Hepatocellular carcinoma (HCC) represents the second most common cause of death from
cancer worldwide, and was responsible for nearly 746 000 deaths in 2012 1]–3]. In patients with cirrhosis, HCC is the most common cause of death. Worldwide, chronic
hepatitis B virus infection remains the major risk factor, with 80Â % of cases occurring
in eastern Asia and sub-Saharan Africa. In most countries, the mortality rate of HCC
approximates the incidence, which is increasing 4]–6]. This is partly due to the rising prevalence of advanced fatty liver disease and
chronic hepatitis C, alongside other risk factors such as hepatitis B infection and
alcohol-related cirrhosis. Some progress has been made with prevention, for example
emerging antiviral agents and vaccination for hepatitis B. However, the vast majority
of HCC cases are associated with fibrosis, and 90Â % of tumours develop in cirrhotic
livers 4], 5], 7]–10]. Furthermore, liver disease severity markers correlate with tumour formation 4]–6], 9], 11]–14]. Currently there are no effective anti-fibrotic therapies available to halt the fibrosis-cirrhosis-HCC
continuum. Patients who present with early disease may benefit from resection, transplantation
or loco-regional therapy, however many are unsuitable for curative treatment due to
advanced malignancy, or the severity of co-existing liver disease. The multi-tyrosine
kinase inhibitor sorafenib is the only available systemic chemotherapy agent with
survival benefit for advanced stage HCC, however its use is limited to those with
well-preserved liver function 11]. Whilst there is scope to optimize our use of existing treatments, for example by
targeting tumours earlier and combining local and systemic approaches, efforts to
broaden our chemotherapy armamentarium have been disappointing. Numerous molecular
therapies with robust preclinical evidence for efficacy have failed to show benefit
in clinical trials. This may in part reflect the abnormal tumour microenvironment,
which acts to support the persistence and growth of cancer cells, and has resulted
in the peri-tumoural stroma and its cellular inhabitants becoming an intense area
of study in the search for efficacious therapies for HCC.
In this review we focus on the complex interplay between hepatic stellate cell (HSC)
biology and hepatocarcinogenesis. The mechanisms by which HSC may facilitate HCC development
and progression are likely to involve diverse biological processes including regulation
of extracellular matrix (ECM) turnover, growth factor and cytokine signalling, promotion
of tumour angiogenesis and immunomodulation. We will discuss how this burgeoning area
of research may yield exciting new therapies for patients with HCC.
Role of the stroma in hepatocarcinogenesis
The stroma is a central component of both hepatic fibrosis and carcinogenesis, and
is a key player in the cellular and molecular mechanisms linking these processes.
It is still unclear, however, whether liver fibrosis specifically promotes HCC, or
if it is merely a wound-healing by-product of chronic hepatic injury and inflammation,
with no direct impact on liver cancer formation 8], 13]–15]. Evidence would suggest the former; the identification of gene signatures from non-tumoural
tissue correlating with late recurrence of HCC, supports the concept of a ‘field effect’
in cancer development 9], 11], 13], 14], 16]–25].
Following liver injury, quiescent HSC become activated to matrix-secreting myofibroblasts
and are the major source of ECM proteins during liver fibrogenesis 8], 13], 26]. As master regulators of the fibrotic matrix, HSC may therefore directly influence
HCC formation via effects on the tumour stroma. Furthermore, it is well established
in other systems that complex intercellular signalling networks exist between tumours
and cancer-associated fibroblasts, contributing to cancer initiation, growth and progression
8], 13], 16]–19], 21]–26]. Tumour secretion of cytokines such as transforming growth factor-? (TGF-?), stimulate
myofibroblast activation leading to profound changes in ECM composition and organization.
