Targeting c-Met as a promising strategy for the treatment of hepatocellular carcinoma

Pharmacological Research 65 (2012) 23–30

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Review

Targeting c-Met as a promising strategy for the treatment of hepatocellular carcinoma Jianjun Gao a,b , Yoshinori Inagaki b,c , Peipei Song b , Xianjun Qu a , Norihiro Kokudo b , Wei Tang a,b,∗ a b c

Department of Pharmacology, School of Pharmaceutical Sciences, Shandong University, Ji’nan, Shandong, China Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan The Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

a r t i c l e

i n f o

Article history: Received 20 September 2011 Received in revised form 15 November 2011 Accepted 16 November 2011 Keywords: c-Met Des-␥-carboxy prothrombin Hepatocellular carcinoma Molecular target therapy Receptor tyrosine kinase

a b s t r a c t Hepatocellular carcinoma (HCC) is a severe condition that is found worldwide. Liver transplantation, surgical resection, and local-regional therapy such as transarterial chemoembolization have made great progress and play a dominant role in HCC management. However, the high frequency of tumor recurrence and/or metastasis after those treatments acquires systematic drug intervention. The approval of sorafenib, an agent that targets receptor tyrosine kinases (RTKs), as the first effective drug for systemic treatment of HCC represents a milestone in treatment of this disease. As a typical member of the RTK family, c-Met represents an intriguing target for cancer therapy. However, the role of the c-Met signal transduction pathway is less unambiguous in HCC pathology, giving rise to concerns about the feasibility of utilizing c-Met targeting approaches for HCC treatment. Recently, studies on des-␥-carboxy prothrombin, an abnormal cytokine secreted by HCC cells, by the current authors and other researchers have highlighted the critical role of c-Met signaling in HCC progression. This review takes a second look at the c-Met signal transduction pathway and discusses the possibility of targeting c-Met as a therapeutic strategy for HCC treatment. © 2011 Elsevier Ltd. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The c-Met expression profile and findings associated with human HCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Increased c-Met expression in some cases of HCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. A high level of c-Met expression indicates increased malignancy of human HCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. c-Met overexpression or excessive activation suggests a poor prognosis for patients with HCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c-Met activating patterns in HCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The HGF-dependent pattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. The HGF-independent pattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The c-Met signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of c-Met signaling in HCC initiation and progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. HGF stimulates or inhibits HCC initiation and growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. DCP may play a critical role in stimulating human HCC progression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Suppressing c-Met expression prohibits the progression of spontaneously developing HCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Developing drugs to target c-Met signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. c-Met kinase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. c-Met adaptor inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future directions and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disclosure of potential conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author at: Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. Tel.: +81 3 5800 9269; fax: +81 3 5684 3989. E-mail address: [email protected] (W. Tang). 1043-6618/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2011.11.011

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J. Gao et al. / Pharmacological Research 65 (2012) 23–30

