New molecular targets for hepatocellular carcinoma: the ErbB1 signaling system

Authors


Correspondence
Matías A. Avila, PhD, Associate Professor of Biochemistry and Molecular Biology, Division of Hepatology and Gene Therapy, CIMA, Universidad de Navarra, Avda, Pio XII No 55. 31008 Pamplona, Spain.
Tel: +34 948 194700
Fax: +34 948 194717
e-mail: maavila@unav.es

Abstract

Hepatocellular carcinoma (HCC) is a major cause of cancer-related deaths. This malignancy is often diagnosed at an advanced state, when most potentially curative therapies are of limited efficacy. In addition, HCC is a type of tumor highly resistant to available chemotherapeutic agents, which leaves HCC patients with no effective therapeutic options and a poor prognosis. From a molecular perspective, HCC is a heterogeneous type of tumor. However, in most cases, HCC emerges on a background of persistent liver injury, inflammation and hepatocellular proliferation, which is characteristic of chronic hepatitis and cirrhosis. Recent studies have revealed that dysregulation of a limited number of growth and survival-related pathways can play a key role in HCC development. The epidermal growth factor receptor (ErbB1) can be bound and activated by a broad family of ligands, and can also engage in extensive cross talk with other signaling pathways. This system is considered as an important defense mechanism for the liver during acute tissue injury; however, accumulating evidences suggest that its chronic stimulation can participate in the neoplastic conversion of the liver. Agents that target the ErbB1 receptor have shown antineoplastic activity in other types of tumors, but their efficacy either alone or in combination with other compounds has just started to be tested in experimental and human HCC. Here, we review the evidences that support the involvement of the ErbB1 in HCC development and that provide a rationale for ErbB1 targeting in HCC prevention and treatment.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths and a malignancy with increasing worldwide incidence (1–3). This disease is more prevalent in Middle Africa and parts of Asia, with more than half of the patients being reported from China. In industrialized countries an increase in HCC incidence and mortality has been observed, being attributed to increased prevalence of hepatitis C virus (HCV) infection. It is predicted that this trend will continue in the coming years due to the high numbers of patients who became infected before the 1990s and the slow progression from HCV infection to HCC, which may take more than two decades (2, 3). Most HCC cases have a very poor prognosis, with a number of new cases per year that almost equals the number of deaths attributed to liver cancer for the same year (4). This dramatic situation stems in part from the high resistance of HCC to available chemotherapeutic agents administered either alone or in combination, the deteriorated condition of the cirrhotic liver on which most of HCC cases develop and the lack of reliable markers that allow early detection of the disease and tumor resection (5).

As mentioned above, epidemiological studies clearly indicate that HCC is strongly associated with chronic liver diseases, including chronic hepatitis and liver cirrhosis (2–4). From this perspective, among other cancers HCC is probably the type of tumor for which the causative agents are better defined. Besides HCV infection, chronic hepatitis B virus (HBV) infection represents a significant risk for HCC development, with a 25–35 times higher risk of HCC incidence than non-infected individuals, while coinfection with HCV and HBV results in a 130fold risk increase (6, 7). Viral infection, together with aflatoxin B1 exposure, may account for over 90% of all HCCs (2), while other risk factors include chronic alcohol consumption leading to alcoholic cirrhosis, hereditary hemochromatosis and obesity accompanied by insulin resistance and non-alcoholic fatty liver disease (8–10).

Like other solid tumors, HCC is characterized by the accumulation of numerous genomic alterations, including aneuploidy and abnormal chromosome numbers, allelic losses, microsatellite instability, epigenetic changes and alterations in gene expression patterns that do not involve structural abnormalities (11–14). These lesions accumulate slowly along the preneoplastic stages and progression of the tumor, and to a certain extent reflect the nature of the etiologic agent, as illustrated by the finding of increased chromosomal instability and p53 mutations in HBV infection (12, 13, 15). Mutations and/or changes in levels of expression have been observed in a number of key genes involved in the control of cell proliferation and survival, such as p53, p73, Rb, mdm2, APC, β-catenin, E-cadherin, p16, gankyrin, the Ras inhibitors RASSF1A and NORE1A, c-myc and cyclin D1, among others (4, 12–17). HCCs are thus molecularly heterogeneous tumors, and no consistent pattern of genetic lesions corresponding to the different stages of tumor development has been found so far.