Therefore, HSC or HSC-secreted products may be either permissive or necessary for
oncogenesis and HCC persistence. In other cancers, the identification of pathways
that the tumour depends upon for growth and proliferation, so-called “oncogenic addiction
loops†has led to revolutionary therapeutic approaches. The landmark discovery of
the protein kinase oncogene BCR-ABL and subsequent development of imatinib, allowed
curative treatment of chronic myeloid leukaemia, and has paved the way for targeted
therapies in other malignancies 27], 28]. Despite extensive genomic profiling of HCC, targeting other non-kinase oncogenes
such as RAS and MYC has proven more challenging. The identification of promising candidate
pathways targeting inhibition of a driving molecular alteration, which is also applicable
in a significant proportion of patients, remains an elusive yet alluring goal 29]. Furthermore, the microenvironment may modulate susceptibility to inhibition of specific
oncogenic pathways. Straussman et al. developed a co-culture system to test the ability of 23 stromal cell types to influence
the susceptibility of 45 different cancer cell lines to 35 therapeutic agents 7]. They demonstrated that stroma-mediated resistance to anti-cancer drugs (especially
targeted agents) is common. In particular, although melanomas expressing mutant BRAF
respond to vemurafenib, hepatocyte growth factor (HGF) secretion by peri-tumoural
stromal cells correlated with resistance to vemurafenib-induced cell death 7], 30], 31]. This illustrates the importance of stroma-derived resistance to chemotherapy, in
many different organs and disease settings. Therefore, in the search for key driver
mutations in HCC, the effect of the microenvironment cannot be underestimated. This
may necessitate combinations of chemotherapeutic agents, to neutralize specific stromal
interactions, resulting in greater overall clinical efficacy.
HSC in HCC
It is well-known that activated HSC infiltrate HCC stroma and peri-tumoural tissue,
and are localised around tumour sinusoids, fibrous septae and the tumour capsule 32]–34]. Activated HSC have also been identified around the periphery of dysplastic nodules
within the liver 35]. Following activation to the myofibroblast phenotype, HSC secrete substantial amounts
of ECM proteins into the stroma. Fibrotic matrix deposition and degradation by HSC
is tightly regulated in the liver. For example, tissue inhibitors of metalloproteinases
1 (TIMP-1) secretion favours scar deposition by inhibiting the endogenous matrix-degrading
activities of various matrix metalloproteinases (MMPs). However, the balance of TIMPs
and MMPs is complex; activated HSC are also a major source of MMP-2 in vitro, elevation of which has been correlated with increased tumoural collagen I, extracellular
remodeling, and HCC progression 12], 36], 37]. Interestingly, the biomechanics of the ECM are also relevant. Differentiation of
primary hepatocytes is inhibited by culture on a stiff collagen gel, with accompanying
promotion of proliferation 38], 39]. In vitro increasing matrix stiffness has also been shown to directly stimulate growth of the
HCC cell lines, HuH-7 and HepG2, and reduce chemotherapy-induced apoptosis 40]. Integrin ?1 signalling was an integral driver of this response, via Fak, Erk, Pkb/Akt
and Stat3 pathways 40]. Furthermore, stromal stiffness is self-perpetuating, causing stellate cell activation,
and therefore further fibrosis 15], 41], 42]. Data in humans support these experimental findings. Ultrasound elastography has
demonstrated that measurements of liver stiffness predict HCC development 43]–46]. Similarly, established HCC demonstrates further increases in matrix stiffness, more
so than the peri-tumoural hepatic parenchyma 47]. The mechanical tension provided by an altered ECM is likely to act on HCC development
and progression via outside-in signalling, for example by integrins, (discussed below)
to support tumour growth and progression. This has also been observed in other malignancies,
such as a mouse model of breast cancer 48]. Hepatocarcinogenesis in the context of cirrhosis, however, is a unique model of
diseased ECM, and an ideal setting to further characterise and potentially target
stromal drivers.
Integrins as mediators of HSC/HCC crosstalk
Consisting of an ?- and ?-subunit, integrins form a family of transmembrane receptors
that ‘integrate’ the extracellular and intracellular environments through binding
ECM and the cytoskeleton 49]. Via transduction of signals between the internal and external cellular domains,
integrins regulate cell adhesion, spreading, migration, proliferation and differentiation
as well as ECM deposition and remodelling 50].