1. Introduction Liver cancer ranked fifth in incidence and third in mortality in terms of the global cancer burden in 2008, according to statistics published by the World Health Organization [1]. Among the diverse, histologically distinct primary hepatic neoplasms, hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for 83% of all cases [2]. Therapeutic approaches including hepatic resection, liver transplantation, and local-regional therapies play a major role in the clinical management of HCC [3,4]. In recent years, biotherapy such as molecule targeted therapy has offered new prospects and attracted a great deal of attention with regard to its use in the standardized treatment of HCC [5–7]. Systemic treatment with sorafenib, a multikinase inhibitor targeting Raf kinase and receptor tyrosine kinases (RTKs) including platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), and c-kit (a receptor specific for stem cell factor), is recommended for patients with more advanced HCC. In addition, several other RTK-targeting drugs such as bevacizumab, erlotinib, gefitinib, lapatinib, cetuximab, sunitinib, and brivanib have been used in clinical trials to treat HCC. These studies illustrate the benefit of targeting protein RTKs to manage HCC. c-Met is a prototypic member of the RTK family. The ligand for c-Met is a growth factor known as hepatocyte growth factor (HGF). The c-Met signaling pathway is involved in diverse cellular responses such as mitogenesis, motogenesis, or morphogenesis depending on the particular cell type and the microenvironment. In circumstances of tissue removal or damage such as liver regeneration or renal and lung injury, c-Met expression is induced as an important mediator in the wound healing and tissue repair processes. Deregulation and activation of c-Met may result in unregulated cell growth and differentiation, thus contributing to malignant transformation [8]. c-Met overexpression or enhanced activation relative to normal tissues has been noted in many human cancers such as gastric, colorectal, pancreatic, lung, head and neck, ovarian, renal, glioma, metastatic melanoma, prostatic, and breast carcinomas [9,10]. Thus far, a c-Met-targeting strategy has had impressive results in preclinical studies of these cancers and a number of such agents have entered the clinical trial phase [11]. Although a high level of c-Met expression was found to correlate with the increased malignancy of human HCC [12–14], the effect of the HGF–c-Met signaling pathway is less unambiguous or may even inhibit the growth of HCC cells in vitro or hepatocarcinogenic in animal models [15–19]. Therefore, there are concerns about the feasibility of using c-Met signaling-targeting therapies to treat HCC, and these concerns may have lead to fewer clinical trials of such therapies thus far. Recently, studies on des-␥-carboxy prothrombin (DCP) by the current authors and other researchers highlighted the role of abnormally triggered c-Met signaling in HCC progression. A second look at the c-Met signaling pathway in HCC carcinogenesis and progression could increase the understanding of HCC pathology and therapeutics. This review provides a systemic look at the role of c-Met in HCC pathology and it discusses the possibility of molecularly targeting c-Met as a potential therapeutic strategy for HCC. An ideal cancer target meets the following criteria: (i) it is relatively specific for cancer cells, meaning that it is not expressed or is expressed at a very low level in normal cells but overexpressed in cancer cells. Meanwhile, its overexpression is associated with malignant biological phenotypes and/or a poor prognosis of patient survival; (ii) it is of efficacy, meaning that it plays an essential role in cancer initiation and progression and inhibition of its expression or activity induces growth suppression and/or apoptosis in cancer cells; (iii) it is “drugable”, meaning that it is an enzyme (e.g. kinase) or a cell surface molecule (e.g. membrane-bound receptor) that can be easily screened for small-molecule inhibitors or that can be

Fig. 1. Relationship between c-Met overexpression and HCC clinicopathologic characteristics and prognosis.

targeted by a specific antibody [20]. The c-Met discussed below would meet some of these criteria as a target for HCC treatment. 2. The c-Met expression profile and findings associated with human HCC 2.1. Increased c-Met expression in some cases of HCC c-Met overexpression, relative to levels in peritumorous liver tissue, is observed in 20–48% of human HCC samples [12,21–25]. Dysregulation of c-Met is associated with various factors (Fig. 1): (i) abnormal induction by endogenous cytokines such as HGF, EGF, IL-1, IL-6, and TNF␣ as well as exogenous factors like hepatitis B virus X protein (HBX) [26,27]; (ii) down-regulated c-Met-targeting microRNAs (miRNAs), including miR-34a, miR-23b, and miR-199a3p [28–30]; (iii) amplified maturation process in carcinogenesis from pro-receptors to mature receptors [31]. Although c-Met is found to be overexpressed in HCC tissues in a subgroup of patients, c-Met as a potential target for anti-HCC therapy may still exist. This speculation may be supported by the following facts. In the case of the best characterized molecular target, the oestrogen receptor, for treatment of breast cancer, only approximately 50% of patients have receptor positive tumors [32]. Similarly, the HER-2/neu receptor is highly expressed in only 20% of breast cancers [33]. Application of these targets inhibitors, e.g. tamoxifen and herceptin, are clinically proven effective against breast cancer. However, the impact of these molecule targeted therapies has been limited by the biology of target expression. A similar problem may develop with the use of c-Met inhibitors in treating HCC. In this regard, the identification of sub-populations who respond to the c-Met targeted therapy may be of critical importance. 2.2. A high level of c-Met expression indicates increased malignancy of human HCC c-Met expression was found to correlate with the clinicopathologic features of HCC (Fig. 1). Generally, HCCs with multiple nodular tumors or high proliferative index show higher c-Met expression [12–14]. Reports provide conflicting findings with regard to characteristics such as tumor size, degree of differentiation, stage, as well as invasion and metastasis [12–14,21,23,25]. However, characteristics like tumor invasion and metastasis and degree of differentiation are more frequently reported to be correlated with c-Met expression. In those studies, the level of c-Met expression was significantly higher in invasive or poorly differentiated HCCs. That said, most