The majority of HCCs slowly unfold in a background of chronic hepatitis and cirrhosis, which can be considered as preneoplastic conditions of the liver. Chronic hepatitis is characterized by persistent inflammation, cytokine and oxidative stress-mediated hepatocyte death and active proliferation of residual hepatocytes to replace the lost parenchyma (4, 12). The capacity of liver cells to activate regenerative and survival mechanisms is outstanding, and likely evolved to protect the liver from the toxic effects of xenobiotic or endogenous compounds that have to be metabolized in this organ (18). Long-standing hepatitis, mainly because of chronic viral infection or alcohol consumption, leads to hepatic cirrhosis characterized by loss of liver function and profound alterations of liver architecture, due to the formation of scar tissue and fibrous septae that encompass regenerative nodules of hepatocytes (4, 19–21). Within these regenerative nodules arise monoclonal cell populations derived from cells that may have accumulated genetic alterations. These nodular lesions can contain phenotypically altered hepatocytes and may progress to dysplastic nodules, with enhanced rates of proliferation and proportionally decreased rates of apoptosis, which have been correlated with increased risk of development of HCC (4, 22–24). Therefore, a pro-inflammatory and proliferative tissue microenvironment is a common characteristic in the preneoplastic liver regardless of the etiology. Moreover, gene expression profiling studies of human HCCs identified two distinct subclasses with differential survival of the patients; the identity of the genes in the group with a poorer prognosis supported the idea that an imbalance between cell proliferation and death is central to HCC progression (17). Active research in the past few years has identified a number of growth factors and signaling pathways involved in the support of cell proliferation and survival from the early stages of hepatocarcinogenesis, and in the autonomous growth and drug resistance of transformed HCC cells. Among these signaling systems, those controlled by insulin-like growth factor (IGF), Wingless (Wnt), hepatocyte growth factor (HGF), transforming growth factor-β (TGFβ) and the epidermal growth factor (EGF) family of growth factors are believed to play a prominent role. Most of these growth factors, receptors and signaling cascades are part of the tightly controlled regenerative and protective natural responses of the liver to acute tissue injury. However, when chronically stimulated or dysregulated through a series of molecular and functional changes, they can contribute to neoplastic transformation and the maintenance of the transformed phenotype of HCC cells (12, 14, 25–29). Interestingly, there is increasing evidence that hepatitis B and C viral proteins can directly interact with cellular components of growth factor signaling cascades. Such interactions can result in a direct oncogenic effect that may act independently or in cooperation with the hyperproliferative response triggered by chronic inflammation and hepatocellular death, and contribute to explain the enhanced risk of hepatocellular transformation observed upon chronic viral infection (30–32).

A detailed knowledge of the key molecular pathways involved in HCC cell growth, survival and metastasis should lead to the development of more effective targeted therapies with greater selectivity and reduced side effects. The above-mentioned growth factor signaling pathways dysregulated in HCC represent prominent targets for specific therapeutic intervention. In fact, different strategies targeting signaling systems like the EGF axis in other solid tumors such as non-small-cell lung carcinomas (NSCLC), pancreatic and colorectal carcinomas have demonstrated promising clinical benefit (33–35). Accumulating preclinical and emerging clinical data obtained with EGF receptor (EGFR) inhibitors suggest that targeting the EGF axis could also be a promising therapy for HCC. In the present article, we review the evidence supporting a role for the EGF signaling system in HCC development and the basis for EGF-targeted therapy in this type of tumor.