In activated HSC downstream integrin signalling, via the focal adhesion kinase (FAK)-phosphatidylinositol
3-kinase (PI3K)-Akt signaling pathway, promotes ECM deposition 51]. Increased ECM stiffness in vitro enhances integrin expression and activity and focal adhesion formation, 48] with subsequent activation of downstream integrin signalling within the hepatocyte
that may nurture the growth and survival of precancerous cells. Matrix stiffness has
been reported to dictate differentiation and chemotherapeutic resistance of human
HCC cell lines, with softer matrices abrogating hepatoma proliferation and stiffer
platforms promoting proliferation 40], 52]. In an elegant in vivo study, cells from the HCC cell line McA-RH7777 were implanted into rats treated with
carbon tetrachloride (CCl4) for varying lengths of time, thereby modelling tumourigenesis on different liver
stiffness backgrounds. Microarray analysis of the tumours demonstrated a positive
correlation between matrix rigidity and tumour angiogenesis 52]. Correlations between collagen expression, integrin expression and tumourigenicity
have also been reported in human HCC and murine HCC models 53], 54]. Characterisation of integrin expression in hepatoma cell lines has revealed a high
degree of heterogeneity in integrin expression 55]. Comparing two clinically relevant mouse models of HCC, platelet-derived growth factor
(PDGF)-C overexpressing and PTEN null mice, Lai et al. demonstrated that each model had a specific pattern of integrin gene expression,
further indicating HCC heterogeneity 54].
The ?1 integrin subfamily has been extensively studied in the context of HCC, and
hepatocarcinogenesis is associated with the enhanced expression of integrins ?1?1,
?2?1 and ?3?1 and the acquisition of a migratory phenotype by hepatocytes 56]–58]. Further, assessment of integrin ?1 expression in human HCC tissues demonstrated
a positive correlation with ECM stiffness, pathological grade and metastasis 59]. Blockade of integrin ?1 in vitro significantly abrogates migration and invasion of HCC cell lines induced by TGF-?1
and epidermal growth factor (EGF) 58], 60]. Conversely, overexpression of integrin ?1 has been reported to enhance HepG2 cell
migration 61]. More recently it has been reported that integrin ?1 is involved in the transduction
of ECM signalling into HCC cells, resulting in the downstream activation of angiogenic
signalling 52]. Utilising a high-stiffness gel to culture HCC cell lines Dong et al. found that vascular endothelial growth factor (VEGF) expression is suppressed by
treatment with an integrin ?1-specific antibody 52]. SERPINA5 (Protein C inhibitor), a member of the serine protease inhibitor superfamily
know to have anti-metastatic and anti-angiogenic effects, 62] is down-regulated in human HCC tissues and further assessment of it’s anti-tumourigenic
activity demonstrated that this was mediated by effects on the fibronectin-integrin
?1 signalling pathway 63]. The relationship between integrin ?1 and ECM stiffness in HCC is further highlighted
in a study where resistance of the HCC cell line, Hep3B, to sorafenib was found to
be mediated by integrin ?1 and its downstream effector JNK 64].
Other integrin subunits, in addition to ?1, have been reported to have key roles in
HCC progression. Fan et al. have reported integrin ?6 expression to strongly correlate with HCC metastasis in
humans 65]. Integrin ?6 overexpression in HCC cell lines (utilising a viral short hairpin RNA-mediated
strategy) revealed that integrin ?6 can form a complex with CD151, a tetraspanin protein
also associated with HCC invasion 65]. Further investigation in vivo indicates that the CD151/?6 complex stimulates the PI3K-Akt signalling pathway leading
to enhanced epithelial-mesenchymal-transition (EMT) of HCC cell lines 65].
Crosstalk between integrins and TGF-? signalling has also been studied in hepatocarcinogenesis.
TGF-? receptor I (TGF-? RI) activation has been reported to promote HCC cell invasiveness
through phosphorylation of the intracellular portion of the ?1 subunit of the ?5?1
integrin via Smad-2 and Smad-3, leading to an inside-out conformational change and
stimulating vascular invasion 66]. Up-regulation of other integrins including ?3?1 and ?6?1 by TGF-?1 has also been
reported, leading to increased tumour invasiveness into surrounding tissues 67]. Furthermore specific crosstalk between fibronectin-binding integrins and TGF-?1
can promote cell cycle progression in HCC cells through activation of c-Src 68]. Crosstalk between integrins, growth factor receptors and ECM proteins including
collagen, have further been shown to alter downstream signal transduction pathways
such as Smad, promoting both hepatocyte proliferation and sustaining HSC activation
69], 70]. TGF-?1 has also been reported to modulate ?5?1 expression and synergistically enhance
integrin-mediated FAK phosphorylation and cell adhesion in the HCC cell line SMMC-7721
71]. Therefore, integrins (via modulation of TGF-? signalling) may render hepatocytes
less sensitive to pro-apoptotic signals in early HCC stages, and more sensitive to
tumourigenic differentiation and metastasis formation in advanced HCC.
