The Function of the HGF/c-Met Axis in Hepatocellular Carcinoma

1Hepatocellular carcinoma (HCC) is the sixth most common cancer and the fourth leading cause of cancer-related death in the world, and its burden is expected to increase in the next few years. The main cause of HCC is chronic liver disease caused by hepatitis virus B and/or C. However, other factors are also related to the occurrence of HCC, including alcohol-related liver disease, obesity, type 2 diabetes, and mellitus-related non-alcoholic fatty liver disease. Only about 40% of patients with early-stage or localized HCC are suitable for potentially curative treatments (surgical resection, liver transplantation, and local radiofrequency ablation) and 20% are suitable for transcatheter arterial chemoembolization (TACE) if in an intermediate stage. Due to lack of early effective diagnosis, around 80% patients are diagnosed with advanced HCC. The prognosis of advanced HCC is poor, the median overall survival (OS) time is roughly 1–2 months and only systemic therapy can improve survival time.

2Systemic treatments with sorafenib or lenvatinib followed by regorafenib, nivolumab, or ramucirumab have been demonstrated to improve survival. Among them, sorafenib [against c- rapidly accelerated fibrosarcoma (c-RAF), b-RAF, vascular endothelial growth factor receptor (VEGFR), c-KIT, and platelet-derived growth factor receptor] was the first drug for first-line treatment of advanced HCC, until the appearance of lenvatinib [against VEGFR/rearranged during transfection proto-oncogene (RET)/fibroblast growth factor receptor (FGFR)]. Second-line treatments have also been designed over the past few years, including regorafenib (antiangiogenesis), nivolumab [against the programed cell death-1 (PD1) immune checkpoint], and ramucirumab (against VEGFR2). For sorafenib used in first-line therapy, results of a randomized, placebo-controlled, phase III clinical trial investigating efficacy of sorafenib in 271 patients with HCC have been reported. The primary endpoint, median OS was 6.5 months for patients with sorafenib and 4.2 months for patients with placebo. Another recent study demonstrated that OS was higher in patients treated with sorafenib (10.7 months) than patients who received the placebo (7.9 months). However, the survival benefit of sorafenib was disappointing in clinical trials, owing to its low response rate and short effective duration, caused by intrinsic and acquired resistance. Therefore, we need to explore the resistance mechanism of sorafenib and novel treatments against HCC, such as c-Met and downstream signaling pathway inhibitors.

3Hepatocyte growth factor (HGF) and its high-affinity receptor, mesenchymal epithelial transition factor (c-Met), are closely related to the onset, progression and metastasis of multiple tumors. The HGF/c-Met axis has a pivotal role in normal liver growth, regeneration, and protection. Inappropriate Met activation promotes the onset, proliferation, invasion, and metastasis of HCC (Figure 1) via canonical or non-canonical pathways to regulate cell proliferation, movement, differentiation, invasion, angiogenesis, and anti-apoptosis. HCC patients with high expression of Met have lower survival time than patients with low or no expression of Met. Moreover, the HGF/c-Met axis is regarded as a tumor aggressiveness and prognosis biomarker of patients with HCC. In addition, c-Met plays a critical role in drug resistance. Therefore, targeting HGF/c-Met axis is one of the most promising therapies for HCC.

4Hepatocyte growth factor was originally discovered in in vitro experiments as a hepatocyte mitogen for primary hepatocytes that promoted the cell motility of epithelial cells. Subsequently, several studies also revealed other effects, such as intensifying cell motility, angiogenesis, immune response, cell differentiation, and anti-apoptosis. Hepatocyte stromal cells or HCC tumor cells can express and release HGF into the tumor microenvironment. HGF binds to its specific receptor, c-Met, which is located on the surface of hepatocytes, in a paracrine or autocrine manner. Moreover, the autocrine and paracrine activation of c-Met play an important role in the development and metastasis in HCC.