ErbB family of receptors and ligands

The EGFR family is composed of four different membrane receptors endowed with tyrosine kinase activity. Besides EGFR, also known as ErbB1, it includes ErbB2, ErbB3 and ErbB4 (36–38). All these proteins share a common structure showing an extracellular ligand-binding domain, a transmembrane domain that makes a single pass through the plasma membrane and an intracellular domain where the tyrosine kinase activity resides (Fig. 1) (39). The tyrosine kinase domain is followed by a carboxy-terminal tail with tyrosine autophosphorylation sites (37–39). This domain is highly conserved among the different members of the family, except for ErbB3, in which key amino acids have been substituted, rendering this receptor devoid of tyrosine kinase activity (38). The extracellular ligand-binding domain of the different ErbB receptors contains two cysteine-rich regions and is less well conserved, in concordance with the differential specificity of these receptors for ligand binding. With the exception of ErbB2, which has no ligand, the ErbB receptors can be bound and activated by a family of growth factors encoded by 11 different genes (36–38). These ligands are characterized by the presence of an EGF-like domain, which dictates receptor-binding specificity, and additional motifs like an immunoglobulin-like domain, heparin-binding sites and glycosylation sites (38). According to their binding specificities ErbB ligands can be classified into three different categories. EGF, amphiregulin (AR), TGFα and epigen (EPG) specifically bind ErbB1, while β-cellulin (BTC), heparin-binding EGF-like growth factor (HB-EGF) and epiregulin (EREG) bind ErbB1 and ErbB4. The neuregulins (NRGs), which can exist in different alternatively spliced isoforms, bind ErbB3 and ErbB4 (Fig. 1). This pattern of ligand/receptor interactions is further complicated by the fact that upon ligand–binding, ErbB receptors can homo- or heterodimerize and cross-phosphorylate each other. In these interactions the ligandless ErbB2 and the kinase-defective ErbB3 receptors also participate; in fact, the most potent heterodimer in terms of triggering proliferating and survival signals is the ErbB2/ErbB3 heterodimer (37, 38). Ligand-binding activates ErbB autophosphorylation in distinct tyrosine residues, thus creating docking sites for several signaling proteins such as Shc, Grb7, Grb2, Crk, phospholipase Cγ (PLCγ) the kinases Src and PI3K, the protein phosphatases SHP1 and SHP2 and the Cbl E3 ubiquitin ligase (37–39). Other signaling proteins, such as phospholipase D (PLD) and the STAT 1, 3 and 5 proteins, do not bind to the ErbB receptors through the C-terminal phosphotyrosines, but are also activated upon ligand binding (39, 40). These interactions trigger intracellular signaling cascades such as the ras/raf/MEK/MAPK pathway (including the activation of ERK and JUN NH2-terminal kinase-JNK), p38 mitogen-activated protein kinase (p38-MAPK), the protein kinase C (PKC) pathway, the PI3K/Akt pathway (which can lead to NF-kB activation) and the STAT pathway (37–41). These pathways, which show a high degree of interaction, activate different transcriptional programs in the nucleus, leading to the expression of a battery of genes involved in cell cycle progression, apoptosis resistance, differentiation, adhesion and cell migration. In spite of the apparent redundancy of ErbB signaling, specificity within this signaling system can be achieved at different levels. Besides the above-mentioned preference of each ligand for a given ErbB receptor (or heterodimerization partner), differences in the preferred intracellular signaling pathway also exist. For instance, upon heterodimerization, the cytoplasmic domain of the kinase-deficient ErbB3 undergoes tyrosine phosphorylation and can recruit PI3K to six different sites, although there is no site for Grb2, PLC or for the Ras-specific GTPase-activating protein (GAP), which in turn can be recruited by ErbB1 (37). Obviously, signaling specificity can also be gained at the physiological level, when we consider the tissue specific and temporal pattern of expression of the different ErbB receptors and ligands.

Figure 1.

 ErbB1 family of receptors and their cognate ligands.