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studies suggest that tumor size is unrelated to c-Met expression. Ke et al. and Xie et al. obtained contradictory findings with regard to tumor stage [14,34]. Ke et al. found that the level of c-Met expression was markedly higher in advanced HCC (TNM stage III or IV), whereas Xie et al. found no significant difference in c-Met expression in early and advanced stages of HCC. Ke et al. studied 520 patients while Xie et al. studied 20, and these small patient populations may have affected the reliability of their results. Taken together, determining the level of c-Met expression in HCC might help to evaluate the status of the disease. c-Met overexpression may hint at active proliferation of tumor cells and the presence of intrahepatic metastasis or multiple nodular tumors. 2.3. c-Met overexpression or excessive activation suggests a poor prognosis for patients with HCC Currently, much work is underway to determine molecular predictors of HCC prognosis [35–38]. Expression of c-Met in HCC tissue is considered to be one of prognostic factors (Fig. 1) [14,39,40]. The overexpression of c-Met in HCC tissue or sustained high level of HGF in serum after hepatectomy is related with early tumor recurrence and metastasis [40]. Patients with a high level of cMet expression HCC usually have a significantly shorter 5-year survival than do patients with low c-Met expression HCC after a curative surgical resection [14,21,39]. Kaposi-Novak reported that a group of HCCs (27%) with potentially activated c-Met signaling were defined based on a c-Met-induced transcription signature [41]. These tumors were characterized by a higher rate of vascular invasion and increased microvessel density. Moreover, a predictive model was established according to c-Met gene signatures and was able to differentiate HCC patients into groups with a good or bad prognosis with 83–95% accuracy [41]. This evidence suggests that overexpression or excessive activation of c-Met in HCC tissues hints at a poor prognosis for HCC patients. Taken together, the above evidence indicates that the c-Met signaling pathway is dysregulated in HCC pathology. The correlations between c-Met expression and HCC clinicopathologic features and prognosis suggest that an enhanced c-Met signaling pathway is involved in HCC development and progression. 3. c-Met activating patterns in HCC 3.1. The HGF-dependent pattern The classical mode of c-Met activation requires the binding of HGF to c-Met. Under physiological conditions, c-Met expression is mainly observed in the epithelial compartment of various tissues, while its ligand HGF is expressed in cells of mesenchymal origin [42]. Accordingly, HGF and c-Met constitute a paracrine signaling system that plays a critical role in the formation of parenchymal organs during embryonic development. In HCC, a somewhat different scenario exists. Intracytoplasmic positivity for HGF was evident in a large number of neoplastic cells in some HCCs, indicating that an autocrine pattern of action of HGF may exist in HCC [13,21].

