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Met as a therapeutic target in HCC: Facts and hopes

  • Silvia Giordano
    Correspondence
    Corresponding authors. Addresses: Department of Oncology, University of Torino, Institute for Cancer Research and Treatment (IRCC), Strada Provinciale 142, 10060 Candiolo (Torino), Italy. Tel.: +39 011 9933233; fax: +39 011 9933225 (S. Giordano). Department of Biomedical Sciences, Unit of Oncology and Molecular Pathology, University of Cagliari, Via Porcell 4, 09124 Cagliari, Italy. Tel.: +39 070 6758345; fax: +39 070 666062 (A. Columbano).
    Affiliations
    Department of Oncology, University of Torino, Institute for Cancer Research and Treatment (IRCC), 10060 Candiolo (Torino), Italy
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  • Amedeo Columbano
    Correspondence
    Corresponding authors. Addresses: Department of Oncology, University of Torino, Institute for Cancer Research and Treatment (IRCC), Strada Provinciale 142, 10060 Candiolo (Torino), Italy. Tel.: +39 011 9933233; fax: +39 011 9933225 (S. Giordano). Department of Biomedical Sciences, Unit of Oncology and Molecular Pathology, University of Cagliari, Via Porcell 4, 09124 Cagliari, Italy. Tel.: +39 070 6758345; fax: +39 070 666062 (A. Columbano).
    Affiliations
    Department of Biomedical Sciences, Unit of Oncology and Molecular Pathology, University of Cagliari, Cagliari, Italy
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Open AccessPublished:September 16, 2013DOI:https://doi.org/10.1016/j.jhep.2013.09.009

      Summary

      Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, and its burden is expected to increase further in the next years. In spite of the advances of classical therapies, such as surgery, transplantation, use of radiofrequency and transarterial embolization, the prognosis of this neoplasm has not considerably improved over the past few years. The advent of targeted therapies and the approval of the systemic treatment of advanced HCC with the kinase inhibitor sorafenib have provided some hope for the future. Even if the molecular mechanisms responsible for the onset and progression of HCC are still largely unknown, new therapeutic targets have recently come to the spotlight. One of these targets is the tyrosine kinase receptor for the Hepatocyte Growth Factor, encoded by the MET gene, known to promote tumor growth and metastasis in many human organs. In this review we will summarize the contrasting results obtained in vitro (in HCC cell lines) and in animal experimental models and we will also try to analyze the reasons for the opposite findings, suggesting that the HGF/MET axis can have either a promoting or a suppressive role in the development of HCC. We will also reconsider the evidence of activation of this pathway in human HCCs and discuss the results of the clinical trials performed with MET inhibitors. The final purpose is to better clarify which can be the role of MET as a therapeutic target in HCC.

      Keywords

      Introduction

      Hepatocellular carcinoma is a leading cause of cancer-related death worldwide, and its burden is expected to increase further in the next years. HCC in men is the fifth most frequently diagnosed cancer worldwide, and the second leading cause of cancer-related death, while in women it is the seventh most commonly diagnosed cancer and the sixth cause of cancer mortality [
      • Jemal A.
      • Bray F.
      • Center M.M.
      • Ferlay J.
      • Ward E.
      • Forman D.
      Global cancer statistics.
      ]. The incidence of HCC varies widely, according to geographic location, and differs among racial and ethnic groups within the same country. These differences in HCC distribution are probably due to variations in exposure to hepatitis viruses and environmental pathogens.
      Despite an improved treatment of viral hepatitis and an increased screening of high-risk patients in developed countries, only around 40% of HCC patients are eligible for potentially curative treatments (resection, transplantation, or local ablation) and 20% for chemoembolization. Around 40% of patients are diagnosed with advanced disease [
      • Llovet J.M.
      • Di Bisceglie A.M.
      • Bruix J.
      • Kramer B.S.
      • Lencioni R.
      • Zhu A.X.
      • et al.
      Design and endpoints of clinical trials in hepatocellular carcinoma.
      ] and thus systemic therapy is indicated for a considerable proportion of patients. The major problems in developing effective therapies for HCC involve the intrinsic chemoresistance of HCC, the pharmacologic problems due to the presence of a diseased liver and the very advanced stage of diagnosis. Unfortunately, the efficacy of traditional chemotherapeutic agents and their ability to produce a significant survival benefit is questionable. In light of these unsatisfactory results, several studies have been performed to elucidate the molecular mechanisms underlying HCC development and progression, in order to identify targets for HCC treatment [
      • Llovet J.M.
      • Bruix J.
      Molecular targeted therapies in hepatocellular carcinoma.
      ].
      Recent progresses in the elucidation of HCC molecular pathways have brought to the clinic the multikinase inhibitor sorafenib (active against c-RAF, b-RAF, vascular endothelial growth factor receptor, c-KIT, and platelet-derived growth factor receptor beta), which has provided survival benefit in patients with advanced HCC and well-preserved liver function [
      • Llovet J.M.
      • Ricci S.
      • Mazzaferro V.
      • Hilgard P.
      • Gane E.
      • Blanc J.F.
      • et al.
      Sorafenib in advanced hepatocellular carcinoma.
      ], and it is now the standard of care for patients with advanced-stage HCC [
      • Llovet J.M.
      • Di Bisceglie A.M.
      • Bruix J.
      • Kramer B.S.
      • Lencioni R.
      • Zhu A.X.
      • et al.
      Design and endpoints of clinical trials in hepatocellular carcinoma.
      ]. However, the benefits obtained from this treatment are still disappointing and, thus, it is mandatory to find alternative effective treatments. Unlike other solid tumors, the specific sequence of genetic events that sustain hepatocarcinogenesis is unknown and, in particular, no genes to which HCC cells are “addicted” have been identified. The concept of oncogene addiction is quite recent and implies that continuous activation of specific oncogenes or inactivation of tumor suppressors is required to drive proliferation and survival of cancer cells [
      • Luo J.
      • Solimini N.L.
      • Elledge S.J.
      Principles of cancer therapy: oncogene and non-oncogene addiction.
      ]. Clinical experience has clearly shown that targeting the genes to which tumor cells are addicted can give significant therapeutic results. This is the case, for example, of Chronic Myelogenous Leukemia (CML), in which the targeted drug Imatinib inhibits the BCR-ABL tyrosine kinase to which leukemic cells are addicted, resulting in long lasting remissions in CML patients.
      Many studies have tried to identify genes or pathways to which HCC cells are addicted but, probably due to the heterogeneity of this illness, no definitive conclusion has been achieved yet. However, some genes have gained interest as possible therapeutic targets in HCC, and among them there is the receptor for Hepatocyte Growth Factor, the tyrosine kinase (TK) encoded by the MET gene [
      • Giordano S.
      • Ponzetto C.
      • Di Renzo M.F.
      • Cooper C.S.
      • Comoglio P.M.
      Tyrosine kinase receptor indistinguishable from the c-met protein.
      ]. Indeed MET plays a role in tumor onset and progression of different tumor types and has recently become a very interesting and studied target. Several strategies to inhibit MET activation are under development, such as tyrosine kinase inhibitors and monoclonal antibodies, and some of them are in advanced phases of clinical trials [
      • Corso S.
      • Giordano S.
      Cell autonomous and non-cell autonomous mechanisms of HGF/MET-driven resistance to targeted therapies: from basic research to a clinical perspective.
      ].
      In this review we will reconsider the existing evidence for a role of MET in sustaining HCC progression and discuss if and how MET can be foreseen as a therapeutic target in this pathology.

