Hepatic Stellate Cells in Hepatocellular Carcinogenesis: Possible Therapeutic Targets?

Cristin Constantin Vere; Alin Gabriel Ionescu; Costin Teodor Streba; Otilia Rogoveanu

doi:10.4172/2161-069X.S12-006

ISSN: 2161-069X

Journal of Gastrointestinal & Digestive System

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Hepatic Stellate Cells in Hepatocellular Carcinogenesis: Possible Therapeutic Targets?

*Cristin Constantin Vere^1,2, Alin Gabriel Ionescu¹, Costin Teodor Streba^1,2 and Otilia Rogoveanu¹**
¹University of Medicine and Pharmacy of Craiova, Romania
²Research Center of Gastroenterology and Hepatology of Craiova, Romania
Corresponding Author :	Costin Teodor Streba Research Center of Gastroenterology and Hepatology Bvd. 1 Mai nr. 66, Craiova 200638, Romania E-mail: costinstreba@gmail.com
Received March 29, 2013; Accepted May 03, 2013; Published May 05, 2013
Citation: Vere CC, Ionescu AG, Streba CT, Rogoveanu O (2013) Hepatic Stellate Cells in Hepatocellular Carcinogenesis: Possible Therapeutic Targets? J Gastroint Dig Syst S12:006. doi:10.4172/2161-069X.S12-006
Copyright: © 2013 Vere CC, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis, following inflammatory processes within the parenchyma. Their activation pathways give way to a cascade of phenomena which are potentially dangerous for the liver metabolism at a cellular level. The changes towards fibrosis pave the way for the evolution of hepatitis into cirrhosis, the most important etiological entity of hepatocellular carcinoma. In this review, we try to cover a few of the basic aspects of the intricate mechanisms that govern the complex activation of HSCs, their involvement in carcinogenesis and how these molecular targets may become valuable in the future therapeutic approaches for primary liver carcinomas.

Keywords

Hepatocellular carcinoma; Hepatic stellate cells; Carcinogenesis; Fibrosis; Antifibrotic therapy

Introduction

Activation of hepatic stellate cells (HSCs) following chronic liver inflammation and injury, is a major phenomenon in the initiation and progression of liver fibrosis - major risk factor for hepatocellular carcinoma (HCC) [1].

Activated HSCs involved in hepatocarcinogenesis by initiating autocrine signaling mediated by transforming growth factor- β (TGF- β) and β-catenin accumulation in the nucleus of neoplastic hepatocytes [2,3]. TGF-β synthesized by activated HSCs stimulates tumor progression of neoplastic hepatocytes and also induce epithelial cell transformation into mesenchymal cells.

One hypothesis regarding the initiation of HCC tumorogenesis concerns the combined effect of several growth factors synthesized by activated HSCs: platelet-derived growth factor (PDGF), fibroblast growth factors (FGF) 1 and 2, as well as the insulin-like growth factor (IGF) [4].

In CHC, HSCs significantly increase the activation of signaling pathways mediated by nuclear factor kappaB (NF-kB) and extracellular regulated kinases (ERK). NF-kB pathways and MAP kinase/ERK are involved in HCC progression by stimulating tumor cell proliferation and inhibition of apoptosis [5]. Several immunohistochemical studies have reported an increase in activated HSCs in the tumor sinusoids, fibrous septa, and tumor capsule [6,7].

Recent studies have highlighted the major role of HSCs both in inhibiting the immune response in the liver and in stimulating neoangiogenesis in patients with chronic viral hepatitis infection [8-11]. It has also been demonstrated experimentally that HSCs are involved in the development of liver metastases as a result of the inflammatory response generated by stimuli from gastrointestinal neoplasms, various carcinomas and malignant melanoma [12]. In vitro studies and murine models based xenografts demonstrated that HSCs can be activated by HCC cells and contribute via growth factors, both in progression and increase aggressiveness in HCC [2,13,14]. Enzan et al., by immunohistochemical studies regarding the involvement of HSCs in carcinogenesis, demonstrated that activated HSC found in both intratumoral and peritumoral stroma stimulate the development of HCC [6,15].

