DDI2012030005445

DDI2012030005445
Viral Hepatitis
Dig Dis 2012;30:445–452
DOI: 10.1159/000341688
Promotion of Hepatocellular Carcinoma
by Hepatitis C Virus
Sandra Bühler Ralf Bartenschlager
Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
Key Words
Hepatitis C virus ⴢ Hepatocellular carcinoma ⴢ Liver cancer ⴢ
Steatosis ⴢ ER stress
Abstract
Persistent infection with the hepatitis C virus (HCV) is a major
global health problem. Around 2–3% of the world’s population are chronically infected, and infected individuals are at
high risk of developing steatosis, fibrosis, and liver cirrhosis.
The latter is a major predisposing factor for the development
of hepatocellular carcinoma (HCC). It is generally accepted
that an inflammatory response triggered by persistent HCV
infection leads to increased cell proliferation and fibrogenesis that in turn promotes cirrhosis and ultimately HCC development. This indirect mechanism of tumor induction
would explain the long incubation period from primary HCV
infection to HCC and the requirement for additional cofactors such as toxins or drugs (most notably alcohol), metabolic liver diseases, steatosis, nonalcoholic liver disease, or
diabetes. With the advent of adequate cell culture systems
for HCV it is, however, becoming increasingly clear that the
virus also contributes directly to HCC formation. Examples
are the continuous induction of stress response or the massive accumulation of intracellular lipids. Moreover, viral proteins can bind to and sequester cell cycle control factors
such as the retinoblastoma protein or the tumor suppressor
DDX3. Thus, HCV-associated liver cancer is most likely pro-
© 2012 S. Karger AG, Basel
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moted by the combined action of long-term chronic inflammation and targeted perturbations of cellular key pathways
involved in metabolic homeostasis as well as cell cycle control.
Copyright © 2012 S. Karger AG, Basel
Introduction
Persistent infection with the hepatitis C virus (HCV)
is a major global health problem. Around 170 million
people are infected with this virus, corresponding to
⬃3% of the world’s population. These people are at high
risk of developing serious liver damage; ⬃20% develop
liver cirrhosis within 20–30 years after infection and people with HCV-associated cirrhosis develop hepatocellular
carcinoma (HCC) in 1–6% of cases per year. Although for
many types of cancer the incidence rates are stable or
even declining, in liver cancer they are rising. In fact,
HCC, which accounts for 70–85% of liver cancers [1], is
the most frequently diagnosed cancer worldwide in men
and the second most frequent cause of cancer death. In
women, it is the seventh most commonly diagnosed cancer and the sixth leading cause of cancer death [2]. Remarkably, HCV infection accounts for a large proportion
of these liver cancers. The prognosis of patients with
HCC is poor and the 1-year survival remains less than
50% in the USA [3].
Ralf Bartenschlager
Department of Infectious Diseases, Molecular Virology
Heidelberg University, Im Neuenheimer Feld 345
DE–69120 Heidelberg (Germany)
Tel. +49 6221 56 4225, E-Mail Ralf_Bartenschlager @ med.uni-heidelberg.de
In the absence of adequate in vivo/animal models,
analyses of HCV-induced pathogenesis are difficult.
Thus, most studies are based on the use of engineered in
vitro culture systems that are inherently prone to artifacts caused, for example, by ectopic overexpression of
single HCV proteins. Moreover, in many studies heterologous cell systems of nonhepatic or nonhuman origin
have been used. Some of these limitations have been overcome with the implementation of more authentic cell culture systems recapitulating the HCV life cycle in parts
(e.g. the replicon system) or in total [reviewed in 4]. The
latter is based primarily on a particular HCV isolate, designated JFH1 (an acronym derived from Japanese fulminant hepatitis), that replicates to exceptionally high levels
in the human hepatoma cell line Huh7. Importantly,
HCV particles produced in these cells are infectious in
vivo (i.e. immunodeficient transgenic mice with human
liver xenografts) and particles isolated from these in vivo
samples are infectious for Huh7 cells [5]. While these are
important achievements, the primary restriction to Huh7
human hepatoma cells is a serious limitation.