      The HGF/MET axis

      The MET proto-oncogene encodes the tyrosine kinase receptor for Hepatocyte Growth Factor (HGF) [
      • Bottaro D.P.
      • Rubin J.S.
      • Faletto D.L.
      • Chan A.M.
      • Kmiecik T.E.
      • Vande Woude G.F.
      • et al.
      Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product.
      ,
      • Naldini L.
      • Vigna E.
      • Narsimhan R.P.
      • Gaudino G.
      • Zarnegar R.
      • Michalopoulos G.K.
      • et al.
      Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded by the proto-oncogene c-MET.
      ]. Upon binding to HGF, MET becomes active and drives a complex biological program, defined as “invasive growth”, resulting from the promotion of several biological activities, such as cell proliferation, cell invasion and protection from apoptosis (for a review see [
      • Birchmeier C.
      • Birchmeier W.
      • Gherardi E.
      • Vande Woude G.F.
      Met, metastasis, motility and more.
      ]). MET-induced invasive growth is physiologically activated during the embryonic development and in adulthood during tissue regeneration. In transformed tissues, the gain of the invasive growth program is advantageous for cancer progression and metastasis. In fact, constitutive MET activation can contribute to several aspects of tumor progression, since it forces neoplastic cells to disaggregate from the tumor mass, erode basement membranes, infiltrate stromal matrices, and eventually colonize new territories to form metastases [
      • Birchmeier C.
      • Birchmeier W.
      • Gherardi E.
      • Vande Woude G.F.
      Met, metastasis, motility and more.
      ].
      Many works have investigated the MET-activated signaling pathways that are shared with many other receptor tyrosine kinases (RTKs), including the MAP Kinase and PI-3 Kinase-AKT pathways, STAT3, RAC1, and the NF-KB pathway (reviewed in [
      • Migliore C.
      • Giordano S.
      Molecular cancer therapy: can our expectation be MET?.
      ]) (Fig. 1).
      Figure thumbnail gr1
      Fig. 1MET-induced signaling pathways. Hepatocyte growth factor (HGF) promotes MET dimerization and activation. The phosphorylation of two tyrosines in the receptor tail creates binding sites for SH2-containing intracellular proteins such as Growth factor Receptor-Bound protein 2 (GRB2), Grb2-Associated Binding protein 1 (GAB1), phospholipase Cγ (PLCγ), Phosphoinositide 3-Kinase (PI3K), and Signal Transducer and Activator of Transcription 3 (STAT3). These pathways originate signals that reach the nucleus and control gene transcription and DNA replication. RAC1-dependent signals influence cytoskeletal organization.
      MET-driven signaling results from pathways directly activated by this receptor, but it can also be modulated by the cross-talk between MET and different membrane receptors, acting in complex interacting networks (Fig. 2). They involve the interaction with adhesive receptors, such as CD44 [
      • van der Voort R.
      • Taher T.E.
      • Wielenga V.J.
      • Spaargaren M.
      • Prevo R.
      • Smit L.
      • et al.
      Heparan sulfate-modified CD44 promotes hepatocyte growth factor/scatter factor-induced signal transduction through the receptor tyrosine kinase c-Met.
      ,
      • Orian-Rousseau V.
      • Chen L.
      • Sleeman J.P.
      • Herrlich P.
      • Ponta H.
      CD44 is required for two consecutive steps in HGF/c-Met signaling.
      ] and the α6β4 integrin [
      • Trusolino L.
      • Bertotti A.
      • Comoglio P.M.
      A signaling adapter function for alpha6beta4 integrin in the control of HGF-dependent invasive growth.
      ], with receptors for semaphorins [
      • Giordano S.
      • Corso S.
      • Conrotto P.
      • Artigiani S.
      • Gilestro G.
      • Barberis D.
      • et al.
      The semaphorin 4D receptor controls invasive growth by coupling with Met.
      ], receptor tyrosine kinases, such as members of the Epidermal Growth Factor Receptor Family (EGFR and HER2) [
      • Jo M.
      • Stolz D.B.
      • Esplen J.E.
      • Dorko K.
      • Michalopoulos G.K.
      • Strom S.C.
      Cross-talk between epidermal growth factor receptor and c-Met signal pathways in transformed cells.
      ,
      • Bergstrom J.D.
      • Westermark B.
      • Heldin N.E.
      Epidermal growth factor receptor signaling activates met in human anaplastic thyroid carcinoma cells.
      ,
      • Fischer O.M.
      • Giordano S.
      • Comoglio P.M.
      • Ullrich A.
      Reactive oxygen species mediate Met receptor transactivation by G protein-coupled receptors and the epidermal growth factor receptor in human carcinoma cells.
      ] and the Vascular Endothelial Growth Factor Receptor (VEGFR) [
      • Lu K.V.
      • Chang J.P.
      • Parachoniak C.A.
      • Pandika M.M.
      • Aghi M.K.
      • Meyronet D.
      • et al.
      VEGF inhibits tumor cell invasion and mesenchymal transition through a MET/VEGFR2 complex.
      ] and, finally, with the pro-apoptotic receptor FAS [
      • Wang X.
      • DeFrances M.C.
      • Dai Y.
      • Pediaditakis P.
      • Johnson C.
      • Bell A.
      • et al.
      A mechanism of cell survival: sequestration of Fas by the HGF receptor Met.
      ]. Even if in vitro data suggest that these cross-talks are not essential for cell survival, they can allow a better integration of the signals present in the extracellular environment. While in physiological conditions these networks are probably redundant, it is likely that these interacting receptors cooperate in promoting tumorigenesis and/or metastasis and in inducing resistance to targeted drugs.
      Figure thumbnail gr2
      Fig. 2MET interplay with other membrane receptors. MET-driven signaling can be modulated by cross talk with different membrane receptors. Interaction with adhesive receptors amplifies the signaling response and controls cytoskeletal reorganization; interaction with plexins (semaphorin receptors) allows HGF-independent MET activation of invasive growth; association with FAS regulates induction of apoptosis; interaction with tyrosine kinase receptors controls their reciprocal activation and (for MET-VEGFR) angiogenesis.
      Data produced by many laboratories provide compelling evidence that HGF/MET signaling plays an important role in the development and progression of tumors. Indeed, (i) cell lines ectopically overexpressing MET or HGF are tumorigenic and metastatic in nude mice, whereas MET down-regulation decreases their tumorigenic potential [
      • Birchmeier C.
      • Birchmeier W.
      • Gherardi E.
      • Vande Woude G.F.
      Met, metastasis, motility and more.
      ]; (ii) MET- or HGF-transgenic mice develop metastatic tumors [
      • Sakata H.
      • Takayama H.
      • Sharp R.
      • Rubin J.S.
      • Merlino G.
      • Larochelle W.J.
      Hepatocyte growth factor/scatter factor overexpression induces growth, abnormal development, and tumor formation in transgenic mouse livers.
      ,
      • Wang R.
      • Ferrell L.D.
      • Faouzi S.
      • Maher J.J.
      • Bishop J.M.
      Activation of the Met receptor by cell attachment induces and sustains hepatocellular carcinomas in transgenic mice.
      ,
      • Takayama H.
      • Larochelle W.J.
      • Sharp R.
      • Otsuka T.
      • Kriebel P.
      • Anver M.
      • et al.
      Diverse tumorigenesis associated with aberrant development in mice overexpressing hepatocyte growth factor/scatter factor.
      ,
      • Liang T.J.
      • Reid A.E.
      • Xavier R.
      • Cardiff R.D.
      • Wang T.C.
      Transgenic expression of tpr-met oncogene leads to development of mammary hyperplasia and tumors.
      ]; (iii) aberrant MET expression (usually overexpression) has been found in many kinds of solid tumors and correlates with poor prognosis [
      • Comoglio P.M.
      • Giordano S.
      • Trusolino L.
      Drug development of MET inhibitors: targeting oncogene addiction and expedience.
      ]; (iv) the unequivocal evidence linking MET and human cancer comes from the presence of germline-activating mutations in patients suffering from hereditary papillary renal carcinomas [
      • Schmidt L.
      • Duh F.M.
      • Chen F.
      • Kishida T.
      • Glenn G.
      • Choyke P.
      • et al.
      Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas.
      ]. Deregulated MET activation in cancer can be due to different molecular alterations such as overexpression, gene amplification, autocrine activation, presence of activating point mutations or downregulation of MET-targeting miRNAs [
      • Corso S.
      • Giordano S.
      Cell autonomous and non-cell autonomous mechanisms of HGF/MET-driven resistance to targeted therapies: from basic research to a clinical perspective.
      ]. While overexpression can make MET activation independent from HGF stimulation, in most cases the ligand is still required for full receptor activation [
      • Michieli P.
      • Basilico C.
      • Pennacchietti S.
      • Maffe A.
      • Tamagnone L.
      • Giordano S.
      • et al.
      Mutant Met-mediated transformation is ligand-dependent and can be inhibited by HGF antagonists.
      ]. This is also true for the receptor forms containing activating mutations that need HGF to fully activate their kinase activity.

