thimme, 2014). Although during the acute phase, patients mount innate and adaptive immune responses, T cell responses vanish (for reasons that are still not understood), often as a prelude to chronicity (Figure 3). During this chronic phase, viremia fluctuates, which may correlate with flares of liver enzymes. Patients mount a non-protective antibody response, targeting either viral antigens that are not part of the virion or epitopes in the envelope proteins that are irrelevant for neutralisation. Moreover, T cell responses become exhausted in the course of infection and remain dampened during chronicity, but can recover (at least to some extent) upon therapeutic elimination of the virus (Martin et al., 2014). Interestingly, patients with a clinically-overt acute infection have a higher chance to clear HCV, supporting the notion that pathogenesis is driven mainly by the immune response.

Pathogenesis of chronic HCV infection

About 20% of persistently HCV-infected individuals develop liver cirrhosis within 20–30 years and once cirrhosis is established, the rate of HCC development is 1–6% per year. As alluded to above, chronic HCV infection induces a persistently-activated immune reaction targeting HCV-infected liver cells, driving hepatocarcinogenesis. In addition to this chronic inflammation, HCV causes massive alterations of infected hepatocytes, including: metabolic reprogramming; prolonged (ER) stress responses; production of intra-hepatocellular ROS; and perturbations of tumour-relevant signalling pathways. In addition, HCV proteins and viral RNA interfere with tumour suppressors, thus contributing to HCC development. In the following sections, we elaborate on these features of HCV infection and their link to hepatocarcinogenesis (Table 2).

Metabolic reprogramming of infected cells by HCV

A hallmark of HCV infection is the induction of hepatosteatosis, which is defined as excessive triglyceride deposition in hepatocytes. Importantly, hepatosteatosis is associated with an increased risk of HCC development and is found in 40–80% of HCV-positive patients that are biopsied (Pekow et al., 2007).

One key player in altering lipid homeostasis is the HCV Core protein, impacting lipid metabolism in several ways: (a) by inhibition of lipid droplet (LD) mobility; (b) by decreasing lipid turnover of HCV core-coated LDs; (c) by inhibition of microsomal triglyceride transfer protein (MTTP) to impede lipid export and degradation; (d) by inhibition of VLDL secretion; (e) by inhibiting peroxisome proliferator-activating receptor (PPAR)-α/γ; (f) by inhibition of diacylglycerol acyltransferase 1 (DGAT-1) (reviewed in Buhler and Bartenschlager, 2011; Chang, 2016). Of note, HCV Core has also been described to be a major HCV-related driver of liver tumour formation. Two independent lines of HCV Core-transgenic mice were found to develop hepatic steatosis as an early histological change, followed by the formation of adenomas with massive fat deposition and ultimately liver tumours resembling early-stage HCC in patients with chronic HCV infection (reviewed in Negro, 2014; Yamane et al., 2013).

LDs are cellular organelles playing an important role in the regulation of intracellular lipid storage and metabolism. They are composed mainly of triacylglycerol and cholesterol esters forming the lipid core surrounded by a monolayer lipid membrane into which distinct proteins of the PAT family are embedded (Perilipin, Adipophilin, TIP47). Of note, high LD numbers and elevated cholesteryl ester content in tumours are considered as marker of cancer aggressiveness (reviewed in Beloribi-Djefaflia et al., 2016). In addition, lipidomic profiling combined with transcriptome/proteome analyses unravelled unexpected and recurrent lipid changes in cancer cells underscoring the important role of lipid metabolism for carcinogenesis.

While the underlying mechanisms are only beginning to emerge, it is clear that for genotype 3 HCV infections, the extent of hepatosteatosis is directly correlated with viraemia of the patient whereas for other genotypes steatosis seems to be more of a metabolic origin. Interestingly, a recent study demonstrated reduced de novo lipogenesis in HCV genotype 3 patients, possibly due to a perturbation of the distal cholesterol biosynthesis pathway or a selective derangement of hepatocyte lipid secretion, resulting in relative hypocholesterolemia (Lonardo et al., 2014). However, the detailed molecular mechanisms and involved viral and cellular determinants are poorly understood.