Liver injury initiates fibrogenesis through signaling molecules released by hepatocytes, inflammatory cells and other cells, especially endothelial sinusoidal and Kupffer cells.

Factors Involved in HSC Activation

Oxidative stress

By generating reactive oxygen species (ROS) plays an important role in hepatic injury and fibrogenesis initiation. Hepatocyte necrosis and apoptosis occur due to oxidation of lipids, proteins and DNA, followed by amplification of the inflammatory response and the onset of fibrogenesis [16].

Hypoxia

It occurs in fibrogenesis by stimulating the release of hypoxia inducible factor (FIH)-1α by CSH. In turn, FIH-1α stimulates cell growth factor vascular endothelial (VEGF), which increases the synthesis of collagen type I by CSH [17].

Chronic liver inflammation

It leads to activation of hepatic Kupffer cells which release proinflammatory cytokines locally, such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) [18,19]. Kupffer cell activation leads to increased NF-kB activity and a subsequent release of pro-inflammatory cytokines, including TNF-α and monocyte-chemoattracting protein-1 (MCP-1), which triggers the activation of HSCs [20]. CSH, in turn, respond to this stimulation by releasing macrophage colony stimulating factor (M-CSF), MCP-1 and IL-6, leading to an increase in acute phase response, accompanied by a further increase in activated macrophages.

Hepatocyte apoptosis

It is involved in fibrogenesis and the pro-inflammatory stimulation through apoptotic bodies phagocytized by the Kupffer cells [21].

Fatty liver fibrogenesis

It occurs indirectly by increasing oxidative stress and hepatocyte susceptibility to enter apoptosis by altering cellular response to injury, stimulating and activating the peroxisome proliferator-activated receptor (PPAR), and by altering the synthesis and leptin signaling [22].

HSC activation

It is composed of two major phases: initiation (preinflammatory stage) and perpetuation, followed by a phase of resolution, where hepatic aggression ceases [23]. Initiation is the emergence of early genetic and structural changes of HSCs, followed by emphasizing their response to cytokine stimulation. Initial paracrine stimulation, including signals from damaged hepatocytes, Kupffer cells and endothelial cells lead to early activation of HSCs and changes in the surrounding extracellular matrix (ECM). Once the activation process is triggered, perpetuating the result of continuous stimulation leads to the maintenance stage of activated HSCs.

HSC Involvement in Carcinogenesis

Mechanisms involved in the initiation and progression of hepatocellular carcinoma intertwine, not being strictly defined. A number of studies have shown that oxidative stress is induced at the molecular level in chronic liver aggression of any etiology, thus fulfilling a major role in fibrogenesis and HCC occurrence [24-28].

HSCs are an important source of reactive oxygen species (ROS) in fibrogenesis [26,29]. In hepatocytes, the main source of ROS is the cytochrome P450 2E1. In both Kupffer cells and HSCs the main sources of ROS are the phagocytic and non-phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [26,30,31].

Phagocytic form of NADPH oxidase synthesized in Kupffer cells intervenes in the defense against bacterial products that reach the liver through the portal system. ROS have proinflammatory effects which sensitize hepatocytes to apoptotic stimuli, thus being involved in fibrogenesis and carcinogenesis. Recent in vitro studies have shown that activated HSCs synthesize the non-phagocytic form of NADPH oxidase and thus demonstrated the involvement of ROS in other HSC activation and fibrogenesis [29-32]. Hence, multiple ROS-generating parenchymal and nonparenchymal cells contribute directly to the formation and activation of pathways involved in either fibrogenesis or carcinogenesis. Several studies in animal models of chronic liver disease showed the major role of cytokines and growth factors in fibrogenesis [33,34] and HCC occurrence [28].