      HGF/MET and liver

      Hepatocyte growth factor was discovered as a mitogenic protein for rat hepatocytes [
      • Nakamura T.
      • Nawa K.
      • Ichihara A.
      Partial purification and characterization of hepatocyte growth factor from serum of hepatectomized rats.
      ], and its cDNA was cloned in 1989 [
      • Nakamura T.
      • Nishizawa T.
      • Hagiya M.
      • Seki T.
      • Shimonishi M.
      • Sugimura A.
      • et al.
      Molecular cloning and expression of human hepatocyte growth factor.
      ]. In 1991, the scatter factor and the tumor cytotoxic factor, fibroblast-derived cell molecules, were shown to be identical to HGF [
      • Furlong R.A.
      • Takehara T.
      • Taylor W.G.
      • Nakamura T.
      • Rubin J.S.
      Comparison of biological and immunochemical properties indicates that scatter factor and hepatocyte growth factor are indistinguishable.
      ,
      • Shima N.
      • Nagao M.
      • Ogaki F.
      • Tsuda E.
      • Murakami A.
      • Higashio K.
      Tumor cytotoxic factor/hepatocyte growth factor from human fibroblasts: cloning of its cDNA, purification and characterization of recombinant protein.
      ]. In the same year, the tyrosine kinase encoded by the MET gene was identified as the receptor for this growth factor [
      • Bottaro D.P.
      • Rubin J.S.
      • Faletto D.L.
      • Chan A.M.
      • Kmiecik T.E.
      • Vande Woude G.F.
      • et al.
      Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product.
      ,
      • Naldini L.
      • Vigna E.
      • Narsimhan R.P.
      • Gaudino G.
      • Zarnegar R.
      • Michalopoulos G.K.
      • et al.
      Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded by the proto-oncogene c-MET.
      ].
      Even if HGF was originally discovered for its mitogenic and motogenic properties, further studies revealed its ability to suppress apoptotic cell death. This cytoprotective action of HGF is responsible for liver protection from tissue damage and suppression of FAS-induced massive apoptosis of hepatocytes [
      • Kosai K.
      • Matsumoto K.
      • Nagata S.
      • Tsujimoto Y.
      • Nakamura T.
      Abrogation of Fas-induced fulminant hepatic failure in mice by hepatocyte growth factor.
      ,
      • Amicone L.
      • Spagnoli F.M.
      • Spath G.
      • Giordano S.
      • Tommasini C.
      • Bernardini S.
      • et al.
      Transgenic expression in the liver of truncated Met blocks apoptosis and permits immortalization of hepatocytes.
      ]. Accordingly, expression of HGF is increased in response to liver injuries, while neutralization of endogenous HGF enhances liver damage. The increased HGF production in these conditions is probably due to recruitment of bone marrow-derived liver sinusoidal endothelial cell progenitor cells [
      • Wang L.
      • Wang X.
      • Xie G.
      • Wang L.
      • Hill C.K.
      DeLeve LD Liver sinusoidal endothelial cell progenitor cells promote liver regeneration in rats.
      ]. The anti-apoptotic role of HGF has been clearly proven in hepatocyte conditional knockout Met mice, which are hypersensitive to liver injury due to treatment with agonistic anti-Fas antibodies, show delayed liver regeneration, and are more prone to liver fibrosis [
      • Huh C.G.
      • Factor V.M.
      • Sanchez A.
      • Uchida K.
      • Conner E.A.
      • Thorgeirsson S.S.
      Hepatocyte growth factor/c-met signaling pathway is required for efficient liver regeneration and repair.
      ,
      • Borowiak M.
      • Garratt A.N.
      • Wustefeld T.
      • Strehle M.
      • Trautwein C.
      • Birchmeier C.
      Met provides essential signals for liver regeneration.
      ,
      • Giebeler A.
      • Boekschoten M.V.
      • Klein C.
      • Borowiak M.
      • Birchmeier C.
      • Gassler N.
      • et al.
      C-Met confers protection against chronic liver tissue damage and fibrosis progression after bile duct ligation in mice.
      ]. Animal studies also showed that Hgf and Met provide essential signals for survival and proliferation of hepatocytes during embryogenesis, since Hgf or Met knockout mice display considerably reduced liver size, due to decreased proliferation and increased apoptosis of hepatocytes [
      • Schmidt L.
      • Duh F.M.
      • Chen F.
      • Kishida T.
      • Glenn G.
      • Choyke P.
      • et al.
      Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas.
      ,
      • Aberger F.
      • Schmidt G.
      • Richter K.
      The Xenopus homologue of hepatocyte growth factor-like protein is specifically expressed in the presumptive neural plate during gastrulation.
      ]. These observations indicate that the HGF/MET axis is critical for liver development, protection and regeneration.