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structure of DCP are similar to those of HGF and are considered to be mandatory for HGF to bind with c-Met [43]. Based on this similarity, DCP could bind with and activate c-Met and thus cause HCC to continue to progress. (ii) Activation through cell attachment. c-Met can be activated in response to cell attachment independent of the ligand. In a mouse model, overexpression of the human wild type c-Met in mice hepatocytes allowed activation of the receptor [44]. Although human c-Met cannot respond to murine HGF, it was enzymatically active in the hepatocytes, and this activity was found to depend upon cell adherence. Since this form of activation can be tumorigenic, cell adherence may be a mechanism of activating c-Met in human HCC [44]. (iii) Activation through crosstalk with other membrane receptors. cMet was found to interact with EGFR and proteinase-activated receptor-2 (PAR-2), which act as c-Met partners in HCC. EGFR activation leads to c-Met constitutive activation. EGFR exists on the cell surface and is activated by binding of its specific ligands EGF and TGF␣ [45]. The association between EGFR and c-Met was found to occur either directly, or via adapter molecules, thus enabling TGF␣ or EGF to phosphorylate c-Met through EGFR [46]. PAR-2 is a G-protein-coupled receptor expressed in HCC cells. The metastatic potential of HCC cells was enhanced when PAR-2 was activated with trypsin or its selective activating peptide, 2-furoyl-LIGRLO-NH2 [47]. PAR-2-initiated HCC cell invasion was mediated by activation of c-Met signaling [47]. (iv) Mutations of c-Met that lock the receptor into an active state. Mutations that alter catalytic activity or substrate specificity may activate RTKs. In HCC, three missense mutations, i.e. K1262R (codon 1262, AAG → AGG; amino acid, Lys → Arg), M1268I (codon 1268, ATG → ATA; amino acid, Met → Ile), and T1191I (codon 1191, ACT → ATT; amino acid, Thr → Ile), in the tyrosine kinase domain have been detected in childhood HCCs [48]. The K1262R and M1268I mutations that occur in the kinase domain of c-Met are located in a specific region that is believed to act as an intramolecular substrate which, in the absence of ligand, functions to inhibit enzymatic activity by blocking the active site [48]. These mutations are surmised to stimulate the kinase activity of c-Met by altering the structure of the intramolecular substrate so that it is constitutively disengaged from the active site [48]. Mutations that cause constitutive activation of c-Met may play an important role in the development of HCC by conferring a selective growth advantage to cells. Thus, various factors might participate in triggering the c-Met signal transduction pathway in HCC. This fact highlights the central role of c-Met in the aberrant activation of the c-Met signal pathway and implies the relatively high efficiency of suppressing the activities of c-Met as well as its downstream signal mediators in inhibiting the c-Met signal transduction in HCC cells. 4. The c-Met signaling pathways

3.2. The HGF-independent pattern In addition to the HGF-dependent pattern, an HGF-independent pattern of c-Met activation also exists in HCC. Mechanisms involved include: (i) Activation by DCP. DCP, secreted from HCC cells, is used as a tumor biomarker in clinical settings because of its high sensitivity and specificity when screening and diagnosing HCC. DCP is an aberrant prothrombin that lacks the ability to interact with other coagulation factors. Two kringle domains in the

c-Met protein is first synthesized in hepatocytes as a single chain precursor (p170met ), and then processed into a mature glycosylated heterodimer receptor (p190met ) that consists of an extracellular ␣ subunit (p50met ) and a transmembrane ␤ subunit (p140met ) [49]. The intracellular ␤ subunit has a protein kinase domain and a docking site for cell-signaling molecules, both of which were considered to contribute to receptor bioactivity [50,51]. In HCC, c-Met signaling may be triggered in association with or independent of ligand binding (Fig. 2). In the event of the former, signal transduction triggered by HGF and DCP can be distinguished.

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Fig. 2. Schematic representation of the c-Met signaling pathway suggested in HCC cells.