TGF-β1 is the main cytokine that is involved in hepatic fibrogenesis, having an important role in activating myofibroblasts [35]. TGF-β, TNF-α and matrix metalloprotease-9 (MMP-9) are synthesized by activated Kupffer cells and fulfill a role in activation, cell proliferation, increased collagen I synthesis and release of retinoids by HSCs [36-38]. Liver inflammation is induced through different mechanisms by other molecules having profibrogenic activity, such as PDGF, which has a strong mitogen effect [39]. Brenner et al. reported increased levels of TGF-β in HCC correlated with decreased accumulation of collagen and its degradation, characteristic changes and liver fibrogenesis [35].

Connective tissue growth factor (CTGF) is involved in fibrogenesis by reshaping MEC due to its ability to stimulate the synthesis of MMP and tissue inhibitor of metalloproteases (TIMPs), thus having the potential to enable synthesis and degradation of MEC . Liu et al. [40] have shown, in animal models of xenografts, increased canonical Wnt signaling pathway activity/β-catenin protein core of hepatitis C virus (HCV) and HCC cells. Following this signaling, CTGF synthesis is enhanced, which accelerates tumor growth, invasion and migration, but not angiogenesis, due to binding of vascular endothelial growth factor (VEGF), and hence its suppression. Human HCC cell line synthesizes high levels of CTGF to form stromal rich tumors [40,41].

As a result of paracrine stimulation of neoplastic cells through TGF-β, cancer associated fibroblasts (CAF) synthetize CTGF [42]. CAF originate from endothelial cells and can trigger their endothelial - mesenchymal transdifferentiation [42]. A recent study demonstrated the involvement of CAF generated by stellate cells in increasing resistance to chemotherapy or radiotherapy associated with pancreatic cancer [43]. By analogy, activated HSCs can be a source of CAF in HCC.

Toll-like receptors (TLRs), TLR2 and especially TLR4, play an important role in identifying endogenous ligands released from damaged cells or apoptosis [44,45].

HSCs inflammatory signaling involved in triggering innate immune response through recognition of TLR4 are identified immunohistochemically on the surface of activated HSCs and Kupffer cells. An animal model study showed a reduction of macrophage infiltration and local relief of experimentally induced liver injury and fibrosis by genetic deletion of TLR4. As a result of blocking the TLR4 ligands, it activates an intracellular signaling pathway that includes the activation of NF-kB [46,47]. TLR activation can be triggered by hepatitis B and C viruses, alcoholic liver disease and nonalcoholic steatohepatitis (NASH), all involved in HCC development [48].

Yu et al. [49] conducted an experimental study on mice with chemically induced HCC by diethylnitrosamine, which showed a decrease in the number of TLR4 and genes responsible for myeloid differentiation 88 (MyD88), but not TLR2 receptor deficiency. In this study, they reported a decrease in the incidence, size and number of chemically induced cancer cells, thus demonstrating the important role of TLR4 in the initiation of hepatocarcinogenesis [50]. Apoptotic hepatocytes following diethylnitrosamine action activates and stimulates TLRs through both myeloid cells - Kupffer cells and HSCs to synthesize proinflammatory cytokines that can initiate the appearance of HCC.

A recent study showed that activated HSCs promote carcinogenesis by increasing the activity of NF-kB mediated signaling pathway [5]. Transcription factors of NF-kB play a major role in regulating the adaptive and innate immune response, inflammation and cell survival [50,51].

Blocking apoptosis leads to initiation of tumor genesis and occurs due to DNA mutations, with NF-kB signaling initiation. The first argument for NF-kB involvement in carcinogenesis is given by encoding a subunit of NF-kB by protein c-rel, a cellular homologue of the v-rel oncogene. These proteins share a domain with Rel homologue that binds to DNA [52]. Oncogenic transformation is favored by increased synthesis of normal Rel proteins. Recent studies have shown that activation of NF-kB is involved in the initiation and progression of HCC. Initiation of carcinogenesis is linked in particular to NF-kB signaling pathway involved in controlling a number of processes such as proliferation, apoptosis, angiogenesis, invasion and metastasis [52,53].