      HGF/MET alterations in human HCC

      On the base of the critical role of the HGF/MET axis in controlling hepatocyte proliferation and apoptosis, many studies have been performed to identify genetic and functional alterations of this signaling system in human HCC (Table 1).
      Table 1Molecular alterations of HGF/MET in human HCC.
      IHC, immunohistochemistry; OS, overall survival; qRT PCR, quantitative Reverse Transcription Polymerase Chain Reaction.
      As previously mentioned, in human tumors MET is activated by gene amplification, overexpression or activating mutations. Takeo’s and Kondo’s [
      • Takeo S.
      • Arai H.
      • Kusano N.
      • Harada T.
      • Furuya T.
      • Kawauchi S.
      • et al.
      Examination of oncogene amplification by genomic DNA microarray in hepatocellular carcinomas: comparison with comparative genomic hybridization analysis.
      ,
      • Kondo S.
      • Ojima H.
      • Tsuda H.
      • Hashimoto J.
      • Morizane C.
      • Ikeda M.
      • et al.
      Clinical impact of c-Met expression and its gene amplification in hepatocellular carcinoma.
      ] search for MET amplification in HCCs revealed only a very low frequency (1 out of 20 and 1 out of 59 HCCs, respectively); however, aneuploidy of chromosome 7 (where both MET and HGF are located) was present in 22/59 patients. A very recent work by Wang et al. [
      • Wang K.
      • Lim H.Y.
      • Shi S.
      • Lee J.
      • Deng S.
      • Xie T.
      • et al.
      Genomic landscape of copy number aberrations enables the identification of oncogenic drivers in hepatocellular carcinoma.
      ] examined the genomic landscape of copy number aberrations in 286 hepatocarcinoma patients and identified recurrently amplified regions with a high level of copy number changes. MET was identified as one of the ten genes in the amplification peak located at 7q31.2, present in 4–5% of the cases. No amplification of HGF, located on the same chromosome but in a different region, was found. Concerning activating point mutations, Guichard and colleagues [
      • Guichard C.
      • Amaddeo G.
      • Imbeaud S.
      • Ladeiro Y.
      • Pelletier L.
      • Maad I.B.
      • et al.
      Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma.
      ], who performed the whole exome sequencing on 24 tumors, did not identify any activating mutation in MET.
      Over the past few years, expression of MET and HGF (produced by stromal components, cancer-associated fibroblasts and endothelium in the tumor mass [
      • Jia C.C.
      • Wang T.T.
      • Liu W.
      • Fu B.S.
      • Hua X.
      • Wang G.Y.
      • et al.
      Cancer-associated fibroblasts from hepatocellular carcinoma promote malignant cell proliferation by HGF secretion.
      ,
      • Ueki T.
      • Fujimoto J.
      • Suzuki T.
      • Yamamoto H.
      • Okamoto E.
      Expression of hepatocyte growth factor and its receptor c-met proto-oncogene in hepatocellular carcinoma.
      ]) has been evaluated in many studies. In small groups of patients (18 and 19, respectively) [
      • Boix L.
      • Rosa J.L.
      • Ventura F.
      • Castells A.
      • Bruix J.
      • Rodes J.
      • et al.
      C-met mRNA overexpression in human hepatocellular carcinoma.
      ,
      • Suzuki K.
      • Hayashi N.
      • Yamada Y.
      • Yoshihara H.
      • Miyamoto Y.
      • Ito Y.
      • et al.
      Expression of the c-met protooncogene in human hepatocellular carcinoma.
      ] Northern blot analysis showed an increase of MET mRNA in 30–40% of HCCs compared to peritumoral tissue. While Boix et al. did not find any correlation with clinical parameters, Suzuki et al. observed an association between MET overexpression and poor-to-moderate differentiation of cancer cells and a non-significant increase in the proliferative activity of tumor cells. By competitive PCR, Noguchi and collaborators [
      • Noguchi O.
      • Enomoto N.
      • Ikeda T.
      • Kobayashi F.
      • Marumo F.
      • Sato C.
      Gene expressions of c-met and hepatocyte growth factor in chronic liver disease and hepatocellular carcinoma.
      ] found that MET expression was increased in some cases of HCC, while HGF was expressed at levels lower than those of the peritumoral tissue. By western blot analysis, Ueki et al. [
      • Ueki T.
      • Fujimoto J.
      • Suzuki T.
      • Yamamoto H.
      • Okamoto E.
      Expression of hepatocyte growth factor and its receptor c-met proto-oncogene in hepatocellular carcinoma.
      ] found MET overexpression in 48% of 62 HCC patients, correlating with an increased incidence of intrahepatic metastases. Patients with high MET HCC had a significantly shorter 5-year survival than patients with low MET HCC (33.5% vs. 80.3%, respectively; p <0.05). However, they did not find any correlation between HGF concentration in the tumor tissue, clinic pathological factors and patient survival. Kiss et al. [
      • Kiss A.
      • Wang N.J.
      • Xie J.P.
      • Thorgeirsson S.S.
      Analysis of transforming growth factor (TGF)-alpha/epidermal growth factor receptor, hepatocyte growth Factor/c-met, TGF-beta receptor type II, and p53 expression in human hepatocellular carcinomas.
      ] analyzed 86 HCCs by immunohistochemistry and found MET overexpression in 20% and downregulation in 32% of cases, while HGF was increased in 33% and decreased in 21% of tumors. Tavian et al. [
      • Tavian D.
      • De P.G.
      • Benetti A.
      • Portolani N.
      • Giulini S.M.
      • Barlati S.
      U-PA and c-MET mRNA expression is co-ordinately enhanced while hepatocyte growth factor mRNA is down-regulated in human hepatocellular carcinoma.
      ] performed RT-PCR on 24 HCCs and found overexpression of MET and under-expression of HGF compared to the corresponding peri-tumoral tissues. HGF and MET levels did not correlate with clinical features, but increased MET was inversely associated with patient survival. Finally, Daveau et al. [
      • Daveau M.
      • Scotte M.
      • Francois A.
      • Coulouarn C.
      • Ros G.
      • Tallet Y.
      • et al.
      Hepatocyte growth factor, transforming growth factor alpha, and their receptors as combined markers of prognosis in hepatocellular carcinoma.
      ] performed quantitative RT-PCR on 15 HCCs and peritumoral tissues and found low levels of HGF in highly differentiated tumors, whereas overexpression of MET was observed in poorly differentiated tumors and in patients with early tumor recurrence.
      As it can be seen by the overall analysis of these works, while most of the studies agree on the decrease of HGF in HCC, it is difficult to draw a definitive picture on the status and role of MET in liver cancer. In fact, there is not only disagreement on the percentage of tumors showing MET overexpression, but opposite results are often present in the literature. Which are the explanations for these discrepancies? One possible reason is the use of different techniques (Northern blot, Western blot, competitive PCR, RT-PCR), which have different sensitivity and different modality of quantification. Moreover, even when the same technique is used, there is no agreement on the adopted scoring system. Another possible reason is that many of these studies have been performed on small groups of patients and, due to the different etiologies of HCC, they could have included unbalanced types of tumors. Most importantly, none of these studies investigated the activation status of MET, which is critical to identify tumors that can benefit from anti-MET drugs. As MET amplification or mutation seem to be very rare, the only criterium to select patients for possible anti-MET therapies is overexpression, but – at the moment – there is no standardized test to identify a level of expression that could render tumor cells “addicted” to MET. This is even more important if we think that most of the studies found decreased HGF levels in HCC, suggesting that MET activation has to be largely ligand-independent, such as that due to very high levels of overexpression. Alternatively, as shown by two studies [
      • Suzuki M.
      • Shiraha H.
      • Fujikawa T.
      • Takaoka N.
      • Ueda N.
      • Nakanishi Y.
      • et al.
      Des-gamma-carboxy prothrombin is a potential autologous growth factor for hepatocellular carcinoma.
      ,
      • Gao J.
      • Feng X.
      • Inagaki Y.
      • Song P.
      • Kokudo N.
      • Hasegawa K.
      • et al.
      Des-gamma-carboxy prothrombin and c-Met were concurrently and extensively expressed in hepatocellular carcinoma and associated with tumor recurrence.
      ], MET could be activated by the binding with des-gamma-carboxy prothrombin (DCP), a well-recognized tumor marker recently exploited due to its high sensitivity and specificity in the screening and diagnosis of hepatocellular carcinoma. DCP is elevated in the serum of 44–81% of HCC patients [
      • Weitz I.C.
      • Liebman H.A.
      Des-gamma-carboxy (abnormal) prothrombin and hepatocellular carcinoma: a critical review.
      ] and was shown to be able to bind MET, causing its autophosphorylation and the proliferation of HCC cells [
      • Suzuki M.
      • Shiraha H.
      • Fujikawa T.
      • Takaoka N.
      • Ueda N.
      • Nakanishi Y.
      • et al.
      Des-gamma-carboxy prothrombin is a potential autologous growth factor for hepatocellular carcinoma.
      ]. Even if further studies are required to draw a final conclusion, it is thus possible that in many HCCs MET activation is not due to HGF but to the autocrine/paracrine production of DCP.
      A different approach to investigate the role of MET activation in human tumors was taken by Kaposi-Novak and colleagues [
      • Kaposi-Novak P.
      • Lee J.S.
      • Gomez-Quiroz L.
      • Coulouarn C.
      • Factor V.M.
      • Thorgeirsson S.S.
      Met-regulated expression signature defines a subset of human hepatocellular carcinomas with poor prognosis and aggressive phenotype.
      ]. Using global gene expression profiling of WT and Met-deficient primary mouse hepatocytes, the authors defined a Met-dependent gene expression signature. To assess the importance of this signature, they applied a comparative functional genomic approach to 242 human HCCs and liver metastases. The analysis revealed that a subset of human HCCs and all liver metastases shared the Met-driven expression signature, which correlated with increased vascular invasion rate, microvessel density and decreased mean survival time of HCC patients. Thus, they concluded that Met-driven expression signature defines a subset of human hepatocellular carcinomas with poor prognosis and aggressive phenotype.