5Originally, in a chemically transformed human osteosarcoma cell line, researchers found the c-Met proto-oncogene and identified it as a fusion gene. It encodes the receptor for the ligand HGF. Several kinds of cells express c-Met, such as epithelial cells, neurons, hepatocytes, and hematopoietic cells. C-Met is a receptor tyrosine kinase (RTK) that is composed of a disulfide-linked heterodimeric complex. The complex is a transmembrane monomer that has five catalytic tyrosines in a cytoplasmic tail with four distinct hotspots. One of five catalytic tyrosine regulates c-Met negatively (Y1003), while the others (Y1234, Y1235, Y1349, Y1356) regulate c-Met positively. Y1003 regulates Cbl-mediated Met lysosomal degradation. Activated Y1234 and Y1235 upregulate kinase activity and result in phosphorylation of the docking site residues Y1349 and Y1356, leading to the recruitment of adaptor proteins and signaling molecules.

6Additionally, protein kinase-c activates S985 to degenerate c-Met. Hotspots are the domains of Met responsible for interaction with HGF. For four hotspots, the first hotspot is located on blades 2–3 of the semaphoring (SEMA) homology domain β-propeller, the known HGF β chain binging site. The second and third hotspot are known as the HGF α chain, localized on blade five of the SEMA domain and immunoglobulin-plexin-transcription factor (IPT) homology domains 2–3, respectively. The fourth hotspot is not previously correlated with the HGF binding site, which is across the plexinsemaphorin-integrin homology domain (PSI)-IPT 1 domains. C-Met, activated by the canonical pathway or the non-canonical pathway is involved in cell proliferation, motility, angiogenesis, invasion, and apoptosis.

7The canonical mode of c-Met activation involves the binding of HGF to c-Met, which induces homodimerization and autophosphorylation of the cytoplasmic domain of c-Met and then triggers downstream signaling pathways, including the mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK), phosphatidylinositol 3 kinase (PI3K), p-38, and the Akt/protein kinase B (PKB) pathways (Figure 2). Many studies have shown that these signaling pathways are shared with other RTKs. Adaptor proteins involved in these signaling pathways include, growth factor bound protein2 (Grb-2), Grb-2 associated binding protein 1 (Gab1), PI3K, signal transducer and activator of transcription 3 (STAT3), SH2-containing inositol 5-phosphatase 1 (SHIP1), phospholipase Cγ (PLCγ), SH2 domain-containing tyrosine phosphatase 2 (SHP2), and Src homology and collagen homology (Shc). These proteins induce hepatocarcinogenesis and contain Src-homology 2 (SH2) domains or phosphotyrosine-binding domains and Src homology 3 (SH3) domains, and are directly or indirectly bound to c-Met.

8C-Met can also be inappropriately activated by other pathways. Deregulated Met activation can induce several types of tumors in humans. (I) Des-γ-carboxy prothrombin (DCP) is secreted from HCC cells and activates c-Met because it contains two structural regions that are similar to HGF. Due to this similarity, DCP can bind to and activate c-Met. Moreover, DCP is used as a tumor screening and diagnostic biomarker owing to its sensitivity and specificity. (II) C-Met is modulated through crosstalk with different membrane receptors, including epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER), Integrin, β- catenin, cluster of differentiation-44 (CD44), intercellular adhesion molecule-1 (ICAM-1), Plexin B1, VEGF-A, insulin receptor (INSR), FAS, Mucin 1 (MUC1), neuropilin (Nrp)-1 and -2, and focal adhesion kinase (FAK) (Figure 2;; ). Although this crosstalk is not necessary for cell survival, it is able to better integrate the signals presented in the extracellular environment. Even if the crosstalk is redundant in physiological conditions, these interacting receptors may collaborate with each other in promoting tumorigenesis and/or metastasis and even cause resistance to target drugs in pathologic conditions. (III) C-Met overexpression drives the receptor activation and is induced by a few factors, including hypoxia, inactivation of tumor suppressor genes, activation of upstream oncogenes and loss of miRNAs. (IV) C-Met mutations can activate the receptor, thereby altering substrate specificity or catalytic activity. The identification of germline activating mutations in hereditary papillary renal carcinomas is unequivocal evidence correlating Met with cancer. (V) C-Met can also be activated by amplification. It has been shown that non-canonical pathways are associated with tumor progression, metastasis and drug resistance in in vivo experiments. (VI) Autocrine Met-induced-activation is due to ectopic Met expression in cells yielding HGF, especially in acute myeloid leukemia. (VII) miRNAs directly degrade messenger RNA or repress translation to regulate gene expression. Deregulated miRNA expression in HCC tissues has been detected. (VIII) Long non-coding RNAs (lncRNAs) could modulate c-Met expression by interacting with miRNAs. (IX) Slug can mediate activation of c-Met in a ligand-independent manner because of increased levels of fibronectin and induced integrin α V function.