Chronic inflammation induced by viral infection appears to be a major predisposing condition for liver cancer. In the case of hepatitis C it is assumed that a persistently activated immune reaction targeting infected liver
cells leads to increased cell proliferation and fibrogenesis,
thus enhancing cirrhosis and HCC development [6, 7].
This indirect mechanism of tumor induction by HCV infection could explain why tumors most often develop
only 10–30 years after primary infection and require additional risk factors such as alcohol consumption, metabolic liver diseases, or diabetes [6]. Nevertheless, there is
increasing evidence that HCV itself, or specific viral proteins, contribute directly to HCC formation. In the following sections, we will focus on some of the possible
direct contributions of HCV to the development of HCC
and the molecular mechanisms underlying this process.
HCV Replication Cycle
Understanding the molecular mechanisms underlying the development of HCV-associated HCC requires
detailed knowledge of the viral replication cycle and the
interaction of HCV with its host cell. With the advent
of robust culture models and molecular biological approaches, the principles by which HCV replicates have
been unraveled (fig. 1) [8–12]. The virus enters the host
cell, which is primarily the hepatocyte, via receptor-mediated endocytosis. It is achieved by interactions between
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Dig Dis 2012;30:445–452
the virus particle and numerous host cell molecules presumably in a consecutive manner. Virus particles are
probably trapped on the surface of the hepatocyte by interaction with glycosaminoglycans and low-density lipoprotein (LDL) receptors that likely interact with the
lipoprotein moiety of the HCV particle. Subsequent interactions involve CD81, scavenger receptor B1 (SR-B1),
claudin-1 and occludin. The efficiency of entry is enhanced by direct or indirect interaction between HCV
and the Niemann-Pick C1-like 1 (NPC1L1) cholesterol
uptake receptor and the epidermal growth factor receptor
(EGFR), respectively [11, 13, 14]. Upon HCV entry, the
viral genome is released into the cytoplasm. This plusstrand RNA has a length of ⬃9,600 nucleotides and contains one long open reading frame that is flanked by two
terminal nontranslated regions that form higher-order
RNA structures. The 5ⴕ nontranslated region contains an
internal ribosome entry site (IRES) mediating the translation of the RNA genome and giving rise to a polyprotein
that is cleaved by host proteases and two viral proteases
into 10 different products. The N-terminal region of the
polyprotein is composed of the structural protein core,
envelope glycoprotein 1 (E1) and E2. These proteins are
the major building blocks of the virus particle. The following two auxiliary proteins are required for the assembly of infectious HCV particles, but it is unclear whether
they are also part of the virus particle: p7, a small hydrophobic protein that may act as an ion channel, and nonstructural protein 2 (NS2), that appears to contribute to
some step of virus formation. The remainder of the NS
proteins is sufficient for replication of the viral genome
[15]. NS3 contains in its N-terminal domain a serine-type
protease that is tightly associated with and activated by
NS4A. The C-terminal NS3 domain contains NTPase/
helicase activity. NS4B is a protein inducing the formation of the so-called membranous web that probably harbors the replicase complex. NS5A is an important regulator of RNA replication and virus assembly and NS5B is
the RNA-dependent RNA polymerase (RdRp), the key
enzyme of viral genome amplification.
HCV RNA is amplified via a minus-strand RNA template and the newly synthesized plus-strand RNA is either used for RNA translation and replication or packaged into progeny HCV particles. Viral replication occurs exclusively in the cytoplasm and leads to massive
rearrangements of intracellular membranes mostly induced by NS4B. The assembly and release process of
progeny virus is closely associated with the lipid metabolism of the cell [reviewed in 9]. As a result, infectious
HCV particles have a unique composition and contain, in
Bühler/Bartenschlager
Fig. 1. Schematic of the HCV replication
cycle. Numbers refer to the individual
steps of the cycle. For details, see text.
MW = Membranous web.
addition to E1 and E2, components of the ‘very LDL’
(VLDL) system, most notably apolipoprotein E (ApoE).
Moreover, HCV particles have a unique lipid composition
that is very distinct from all other viruses analyzed so far
and from the human liver cells in which the virus replicates.