      In vitro preclinical data of HGF/MET inhibition/overexpression

      Many in vitro studies have evaluated the effect of the activation of the HGF/MET axis in HCC cell lines. Interestingly, the first studies aimed at demonstrating a pro-proliferative role of HGF in liver tumor cells, as observed in normal hepatocytes, showed a marked inhibition of cell growth [
      • Shiota G.
      • Rhoads D.B.
      • Wang T.C.
      • Nakamura T.
      • Schmidt E.V.
      Hepatocyte growth factor inhibits growth of hepatocellular carcinoma cells.
      ,
      • Ogasawara H.
      • Hiramoto J.
      • Takahashi M.
      • Shirahama K.
      • Furusaka A.
      • Hiyane S.
      • et al.
      Hepatocyte growth factor stimulates DNA synthesis in rat preneoplastic hepatocytes but not in liver carcinoma cells.
      ]. Growth inhibition was also observed in HCC cells stably transduced with HGF. However, HGF stimulation resulted in increased invasive properties of tumor cells [
      • Ogasawara H.
      • Hiramoto J.
      • Takahashi M.
      • Shirahama K.
      • Furusaka A.
      • Hiyane S.
      • et al.
      Hepatocyte growth factor stimulates DNA synthesis in rat preneoplastic hepatocytes but not in liver carcinoma cells.
      ,
      • Nakanishi K.
      • Fujimoto J.
      • Ueki T.
      • Kishimoto K.
      • Hashimoto-Tamaoki T.
      • Furuyama J.
      • et al.
      Hepatocyte growth factor promotes migration of human hepatocellular carcinoma via phosphatidylinositol 3-kinase.
      ]. The biochemical mechanisms responsible for the concomitant anti-proliferative and pro-invasive role of HGF in HCC cells are not fully elucidated. Shirako et al. [
      • Shirako E.
      • Hirayama N.
      • Tsukada Y.
      • Tanaka T.
      • Kitamura N.
      Up-regulation of p21CIP1 expression mediated by ERK-dependent and -independent pathways contributes to hepatocyte growth factor-induced inhibition of HepG2 hepatoma cell proliferation.
      ] have shown in HepG2 cells that the growth inhibitory activity of HGF requires a strong activation of ERK and up-regulation of p16 and p21 expression, which contributes to the suppression of Cdk2 activity.
      On the other hand, different studies have shown that MET down-regulation or inhibition interferes with both cell growth and cell invasiveness [
      • Zhang S.Z.
      • Pan F.Y.
      • Xu J.F.
      • Yuan J.
      • Guo S.Y.
      • Dai G.
      • et al.
      Knockdown of c-Met by adenovirus-delivered small interfering RNA inhibits hepatocellular carcinoma growth in vitro and in vivo.
      ,
      • Salvi A.
      • Arici B.
      • Portolani N.
      • Giulini S.M.
      • De P.G.
      • Barlati S.
      In vitro c-met inhibition by antisense RNA and plasmid-based RNAi down-modulates migration and invasion of hepatocellular carcinoma cells.
      ,
      • Xie B.
      • Xing R.
      • Chen P.
      • Gou Y.
      • Li S.
      • Xiao J.
      • et al.
      Down-regulation of c-Met expression inhibits human HCC cells growth and invasion by RNA interference.
      ]. The reasons for the different behavior of HGF in normal vs. transformed hepatocytes and for the similar effects caused by HGF stimulation and MET inhibition in HCC remain elusive. One possibility could be the availability of other ligands able to activate MET in the liver. Suzuki et al. [
      • Suzuki M.
      • Shiraha H.
      • Fujikawa T.
      • Takaoka N.
      • Ueda N.
      • Nakanishi Y.
      • et al.
      Des-gamma-carboxy prothrombin is a potential autologous growth factor for hepatocellular carcinoma.
      ] identified such a molecule in des-gamma-carboxy prothrombin. DCP contains two kringle domains similar to those of HGF, required for HGF binding to MET. They showed that DCP binds MET and that this interaction promotes proliferation of HCC cell lines and induces JAK1/STAT3 activation, without affecting the Raf/MAPK and the PI3K/AKT pathways. This study thus suggests that the HCC cells can autocrinally activate MET signaling by producing DCP, whose expression is a bad prognostic indicator. If this holds true, it remains to be explained why the biological effects of DCP/MET interaction are opposite to those elicited by HGF/MET binding in the same cells.
      As mentioned above, several studies have shown that MET can interact with different tyrosine kinase receptors; in particular, a functional cross-talk has been demonstrated between MET and the different members of the EGFR family [
      • Jo M.
      • Stolz D.B.
      • Esplen J.E.
      • Dorko K.
      • Michalopoulos G.K.
      • Strom S.C.
      Cross-talk between epidermal growth factor receptor and c-Met signal pathways in transformed cells.
      ,
      • Bergstrom J.D.
      • Westermark B.
      • Heldin N.E.
      Epidermal growth factor receptor signaling activates met in human anaplastic thyroid carcinoma cells.
      ,
      • Fischer O.M.
      • Giordano S.
      • Comoglio P.M.
      • Ullrich A.
      Reactive oxygen species mediate Met receptor transactivation by G protein-coupled receptors and the epidermal growth factor receptor in human carcinoma cells.
      ]. Indeed, Met associates with EGFR in tumor cells, and this association results in MET phosphorylation in the absence of HGF [
      • Jo M.
      • Stolz D.B.
      • Esplen J.E.
      • Dorko K.
      • Michalopoulos G.K.
      • Strom S.C.
      Cross-talk between epidermal growth factor receptor and c-Met signal pathways in transformed cells.
      ]. It can thus be hypothesized that MET phosphorylation in HCC cells could also be due to constitutive activation of EGFR family members, whose alterations in liver cancers have been reported [
      • Llovet J.M.
      • Bruix J.
      Molecular targeted therapies in hepatocellular carcinoma.
      ]. The demonstration of a functional and biologically meaningful cross talk between MET and EGFR in HCC cells would pave the way to the use of combination therapies targeting both receptors, which are already in clinical trials in other tumor types.