9The HGF/c-Met axis has an important role in cellular behaviors, such as cell proliferation, migration, survival, morphogenesis and the epithelial–mesenchymal transition (EMT). Moreover, it also is essential for liver formation, growth, regeneration, protection, and angiogenesis during embryonic development and in adulthood after injury. In HGF and c-Met knockout mice, mice are embryonic lethal due in part to impaired liver formation. After partial hepatectomy, HGF expression levels increased quickly in rodents, and mice that conditionally inactivate c-Met in mature hepatocytes show insufficient liver regeneration. However, the aberrant activation of HGF/c-Met signaling pathways, such as c-Met over-expression, amplification, binding to other ligands or abnormally high HGF levels, leads the initiation and progression of tumors, such as non-small cell lung cancer, HCC, colon cancer, renal caner, and breast cancer. Furthermore, both the canonical and non-canonical signaling pathways require the dimerization and autophosphorylation of c-Met. Therefore, c-Met is the key factor in the HGF/c-Met signaling pathways. C-Met and its downstream signal mediators are promising targets in treating patients with advanced HCC.

10Numerous in vivo and in vitro studies have demonstrated that HGF/c-Met play a critical role in the development of various human cancers (renal, lung, liver, breast, colon, thyroid, ovarian, and pancreas). HGF/c-Met signaling pathways are uncontrolled in human cancer via overexpression of HGF or c-Met, gene amplification, mutational activation of c-Met, down-regulation of Met-targeted miRNA, binding to other ligands, autocrine signaling, or abnormally high HGF levels. Deregulated activation of c-Met contributes to a few aspects of tumor progression, such as inducing neoplastic cells to disaggregate from the tumor mass, eroding basement membranes, infiltrating stromal matrices, and finally colonizing new tissues to form metastases. Here we mainly discuss the HGF/c-Met axis in HCC.

11Chronic liver diseases such as cirrhosis and hepatitis B or C are triggers of HCC. There is a complicated interplay between HCC, chronic liver diseases and c-Met. Liver diseases reduce hepatocytes and increase the need for hepatocyte proliferation, thereby promoting up-regulation of c-Met and/or HGF. The increasing c-Met levels induce hepatocyte proliferation, regeneration, and survival during liver repair and delay the development of liver diseases by repressing chronic inflammation and the progression of fibrosis. Although it is potentially beneficial for liver diseases, increased c-Met activity can initiate, drive or promote the progression of HCC. Reversely, a knockout of the c-Met increased chemically-mediated HCC initiation but did not affect phenobarbital-induced HCC promotion. Moreover, the intact and normal HGF/c-Met signaling is elementary for sustaining normal redox homeostasis and could suppress tumor in the N-nitrosodiethylamine-induced HCC. Additionally, c-Met may induce VEGF-A expression, which can enhance tumor angiogenesis.