HSC growth factor signalling
HSC have been shown to favour HCC tumourigenicity, potentially as a result of a change
in their secretory phenotype upon activation. In vitro studies, using conditioned media from activated HSC, have consistently reported increased
proliferation, migration and invasion of tumour cells 72]–74]. Isolation and subsequent co-culture of human intratumoural HSC with hepatoma cell
lines enhanced their viability and migratory capacity 72]. Furthermore, co-transplantation with HCC cells into nude mice promoted tumour formation
and growth 75]. Utilising both co-culture and conditioned media from primary human HSC Giannelli
and colleagues determined Laminin-5 to be a mediator of HSC-induced HCC migration
via its activation of the MEK/ERK pathway 76]. This is supported by in vivo experiments, in which co-transplantation of murine activated HSC with murine HCC
cells (H22 line) into immunocompetent mice resulted in significantly larger tumour
volumes 73]. Furthermore, implantation of human HCC cell lines (PLC and Hep3B) into nude mice
did not form tumours unless activated HSC were concurrently implanted 72]. HepG2 cells did form tumours when implanted alone, however tumour growth was more
rapid when co-transplanted with activated HSC 72]. Activated HSC secrete a broad range of growth factors including HGF, TGF-?, fibroblast
growth factor (FGF), EGF, VEGF and insulin-like growth factor (IGF). The following
sections discuss how these growth factors are involved in HCC pathogenesis.
Hepatocyte Growth Factor
HGF is expressed by HSC and myofibroblasts, 77], 78] and is a highly potent hepatocyte growth factor regulating cell proliferation, migration,
survival and angiogenesis 79]–82]. As such it is widely regarded as a key factor for tumour cell invasion and metastasis
83]. HGF binding to its receptor, c-MET, induces receptor homodimerization and a subsequent
phosphorylation cascade. A transmembrane receptor tyrosine kinase, c-MET is found
in 20-48 % of HCCs, 84]–86] and has been shown to be expressed by multiple HCC cell lines 72]. Correlations between increased c-MET and HCC tumour size or invasiveness of HCC
have been reported in some studies 87], 88]. c-MET overexpression is also associated with a reduced five-year HCC survival, and
a c-MET-regulated expression signature has been reported to define a subset of patients
with poor prognosis and an aggressive phenotype 89], 90]. Within HCC tumours, activated HSC have been found to initiate signalling pathways
downstream of c-MET, including NF-?B and ERK leading to tumour proliferation and migration
72], 91].
The pro-tumourigenic activity of fibroblast-secreted HGF has also been reported in vitro. Conditioned media from isolated and activated HSC, pre-incubated with anti-HGF antibodies,
was found to abrogate the proliferative and migration-inducing effects on HCC cell
lines, seen in non-treated conditioned media 72]. This has also been demonstrated in cancer-associated fibroblasts (CAF) isolated
from HCC, where treatment of CAF-conditioned media with an anti-HGF antibody significantly
reduced HCC proliferation in Hep3B and MHCC97L cell lines 74]. Moreover, a HGF/c-MET specific antagonist, NK4, has been found to inhibit markedly
the fibroblast-induced invasion of cancer cells, both in vitro and in vivo,92]–94] although this has yet to be translated into the clinical setting. A murine model
of HCC with similarities to the human disease was recently developed, in which progressive
fibrosis and cirrhosis, initiated by ectopic expression of PDGF-C, precedes hepatocyte
dysplasia and eventual HCC development 95]. Analysis of these PDGF-C transgenic mice demonstrated that expression of hepatic
HGF and its receptor were elevated at the time point at which dysplastic foci are
present, further suggesting a pro-tumourigenic role for HGF. Activation of HGF/c-MET
signalling has also been shown to enhance HCC chemoresistance. Conditioned media from
the activated HSC cell line LX-2 enhanced resistance of the HCC cell line Hep3B to
the chemotherapeutic agent cisplatin, an effect mediated by HGF 96]. Tumour cells may also potentiate pro-metastatic c-MET signalling via an autocrine
mechanism involving TIMP-1, leading to downstream expression of metastasis-promoting
genes 97], 98].
However, HGF signalling is not unidirectional. A high level of bi-directional crosstalk
between tumour cells and stromal cells, in particular fibroblasts, has been reported.