All the ErbB ligands are synthetized as transmembrane precursors that are proteolytically processed to release the soluble growth factor from the surface of the cell (42). The enzymes that mainly mediate the shedding of the different ErbB ligands have been identified as members of the ADAM family (a disintegrin and metalloprotease), which are also membrane-anchored proteins endowed with metalloprotease activity (43, 44). Different members of the numerous ADAM family have been involved in ErbB ligand cleavage, including ADAM 9, 10, 12, 15, 17 and 19 (44). However, ADAM17, also known as tumour necrosis factor-α (TNFα)-converting enzyme or TACE, is believed to play a central role (43). This metalloprotease can cleave the AR, TGFα and HB-EGF membrane-anchored precursors, and interestingly it can also cleave several membrane receptors including ErbB4 and the HGF receptor Met (45). The complexity of ErbB signaling is further increased by recent findings showing that the carboxy-terminal cell-associated remnant of ligands generated upon precursor cleavage can translocate to the nucleus and interact with transcriptional regulators. This has been cogently demonstrated for the carboxy-terminal fragment of HB-EGF, which can enter the nucleus, where it binds and promotes the nuclear export of the PLZF transcriptional repressor, resulting in increased expression of cyclin A and cell cycle progression (46).

Taking into account all these considerations, ErbB ligands can function either in a juxtacrine manner, when they are still attached to the cell surface, or in an autocrine or paracrine manner after being released from the cell surface (47–49). As previously stated, autocrine or paracrine signaling by ErbB ligands needs the proteolytic release of the soluble growth factor by ADAM-type metalloproteases. Therefore, the regulation of ADAM activity is an important step in the modulation of ErbB signaling, and adds another layer of complexity to the system. Most ADAMs contain predicted signaling motifs in their cytoplasmic domains such as phosphorylation sites and proline-rich regions that can interact with SH3 domains (43). Certain G-protein-coupled receptors (GPCRs) have been shown to activate ErbB-mediated signal transduction in the absence of a direct interaction of the GPCR ligand with the ErbB (43, 44, 50). This event is known as ErbB receptor transactivation, and has been described for a variety of GPCRs agonists such as thrombin, carbachol, lysophosphatidic acid, endothelin-1 and angiotensin II (50). The signaling between GPCRs and ADAM activation is not completely known; the involvement of PKC, reactive oxygen species, Src kinases and calcium ions that in turn activate Ser/Thr or Tyr ADAM phosphorylation has been proposed (Fig. 2) (44). The GPCR/ADAM/ErbB axis is thought to play a critical role in stimulating cell growth and progression of several carcinomas, including breast, lung and prostate carcinoma (50, 51). Although data on the implication of this pathway in HCC are lacking, the relevance of ErbB signaling for HCC development, together with the expression of certain GPCR receptors and ADAM 17 in liver tumor cells, suggests a potential role for ErbB receptor transactivation in liver cancer.

Figure 2.

 Cross talk between the ErbB1 receptor and other signaling systems relevant to Hepatocellular carcinoma (HCC) development.

ErbB signaling system in liver injury and HCC development

As mentioned previously, upon tissue injury the liver triggers a powerful defensive response aimed at the protection of the organ and at the restoration of the lost parenchymal mass (18, 19). This is a complex response mediated by a network of cytokines, comitogens and growth factors in a coordinate multistep process. There are at least two growth factor signaling systems that appear to be critically involved in liver regeneration: the HGF and its receptor c-Met, and the ErbB axis (52). In fact, the hepatocytes of the mature liver express the highest levels of ErbB1 of any non-transformed cell (53), and ErbB1 ligands such as EGF, TGFα, AR, HB-EGF and EREG display a potent mitogenic effect on isolated and cultured hepatocytes (54–58). The expression of the ErbB ligands HB-EGF, TGFα EREG and AR is increased shortly after tissue removal in the experimental model of liver regeneration after two-thirds partial hepatectomy (PH) (55, 56, 59, 60). The relative contribution to liver regeneration of most of these ligands has been established in the corresponding knockout mice. Although none of these genetically modified mice showed a complete blockade of DNA replication or cell proliferation after PH, in agreement with the idea that no single gene can be considered essential for liver regeneration (61), different phenotypes were observed. Mice with homozygous deletion of TGFα were the first to be tested, and showed essentially normal liver regeneration after PH, which was attributed to the compensatory effect of the other EGFR ligands (62). A similar observation was recently made in EREG knockout mice (60), while HB-EGF-deficient animals showed delayed hepatocyte DNA synthesis after PH (63). A more prominent phenotype was noticed in mice with homozygous deletion of AR, in which a substantial reduction and delay in DNA synthesis was observed (57). Currently, there is no clear mechanistic explanation for the putatively different role played by the various EGFR ligands that are upregulated during liver regeneration. The differential interplay of these growth factors with other ErbB receptors besides ErbB1, such as ErbB2 and ErbB4, has been invoked (52); however, the expression of these two receptors is not consistently detected in normal or regenerating rodent liver (53). A differential expression and interaction of these growth factors in other liver cell types, such as macrophages or stellate cells (64), or the generation of distinct growth factor-specific intracellular signals elicited by the carboxy-terminal fragments produced after ADAM-mediated cleavage of membrane-bound precursors, could be important and deserve further consideration (47, 65).