12As mentioned above, c-Met is aberrantly activated by gene amplification, overexpression, mutation, binding to other ligands, autocrine signaling or abnormally high HGF levels in cancer. However, according to the study by, Met amplification was at a very low frequency in HCC (one-twentieth). In the study by Kondo, the amplification frequency is 159th. Concerning activating Met kinase domain mutations, Park and Di Renzo and Lee and Aebersold observed three missense mutations in childhood HCC (K1262R, M12681, T11911, respectively). Mutations in the Casitas B-cell lymphoma (Cbl)-binding domain are demonstrated to be oncogenic because binding of Cbl to Y1003 causes Met ubiquitination that is vital to maintenance of physiological Met activation and prevention of continued activation of Met. Under normal conditions, activated Met is rapidly removed from the cell surface by ubiquitination, and then targets the lysosomal degradation chamber. More and more evidence shows that the ubiquitination of RTK is the key to its lysosomal degradation and recruitment of the ubiquitin protein ligase Cbl family is required for ligand-induced degradation of many RTKs. Moreover, the phosphorylation of Y1003 provides a direct docking site for the SH2-like crystal structure of Cbl (TKB) domain of Cbl ubiquitin ligase and is required for ligand-dependent ubiquitination and Met receptor degradation. Therefore, Y1003 mutations in the cbl-binding region can lead to the continued activation of Met and its downstream signaling pathways, and even induce cancer. Furthermore, mutations in the juxtamembrane domain led to tumorigenesis in an in vitro trial. Levels of c-Met were higher (or showed overexpression) in 20–48% of HCC samples than levels in peritumoral liver tissue. Over-expression of c-Met occurs more often than mutation and amplification. While not all HCC are related to HGF or c-Met overexpression, HCC patients with c-Met overexpression have poor prognosis. The expression of HGF is decreased in HCC, but is increased in peritumoral liver tissue. The increased secretion of HGF in the peritumoral liver tissue may be due to the increased release of HGF from hepatic stellate cells to the peritumoral liver tissue. While the decreased secretion of HGF in HCC tissue may be due to the HGF from HCC cells directly bounding to c-Met through autocrine pathway.

13Additionally, microRNAs (miRNAs) and suppressor of cytokine signaling 1 (SOCS1) can control hepatocarcinogenesis by regulating HGF/c-Met., have demonstrated that SOCS1 overexpression can inhibit HCC cells proliferation and migration by attenuating HGF-induced phosphorylation of c-Met, Gab1, and ERK1/2. MiR-181a can inhibit hepatocarcinogenesis through repressing activation of c-Met. Therefore, up-regulation of oncogenic miRNA induces HCC progression.

14In addition to the above, cooperation of the HGF/c-Met pathway with MUCI or β-catenin can induce hepatocarcinogenesis. demonstrated that loss of axis inhibition protein (Axin1) cooperated with c-Met to cause HCC in mice. Similarly, loss of β-Catenin also exacerbated hepatocarcinogenesis driven by Met and oncogenic β-catenin.

15A study carried out by Kaposi-Novak has found that a Met-regulated expression signature correlated vascular invasion rate and decreased mean survival time and microvessel density in a subset of human HCC and liver metastases. did a trial by using human Met transgenic mice to understand how ligand-independent activation of RTKs affects tumorigenesis. It was found that transgenic mice developed HCC, which subsided when the transgene was inhibited, which showed that Met over-expression induced tumorigenesis without HGF. The HGF/c-Met axis can also induce onset of HCC by promoting angiogenesis. In summary, although there are many ways to activate the c-met signaling pathway to induce the occurrence of HCC, c-met expression and activation are indispensable. Therefore, c-met is a therapeutic target that is worthy of research, and there are still many mechanisms of how HGF/c-Met signaling mediates tumorigenesis in HCC that we need to explore.

16Besides onset, the HGF/c-Met axis is also involved in proliferation of HCC. In 2005, to investigate the effects of c-Met expression on HCC cell growth used an adenovirus-delivered small interfering RNA (siRNA) method to observe the knockdown of c-Met on tumorigenic growth of HCC in in vitro and in vivo trials In the in vitro trial, compared with adenovirus alcohol dehydrogenase (AdH1)-null or mock-infected cells, proliferation of MHCC97-L cells, which had high c-Met expression, were inhibited by adenovirus AdH1-siRNA, and c-Met expression also decreased. The MHCC97-L cells were arrested at G1-G0 phase. In the in vivo study, the proliferative indices of adenovirus AdH1-siRNA/Met-injected mouse tumors were lower (23.4%) than the adenovirus AdH1-null injected tumors (69.8%) and mock-injected tumors (72.8%). C-Met expression was obviously reduced by adenovirus AdH1-siRNA/Met injection. In addition, some studies have found lncRNAs can promote HCC cells proliferation, migration, and even invasion. According to the study of Zhang, lncRNA FLVCR1-AS1 sponges miR-513c and increases c-Met expression in HCC cells, which induces HCC progression. In another study, has demonstrated that lncRNA HULC promotes HCC progression by inhibiting miR-2052 expression and activating c-Met signaling pathway. However, another study found that HGF plays a crucial role in HCC proliferation induced by cancer-associated fibroblasts from HCC (H-CAFs) in in vitro and in vivo trials. Tumor volume growth was consistent with HGF production. Furthermore, the effect of H-CAF conditioned medium on proliferation of HCC cells was significantly reduced by anti-HGF. Therefore, according to these studies, HGF/c-Met can induce proliferation of hepatocellular cells.