Pathogenesis of Chronic Hepatitis C: Implications
for HCC Development
Since HCV replicates exclusively in the cytoplasm
without integration of the viral genome into the host cell
chromosome, persistence can only be achieved by continuous viral replication. HCV also triggers an immune
response that is, however, inefficient; it does not clear the
virus, but sustains a chronic inflammation. This is exemplified, among other things, by: (i) the correlation between the strength of the immune response and clinical
Promotion of Hepatocellular Carcinoma
by HCV
manifestation of hepatitis C, (ii) the lack of correlation
between the level of viremia and the course of diseases,
and (iii) the observation that patients treated with ribavirin monotherapy normalize liver histology even though
viremia is not affected. Thus, an inefficient immune response unable to eradicate HCV appears to sustain a
chronic inflammation (fig. 2). This in turn likely increases cell turnover and thus the chance for manifestation of
alterations affecting the control of cell growth. Such alterations are promoted by various conditions including
risk factors of DNA damage (e.g. aflatoxins), cirrhosis
(e.g. alcohol consumption), or steatosis (e.g. diabetes) [for
detailed reviews, see 16–19]. Apart from these indirect
and host-determined effects, it is becoming increasingly
clear that the virus itself also contributes to tumor progression (fig. 2, table 1). For instance, it has been shown
that the core protein binds to and sequesters the tumor
suppressor protein DDX3 [20]. Along the same lines, it
has been convincingly demonstrated that nonstructural
Dig Dis 2012;30:445–452
447
Fig. 2. Schematic representation of the dif-
ferent conditions promoting the development of HCV-associated liver cancer. Proliferation of hepatocytes is affected by
chronic inflammation. Control of the cell
cycle (black circle) is affected by multiple
external cues that depend either on the
host (grey box) or viral infection (black
box). In addition, two HCV proteins (core
and NS5B) might affect the cell cycle directly by sequestration of cell cycle control
factors. For further details, see text.
protein 5B (NS5B), which is the RNA-dependent RNA
polymerase, binds to and sequesters the retinoblastoma
protein (Rb) [21]. Although experimental proof so far is
missing, it is tempting to speculate that DDX3 or Rb sequestration by core protein or NS5B, respectively, might
affect cell cycle control and thus contribute rather directly to tumor progression.
Another viral protein that received much attention in
this context is NS5A. It is a highly phosphorylated protein
required for HCV RNA (table 1) replication and virus
production. NS5A is composed of an amino-terminal
amphipathic ␣-helix that tethers the protein to intracellular membranes, an RNA-binding domain 1 that forms
homodimers, and two natively unfolded protein domains
that can form multiple complexes with host cell proteins
[22]. It is this structural property of domains 2 and 3 that
explains why so many NS5A-interacting host cell factors
have been identified, in most cases, in rather artificial
systems such as the yeast-two-hybrid system. Moreover,
in several cases artificially truncated NS5A variants have
been used that display biochemical properties not found
with the authentic full-length proteins. Thus, even though
some of the cellular NS5A interactants have been confirmed in more authentic cell-based replication systems,
in most instances this is not the case and, therefore, the
physiological relevance of NS5A for alterations of cellular
homeostasis and the contribution of this protein to tumor
formation remain to be determined.
With the advent of authentic cell culture systems it is
becoming clear that, apart from individual viral proteins,
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Dig Dis 2012;30:445–452
HCV infection and replication per se induces numerous
cellular and metabolic alterations that likely contribute to
tumor progression. Examples are the induction of oxidative and ER stress, the enhanced production and secretion of TGF-␤, and the enhanced accumulation of lipids.
Two of these alterations will be described below. The
reader interested in more details is referred to excellent
recent reviews [16, 17, 23, 24].