Long-term stress response

Another contributor to HCV-associated HCC development is the stress response triggered by the virus. HCV proteins accumulate on the surface of ER-derived membranes where most steps of the viral replication cycle occur. High abundance of HCV proteins (106 molecules per cell) (Quinkert et al., 2005) accumulating at the ER membrane can trigger an unfolded protein response (UPR). The UPR is a transcriptional cascade affecting multiple cellular pathways such as lipid metabolism, expression of chaperones, or autophagy with the aim to restore ER homeostasis. Moreover, HCV induces a profound IFN response, increasing the expression of IFN-stimulated genes (ISG) to which protein kinase R (PKR) belongs. This protein kinase senses viral RNA and, via phosphorylation of the translation factor eIF2α, shuts down RNA translation, concomitant with the formation of stress granules, which are transient storage sites of RNA to be reused for protein synthesis when stress phases cease. Interestingly, HCV infection, in conjunction with PKR, causes an oscillation of RNA translation arrest as deduced from the dynamics of stress granule formation and dissolution in infected cells (Ruggieri et al., 2012). Of note, the frequency of oscillation correlates with long-term survival of infected cells, consistent with the fact that prolonged phases of translation arrest trigger apoptosis. Thus, by exploiting the dynamic of translation arrest and reinstalment, HCV appears to prolong the survival of infected cells and thus, viral persistence (Ruggieri et al., 2012).

Oxidative stress

HCV proteins appear to induce ROS in hepatocytes (reviewed in Ivanov et al., 2013). It was found that HCV Core protein can localise to outer mitochondrial membranes and perturb intracellular calcium homeostasis by increasing Ca2+ influx into mitochondria. This is mediated by activation of the Ca2+ uniporter, leading to the failure of the electron transport chain and the production of ROS. In addition, Core and NS5A can also deplete ER Ca2+ stores via the induction of a passive leak of Ca2+ ions and inhibition of sarco/endoplasmic reticulum Ca2+-ATPase, respectively (Gong et al., 2001; Benali-Furet et al., 2005). While these observations have been made with in vitro systems, induction of ROS by HCV appears to be of relevance also in vivo. First, HCV core-transgenic mice show an increased accumulation of ROS that correlates with HCC development (Moriya et al., 1998, 2001); second, markers for acute intracellular oxidative stress are expressed at elevated levels in patients with chronic hepatitis C (Sumida et al., 2000).

An additional mechanism by which HCV induces ROS is the production of superoxide anions by NADPH oxidases (Noxes), which is mediated by activation of the Nox family members Nox1, -2 and -4 (Bureau et al., 2001; Boudreau et al., 2009; de Mochel et al., 2010). Nox1 and -4 activity is also significantly enhanced in HCV-infected human liver samples (de Mochel et al., 2010). The underlying mechanisms are not yet clear, but it was shown that HCV Core protein induces NOX4 via TGF-β (Boudreau et al., 2009). Moreover, Smirnova and colleagues showed that also NS5A is able to induce Nox1 and -4 and in addition cytochrome P450 2E1, thus contributing to ROS production (Smirnova et al., 2016).

HCV-induced alterations of cell survival, mitosis and differentiation pathways

The HCV phosphoprotein NS5A lacks enzymatic activity, but instead fulfils multiple functions in the viral replication cycle by recruiting numerous host cell factors (Lohmann, 2013). This promiscuity of NS5A suggests that it also plays an important role in the pathogenesis of chronic hepatitis C, reportedly affecting diverse cellular pathways, such as: apoptosis, signal transduction, cell cycle regulation, transcriptional activation and transformation. The most convincing link between NS5A and HCC is the interaction with and activation of class I phosphoinositide 3-kinases (PI3K), which has been identified in two independent studies (He et al., 2002; Street et al., 2005). PI 3-kinases are involved in numerous cellular functions, including cell growth, proliferation, differentiation, motility, survival and intracellular trafficking. Many of these functions relate to the ability of class I PI3-kinases to activate protein kinase B (also known as Akt) that is recruited by phosphatidylinositol 3,4,5 trisphosphate to the inner leaflet of the plasma membrane. Akt is then phosphorylated, and thus activated, by phosphoinositide dependent kinase 1 (PDK1). This PI3K/AKT signalling pathway plays an important role in cell proliferation and survival. Notably, Akt phosphorylation also impacts on β-catenin signalling that is critically involved in HCC development. Hence, PI3K activity contributes significantly to cellular transformation and the development of cancer. In the case of HCV, NS5A interacts with and activates PI3K and Akt, ultimately leading to the accumulation of β-catenin and elevated levels of β-catenin-dependent transcription.