      In vivo preclinical data of MET/HGF manipulation

      The critical role of the HGF/MET axis in development was well illustrated by the effects exerted by knock-out of either Met or Hgf in mice. Hgf- and Met-null mutant embryos fail to complete development and die in utero [
      • Schmidt C.
      • Bladt F.
      • Goedecke S.
      • Brinkmann V.
      • Zschiesche W.
      • Sharpe M.
      • et al.
      Scatter factor/hepatocyte growth factor is essential for liver development.
      ,
      • Uehara Y.
      • Minowa O.
      • Mori C.
      • Shiota K.
      • Kuno J.
      • Noda T.
      • et al.
      Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor.
      ]. The mutation affects the embryonic liver, which is reduced in size and shows extensive loss of parenchymal cells.
      Several works examined the effects of genetic inactivation or overexpression of either Met or Hgf on hepatocarcinogenesis. The published results, however, are discordant and the reasons for these discrepancies are not yet clear (Table 2).
      Table 2In vivo manipulation of HGF/MET.
      DEN, Diethylnitrosamine; TGFα, Transforming Growth Factor α; 3′-Me-DAB, 3′-Methyl-4-dimethylaminoazobenzene.
      Two works examined the effect of liver-specific Met knock out in hepatocarcinogenesis [
      • Takami T.
      • Kaposi-Novak P.
      • Uchida K.
      • Gomez-Quiroz L.E.
      • Conner E.A.
      • Factor V.M.
      • et al.
      Loss of hepatocyte growth factor/c-Met signaling pathway accelerates early stages of N-nitrosodiethylamine induced hepatocarcinogenesis.
      ,
      • Marx-Stoelting P.
      • Borowiak M.
      • Knorpp T.
      • Birchmeier C.
      • Buchmann A.
      • Schwarz M.
      Hepatocarcinogenesis in mice with a conditional knockout of the hepatocyte growth factor receptor c-Met.
      ]. The loss of Met signaling in hepatocytes increased rather than suppressed tumor initiation by DEN (N-nitrosodiethylamine), as the animals developed significantly more and bigger tumors and with a shorter latency, compared to controls. The authors suggested that this was probably due to the loss of the physiological role of Met in maintaining normal redox homeostasis in the liver and, thus, the lack of this activity resulted in a tumor suppressive role of Met in this context. On the contrary, a protumorigenic role was observed when Met was hydrodynamically transfected in the liver or in Met transgenic mice [
      • Tward A.D.
      • Jones K.D.
      • Yant S.
      • Kay M.A.
      • Wang R.
      • Bishop J.M.
      Genomic progression in mouse models for liver tumors.
      ].
      In the same manner, both stimulatory and inhibitory effects of exogenous administration of HGF on carcinogen-treated rats have been reported [
      • Ogasawara H.
      • Hiramoto J.
      • Takahashi M.
      • Shirahama K.
      • Furusaka A.
      • Hiyane S.
      • et al.
      Hepatocyte growth factor stimulates DNA synthesis in rat preneoplastic hepatocytes but not in liver carcinoma cells.
      ,
      • Liu M.L.
      • Mars W.M.
      • Michalopoulos G.K.
      Hepatocyte growth factor inhibits cell proliferation in vivo of rat hepatocellular carcinomas induced by diethylnitrosamine.
      ,
      • Yaono M.
      • Hasegawa R.
      • Mizoguchi Y.
      • Futakuchi M.
      • Nakamura T.
      • Ito N.
      • et al.
      Hepatocyte growth factor enhancement of preneoplastic hepatic foci development in rats treated with diethylnitrosamine and N-ethyl-N-hydroxyethylnitrosamine.
      ]. In fact, while injection of HGF to rats initiated with DEN and promoted with N-ethyl-N-hydroxyethylnitrosamine significantly increased the development of preneoplastic foci [
      • Yaono M.
      • Hasegawa R.
      • Mizoguchi Y.
      • Futakuchi M.
      • Nakamura T.
      • Ito N.
      • et al.
      Hepatocyte growth factor enhancement of preneoplastic hepatic foci development in rats treated with diethylnitrosamine and N-ethyl-N-hydroxyethylnitrosamine.
      ], Liu et al. observed a strong inhibitory activity of HGF in rat liver tumors induced by DEN [
      • Liu M.L.
      • Mars W.M.
      • Michalopoulos G.K.
      Hepatocyte growth factor inhibits cell proliferation in vivo of rat hepatocellular carcinomas induced by diethylnitrosamine.
      ]. Finally, Ogasawara et al. found that HGF promotes proliferation of preneoplastic hepatocytes but does not affect the growth of liver carcinoma cells in 3′-Me-DAB-treated rats [
      • Ogasawara H.
      • Hiramoto J.
      • Takahashi M.
      • Shirahama K.
      • Furusaka A.
      • Hiyane S.
      • et al.
      Hepatocyte growth factor stimulates DNA synthesis in rat preneoplastic hepatocytes but not in liver carcinoma cells.
      ].
      Reports obtained in HGF transgenic mice are conflicting as well. A pro-tumorigenic role for HGF was clearly suggested by Sakata et al. [
      • Sakata H.
      • Takayama H.
      • Sharp R.
      • Rubin J.S.
      • Merlino G.
      • Larochelle W.J.
      Hepatocyte growth factor/scatter factor overexpression induces growth, abnormal development, and tumor formation in transgenic mouse livers.
      ], who generated Hgf transgenic mice (under the control of the mouse metallothionein gene promoter); they found that in adult transgenic animals, liver weight (as a percentage of total body weight) was at least twice the weight of wild-type mice and the DNA labeling index of hepatocytes was significantly increased. Moreover, this proliferative stimulus triggered the formation of hepatocellular adenomas and/or carcinomas in most transgenic mice older than 1.5 years. In contrast, overexpression of a human HGF cDNA under the control of the albumin promoter did not induce HCC [
      • Shiota G.
      • Wang T.C.
      • Nakamura T.
      • Schmidt E.V.
      Hepatocyte growth factor in transgenic mice: effects on hepatocyte growth, liver regeneration and gene expression.
      ]. These different results can be due to at least two reasons: (i) the level of Hgf expression achieved in the serum through the use of the metallothionein promoter was 2–5-fold higher compared to that obtained with the albumin promoter; (ii) Sakata used a mouse Hgf cDNA instead of the human HGF cDNA utilized by Shiota; it is known that the cross interaction between human HGF and mouse Met is not adequate to promote the optimal activity of the receptor.
      A pro-tumorigenic role of HGF was found in transgenic mice also by Horiguchi et al. [
      • Horiguchi N.
      • Takayama H.
      • Toyoda M.
      • Otsuka T.
      • Fukusato T.
      • Merlino G.
      • et al.
      Hepatocyte growth factor promotes hepatocarcinogenesis through c-Met autocrine activation and enhanced angiogenesis in transgenic mice treated with diethylnitrosamine.
      ], who observed accelerated DEN-induced hepatocarcinogenesis, often accompanied by abnormal blood vessel formation. Thirty-three percent of males and 23% of female transgenic mice developed hepatocellular carcinoma, while none of the wild-type mice developed HCC. Since the authors also detected enhanced Met kinase activity in most of these tumors, they concluded that HGF promotes hepatocarcinogenesis through the autocrine activation of the HGF/MET signaling pathway, in association with stimulation of angiogenesis.
      On the contrary, an inhibitory role of HGF in liver carcinogenesis was demonstrated in double transgenic animals, where the Hgf transgene inhibited hepatocarcinogenesis in mice overexpressing c-myc [
      • Santoni-Rugiu E.
      • Preisegger K.H.
      • Kiss A.
      • Audolfsson T.
      • Shiota G.
      • Schmidt E.V.
      • et al.
      Inhibition of neoplastic development in the liver by hepatocyte growth factor in a transgenic mouse model.
      ] and transforming growth factor alpha (TGFα) [
      • Shiota G.
      • Kawasaki H.
      • Nakamura T.
      • Schmidt E.V.
      Characterization of double transgenic mice expressing hepatocye growth factor and transforming growth factor alpha.
      ].
      Therefore, in light of these results, the issue of the pro-tumorigenic role of HGF and MET in the liver remains to be resolved.