17The high lethality of HCC results from primary tumors invading and migrating to other tissues. This process begins with tumors invading blood vessels and subsequently migrating into intrahepatic and extrahepatic tissues. Tumor metastasis is a complicated multistep process and invasion is a major element of this process, which includes damage of basement membranes and proteolysis of the extracellular matrix (ECM). Several studies have reported that overexpression levels of HGF or c-Met in HCC correlate with incidence of invasion and metastasis and suggest that HGF/c-Met signaling had a crucial role in the invasion and metastasis of HCC cells. HGF/c-Met signaling pathways are involved in HCC cell invasion via HGF-induced c-Met phosphorylation, AKT phosphorylation, nuclear factor-κB (NF-κB) activation, and matrix metalloproteinase-9 (MMP-9) expression.

18To investigate the invasion and metastasis effect of HGF/c-Met signaling in HCC, Liu and colleagues conducted a study using two different HGF-treated HCC cell lines, Hep3B and HepG2. The Hep3B HCC cell line was p53 deficient and overexpressed c-Met after treatment with HGF. Loss of p53 expression reinforced HGF/c-Met signaling, which promoted invasion and metastasis by upregulating Snail expression. found that overexpression of c-Met induces cell invasion. Other studies have also reported that peritumoral stromal neutrophils and mesenchymal cells secrete high levels of HGF, which drove high rates of proliferation, invasion and metastasis in HCC by promoting the EMT. Moreover, phenotypic analysis validated that mixed-lineage leukemia (MLL), an epigenetic regulator, interacts with HGF/c-Met signaling to induce invasion and metastatic growth of HCC cell lines.

19The liver is an organ filled with blood vessels that rely on angiogenesis for cellular regeneration. Likewise, angiogenesis plays a critical role in tumor growth, invasion and metastasis. The angiogenic balance between proangiogenic and antiangiogenic factors maintains normal angiogenesis. However, the balance in HCC is disordered due to excessive angiogenic factors that are secreted by tumor cells, endothelial cells and pericytes. Many angiogenic factors, such as VEGF-A, HGF, transforming growth factor (TGF) and epidermal growth factor (EGF), demonstrated elevated expression levels in HCC tumors, Moreover, these factors induce angiogenesis through a number of mechanisms, one of them is via the HGF/c-Met signaling pathway. Several studies have reported that the HGF/c-Met axis induces angiogenesis and cell growth through interaction with the VEGF and VEGFR pathway and decreasing expression of thrombospondin-1.

20Although new treatments have been used in HCC patients and provided possible cures, the long-term survival rate is still poor due to late diagnosis and high recurrence. Therefore, sensitive and specific diagnostic or prognostic biomarkers are urgently needed. Although the HGF/c-Met axis is an emerging study target, the possibility of its use in diagnosis and prognosis has been studied in addition to its mechanism in HCC.

21In the study by, HCC patients had significantly increased serum levels of HGF than patients with chronic viral hepatitis C and cirrhosis. Thus, the serum HGF concentration may be helpful as a tumor biomarker for HCC. Likewise, and also identified serum HGF level as a potential diagnostic. However, demonstrated that the individual diagnostic performance of HGF was inadequate. Although the concentration of HGF is obviously higher in patients with alcoholic liver cirrhosis than in healthy humans, there is no significant difference in HGF serum level between cirrhosis patients with HCC and cirrhosis patients without HCC. It may be due to the reduction of hepatocytes in cirrhosis patient, which promotes the secretion of HGF and then activates c-Met to increase hepatocytes proliferation. Moreover, the diagnosis sensitivity of HGF was very high (90.62%) but the specificity was very low (25.81%). In conclusion, although the independent use of HGF for diagnosis is controversial, HGF is useful when combined with other diagnostic markers.