Following HGF binding, the intracellular tyrosine kinase domain of c-Met is highly phosphorylated at two tyrosine residues (Tyr1234 and Tyr-1235) that are essential for the catalytic activity of the enzyme [51]. Phosphorylation also occurs at two tyrosine residues (Tyr-1349 and Tyr-1356) located in the carboxyl-terminal region of the ␤-subunit [50]. Upon phosphorylation, this domain acts as a multifunctional docking site and binds numerous srchomology-2 domain (SH2 domain)-containing effectors such as the Grb2, STAT3, PI3K, and PLC␥1 [50]. The docking motif may also be associated with Gab1, a multi-adaptor protein that provides binding sites for those molecules containing the SH2 domain [52]. Gab-1 interaction with c-Met has been found to play a critical role in sustained signaling through its conjugated key adaptors and signaling proteins [53]. Downstream of the c-Met signaling pathway, ERK and PI3K signal transduction pathways, which regulate cell proliferation, survival, and invasion, are activated [54]. The manner of activation of c-Met by DCP differs from that by HGF. Tyrosines-1234 and -1235 in the tyrosine kinase domain and tyrosines-1349 and -1356 in the multifunctional docking site are all phosphorylated when c-Met is activated by HGF. However, following DCP binding phosphorylation occurs only in those tyrosine residues located in the kinase activation loop (Tyr-1234/1235) [43]. Accordingly, DCP has been found to induce signal transduction along the JAK1-STAT3 pathway without activating ERK or PI3K pathways [43]. Following stimulation, STAT3 regulates a range of cellular processes as diverse as cell proliferation, differentiation, and apoptosis.

5. Role of c-Met signaling in HCC initiation and progression 5.1. HGF stimulates or inhibits HCC initiation and growth As the only natural ligand of c-Met, HGF is considered to be a potent mitogen for hepatocytes and various epithelial cells [55]. However, reports have ascribed various roles to HGF in hepatocyte malignant transformation and HCC growth. Exogenous administration of HGF to carcinogen-treated rats resulted in both HCC stimulation and inhibition [15,16,56]. There are also conflicting reports in HGF transgenic mice. Under the control of the mouse metallothionein gene promoter, liver tumors have been induced in mice harboring full-length mouse HGF cDNA [17]. In contrast, the HGF transgene appeared to inhibit hepatocarcinogenesis in bitransgenic mice coexpressing c-myc [18]. Other studies indicated that the effect of HGF on the growth of HCC cells depends on the dose given. Heideman et al. reported that the growth of the HCC cell lines HepG2, Hep3B, and HuH7, which expressed c-Met but not HGF mRNA, was significantly stimulated by exposure to HGF at low doses (below 2.5 ng/mL) although it was markedly inhibited at high doses (more than 10 ng/mL) [19]. With regard to HepG2 cell line, HGF at a dose 50 ng/mL even elicited a low level (approximately 20%) of cell apoptosis [57]. All these studies suggested a dual role of c-Met signal transduction pathway in HCC pathology. Thus far, mechanisms underlying the complicated biological effects of HGF on HCC cells were investigated and two models were revealed from different perspectives, which might help