The expression and role of certain ErbB ligands have also been examined in experimental models of acute and chronic liver damage, as well as in the liver of cirrhotic patients. For instance, it has been shown that the hepatic expression of HB-EGF, TGFα and AR is increased in mouse liver upon acute CCl4 intoxication (55, 66, 67), while that of AR, EREG and TGFα is also upregulated in the clinically relevant model of Fas-mediated liver injury (67). Interestingly, the expression of AR, which, as opposed to other ErbB ligands, is undetectable in the healthy liver, was readily induced upon CCl4 administration or Fas ligation (57, 67). Moreover, after challenge with a lethal dose of the Fas agonistic antibody Jo2, AR knockout mice displayed an enhanced death rate when compared with wild-type animals (67). Together with the impaired liver regenerative response of these mice after PH, these observations support a non-redundant role for AR among other ErbB ligands as an endogenous defense mechanism of the liver (52).

Besides the information gathered in knockout mice, a number of studies have demonstrated the significant hepatoprotective and proregenerative potential of the ErbB axis. Evidence has been collected using different experimental approaches, including for instance transgenic overexpression (TGFα and HB-EGF) (68, 69), adenoviral gene transfer (HB-EGF) (70) or direct intraperitoneal administration of the recombinant growth factor (EGF and AR) (67, 71), in models of acute liver injury and regeneration. These observations underscore the important function of the EGFR system in liver defense against injury, and suggest their potential therapeutic application to prevent acute liver damage.

A common observation made in the different models of acute liver injury is that upon cessation of the noxious stimuli, the expression of the different ErbB ligands returns to the levels found in normal liver. However, persistent tissue damage results in sustained overexpression and overstimulation of the EGFR pathway, which, as we introduced above, is thought to be an important step toward development of liver cancer. In this respect, overexpression of the ligands AR, TGFα and HB-EGF has been detected in experimental models of chronic liver injury as well as in liver samples obtained from cirrhotic patients (57, 72–75). Similarly, examination of human liver tumor tissues demonstrated the overexpression of TGFα, BTC, HB-EGF and AR in a high percentage of the cases (between 60% and 100% of the samples examined, depending on the growth factor) (75–78). Increased levels of ErbB1 and ErbB3 expression are also a common finding in human HCC tissue. This has been associated with more aggressive tumors, enhanced cell proliferation, and in poorly differentiated HCCs ErbB1 overexpression has been correlated with the occurrence of intrahepatic metastasis and poor survival (79–81). Interestingly, the expression of ADAM17 has recently been reported to be elevated in human HCC and liver cirrhosis (78). Besides these correlations, experimental studies have provided more direct evidence of the actual implication of deregulated ErbB signaling in HCC development, and in the maintenance of the transformed phenotype of liver tumor cells. For instance, mice transgenic for TGFα or EGF show a high tendency to develop HCC (82, 83), while hepatocarcinogen treatment of TGFα null mice results in smaller tumors than in wild-type animals (62). Work carried out in human HCC cell lines has consistently shown increased ErbB1 expression and a proliferative response upon treatment with ErbB1 ligands such as EGF, TGFα, HB-EGF and AR (78, 84). Furthermore, AR silencing by small interfering RNAs in HCC cells results in reduced constitutive ErbB1 signaling, inhibition of cell proliferation and increased apoptosis in response to TGFβ or cytotoxic drugs (78). Similar observations have been obtained when HCC cells were treated with specific inhibitors of ErbB1 tyrosine kinase activity or ErbB1 neutralizing antibodies as will be discussed later (85–88). All in all, these findings support the existence of active autocrine loops in HCC potentially involving different members of the ErbB family, as has been observed in other tumor types (89). These loops can be activated early during the premalignant stages of HCC development, and later on some liver tumors may remain dependent on ErbB-mediated oncogenic autocriny. This phenomenon fits well with the emerging concept of ‘oncogene addiction,’ which is gaining increased recognition, and provides further rationale for targeted therapy in HCC (90).