TNF-α, an important trigger activation of NF-kB, is synthesized by macrophages and activated HSCs and plays a major role in inflammation and is also an accelerator of cell proliferation factor [53]. Once activated, NF-kB is involved in controlling the synthesis of a high number of antiapoptotic factors such as cIAPs, c-FLIP and BclX, play a role in blocking cancer cell apoptosis [54].

Both in vivo and in vitro studies have demonstrated the role of NF-kB inhibition of activated HSC apoptosis through a mechanism involving blocking JNK signaling cascade and AP-1 pathway responsible for modulating apoptosis [55,56].

Damaged liver tissue, activated HSCs proliferate following PDGF stimulation, achieved through mitogen activated proteinkinases (MAPKs) of JNK, ERK and p38 types. JNK and ERK activation induces proliferation of activated HSCs, while p38 activation inhibits their proliferative response [33,57].

By phosphorylation of a number of genes associated with carcinogenesis, JNK plays an important role in both the initiation and progression of HCC, by promoting the synthesis of several angiogenic factors. VEGF stimulates endothelial cell proliferation and migration; its synthesis by activated HSCs is also mediated by the activation of JNK [58,59].

Epithelial-Mesenchymal Transformation (EMT) can occur at hepatic level. Hepatocellular EMT was identified both in patients and in animal models by showing epithelial markers synthesized by HCC cells. In the case of well-differentiated HCC, E-cadherin was identified immunohistochemically in both tumor hepatocyte membrane and in those noncancerous from adjacent parenchymal tissue. In the case of poorly differentiated HCC, E-cadherin is localized in the cytoplasm or is frequently absent. Impaired E-cadherin complexes/β-catenin from the cell membrane is characteristic of hepatocellular EMT [60]. Decreased synthesis of E-cadherin is associated with nuclear translocation of β-catenin, with a significant correlation with intrahepatic metastasis and unfavorable prognosis in patients with HCC [60]. Activated HSCs are indirectly involved in initiating hepatocellular EMT through autocrine signaling mediated by TGF-β and β-catenin accumulation in the nucleus of neoplastic hepatocytes [2].

HSCs activated due to signaling from tumor cells, stimulates angiogenesis by increasing VEGF synthesis [61,62]. In angiogenesis associated with tumor progression of HCC, in addition to VEGF synthesis by activated HSCs, also intervenes VEGF synthesized by malignant transformed hepatocytes [63]. Cells involved in tumor progression by affecting EMC remodeling and angiogenesis by inducing angiopoietin secretion [61,62]. Local hypoxia activates HSC by stimulating the release of hypoxia inducible factor (FIH)-1α, thus triggering fibrogenesis. In turn, FIH-1α stimulates VEGF synthesis, leading to an increase in type I collagen synthesis by activated HSCs [17], enhancing angiogenesis [64].

Activated HSCs synthesize and secrete laminin-5 (Ln-5), thus activating the signaling pathway MEK/ERK which has a role in stimulating migration of HCC cells. In vitro experiments have demonstrated the involvement of HSCs in HCC cell proliferation and migration through proteins and growth factors synthesized by them, involved in activation of ERK signaling pathway [5,65].

A recent immunohistochemical study showed the role of activated HSCs in HCC progression, their presence in peritumoral tissue being correlated with increased vascular invasion and aggressive forms of HCC [66]. CSH are involved through several mechanisms modulating the invasive phenotype of HCC cells. The first mechanism is the synthesis and release by activated HSCs of Ln-5, this isoform of laminin promoting strong adhesion as well as tumor invasion and migration [65].

Another mechanism by which activated HSCs induce the appearance of a tumor with an aggressive phenotype and higher invasiveness is through secretion of Ln-5 which triggers the TEM of HCC cells [67]. Ln-5 is involved in carcinogenesis through its G4-5 domain, as demonstrated in a study aimed at the MEK/ERK activation pathway in squamous cell carcinoma [68].

The Role Of Inhibition and/or Induction of Apoptosis CSH and Neoadjuvant Therapy in Prophylaxis of HCC

Reducing oxidative stress, which triggers activation of HSCs, is a potential therapeutic target in preventing fibrogenesis and thus HCC occurrence. An experimental study on animal model with liver cirrhosis induced by common bile duct ligation, reported a reduction of liver fibrosis by reducing oxidative stress after administration of IGF-1, an important regulator of the intermediary metabolism [69].