Nakamura and colleagues have reported the expression of HGF inducers in several carcinoma
cell lines, including squamous cell carcinoma, human epidermoid carcinoma, human non-small
cell lung cancer cells, human cholangiocarcinoma cells, and SBC-3 human small cell
lung carcinoma cells 99]. These HGF inducers include interleukin (IL)-1?, FGF, PDGF and TGF-? and were reported
to up-regulate HGF expression by stromal fibroblasts 99], 100]. Taken together, these studies highlight that HGF and aberrant c-MET signalling have
a critical role in mediating the bi-directional crosstalk between HSC and tumour cells
during hepatocarcinogenesis.
Transforming growth factor-?
The large latent TGF-? complex is secreted by most cell types, including human HSC
and hepatocytes 101], 102] and fixed in the ECM by transglutaminase-dependent linkage of latent TGF-? binding
protein to fibronectin and other ECM proteins, forming a reservoir of latent TGF-?.
In the context of HCC, it has been suggested that defective TGF-? signalling promotes
tumourigenesis secondary to reduced responsiveness to the anti-proliferative effects
of TGF-? signalling 103], 104]. However, TGF-? appears to exhibit multiple roles in HCC pathogenesis. Tumour-suppressor
functions are observed in the early stages of liver damage and regeneration, whereas
during cancer progression, TGF-? may exacerbate tumour invasiveness and metastatic
behavior 105]. It has further been demonstrated that TGF-? and PDGF signaling crosstalk supports
EMT and is crucial for tumour growth and the acquisition of an invasive phenotype
106].
The survival and malignancy of HCC cell lines, including Huh7 and HepG2, have been
reported to require autocrine TGF-? signalling, with exogenous TGF-? leading to growth
inhibition of HCC cells 107]. Utilising HCC cell lines, Meindl-Beinker et al. revealed a heterogeneic response to TGF-?, reflective of different stages and mechanisms
of disease. Variation between cell lines in their endogenous TGF-? and Smad7 levels,
and their transcriptional activity of Smad3, was related to the maintenance of TGF-?
cytostatic activity. In particular, the Hep3B, HepG2 and PLC hepatoma cell lines were
found to have low TGF-? and Smad7 levels and strong Smad3 transcriptional activity
and were thus sensitive to TGF-? cytostatic activity, representative of the early
stages of chronic liver disease 108]. In an analysis of TGF-? gene expression in HCC patients, Coulouarn et al. reported that those tumours displaying an invasive phenotype and increased recurrence
were characterized by a late TGF-? signalling signature, with transcriptional activation
of genes associated with matrix remodelling and cell adhesion 109].
Therefore, as the role of TGF-? in HCC pathogenesis appears to be highly context-dependent,
exhibiting both pro- and anti-tumoural activity, it is highly unlikely that pan-TGF-?
blockade will provide a useful therapeutic avenue in HCC treatment. More selective
strategies to interfere with TGF-? signalling, perhaps even at a cell-specific level,
will likely be required to modulate this signalling pathway for therapeutic gain in
the context of HCC.
Epiregulin
The gut microbiome is increasingly recognized as a powerful modulator of fibrosis,
cirrhosis, and infectious complications in chronic liver disease. Much interest is
currently focused on the translocation of bacterial pathogen-associated molecular
patterns (PAMPs), which activate inflammatory responses through Toll-like receptors
(TLRs). Recently Dapito et al. demonstrated that Tlr4mut mice (harbouring non-functional TLR4) that received diethylnitrosamine (DEN) and
CCl4 show 80-90Â % reduction in HCC tumour size and number, compared with mice expressing
wild-type TLR4 110]. Gut sterilisation significantly reduced this effect whereas LPS treatment enhanced
it, suggesting a role for the LPS-TLR4 pathway in promotion of hepatocarcinogenesis.
Interestingly, alongside hepatocytes, HSC were identified as candidates for TLR4-dependent
tumour promotion in the chronically injured liver. LPS and the gut microbiome were
found to induce HSC activation, resulting in production of the mitogens HGF and epiregulin,
which likely act on malignant hepatocytes. Epiregulin is a member of the EGF family,
and results in EGF receptor and human epidermal growth factor receptor 2 activation
during early stages of DEN/CCl4 carcinogenesis, whereas it reduces hepatocyte apoptosis by NF-KB nuclear translocation
during later stages 110], 111]. This suggests that there may be merit in evaluating whether long-term antibiotic
treatment confers any protection against HCC development. This could initially be
investigated by following up patients with cirrhosis on long-term prophylaxis for
spontaneous bacterial peritonitis or encephalopathy, although identifying a comparable
control group may prove challenging.