Another important pathway that appears to be altered by HCV Core protein is the TGF-β pathway. By interacting with Smad3, HCV Core may disrupt TGF-β dependent signalling required to regulate cell proliferation and apoptosis (Cheng et al., 2004; Pavio et al., 2005).

Interference with tumour suppressors by HCV

In addition to the indirect effects of HCV on HCC formation described above, viral proteins and the RNA genome directly interact with and block several tumour suppressors. Best studied are the interference with the retinoblastoma tumour suppressor protein (Rb), the epidermal growth factor receptor (EGFR), the DEAD-box RNA helicase (DDX3), p53 and microRNA (miR)-122.

The Rb protein is a common target for oncogenic DNA viruses and its downregulation promotes cell cycle progression. It has been shown that the HCV NS5B protein, which is the RNA-dependent RNA polymerase, binds to Rb and thereby targets Rb for degradation via the ubiquitin-proteasome pathway. The resultant lower abundance progresses cell cycle transition from G1- to S-phase, thus stimulating hepatocellular proliferation (Munakata et al., 2005; 2007). In addition to NS5B, the HCV Core protein also inhibits Rb and affecting the mitotic spindle complex, thus resulting in polyploidy (Machida et al., 2009).

EGFR (Lupberger et al., 2011) and its signalling pathway HRas (Zona et al., 2013) are involved in HCV entry, but in addition, EGF-mediated signalling contributes to HCC development. It was shown that HCV enhances EGFR expression in HCV-infected patients (Zona et al., 2013) and that inhibition of the tyrosine kinase function of the EGFR by the cancer drug erlotinib attenuates the development of HCC in mouse models (Fuchs et al., 2014).

DDX3 is a DEAD-box RNA helicase that interacts with the HCV Core protein (Owsianka and Patel, 1999). While DDX3 plays an important role in HCV replication and virus production (Ariumi et al., 2007; Li et al., 2013), it also appears to be a tumour suppressor: DDX3 can inhibit colony formation in various cell culture assays and to up-regulate the p21waf1/cip1 promoter, which is crucial for regulation of the cell cycle (Chao et al., 2006). Moreover, DDX3 can interact with the translation initiation factor eIF4E and inhibit cap-dependent RNA translation. The perturbation of both pathways, p21waf1/cip1 and eIF4E, could explain the anti-proliferative effect of DDX3. In this respect, HCV Core might overcome DDX3-mediated cell growth arrest by sequestration of this tumour suppressor to LDs, thus contributing to HCC development. Both DDX3 and the related RNA helicase DDX5 appear to be implicated in the pathogenesis of HCV-related liver diseases. DDX3 expression is deregulated in HCV-associated HCC (Chang et al., 2006; Chao et al., 2006), and single-nucleotide polymorphisms in the DDX5 gene are associated with an increased risk of advanced fibrosis in patients with chronic hepatitis C (Huang et al., 2006a).

The tumour suppressor protein p53 is a central cell cycle regulator and one of the most frequently mutated genes in human tumours (reviewed Lee, 2015). In vitro and mouse model-based studies indicate several molecular mechanisms how HCV might target p53 (reviewed in McGivern and Lemon, 2011). One study, for example, described an essential role of p53 in the host defence against HCV (Dharel et al., 2008). Moreover, several HCV proteins have been reported to interact with p53 (Otsuka et al., 2000; Lan et al., 2002). However, the precise role of p53 in the development of HCV-associated carcinogenesis is still ill understood.