Cross talk between the ErbB1 system and other signaling pathways relevant to HCC development

We have previously indicated that GPCRs can transactivate the ErbB1 receptor through activation of ADAM metalloproteinases and shedding of different ErbB ligands. Multiple GPCR ligands acting through different members of the ADAM family have been shown to promote the release of mature growth factors and to activate downstream ErbB signaling (40, 50). The mechanisms behind ADAM activation by GPCRs have not been completely elucidated, but they may involve ADAM protein phosphorylation by PKC activation, fluctuations in intracellular Ca2+ or generation of reactive oxygen species (ROS;(44) Fig. 2). An interplay between GPCRs and ErbB signaling seems to be widespread; in fact, HB-EGF, TGFα and AR have been shown to undergo GPCR-stimulated release in a variety of tumor cell lines, leading to tumor cell migration and invasion (50, 91, 92). Although these studies did not involve HCC cell lines, it is tempting to speculate that liver cancer cells will not be an exception as some of these GPCRs are expressed in hepatocytes. In fact, recent observations indicate that angiotensin II and lysophosphatidic acid can activate Erk1/2 phosphorylation through transactivation of the ErbB1 receptor in a rat liver cell line, although the identity of the ErbB1 ligand or ligands involved was not established (93). However, usage of the ErbB system as a ‘signaling hub’ seems not to be restricted only to GPCRs. Accumulating evidences suggest that ErbB receptors serve as points of convergence for growth-regulatory signals arising from many extracellular stimuli (94). These include inflammatory cytokines such as interleukin-1β (IL-1β), IL-8 and TNFα, and also involve the participation of a metalloproteinase activity (95–98). Indeed, ErbB1 transactivation has been demonstrated to mediate the proliferative activity of TNFα in a hepatocyte cell line through the ADAM17-mediated release of TGFα (97), and also TNFα-induced metastatic properties in HCC cells (98). More recently, fibronectin, a glycoprotein that is upregulated and functionally involved in liver fibrosis and HCC development, has been shown to transactivate ErbB1 in HCC cells. This interaction involved the fibronectin receptor integrin αv and the ADAM-mediated release of an unidentified ErbB1 ligand, and was essential to convey the proliferative and invasive stimuli elicited by fibronectin in HCC cells (99). Cross talk between the IGF pathway and ErbB1 has also been demonstrated in various cellular backgrounds by showing that activation of IGF-1 receptor (IGF-1R) leads to the shedding of different ErbB1 ligands including AR, TGFα and HB-EGF (94, 100) (Fig. 2). Given the relevant role attributed to the IGF-II/IGF-1R axis in HCC development and its implication in the resistance toward ErbB1-targeted therapy (27, 100, 101), the interaction between these two systems in liver carcinogenesis must be of significance. In support of this, it has recently been reported that the proliferative effect of IGF-II on human HCC cell lines requires ErbB1 activation through the autocrine/paracrine release of AR (102) (Fig. 2).

An interplay between the ErbB1 receptor and other structurally distinct receptors may take place at a variety of levels, not always involving the shedding of ErbB1 ligands. An example of special relevance regarding liver cancer progression and metastasis is the integral role played by the ErbB1 receptor in HGF-mediated hepatocyte proliferation (103). EGF and TGFα were found to stimulate c-Met phosphorylation and nuclear accumulation of β-catenin through ErbB1 activation in hepatocarcinoma cells. Interestingly, c-Met stimulation involved the production of reactive oxygen species by membrane-bound NADPH oxidases upon ErbB1 binding and activation (104).