22Some studies also suggested that the HGF/c-Met axis has prognostic value for patients with HCC. Overexpression of c-Met correlated with decreased 5-year survival in patients with HCC. In addition, the Met-driven expression signature defines a subset of HCC which has poor prognosis and an aggressive phenotype. has found that inoperable patients with HCC had higher levels of serum HGF than healthy humans due to the impaired clearance of HGF. Serum HGF concentration was negatively correlated with long-term survival time in HCC patients. Additionally, a serum HGF level of 1.0 ng/mL or more indicated a serious prognosis in patients with HCC. Nevertheless, another study showed that c-Met was not an independent prognostic factor of HCC for OS and cumulative recurrence, but the combination of c-Met/CD151 was. Meanwhile, an other study also suggested that the prognostic value of c-Met is contradictory. By univariate analysis, c-Met overexpression was significantly correlated with clinicopathological factors, but not with multivariate analysis. Furthermore, c-Met overexpression was not identified to be obviously correlated with OS rates in this study. The reason for the difference in these studies may be the small number of surveyed people or the different technique and scoring system. Thus, the role of HGF and c-Met as prognostic factors for HCC needs to be explored further in the future. Though, the combination of HGF and c-Met with other biomarkers may be useful in predicting the prognosis of HCC.

23As mentioned above, there are five therapies that can prolong the expected lifespan of patients with HCC including, surgical resection, liver transplantation, local ablation, TACE and sorafenib. Only 40% percent of patients with early stage HCC are eligible for potentially curative treatments (surgical resection, transplantation, local ablation), which prolong median survival times over 60 months. For patients with intermediate-stage HCC, TACE can improve estimated median survival by 26 months. However, a large proportion of HCC patients are diagnosed at advanced stage and only systemic treatment with sorafenib can extend OS from 6 to 11 months. Thus, sorafenib is regarded as the first-line treatment in advanced HCC patients with a manageable adverse event. Nevertheless, in the past few years, seven randomized phase III clinical trials, which tested other first-line and second-line treatments, in intermediate-stage or advanced-stage HCC patients have not found any obvious OS benefits. Moreover, the intrinsic or acquired resistance of sorafenib is the major obstacle in treatment. Thus, based on the understanding of the role of the HGF/c-Met signaling pathway and the uniqueness of c-Met in HCC, more and more therapeutic strategies target c-Met and the interaction between c-Met and downstream signaling mediators instead of the interaction between HGF and c-Met because of the varying activation of c-Met.

24So far, there are six Met inhibitors developed and they have been tested in 10 HCC clinical trials. Small molecular kinase inhibitors can block phosphorylation of the catalytic domain in the receptor by competitive or non-competitive antagonism of the ATP binding site, thereby preventing the recruitment of signal transducers and mediators, and thus impeding the transmission of downstream signals. Anti-c-Met agents can be categorized into three types: selective c-Met tyrosine kinase inhibitors (TKIs), multi-targeted TKIs including against c-Met, and monoclonal antibodies against HGF or c-Met. A lot of preclinical studies have demonstrated the feasibility of HGF/c-Met as targets for the treatment of patients with HCC. For instance, AMG 337, a potential and highly selective small molecule Met kinase inhibitor, significantly decreases tumor growth of Met-high-expression and Met-amplified HCC cell lines in in vitro and in vivo trials. Additionally, Indo5, selectively abrogating HGF-induced c-Met pathway activation and brain-derived neurotrophic factor (BDNF)/nerve growth factor (NDF)-induced Trks signaling activation, significantly inhibits HCC tumor growth in xenograft mice. Moreover, PHA665752 supressed cell proliferation, and induced apoptosis in MHCC97-L and MHCC97-H cells which overexpress c-Met through blocking phosphorylation of c-Met and downstream PI3K/Akt and MAPK/Erk pathways. PHA665752 also repressed MHCC97-L and MHCC97-H tumor growth in xenograft models.