Induction of Stress Response and Impact on Viral
Persistence and Cell Survival
It is known that HCV infection triggers various stress
responses [reviewed in 23, 25]. Recently, we were able to
show that in cells treated with interferon (IFN)-␣, HCV
potently induces the highly dynamic assembly and disassembly of cytoplasmic stress granules (SGs). These structures represent sites of polyA+ mRNA sorting and storage. Unlike P-bodies, mRNAs in SGs are not degraded
but rather stabilized (‘stored’) within this compartment
[26]. By employing live-cell imaging techniques we characterized the mechanism by which HCV triggers SG formation and dynamics and found that SG assembly and
disassembly correlates with phases of stalled and active
RNA translation, respectively. Importantly, SG formation correlates with delayed cell division and prolonged
cell survival, suggesting that oscillation of HCV-induced
stress response contributes to persistence by suppressing
cell death [48]. While it is likely that this strategy faciliBühler/Bartenschlager
Table 1. HCV proteins and their possible contribution to pathogenicity and liver cancer development
HCV protein
Role in the replication cycle
Possible role in pathogenicity
Core
Nucleocapsid
Insulin resistance/steatosis/oxidative stress
Interference (direct or indirect) with p53, p73, and pRb
Interference with host cell signaling pathways
(NF-␬B, Raf1/MAPK, Wnt/␤-catenin)
Interference with TGF-␤ signaling
Transcriptional activation of cellular genes
NS2
Protease/assembly
Modulation of apoptosis
NS3
Protease/helicase
Interference with innate immune response
Interference with NF-␬B
Interference with p53
NS4B
Induction of ER stress
NS5A
Interference with protein ubiquitination
Inhibition of PKR activity
Induction of oxidative stress
Modulation of transcription of cellular genes
Activation of signaling pathways (STAT-3, NF-␬B, etc.)
Activation of phosphatidyl inositol-4-kinase
Accumulation of ␤-catenin by indirect mechanisms
NS5B
Sequestration of Rb
tates persistent infection by HCV, it remains to be determined whether this indirect mechanism of suppression
of cell death plays a role in HCV-associated liver cancer.
Association of HCV Infection with Steatosis and
Development of Liver Cancer
One of the hallmarks of HCV infection is the induction
of steatosis, which is a condition that contributes to tumor
development. In fact, using the JFH1-based cell culture
system we observed a ⬃3-fold higher amount of lipids in
HCV-infected Huh7 cells versus noninfected control cells.
This elevated lipid synthesis might be brought about,
among other things, by the induction of a massive ER
stress response that in turn could activate the SREBP (sterol regulatory element-binding protein) pathway. SREBP
is an ER-resident inactive transcription factor that under
conditions of ER stress is transported to the Golgi. There
SREBP is cleaved by proteases liberating a cytosolic protein fragment that is transported into the nucleus and that
activates transcription of genes required for the synthesis
of cholesterol and for fatty acid homeostasis [27].
Why does HCV cause such profound alterations of intracellular lipid pools? On one hand, enhanced lipid synPromotion of Hepatocellular Carcinoma
by HCV
thesis appears to promote viral RNA replication and
virus production; on the other hand, it might reflect a
‘collateral damage’ caused by viral exploitation of key enzymes involved in lipid synthesis and intracellular lipid
storage. With respect to the first scenario, one has to keep
in mind that HCV replication occurs on ER-derived
membranes. Thus, efficient assembly of viral replication
complexes requires the rearrangement of existing membranes but probably also their novel synthesis. Early studies by Egger et al. [28] described that profound membrane
alterations are mainly induced by NS4B. Overexpression
of this highly hydrophobic protein induced the formation
of single membrane vesicles forming clusters and appearing as a ‘membranous web’. Subsequent studies suggest
that this web is the site of HCV RNA replication [29]. Using electron microscopy and tomography in combination
with 3D reconstructions, we observed in HCV-infected
human hepatoma cells numerous membrane alterations
[Romero-Brey et al., unpubl. data]. Most prominent were
double membrane vesicles that were most abundant and
that might be sites of RNA replication. In addition, at late
stages after infection we detected multi-membrane vesicles that probably reflect cellular stress-induced reactions
including autophagic responses.
Dig Dis 2012;30:445–452
449
The interaction of the HCV core protein with lipid
droplets (LDs), which are intracellular storage organelles
for neutral lipids (mainly triacylglycerol and/or cholesterol esters) [reviewed in 30–32], is another example illustrating how this virus alters lipid homeostasis of the
cell. We and others could demonstrate that assembly of
HCV particles is tightly linked to LDs, which are sites
where structural and nonstructural proteins accumulate
during assembly [12, 33–35]. Importantly, HCV core protein impacts lipid metabolism in several ways: (i) by inhibition of LD mobility, (ii) by decreasing the lipid turnover
in core-coated LDs, (iii) by inhibition of MTP (microsomal triglyceride transfer protein), (iv) by inhibition of
VLDL secretion, and (v) by inhibition of DGAT-1 (diacylglycerol acyltransferase 1) [36–41]. These alterations
appear to play important roles in pathogenesis. For instance, in an elegant recent study it was shown that mice
lacking DGAT-1 are protected from HCV core-induced
lipid accumulation [42]. This could be ascribed to a decrease in lipid turnover in core-coated LDs. Thus, HCV
promotes steatosis both by decreasing lipid mobilization
and by reducing lipid release via interference with VLDL
secretion.