Of particular relevance and complexity is the interaction between cyclooxygenase-2 (COX-2) and the ErbB system in HCC. As mentioned before, liver tumors arise on a background of chronic inflammation and hepatocellular injury. In this context, it has been reported that the expression of COX-2 in non-tumorous tissue correlates with the degree of inflammation, and is consistently found in dysplastic nodules and early HCC (26, 105). Accumulating experimental evidences point to a role for COX-2 upregulation in HCC development; these include the antitumoral properties of COX-2 inhibitors and the potent effects of COX-2-generated prostaglandin E2 (PGE2) on HCC cell growth, migratory properties and invasiveness (105, 106). Cross talk with the ErbB1 system can occur at different levels (Fig. 2). Recent studies have revealed that COX-2-derived PGE2 transactivates ErbB1 in human HCC cells, and that this effect is mediated by the prostaglandin EP1 receptors involving the tyrosine kinase c-Src (107) (Fig. 2). Moreover, in this work it was also demonstrated that PGE2 also induced the phosphorylation of c-Met in an ErbB1-dependent manner, which further supports the central role played by the ErbB1 system in the coordination of signals relevant to HCC development. Interestingly, we have recently demonstrated that PGE2 can upregulate the expression of AR in cultured hepatocytes (57), while others have reported that EGF can induce the expression of COX-2 in human HCC cell lines (107). Together, these observations show the existence of an extensive cross talk and positive feedback between these two signaling systems, and suggest that simultaneous targeting of both COX-2 and ErbB1 could result in synergistic antitumor effects with reduced toxicity.

ErbB1 targeted therapy for the treatment of HCC

As described above for HCC, malignant transformation involving ErbB1 dysregulation has been observed for many human cancers. Alterations in the ErbB1 system may involve overexpression of this receptor, activating mutations, alterations in the dimerization process or activation of autocrine growth factor loops (33). Based on these observations, different antineoplastic strategies targeting the ErbB1 network have been devised. These include small-molecule inhibitors (TKIs) that compete with adenosine triphoshate (ATP) binding to the tyrosine kinase domain of the receptor (gefitinib, erlotinib), and monoclonal antibodies (MoAbs) that compete with the binding of activating ligands to the extracellular domain of the receptor (cetuximab; (33, 34) Fig. 3). Significant differences exist in the mechanism of action and specificity of these two types of agents. For instance, MoAbs like cetuximab bind to the ErbB1 receptor with much higher affinity than the natural ligands, and upon binding ErbB1 is internalized and degraded without receptor phosphorylation and activation, resulting in inhibition of downstream signaling pathways (108). However, ErbB1 TKIs block receptor tyrosine kinase activity in a reversible fashion through binding at the ATP-binding site located in the intracellular kinase domain. As opposed to ErbB1-targeted antibodies, TKIs do not induce ErbB1 internalization or degradation, and although these TKIs are designed as specific inhibitors of ErbB1, they may also inhibit other receptor tyrosine kinases. Over the past few years, clinical experience on the use of these agents in the treatment of solid tumors has accumulated. As mentioned previously, antitumor activity has been shown in colorectal carcinoma, squamous cell carcinomas of the head and neck, NSCLC and renal carcinoma (33,34), however data on their application for the treatment of HCC are still scarce. Several ongoing Phase-II trials are testing the efficacy of ErbB1 TKIs and MoAbs, either alone or in combination with each other or with other targeted agents (National Cancer Institute, http://www.cancer.gov); however, their observations have not yet been published. Nevertheless, preliminary data from a recent Phase-II study of erlotinib in patients with advanced HCC showed clinical benefit manifested by disease control (108).

Figure 3.

 ErbB1 targeting strategies and potential mechanisms of resistance in Hepatocellular carcinoma (HCC).