Another therapeutic target in preventing carcinogenesis is the blocking cytokine signaling pathways mediated by cytokine receptor antagonists. Deepening the role of growth factors in fibrogenesis and carcinogenesis studies Trigger antagonists of cytokines and their receptors. Highlighting the importance of cytokines in the pathogenesis of proliferative fibrosis that occur in signaling pathways of HSCs, and PDGF, fibroblast growth factor, TGF-α and response to tyrosine kinase receptors led to the emergence of inhibitors that block these signaling pathways. Thus, several inhibitors were discovered, such as the gamma-linolenic acid, lipo-oxygenaze and PPAR gamma receptor [70,71].

Inhibitory effects of cytokines interferon-γ and HGF on HSC were observed in experimental animal models, where the degree of activation of HSCs was significantly reduced [72]. HGF’s antifibrotic mechanism is uncertain, but it appears to act by inhibiting the activity of TGF-β1 [73].

A number of studies have highlighted the major role of signaling pathways mediated by TGF-β and PDGF both in tumorogenesis and in fibrogenesis and subsequent activation of HSCs [74]. Mikula et al. reported a decrease in liver fibrosis and tumor progression by inhibiting cell signaling pathway mediated by TGF-β between hepatocytes and activated HSCs with an antagonist Smad7 [2]. A recent study showed a decrease in fibrosis and tumorogenesis after blocking signaling pathways mediated by PDGF and TGF-β from the CSH activated by a tyrosine kinase inhibitor PTK/ZK [75].

Okuno et al. observed in an animal model that administering camostat mesylate lowers activated TGF-β, followed by a reduction in the progression of liver fibrosis [76]. A further study, conducted on mice, showed that imatinib mesylate, a tyrosine kinase receptor inhibitor, causes a reduction of hepatic fibrosis by a significant decrease in proliferation and migration of HSCs induced by PDGF-BB, and a decrease in both α -SMA and synthesis of α 2 - (I)-procollagen mRNA in activated HSCs [77].

Blocking the signaling pathway mediated by NF-kB is another potential therapeutic target in preventing carcinogenesis. Pentoxifylline is a methylxanthine derivative with antifibrotic properties that lowers α I collagen synthesis by activated HSCs by inhibiting degradation of I kappa b α, which in turn blocks the activation of nuclear factor kappa-B (NF-kB) [78]. Anan et al. showed that bortezomib, a protease inhibitor, induces apoptosis of HSCs by blocking NFκB activity due to increased half-life of its inhibitors [79].

Apoptosis is the main mechanism responsible for the reduction of activated HSC during liver lesion healing [80]. Several mediators of apoptosis such as Fas/FasL, TNF receptors and Bcl/Bax were identified in the HSC, so a possible therapeutic target could address through these mediators triggering apoptosis [81,82].

Activated HSCs synthesize an excess of TIMP-1 and TIMP-250 which inhibits interstitial colagenases, reducing degradation of MET and its buildup. TIMP-1 also exerts an antiapoptotic effect on activated HSCs [83].

TIME antagonists represent a therapeutic target aimed at inhibiting the synthesis of collagen I and triggering apoptosis activated HSCs. TIMP-1 plays an important role in HSC survival by blocking apoptosis of these cells directly. TIME antagonists induce apoptosis of HSCs, leading to a decrease in liver fibrosis and a decreased risk of HCC [84].

Natural killer cells (NK), in addition to their role in innate immune response, are involved in limiting liver fibrosis by neutralization of activated HSCs [85,86] and antifibrotic cytokine release and INF α and γ [72,87].

HSC apoptosis was obtained in the absence of oxidative stress in an animal model following administration glitoxin, a fungal metabolite, through mitochondrial cytochrome c release and caspase-3 activation and ATP depletion, responsible for reducing fibrogenesis [88].