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explain the apparently paradoxical activity of HGF on the growth of HCC. In the first model, the activation of ERK by HGF is involved in both the stimulation and inhibition of HCC cell proliferation. More specifically, the level of ERK activation determines the opposing proliferative response: strong activation is required for the inhibition of proliferation, whereas weak activation leads to the stimulation of proliferation [58]. It was revealed that a high level of expression of the adaptor Gab1, which could be seen in some HCC cell lines such as HepG2, is prone to over-activate ERK via HGF through c-Met signaling pathway [59]. The reason why strong activation of the ERK pathway leads to HGF-induced cell cycle arrest is the up-regulation of the cyclin-dependent kinase (Cdk) inhibitor p16 [60,61]. The strong activation of ERK by HGF induces the activation of a member of the Ets family of transcription factors that up-regulates the expression of p16. The p16 protein forms a complex with Cdk4, leading to the redistribution of p21 and p27 from Cdk4 to Cdk2. The association of p21 and p27 with Cdk2 represses Cdk2 activity, resulting in hypophosphorylation of pRb. The hypophosphorylated pRb eventually causes cell cycle arrest at G1 [60,61]. Since different doses of HGF elicited varying degrees of activation of the c-Met–ERK signal transduction [58], one explanation for the dual role of HGF on the growth of HCC cells might be that a low dose of HGF induced a weak activation of ERK which stimulated the growth of HCC cells, while the overactivation of ERK by a high dose of HGF achieved the opposite effect instead. Zarnegar and co-workers depicted another clear scenario in which a cross-talk between c-Met and Fas, a cell surface death receptor, exists in hepatoma cell lines HepG2 and Hepa1-6 [57]. According to their studies, c-Met and Fas physically and naturally associated with each other while dissociated by addition of a high dose of either HGF (50 ng/mL) or Jo2 antibody (500 ng/mL), an agonist of Fas. However, low concentrations of HGF (1–5 ng/mL) or Jo2 antibody (up to 20 ng/mL) were not effective in dissociating cMet from Fas. Since the monomeric Fas with a fraction of 90% was masked by the ␣ chain of c-Met in their preexisting complex, high doses of HGF competed with Fas in binding with c-Met and thus released the monomeric Fas, which gave advantage of monomeric Fas self-aggregating/trimer clustering and initiating apoptosis. This model vividly explained why HGF effectively induces hepatoma cells proliferation at a low dose while elicits apoptosis at a high dose in vitro. From these studies, we are tempted to reason that the growth inhibition or apoptosis induction effects of HGF on HCC cells occurs in an extreme situation, that is, cells are exposed to a concentration of HGF far higher than that in physical condition. In fact, HGF was not expressed or activated in the normal liver and was often absent in human HCC tissues [13,21,62,63]. Even in HCC tissues that were positive for HGF the HGF signal was quite homogeneous and was not correlated with the histological grade or other morphological features [13]. Thus, HGF–c-Met pathway is hardly likely to play a negative role in the development of spontaneously formed HCC. Furthermore, a close relationship between c-Met expression and the cell proliferation index was noted in these HCC tissues [13]. This seems to suggest that c-Met, independent of the native liver HGF, is the most active modulator of HCC cell proliferation. In other words, the activation of c-Met signal transduction pathway seems to depend on other factors except HGF in human HCC initiation and progression. 5.2. DCP may play a critical role in stimulating human HCC progression Among HGF-independent forms of c-Met activation, the role played by DCP has recently attracted attention. Elevated serum