In vivo Studies on the Role of HCV in Promoting
Liver Cancer
Studies of the role of HCV in promoting liver cancer
development are limited by the lack of an HCV-permissive and immunocompetent animal model. Apart from
humans, only chimpanzees are susceptible to HCV infections, but owing to ethical concerns and high costs their
use is virtually impossible. To overcome these limitations,
two alternative attempts are pursued: first, the generation
of transgenic animals expressing HCV protein(s), and
second, the development of an immunocompetent and
HCV-permissive mouse model. With respect to the first
approach, most studies have focused on transgenic mice
expressing constructs containing the core protein or
NS5A. It appears that, depending on the used mouse
strain, expression construct, or experimental conditions,
steatosis and occasionally HCC develop [reviewed in 16].
While these results support the notion that HCV proteins
can promote tumor development, with one exception,
only polyprotein fragments or isolated protein (core or
NS5A) have been expressed and tumors have only been
found in the C57BL/6 mouse background. Thus, the relevance of these observations for infections in humans remains to be determined.
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Dig Dis 2012;30:445–452
With respect to the second approach, in the last few
years several transgenic mouse models have been established that are, however, immunodeficient and rely on the
transplantation of human liver xenografts [43–45]. The
first model, originally described by Kneteman and coworkers used a upa/SCID mouse strain that expresses the
toxic urokinase plasminogen activator (upa) selectively in
the liver [43]. Soon after birth, the upa transgene is expressed and a high proportion of mouse hepatocytes are
destroyed. During this phase, animals can be rescued by
transfer of primary human hepatocytes that migrate into
the liver and form human liver cell ‘nests’ in an otherwise
murine liver architecture. Engrafted human hepatocytes
can be infected productively with HCV, but since these
animals are immunodeficient to avoid graft rejection,
this system is not useful for studies of HCV-associated
carcinogenesis. An improvement of this mouse model
was recently described by Washburn et al. [46]. They generated AFC8-hu HSC/Hep mice by co-transplanting human CD34+ hematopoetic stem cells (HSC) and hepatocyte progenitors (Hep) into mice that were transgenic for
caspase 8 that was expressed under control of the albumin receptor promoter (ACF8). Expression of caspase 8
in liver cells is toxic, thus creating ‘space’ for transplantation of human liver cells. The authors could show that
these AFC8-hu HSC/Hep mice supported HCV infection
in the liver and mice developed an HCV-specific T cell
response that was, however, very weak [46].
More recently, Dorner et al. [47] established a genetically humanized mouse model. By adenoviral transfer of
the HCV entry factors CD81 and occludin into mouse
liver cells, the animals became infectable but did not support HCV replication to a directly detectable level. To
overcome this limitation, the authors made use of the
Rosa26-Fluc mouse strain that expresses an inactive luciferase reporter gene that can be activated upon expression of the CRE recombinase. Upon infection of these
animals with an engineered HCV expressing this recombinase, a small but detectable fraction of inoculated liver
cells started to fluoresce, indicating infection of the cells
and expression of the (HCV-encoded) CRE recombinase.
Although this mouse model is still very inefficient, for the
first time HCV replication in an inoculated immunocompetent transgenic mouse was detected. It is obvious
that much more work will be required to improve this
mouse model to a level that supports the complete HCV
replication cycle, but only with the availability of such an
immunocompetent in vivo system will it become possible
to study the contribution of (persistent) HCV infection to
the development of liver cancer.
Bühler/Bartenschlager
Acknowledgement
Disclosure Statement
Work in the authors’ laboratory was supported by the Deutsche
Forschungsgemeinschaft (Transregio 77, TP1; FOR1202, TP1).
The authors declare that no financial or other conflict of interest exists in relation to the content of the article.
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