      MET as an anti-angiogenic target

      Several experimental works showed, in vitro and in vivo, that the HGF/MET axis promotes angiogenesis. HGF is an angiogenic factor able to activate MET in endothelial cells, in part through a direct effect [
      • Bussolino F.
      • Di Renzo M.F.
      • Ziche M.
      • Bocchietto E.
      • Olivero M.
      • Naldini L.
      • et al.
      Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth.
      ], in part by inducing Vascular Endothelial Growth Factor (VEGF) production and decreasing thrombospondin-1 expression [
      • Zhang Y.W.
      • Su Y.
      • Volpert O.V.
      • Vande Woude G.F.
      Hepatocyte growth factor/scatter factor mediates angiogenesis through positive VEGF and negative thrombospondin 1 regulation.
      ]. Moreover, very recently, it has been shown that VEGFR and MET physically interact and that VEGF enhances recruitment of the protein tyrosine phosphatase 1B to a MET/VEGFR2 heterocomplex, thereby suppressing HGF-dependent MET phosphorylation and activation [
      • Lu K.V.
      • Chang J.P.
      • Parachoniak C.A.
      • Pandika M.M.
      • Aghi M.K.
      • Meyronet D.
      • et al.
      VEGF inhibits tumor cell invasion and mesenchymal transition through a MET/VEGFR2 complex.
      ]. Consequently, VEGF blockade restores and increases MET activity.
      Following the initial observations that anti-angiogenic molecules could function as potent tumor suppressors in mice, several therapeutic strategies have attempted to interfere with tumor growth by suppressing neo-angiogenesis. However, it has become clear that anti-angiogenic therapies induce tumor hypoxia that, in turn, allows for selection of more aggressive tumor cells. MET transcription is induced by the hypoxia-inducible factor HIF-1α in tumor cells [
      • Eckerich C.
      • Zapf S.
      • Fillbrandt R.
      • Loges S.
      • Westphal M.
      • Lamszus K.
      Hypoxia can induce c-Met expression in glioma cells and enhance SF/HGF-induced cell migration.
      ,
      • Pennacchietti S.
      • Michieli P.
      • Galluzzo M.
      • Mazzone M.
      • Giordano S.
      • Comoglio P.M.
      Hypoxia promotes invasive growth by transcriptional activation of the met protooncogene.
      ], and MET expression was found to be upregulated following anti-angiogenic therapies [
      • Rose S.D.
      • Aghi M.K.
      Mechanisms of evasion to antiangiogenic therapy in glioblastoma.
      ]. Thus, antiangiogenic therapies can interfere with MET activation in a dual way: (i) by promoting hypoxia-induced MET-expression and (ii) by promoting MET activity as a consequence of VEGF blockade.
      Some studies have evaluated the effect of the simultaneous inhibition of MET and VEGFR pathways in HCC. Treatment of SK-HEP1 cells with foretinib (an oral multikinase inhibitor targeting MET, RON, AXL, TIE-2, and VEGFR2 receptors) resulted in growth inhibition, G2/M cell cycle arrest, reduced colony formation and blockade of HGF-induced cell migration [
      • Huynh H.
      • Ong R.
      • Soo K.C.
      Foretinib demonstrates anti-tumor activity and improves overall survival in preclinical models of hepatocellular carcinoma.
      ]. In animal models of HCC, foretinib potently inhibited tumor growth and inhibition of angiogenesis correlated with inactivation of VEGFR2/MET signaling pathways. Another study was performed on 20 HCC patients, treated with tivantinib (a small kinase MET inhibitor) plus sorafenib [
      • Martell R.E.
      • Puzanov I.
      • Ma W.W.
      • Santoro A.
      • Dy G.K.
      • Goff L.W.
      • et al.
      Safety and efficacy of MET inhibitor tivantinib (ARQ 197) combined with sorafenib in patients (pts) with hepatocellular carcinoma (HCC) from a phase I study (abstract).
      ]. Overall response rate and disease control rate were 10% and 70%, respectively. Best response was 1 complete response (CR), 1 partial response (PR), and 12 stable diseases (SD). Among 8 patients previously treated with VEGFR inhibitors (6 sorafenib; 1 sunitinib; 1 sorafenib plus sunitinib), best response was 1 CR, 1 PR, and 3 SD. This study thus suggests that the combination of tivantinib plus sorafenib may have therapeutic potential in HCC patients, including those pretreated with VEGFR inhibitors.

      The HGF/MET axis as a clinical target in HCC

      Although there are no approved anti-Met agents, a number of MET/HGF inhibitors have been or are currently under study in trials in cancer patients [
      • Corso S.
      • Giordano S.
      Cell autonomous and non-cell autonomous mechanisms of HGF/MET-driven resistance to targeted therapies: from basic research to a clinical perspective.
      ]. Different molecules like monoclonal antibodies (mAbs) against HGF or MET and specific/non-specific MET inhibitors are now available, but most of them are still at early stages of clinical development (Fig. 3); the data reported so far have shown some clinical benefits in patients with various tumor types.
      Figure thumbnail gr3
      Fig. 3Strategies to target MET signaling. The central panel shows the physiological activation pathway of MET: pro-HGF is converted into mature HGF that binds to the receptor and induces its dimerization and activation. Schematic MET structure: sema domain: yellow rectangle; MET-related sequence: orange rectangle; tyrosine kinase domain (purple); (P): tyrosines of the intracellular docking site. The left side of the figure shows the therapeutic strategies utilized in preclinical studies. The upper square illustrates molecules that act as HGF antagonists (NK4, uncleavable pro-HGF and decoy MET). Geldanamycin induces MET ubiquitination and proteasomal degradation. Specific inhibitors (SH2 competitors and inhibitors of specific downstream molecules, such as Src, PtdIns3K, MAPK or STAT3) block critical transducers. The lower square shows the mechanisms aimed at silencing MET or HGF expression (antisense oligonucleotides, ribozymes and RNAi). The right side illustrates MET/HGF inhibitors in clinical trials. Anti-HGF and anti-MET antibodies prevent ligand-induced activation. Many non-selective and selective small molecules inhibit MET kinase activity. Red lines indicate the site of action of each drug.
      Concerning HCC, three TKIs have been used in Phase II clinical trials: tivantinib, foretinib and cabozantinib (Table 3).
      Table 3Clinical trials targeting MET in HCC.
      SD, Stable disease; mPFS, medium Progression Free Survival; mOS, medium Overall Survival; TKIs, Tyrosine Kinase Inhibitors; mTTP, medium Time To Progression; CR, Complete Response; PR, Partial Response; RR, Response Rate; DCR, Disease Control Rate.
      Tivantinib, a staurosporine derivative showing promising activity in a variety of phase I/II clinical trials, was initially reported as a selective, non-ATP competitive MET inhibitor [
      • Munshi N.
      • Jeay S.
      • Li Y.
      • Chen C.R.
      • France D.S.
      • Ashwell M.A.
      • et al.
      ARQ 197, a novel and selective inhibitor of the human c-Met receptor tyrosine kinase with antitumor activity.
      ]. This is the drug at the most advanced stage of clinical testing in HCC; in fact, a Phase III trial (METIV) is ongoing, on the basis of the results obtained in a phase II, double-blind, placebo-controlled clinical trial [
      • Santoro A.
      • Rimassa L.
      • Borbath I.
      • Daniele B.
      • Salvagni S.
      • Van Laethem J.L.
      • et al.
      Tivantinib for second-line treatment of advanced hepatocellular carcinoma: a randomised, placebo-controlled phase 2 study.
      ]. This trial assessed the efficacy of tivantinib in patients with advanced-stage HCC and Child-Pugh A cirrhosis, who had progressive disease during first-line therapy (72 patients were treated with tivantinib and 36 were assigned to the placebo arm). The primary end-point was time to progression; the trial also included the immunohistochemistry assessment of MET expression in the tumors. Time to progression was longer for patients in the tivantinib arm than for those in the placebo arm (1.6 months vs. 1.4 months; p = 0.04). The results were more positive in patients with high expression of MET, showing a median time to progression of 2.7 months vs. 1.4 months of the placebo arm. Although these results seem encouraging, two recent works [
      • Katayama R.
      • Aoyama A.
      • Yamori T.
      • Qi J.
      • Oh-Hara T.
      • Song Y.
      • et al.
      Cytotoxic activity of tivantinib (ARQ 197) is not due solely to c-MET inhibition.
      ,
      • Basilico C.
      • Pennacchietti S.
      • Vigna E.
      • Chiriaco C.
      • Arena S.
      • Bardelli A.
      • et al.
      Tivantinib (ARQ197) displays cytotoxic activity that is independent of its ability to bind MET.
      ] question the mechanism of action of the drug, as they show that tivantinib acts on microtubule dynamics independently of MET and thus it behaves more as a cytotoxic rather than a targeted drug. In fact, the authors of these two papers provided evidence that (i) tivantinib inhibits the growth of both MET-dependent and independent cancer cells, (ii) it is active on cells not expressing MET, (iii) it does not inhibit MET activation in addicted cells. Altogether these results put a strong caveat on the real ability of tivantinib to specifically target MET. This does not imply that the drug is ineffective, but rather that it may act through a mechanism different from the one hypothesized. In the past, other “targeted drugs” turned out to be “cytotoxic drugs”. A notable example is iniparib, which gave positive results in triple-negative breast cancer in a phase II trial [
      • O’Shaughnessy J.
      • Osborne C.
      • Pippen J.E.
      • Yoffe M.
      • Patt D.
      • Rocha C.
      • et al.
      Iniparib plus chemotherapy in metastatic triple-negative breast cancer.
      ], but later on turned out not to be a PARP (poly-(ADP-ribose) polymerase) inhibitor but rather a cytotoxic agent [
      • Liu X.
      • Shi Y.
      • Maag D.X.
      • Palma J.P.
      • Patterson M.J.
      • Ellis P.A.
      • et al.
      Iniparib nonselectively modifies cysteine-containing proteins in tumor cells and is not a bona fide PARP inhibitor.
      ,
      • Patel A.G.
      • De Lorenzo S.B.
      • Flatten K.S.
      • Poirier G.G.
      • Kaufmann S.H.
      Failure of iniparib to inhibit poly(ADP-Ribose) polymerase in vitro.
      ]. Overall, the results of the study by Santoro et al. should be taken with caution, while waiting for the results of the Phase III METIV trail, enrolling only HCC patients with high MET expression to be treated with Tivantinib.
      Foretinib is an oral multikinase inhibitor targeting MET, RON, AXL, TIE-2, and VEGFR2. Yau et al. reported the results of a phase I/II trial (MET111645), evaluating oral foretinib as first line therapy in advanced Asian HCC patients [
      • Yau T.
      • Sukeepaisarnjaroen W.
      • Chao Y.
      • Yen C.
      • Lausoontornsiri W.
      • Chen P.
      • et al.
      A phase I/II study of foretinib, an oral multikinase inhibitor targeting MET, RON, AXL, TIE-2, and VEGFR in advanced hepatocellular carcinoma (HCC).
      ]. Thirty-nine patients were enrolled and thirty-eight were evaluable for efficacy. The primary endpoint was safety and tolerability at the maximum tolerated dose and the secondary endpoint included antitumor activity (objective response rate, disease stabilization rate, time to progression and overall survival). The overall response rate was 24%, disease stabilization rate 79% and the median time to progression was 4.2 months. The most common adverse effects were hypertension (36%), decreased appetite (23%), and pyrexia (21%). The most common serious adverse effects were hepatic encephalopathy (10%) and ascites (8%).
      Cabozantinib is an oral inhibitor of MET, VEGFR, and RET. A phase II randomized discontinuation trial was performed by Verslype et al. [
      • Verslype C.
      • Cohn A.L.
      • Kelley R.K.
      • Yang T.
      • Su W.
      • Ramies D.A.
      • et al.
      Activity of cabozantinib (XL184) in hepatocellular carcinoma: Results from a phase II randomized discontinuation trial (RDT).
      ] on 41 patients with advanced HCC and Child-Plugh cirrhosis. After 12 weeks, only patients with a partial response were maintained on open-label cabozantinib, while patients with stable disease were randomized to either cabozantinib or placebo. The primary endpoint during the randomization phase was overall response rate. Median PFS from the first day of study was 4.2 months. Two out of 36 patients evaluable for tumor assessment at 12 weeks achieved a confirmed PR. One more patient randomized at week 12 achieved a PR at 18 weeks. Twenty eight out of 36 patients (78%) with ⩾1 post-baseline scan had tumor regression. The overall disease control rate at week 12 was 68%. A reduction of alpha fetoprotein (AFP) >50% in patients with elevated AFP at baseline was observed in 10/26 patients (38%). Interestingly, previous treatment with sorafenib did not influence PFS.
      At the moment, another anti-MET drug, INC280 by Novartis, is in Phase II clinical trial, in HCC patients with MET pathway dysregulation.