HSC and angiogenesis
Angiogenesis has a critical role in HCC initiation, progression and metastasis, as
reflected by the efficacy of sorafenib, which targets this process. The rapid growth
pattern of malignant hepatocytes requires new vessel formation, stimulated by multiple
pro-angiogenic factors. This pro-angiogenic environment in turn supports tumour progression
and metastasis. The relevance of tumour vascularity is reinforced by the observation
that VEGF expression progressively increases from low-grade dysplasia to early-stage
HCC 112]. VEGF overexpression is also associated with high tumour grade, and vascular and
portal vein invasion 113]–117]. Furthermore, raised plasma VEGF and angiopoietin 2 (Ang-2) are independent predictors
of poor prognosis in advanced HCC 118].
HSC are known to secrete VEGF as well as other angiogenic factors including PDGF,
MMPs, FGF, TGF-?1, EGF, angiopoietin-1 (Ang-1) and Ang-2 119]–121]. Upon activation, HSC express multiple smooth muscle cell markers, suggesting they
may act like pericytes during angiogenesis 122], 123]. They also express angiogenic growth factor receptors, such as VEGF receptor, PDGF
receptor and Tie-2 124]–126]. In liver injury and HCC, this facilitates reciprocal signalling between HSC and
endothelial cells or malignant hepatocytes and contributes towards a pro-angiogenic
microenvironment. VEGF secretion by HSC can be hormonally induced by leptin, or by
physical stress such as hypoxia, and is upregulated in HCC 120], 124], 127]. VEGF receptor upregulation also occurs during HSC activation, resulting in increased
mitogenesis in response to VEGF 13].
Conditioned media from HCC cells can activate HSC and stimulate VEGF production. Coulouarn
et al. co-cultured LX2 cells with HepRG HCC cells, and analysis of differential gene expression
identified a gene network linked to VEGFA and MMP9 128]. This was shown to promote angiogenesis, as conditioned medium from LX2-HepaRG coculture
(but not LX2 or HepaRG medium alone) induced tubule complex formation by primary human
umbilical vein endothelial cells. A gene signature of this cross-talk correlated with
poor prognosis and metastasis in humans 128].
Lin et al. have also shown increased angiogenesis by activated HSC in vitro using a murine HCC cell line (H22) and rat colon microvascular endothelial cells
129]. They went on to demonstrate in vivo, using an orthotopic HCC model, that activated HSCs promote tumour vascularisation
via increased VEGF and possibly PDGF secretion.
Of particular interest in HCC is the interaction between malignant hepatocytes, endothelial
cells and activated HSC. Torimura et al. characterised expression of Ang-1, Ang-2 and Tie2 receptors in HCC cell lines (HLE
and HuH-7) and human HCC cases 130]. They concluded angiopoietin-Tie2 signalling in the vascular wall may act in favour
of vessel remodelling in HCC. Ang-2 production by hepatoma cells, HSC and smooth muscle
cells binds Tie2 (on HSC, smooth muscle and endothelial cells) and destabilises connections
between endothelial cells, perivascular support cells and ECM. This allows exposure
to VEGF, which in these relatively hypoxic conditions, is upregulated. Proliferation
of endothelial cells ensues, allowing neovascularization and further tumour growth.
Recently, it has been shown that metformin inhibits angiogenesis in vitro, in an HCC (HepG2 line) and HSC (LX2) co-culture system 131]. This was associated with reduced VEGF production. It was postulated that metformin
was acting via AMPK activation, and specifically targeting HSC in this model. Indeed,
inhibition of AMPK on LX2 cells (but not on HepG2 cells) using siRNA did restore VEGF
levels and abrogate metformin’s anti-angiogenic effect. Metformin would seem a promising
candidate for human HCC treatment, but unfortunately retrospective data would suggest
a lack of survival benefit 132]. However, considering the well-established tolerability of metformin, its potential
HSC-mediated effect on angiogenesis merits further investigation.