MicroRNAs are small double stranded non-coding RNAs acting as negative regulators of protein synthesis. RNA virus replication highly depends on their function to manipulate host cell metabolism (Scheel et al., 2016). One well-known example is miR-122, which is expressed in the liver to high abundance and essential for HCV replication (reviewed in Rupp and Bartenschlager, 2014). Initial hope on the therapeutic use of miR-122 antagonists to treat HCV infection was dashed by the discovery of miR-122 being a tumour suppressor (Hsu et al., 2012; Tsai et al., 2012). Interestingly, analyses of HCC samples revealed reduced expression of miR-122 (Kutay et al., 2006; Bai et al., 2009; Coulouarn et al., 2009) fostering the development of novel treatment concepts of HCC by upregulation of miR-122 expression (Song et al., 2013; He et al., 2015a; Xu et al., 2016). In case of HCV it was shown that miR-122 binds to the HCV RNA with two binding sites close to the 5′ end of the genome being most important for viral replication (Jopling et al., 2005). Of note, Luna and colleagues recently described the sequestration of miR-122 by the HCV genome resulting in a de-repression of miR-122 target genes and creating an environment that might be favourable for carcinogenesis (Luna et al., 2015). Although this is a plausible model, the relevance of miR-122 sequestration in vivo remains to be determined.

Limitations to the study of HBV- and HCV-associated pathogenesis

While much has been achieved in determining HBV- and HCV-associated factors that lead to HCC, numerous systemic factors related to the available molecular tools limit research in this area. For instance, few models replicate the decades-long chronic inflammation associated with almost all HCCs and true long-term HBV and HCV in vitro and in vivo models remain limited and/or impractical. Further, the majority of HBV and HCV research are limited to a single virus clone. For example, the genotype D HBV clone sequenced by Galibert and colleagues (Galibert et al., 1979) in the late 1970s is still used (almost exclusively) for molecular and cellular studies of HBV replication and pathogenesis. This lack of diversity neglects clinically-reported HBV genotype-dependent differences [e.g. in pathogenesis and HCC risk (Yang et al., 2008b; Tong and Revill, 2016)]. Likewise, in case of HCV, most studies are based on the genotype 2a strain JFH-1 that is unique and differs in important aspects from other HCV genotypes. However, increasing availability of novel molecular tools such as the Sleeping Beauty transposon system for rapidly generating stable HBV-expressing cell lines (Wu et al., 2016) or new functional HCV isolates belonging to clinically relevant genotypes (Bukh, 2016) enable simplified circumvention of these limitations.

Limitations of current in vitro models

Experiments in HBV and HCV research have generally used monoculture in vitro systems, which can limit the interpretation of results and their translation to a true physiological environment. In particular for HCC initiation (as we have summarised here), hepatitis virus-associated pathogenesis involves not only hepatocytes, but the concerted participation of multiple cell types, such as macrophages (both resident and infiltrating), endothelial cells, stellate cells and bipotent progenitor cells within the liver. Some of these interactions can be explored using co-culture models (e.g. with cell lines derived from macrophages or endothelial cells) and single-cell analysis, though these require specialised expertise to carry out.

Further, even with co-culturing, cell lines may not display the same properties as cells in situ. For example, of particular concern for HCC-related research, cell lines used that support HBV and HCV replication are generally derived from already transformed cells, with the majority being of HepG2 or Huh7 hepatoma-derived lineages. Also, quiescent hepatic stellate cells have not been successfully passaged in cell culture, leading to difficulties in studying the activation of the fibrotic response that is a major factor in HCC initiation and progression. Some of these limitations have been addressed using the bipotent hepatic progenitor cells HepaRG (at the cost of decreased HBV infection rate, non-permissiveness for HCV, and long differentiation periods) and cells isolated from primary tissue (at the cost of limited availability). Recently, inducible pluripotent stem cells and inducible embryonic stem cells have become available providing more physiologically accurate in vitro model systems. These cell systems hold great promise to decipher the physiologically-relevant principles underlying the direct contributions of HBV and HCV to liver pathogenesis.

Finally, current in vitro systems are generally conducted on adherent monolayers, where the three-dimensional (3D) liver architecture is not reproduced. Many signalling pathways involved in HCC initiation require the polarisation of hepatocytes and their appropriate 3D arrangements (Gissen and Arias, 2015; Zeigerer et al., 2017). Further, it is likely that some of the genes that are differentially expressed in the zones of the liver lobule (up to 50% of the hepatocyte transcriptome; Halpern et al., 2017) may be involved in HCC development, but this diversity is not likely to be represented in a homogeneous two-dimensional (2D) culture system. Thus, 2D monocultures have limited accuracy in reproducing signalling pathways that occur in both pre-cancerous and tumour tissues within their 3D architecture. Novel liver organoid co-culture systems that form physiological features (such as hepatocyte polarisation and biliary ducts) provide a potential answer to these limitations (Huch et al., 2015; Mazza et al., 2015), but the ability and reproducibility in supporting hepatitis virus replication remains to be shown.