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DCP levels were found in 44–81% of HCC patients [64]. A high level of serum DCP was associated with large tumor size, portal vein invasion, and intrahepatic metastasis [36,65,66]. Studies demonstrated that DCP is not only a biomarker of HCC but that it probably participates in the progression of the disease. We previously observed the stimulated proliferation of HCC cell lines HepG2 and SMMC-7721 upon exposure to DCP both in vitro and in vivo [67]. Consistent with our results, Suzuki and colleagues also found the growth stimulation effect of DCP on HCC cells [43]. According to their report, levels of DNA synthesis in HCC cells were significantly enhanced by adding purified DCP and this enhancement was more marked in non-DCP-producing cell lines than in DCP-producing cell lines. The researchers also analyzed the mechanism of this phenomenon and found that DCP bound with c-Met and stimulated JAK1-STAT3 pathway. This is important, because STAT3 signaling is generally considered to be involved in carcinogenesis and cancer development, including HCC [68]. On the other hand, we found that SU11274, a c-Met inhibitor, neutralized the activation of HCC cell growth resulting from the addition of DCP [69]. Based on these results, we reason that the growth stimulating effect of DCP on HCC cells is probably mediated by activating c-Met-JAK-1-STAT3 signal transduction pathway. It is worth noting that c-Met signal transduction triggered by DCP occurs without activating ERK-MAPK signaling pathway. Since the over-activating ERK may elicit growth inhibiting effect on HCC cells according to the first model mentioned above, the uniqueness of c-Met signal transduction induced by DCP ligation may lessen the possibility that DCP acts as an inhibitive factor in HCC progression. That said, whether DCP would compete with the death receptor Fas in combining c-Met and thus release the monomeric Fas is currently unknown. However, the growth inhibition effect of DCP on HCC cell lines including HepG2 has thus far not been observed by us and other researchers, even cells were exposed to DCP at a dose up to 160 ng/mL in vitro [43,67,69]. These results implied that although the structure of DCP contains two kringle domains that are similar to those of the HGF, the conformation changes of c-Met induced by DCP or HGF binding are somewhat different, which were evidenced by the fact that they bound cMet but caused different c-Met autophosphorylation pattern. We speculate that the specific binding of DCP on c-Met may not dissociate the complex of c-Met and Fas, thus is not likely to release the monomeric Fas and induce cell apoptosis. Although this hypothesis still needs further verification, the current evidences suggest that DCP may be important activator of c-Met signal transduction pathway and play a critical role in stimulating human HCC progression. 5.3. Suppressing c-Met expression prohibits the progression of spontaneously developing HCC Inhibition of c-Met expression always leads to prohibition of the progression of spontaneously developing HCC. Knockdown of c-Met by antisense RNA or RNA interference strategies significantly inhibited the in vitro and in vivo growth of HCC cells with high levels of c-Met expression [28,29,70–72]. The migratory and invasive properties of these cells were also suppressed when c-Met expression decreased. miRNAs are small RNA molecules that are approximately 22 nucleotides long and negatively control their target gene expression posttranscriptionally [73]. miRNAs targeting c-Met, including miR-34a, miR-23b, and miR-199a-3p, were found to be down-regulated in HCC tissues [28–30]. Restoring the levels of these miRNAs in HCC cells significantly inhibited negative cell properties. This evidence indicates the feasibility of targeting the c-Met signaling pathway to treat HCC.

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[69]. SU11274 suppressed the growth of HepG2, HuH7, and HLE hepatoma cells with similar IC50 values at 6–8 ␮mol/L in vitro. Expression of phosphorylated c-Met gradually decreased in a dosedependent manner with SU11274. Expression of phosphorylated ERK also decreased in a dose-dependent manner with SU11274. In addition, studies by the current authors found that SU11274 not only suppressed the growth of HepG2, HuH7, and HLE hepatoma cells but also neutralized the increased growth of these cells induced by simultaneous addition of DCP [69]. Thus, targeting cMet, and inhibiting kinase activity in particular, might be a rational strategy to design anti-HCC drugs that interfere with c-Met signal transduction.

6.2. c-Met adaptor inhibitors Fig. 3. c-Met targeting strategies proposed for HCC treatment.

6. Developing drugs to target c-Met signaling Based on knowledge of the c-Met signal transduction pathway, several strategies have been designed to intervene in the pathway at different levels: (i) blocking ligand and receptor interaction, (ii) inhibition of c-Met kinase activity; and (iii) inhibition of c-Met and adaptor interaction. However, as previously mentioned, the activation of c-Met in HCC may occur via various factors besides HGF. As an abnormal cytokine found in certain patients with HCC, DCP may activate c-Met and promote both the growth and metastasis of HCC cells in an autocrine manner. This feature may distinguish HCC from other solid tumors. Considering the uniqueness of c-Met signaling in HCC, therapeutic intervention in c-Met and in the interaction between c-Met and downstream signaling molecules may be a rational strategy for HCC management. Small-molecule inhibitors have emerged as a key approach in this regard. Advances in this category of agents are summarized below (Fig. 3).