Some of the factors mediating crosstalk between HSC and HCC are summarised in Fig. 1.
Fig. 1. Crosstalk between HSC and HCC. HSC-secreted factors such as HGF may promote hepatocarcinogenesis.
Similarly, HCC signalling results in further HGF production from activated HSC. TGF-?
demonstrates both tumour-suppressive and tumour-promoting functions, depending on
context. HSC produce angiogenic cytokines, supporting new vessel growth. HCC cells
contribute to angiogenic signalling, and HSC also possess receptors for some of these
factors. Gut-derived LPS induces HSC activation, resulting in epiregulin and HGF production,
with mitogenic effects on HCC
HSC and immunomodulation
Tumour immune evasion is now regarded as a hallmark of cancer progression and is therefore
a very active area of research. One mechanism by which tumours evade the immune response
is through the augmentation of the numbers and activity of immunosuppressive cells,
at both the tumour site and within lymphoid organs 133]. Such cells include regulatory T-cells (Tregs) and myeloid-derived suppressor cells
(MDSC). Increased levels of Tregs within peripheral blood and tumours have been reported
in human HCC cases, and have further been shown to suppress anti-tumour immune responses
in addition to promoting angiogenic remodeling 134]–136]. Further, intratumoural Treg accumulation has been reported to correlate with disease
progression and poor prognosis 137]. MDSC are defined by the markers CD11b and Ly6-C/G and have been found in the tumour,
lymph nodes and blood, suppressing cellular responses to cancer cells 138].
The immunosuppressive activities of HSC have only recently been recognised with studies
demonstrating, both in vitro and in vivo, that activated HSC are able to strongly suppress T-cell responses. Investigation
into the divergent immunomodulatory activity of quiescent and intratumoural HSC has
revealed that, in vitro, intratumoural HSC induce T-cell hyporesponsiveness, an effect not seen with quiescent
HSC 139]. Moreover, in an orthotopic rat model of HCC, intratumoural HSC number strongly correlated
with T-cell apoptosis and lung metastatic nodules 140]. Although a direct interaction was not reported, this does suggest an additional
role for HSC in HCC metastasis via an immunosuppressive mechanism.
Co-transplantation of HCC cells and HSC into immunocompetent mice promoted HCC proliferation
and enhanced tumour angiogenesis, in association with inhibition of lymphocyte infiltration
and apoptosis of infiltrating monocytes 73]. In an orthotopic model of HCC, activated HSC in tumour-bearing mice significantly
increase Treg and MDSC populations in the spleen and tumour stroma 141]. An increase in tumour vascular and lymphatic vessel density was also reported in
those tumours co-transplanted with HSC.
Investigation into the mechanisms underlying HSC immunomodulatory effects in HCC has
demonstrated that this may be mediated via upregulation of human B7 homolog 1 (B7-H1;
programmed death ligand 1 (PDL-1)) on tumoural HSC 142]–144]. B7-H1 can act as both receptor and ligand and has immunosuppressive functions such
as promoting activated T-cell apoptosis and inhibiting T-cell-mediated tumour cell
apoptosis 1], 145], 146]. Its counter-receptor, PD-1, is expressed on activated, but not resting, T-cells,
B-cells and monocytes 2]. B7-H1/PD-1 signaling has been reported to promote Treg cell induction and immunosuppressive
function through the down-regulation of mTOR and AKT phosphorylation 147], 148]. In vitro experiments involving incubation of T-cells with anti-B7-H1 monoclonal antibody resulted
in a significant reduction in HSC immunomodulatory activity and HCC migration and
invasion 139].
Three monoclonal antibodies against PD-1, and one against B7-H1 have been developed
and promising Phase 1 data has been reported 149]. In one study, varying degrees of tumour regression were found in colon, renal and
lung cancers and melanoma and a significant increase in tumour lymphocyte infiltration
was noted 150]. This has been extended to a second clinical trial where responses were seen in 16
out of 39 patients with advanced melanoma 151]. These early clinical studies further demonstrated encouraging safety data. In the
context of HCC, a Phase 1, dose escalation study investigating the effects of anti-PD-1
therapy is currently underway in patients with advanced HCC (NCT01658878), however
results have yet to be reported. Some of the immunomodulatory effects of HSC in HCC
are summarised in Fig. 2.