Limitations of current in vivo models

The major limitation in studying HBV- and HCV-associated liver cancer is the inability of these viruses to replicate in immune-competent mouse models. Therefore, more artificial approaches have been used such as over-expression models to determine, e.g. the oncogenic potential of particular viral components (e.g. HCV Core, HBx and HBs mutants, as mentioned above). However, these are likely to be poor representations of physiological conditions as over-expression or altered molar ratio of viral proteins can prevent normal secretion and cause unnatural mislocalisation, aggregation, or activation of the cellular UPR. Indeed, in immunocompetent models expressing the entire HBV genome under native promoters (both transgenic animals and long-term experiments after transduction) show no signs of liver pathology or premalignant changes, even years after virus expression (Guidotti et al., 1995; Huang et al., 2006b; Dion et al., 2013; Yang et al., 2014).

True infection-based small-animal models are only available for non-human HBVs, but either do not exhibit extensive liver pathology [e.g. Pekin ducks infected with duck HBV display limited hepatic fibrosis (Marion et al., 1984)] or display pathologies that do not translate well to human HCCs (e.g. woodchucks infected with WHV develop HCC driven via insertional mutagenesis of Myc genes by viral DNA, by 4 years’ post-infection without concomitant cirrhosis (Popper et al., 1987; Wei et al., 1992a,b; Bruni et al., 1999). Since the discovery of NTCP as the HBV receptor, development of novel infection models have been begun (including transgenic mice, cynomolgus macaques and pigs), though some unknown restriction factors remain in some of these models (Li et al., 2014; Lempp et al., 2017). Likewise, in the case of HCV transgenic mice expressing human versions of the entry factors have been established, but robust replication in these animals has not been achieved (Dorner et al., 2013). Many models based on immunocompromised mice with human liver xenografts (in some instances, with human haematopoietic stem cells to reconstitute the immune system) have been shown to support hepatitis virus infection (Meuleman et al., 2005; Kosaka et al., 2013; Bility et al., 2014; Giersch et al., 2015; Strick-Marchand et al., 2015; Billerbeck et al., 2016; Winer et al., 2017), but these remain cost-, time- and labour-intensive. Moreover, virus replication and liver pathologies in these animals are limited.

Concluding remarks

Chronic infection with HBV and HCV alters multiple cellular aspects that lead to hepatocarcinogenesis, though no single factor may result in tumour formation. These mechanisms do not appear to be due to a simple oncogene (as is the case with other virus-associated cancers), but instead are more subtle, involving concerted and cumulative actions that drive HCC initiation and progression over decades. These virus-associated molecular drivers of hepatocarcinogenesis can be viewed through an evolutionary lens (Tu et al., 2015a). Chronic hepatitis virus infections change the microenvironment of the liver by contributing to (1) variation in the hepatocyte population, via DNA mutations and genomic instability and (2) constant liver cell turnover that is driven by chronic inflammation. The result of these alterations is the selection for hepatocytes that can survive the constant selective pressure exerted by the immune response by, e.g. heightened immune evasion or suppression of cell death signals and the emergent clonal expansion of hepatocytes with these survival advantages. These alterations in the liver microenvironment are likely to drive intrahepatic evolution of hepatocytes towards a pro-oncogenic state.

Progression in developing new model systems is key to understanding, prevention of and therapeutics for hepatitis virus-associated hepatocarcinogenesis. Poetically, this has been achieved (and will be further extended) through concerted and cumulative actions of basic molecular, virus, cellular, animal model and clinical research. Together, future work will drive towards deeper knowledge of this deadly and highly complex disease and the development of therapeutic strategies against it.

Acknowledgements

Work in the authors’ laboratory was supported by the Deutsche Forschungsgemeinschaft (TRR179, TP9 to R.B.).

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