While clinical efficacy awaits confirmation, preclinical evidences support the notion that the growth of HCC can be significantly quelled by ErbB1-targeted agents. In this regard, it has been shown that treatment of cultured human HCC cells with gefitinib or erlotinib results in ErbB1 TK activity downregulation, growth inhibition, apoptosis and cell cycle arrest (86, 87). Similar observations were made with cetuximab, and interestingly the effects of this MoAb were potentiated when combined with either erlotinib or cytotoxic agents such as doxorubicin (89). The in vivo effects of ErbB1 TKIs on HCC have also been tested. Gefitinib treatment was reported to prevent HCC development in an experimental model of HCC in rats (72), to significantly inhibit intrahepatic metastasis in a mouse model of orthotopic HCC implantation (85) and to reduce HCC-induced angiogenesis (109). It is important to notice that previous studies in NSCLC found that activating mutations in the kinase domain of ErbB1 closely correlated with the clinical response to gefitinib (33); however, such mutations have not been found in human HCC samples (110, 111). In light of the growth-inhibitory effects observed for gefitinib on human HCC cell lines, this may suggest that constitutive activation of a ‘wild-type’ ErbB1 is essential for HCC cell proliferation and/or that alternative mechanisms also contribute to the observed activity of TKIs.

Data collected in both in vitro and in vivo studies have also made it clear that the action of these ErbB1-targeted agents can meet a certain degree of resistance, and that combination with other targeted compounds may improve the clinical outcome. For instance, simultaneous blockade of the IGF1-R or COX-2 has been shown to enhance the antiproliferative effects of gefitinib and erlotinib, respectively (87, 102). Such findings underscore the role that pathways that extensively cross talk with the ErbB1 axis may play in liver cell transformation and in compromising the efficacy of ErbB1 targeting. In fact, the development of acquired resistance to ErbB1-targeted drugs is a major concern in targeted cancer therapy, and several molecular mechanisms have been invoked. Taking into account the experience gathered in other types of tumors, these mechanisms include the autocrine or paracrine production of ligands, receptor mutations, the constitutive activation of downstream pathways and the activation of alternative pathways to maintain cell survival (112). As mentioned previously, some traits such as the overproduction of ErbB1 ligands (such as AR), the activation of alternative receptor tyrosine kinases (i.e., COX-2 and the IGF1-R axis) and more importantly the constitutive activation of downstream oncogenic pathways (like the PI3K/Akt pathway due to reduced expression of the PIP3 phosphatase PTEN) have been observed for HCC (28) (Fig. 3). Simultaneous pharmacological intervention on these mechanisms is thus of primary importance, and can be central to achieve sustained clinical response. The challenge remains to find drugs that target pathways that are central to physiologic processes, like PI3K, in a tumor-specific fashion and without causing major systemic toxicity. Alternative approaches targeting downstream effectors of the PI3K pathway, such as the mammalian target of rapamycin (mTOR) (112, 113), may prove safer and also effective in overcoming resistance to ErbB1-targeted therapies.

Conclusion

HCC is a very difficult to treat neoplasm, rarely amenable to conventional therapeutic approaches. The identification of cell-signaling pathways critical for HCC development is therefore of primary importance for the design of novel antineoplastic strategies. The experimental evidences reviewed in this article point to the ErbB1 system as a critical signaling module involved in liver resistance against acute tissue injury, but also in the promotion of HCC cell growth and survival. Interfering with the activity of ErbB1 using ErbB1-targeted molecules may therefore prove effective in the prevention and treatment of human HCC. Nevertheless, similar single-agent approaches to the treatment of other solid tumors have met with limited success. Building on these previous experiences, the simultaneous targeting of ErbB1 and other growth-promoting pathways by combination therapy, and its effects on the response of HCC to conventional cytostatics, warrant further investigation and should be tested in the clinical setting.

Acknowledgements

Work in the authors' laboratory is supported by the agreement between FIMA and the ‘UTE project CIMA’. Grants C03/02 and G03/015 were from Instituto de Salud Carlos III. Grants FIS PI040819 PI051098, RD 06/0020/0061 and CP04/00123 were from Ministerio de Sanidad y Consumo. Grant Ortiz de Landazuri was from Gobierno de Navarra. Grant SAF 2004-03538 and the Torres Quevedo Program from Ministerio de Educación y Ciencia.

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