Fig. 2. Immunomodulatory effects of HSC in HCC. Intratumoural HSC (iHSC) promote HCC progression
through i) an increase in Treg cell induction and immunosuppressive function and ii)
upregulation of B7-H1 on iHSC resulting in increased ligation of its receptor (PD-1)
on activated T-cells, leading to increased apoptosis of activated T cells with subsequent
inhibition of T-cell-mediated tumour cell apoptosis. This results in HCC immunotolerance
and a permissive environment for tumour growth. PD-1, programmed death ligand; B7-H1
human B7 homolog 1; ECM, extracellular matrix
Therapeutic approaches to targeting HSC and HSC signalling
HSC represent a small percentage of cells within the liver, and specific therapeutic
targeting of HSC remains challenging. Recently, transgenic mice have been developed
that allow reliable fluorescent labeling or genetic manipulation in HSC and myofibroblasts
152], 153]. These transgenic mice will hopefully prove useful not only in elucidating the molecular
mechanisms in HSC that regulate the stroma-HCC interface, but also in facilitating
the identification of rational, new therapeutic targets in hepatocarcinogenesis.
If a targetable, HSC-dependent pathway driving hepatocarcinogenesis is identified,
cell-specific therapy is conceivable, albeit not entirely straightforward. ECM homeostasis
is a key physiological process and modifying HSC functions may impair this, with potential
for severe adverse effects. Practically, delivering drugs to HSC is hindered by a
lack of multiple transport receptors and endocytic capacity. Furthermore, candidate
compounds may include siRNA and cytokines, which have a short half-life in plasma
following systemic administration, hindering therapeutic efficacy 154].
To overcome these problems, a number of groups have explored active targeting of HSC
to deliver therapeutic compounds. This involves coupling the selected compound to
a carrier possessing a specific receptor-binding ligand, or an antibody.
Carriers recently employed have included an antibody to the synaptophysin receptor
on HSC, and a liposome specific to the vitamin A receptor on HSC 155], 156]. Furthermore Poelstra et al. have used proteins substituted with a sugar moiety that binds the mannose-6-phosphate-IGFII
receptor 157]. They have also utilised a peptide that binds the PDGF receptor-?, 158] to deliver a protein or an adenovirus to HSC 159], 160]. An RGD-peptide which binds to RGD-binding integrins has also been used to create
a carrier that accumulates in HSC 161], 162]. Of note, the carrier molecules used must fit strict criteria such as low immunogenicity,
and high stability, biocompatibility and selectivity, if they are to translate into
clinical practice. Moreover, the target receptors on HSC should be selectively expressed
and ideally upregulated during disease activity. A further challenge is the requirement
for endocytosis of the construct following target receptor binding. This can be particularly
problematic in the case of biological therapeutics, which usually fail to withstand
the endosomal degradation process.
With these challenges in mind, Bansal et al. subsequently developed a recombinant protein construct to deliver interferon gamma
(IFN?) to HSC 163]. This elegant system transported the signalling moiety of IFN? to the PDGF-receptor
with a carrier molecule that was simplified and miniaturised. They found that IFN?
could be effectively delivered to human HSC in vitro, and to mouse HSC in vivo. Furthermore, the targeted fusion proteins were shown to ameliorate hepatic fibrosis
in CCl4-treated mice 163]–165]. This suggests that directing a cytokine to HSC is a feasible and potentially tractable
therapeutic approach, both in the context of developing new treatments for patients
with liver fibrosis, as well as HCC. Therapeutic approaches to targeting HSC are summarised
in Fig. 3.
Fig. 3. Therapeutic approaches to targeting HSC. HSC have been targeted by coupling a compound
to a carrier possessing either a HSC-specific receptor-binding ligand or an antibody.
Carriers utilised include: a monoclonal human single chain antibody (scAb) fragment
to synaptophysin 155]; a sugar moiety that binds the mannose-6-phosphate (M6P) insulin-like growth factor
receptor 157]; a liposome specific to the vitamin A (retinol-binding protein) receptor 156]; PDGF?-peptide 160]; PDGF? receptor recognising peptide (PPB) 164]; an RGD peptide bound to a liposome or coupled to human serum albumin (HSA) 159], 162] scAb Fv, single chain antibody variable fragment; PEG, polyethylene glycol; pCVI,
10 cyclic peptide moieties that recognise collagen type VI receptors