      Conclusions

      HGF was originally discovered for its ability to promote growth of hepatocytes and thus, from the beginning, its involvement in the development and progression of liver tumors was considered quite likely. However, the studies performed both in vitro – in HCC cell lines – and in animal models gave contradictory results, suggesting either an oncogenic or a suppressive role in liver cancer for the HGF/MET axis. The reasons for the observed differences are not clear. It can be hypothesized that MET overactivation can indeed promote signaling of HCC cells, but it is not clear how this receptor can be activated in tumor cells, since almost all the data show that HGF-mediated MET activation leads to inhibition of tumor growth. Several mechanisms of HGF-independent MET activation have been described in different conditions, but they have not been investigated in the context of liver tumors, with the exception of the possible role of des-gamma-carboxiprotrombin as an alternative MET ligand. Concerning the studies that have shown that the loss of HGF/MET signaling can accelerate chemically-induced carcinogenesis, it is possible – as suggested by Takami – that MET-mediated signaling is necessary to maintain normal redox homeostasis in the liver, leading to the identification of an oncosuppressive role for this gene [
      • Takami T.
      • Kaposi-Novak P.
      • Uchida K.
      • Gomez-Quiroz L.E.
      • Conner E.A.
      • Factor V.M.
      • et al.
      Loss of hepatocyte growth factor/c-Met signaling pathway accelerates early stages of N-nitrosodiethylamine induced hepatocarcinogenesis.
      ].
      The clinical data obtained with MET inhibitors in HCC are not very encouraging either. A further complication to the scenario is that the best results have been obtained with a drug whose specific activity against MET has recently been questioned [
      • Katayama R.
      • Aoyama A.
      • Yamori T.
      • Qi J.
      • Oh-Hara T.
      • Song Y.
      • et al.
      Cytotoxic activity of tivantinib (ARQ 197) is not due solely to c-MET inhibition.
      ,
      • Basilico C.
      • Pennacchietti S.
      • Vigna E.
      • Chiriaco C.
      • Arena S.
      • Bardelli A.
      • et al.
      Tivantinib (ARQ197) displays cytotoxic activity that is independent of its ability to bind MET.
      ].
      The main problems of the performed clinical studies are the relatively small number of patients recruited and the lack of selection for patients displaying activation of the MET signaling pathway. The experience obtained from the studies performed with different targeted drugs in other tumors has taught that they are effective almost exclusively in cells addicted to the gene targeted by the drug. If we look, for example, at non-small cell lung cancer, EGFR inhibitors are effective when EGFR mutations are present, while crizotinib treatment is very active only in patients with ALK translocation. Since these latter patients are 3% of total NSCLCs, a study with crizotinib on the whole population of NSCLC patients would securely lead to negative results, with the risk of leaving apart a drug that could be active on a selected population. A similar situation can occur in the case of HCC, where patient selection based on the activation of the MET pathway, a crucial step in targeted therapy, has never been done. Activation due to gene amplification or activating mutations is probably infrequent, since studies evaluating those alterations in the whole genome have revealed rare anomalies in the MET gene. The situation is more complex when overexpression is considered. Some studies have indeed revealed different degrees of overexpression, but the results are very heterogeneous and cannot be compared. Moreover, no proof has been given that the observed overexpression can lead to constitutive MET pathway activation. These evaluations, however, open new questions because, with the currently available IHC protocols, there are no validated techniques to detect MET phosphorylation and HGF expression in tumor samples.
      Figure thumbnail fx4

      Financial support

      This work was supported by Associazione Italiana Ricerca sul Cancro (AIRC, Grants IG-11821 to AC and IG-11819 to SG).

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      Acknowledgments

      We thank Dr. F. Natale for editing the manuscript.

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