There are two types of characteristics of c-Met that distinguish it from other RTKs. First, there is a unique multi-substrate docking site at the C-terminal region of the receptor [76]. Second, the adaptor protein Gab-1, which is unique to c-Met, has been found to mediate most biologically relevant c-Met-dependent signals [50]. Gab1 coupling to the c-Met receptor requires binding to the SH3 domains of adaptor Grb2, which binds the c-Met via its SH2 domains. Agents blocking either the SH2 or the SH3 domains of Grb2 interfere with c-Met/Gab1 interaction [52]. Those traits of the c-Met signal pathway might enable the design of c-Met-specific inhibitors. Thus far, the SH2 domain of the adaptor protein Grb2 has been successfully targeted based on its unique structure among SH2 domains [77,78]. C90, a small selective antagonist of Grb2, potently blocked cell motility and matrix invasion in gastric cancer cells in vitro and it reduced the metastatic spread of primary solid tumors generated from human prostate adenocarcinoma cells in vivo [79,80]. The efficacy of compounds in this class in HCC model needs further studies in the future.

7. Future directions and conclusions 6.1. c-Met kinase inhibitors Small-molecule kinase inhibitors could prevent the phosphorylation of the catalytic domains within the receptor, thus precluding recruitment of signal transducers and mediators and thereby impeding downstream signal propagation. Most of these smallmolecule kinase inhibitors competitively antagonize occupancy of the intracellular ATP binding site to prevent kinase domain phosphorylation. However, compound ARQ197 binds to a region of c-Met outside of the ATP binding site and impairs kinase activation in a non-ATP competitive manner [74]. Thus far, a number of small-molecule kinase inhibitors targeting c-Met have entered clinical trials of various cancer treatments. These candidates include GSK1363089, ARQ197, MK2461, MP470, SGX523, JNJ38877605, MGCD265, XL184, AMG208, PF04217903, BMS777607, E7050, PF02341066, and MK8033. Among these candidates, GSK1363089 and ARQ197 are being studied clinically to evaluate their potential in HCC treatment. GSK1363089 is a multikinase inhibitor with potent activity against c-Met and VEGFR and is currently in a phase I/II trial to assess its safety and tolerability in patients with advanced HCC. ARQ197 is reported to be highly selective for c-Met and has an IC50 of 50 nM in vitro [74]. A completed phase Ib clinical study of ARQ197 in patients with HCC and cirrhosis found no worsening of liver function, confirming the manageable safety profile of the molecule and providing preliminary evidence of its efficacy [75]. We recently found that SU11274, a c-Met kinase inhibitor, suppressed HCC cell growth by inhibiting the activation of c-Met

An important challenge in translating preclinical studies into effective clinical therapies is identifying those patients who are most likely to benefit from molecular targeted agents. Two strategies are currently used to select patients for c-Met-targeted therapies. First is identifying the effectiveness of c-Met inhibitors against known c-Met mutants. For example, PF02341066 is more effective than PF04217903 against the Y1230C mutation. A phase II trial of GSK1363089 is current comparing patients with papillary renal-cell carcinoma and germline or somatic c-Met mutations, cMet gene amplification, or trisomy of chromosome 7 to individuals without those features but with an otherwise histologically similar tumor phenotype. Since the active mutants of c-Met are also found in HCC, future trials are likely to follow this trend where possible. Second is the immunohistochemical analysis and immunoassay of tissue extracts. The former provides important spatial and morphological information, while the latter provides more precise, absolute measurements of c-Met content and phosphorylation. Theoretically, HCC with relatively greater c-Met phosphorylation might respond better to therapy, although this point warrants further investigation. The wealth of basic knowledge about c-Met signaling biology in HCC has enabled an accurate assessment of the pathway’s oncogenic potential and provided the insight needed to develop potent and selective c-Met inhibitors for use in HCC treatment. However, identifying the subclass of patients with c-Met signalingdependent HCCs is of special importance in predicting drug efficiency and reducing side effects.

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