25Also, several clinical trials are being carried out in HCC patients using c-Met inhibitors (Table 1), including cabozanitinib, foretinib, cobazitinib, gefitinib, crizotinib, MSC2156119, AZD4547, MK2461, and INC280. Among them, cabozanitinib is undergoing randomized phase III clinical trials. In the latest clinical trial, OS and progression-free survival (PFS) in months was calculated for Phase 2 of Tepotinib 500 mg (5.55 and 3.22 months, respectively) (NCT02115373). In a randomized phase II study of axitinib, axitinib combined with best supportive care (BSC) did not improve OS versus placebo combined with BSC (NCT01210495). Nevertheless, axitinib combined with BSC led to a significant prolongation of PFS and time to tumor progression (TTP) and an increase of clinical benefit rate (CBR), and the toxicity of patients with advanced HCC is acceptable.

26However, there is currently no effective method for treating HCC based on traditional monotherapy with TKIs. In a previous phase II randomized controlled clinical study, Tivantinib, a highly selective c-Met inhibitor, improved median time to progression and OS time in patients with Met-high advanced HCC compared with placebo. However, in a randomized phase III double-blind clinical trial, tivantinib did not improve OS time in patients with Met-high advanced HCC treated with sorafenib compared with placebo (NCT01755767). Despite the failure of tivantinib in a phase III clinical trial to achieve its primary endpoint, we could not deny the role of c-Met inhibitors in the treatment of liver cancer. The reason for this failure, in my opinion, may be due to tivantinib being a non-selective c-Met inhibitor rather a selective c-Met inhibitor. The cytotoxicity against many HCC cell lines of tivantinib was unrelated to c-Met expression but related to inhibiting microtubule assembly and Glycogen Synthase Kinase-3 alpha (GSK3a) and beta (GSK3b) in other studies. Inhibition of non-c-Met targets may enhance the antitumor activity of non-selective c-Met inhibitors, but is also correlated with increased toxicity and limits the dose so that inhibitors cannot effectively suppress c-Met. Furthermore, the increased toxicity and limitation of potential benefit may outweigh the enhanced antitumor activity because of inhibition of multiple targets. Moreover, we cannot attribute the anti-tumor effect of non-selective c-Met inhibitors to the inhibition of c-Met. Thus, selective c-Met inhibitors may be a better choice for treatment in patients and more studies are needed to identify the reason for phase II clinical trial failure and the feasibility of using c-Met targeting therapies.

27Fortunately, using potential miRNAs for suppressing aberrant c-Met signal is an emerging and promising therapy strategy that bypasses traditional approaches (Table 2). miRNAs are small non-coding RNAs and regulate gene expression by resolving mRNA or suppressing translation. Previous studies have found that miRNA expression in cancer tissue is different from normal tissues. miRNAs can not only inhibit tumor growth, proliferation, invasion, and metastasis but also induce many kinds of tumors. Herein, we mainly focus on the role of miRNAs in HCC. For example, miR-101 suppresses the proliferation and migration of HCC cells and tumors through targeting HGF/c-Met, Girdin, SOX9 and TGF-β in in vitro and in vivo trials. miRNA-206 targets c-Met and cyclin-dependent kinase 6 (Cdk6) to suppress development of HCC in mice. Also, miR-26a inhibits tumor growth, metastasis and angiogenesis of HCC via targeting HGF-induced c-Met signaling pathways and FBXO11, ST3GAL5 signaling pathways. MiR-93 induces HCC cell proliferation and invasion by activating c-Met/PI3K/Akt signaling pathways and targeting PDCD4 and TIMP2. Thus, we can design miRNA mimics to overexpress miRNAs that down-regulate c-Met signaling pathways, and design miRNAs antagonists to inhibit miRNAs that up-regulate c-Met signaling pathways. Moreover, one of the major advantages of miRNA therapy is simultaneously targeting multiple effectors in several signaling pathways involved in tumorigenesis.

28Although c-Met inhibitors show a survival benefit for advanced HCC, there has also been some toxicity and adverse effects demonstrated. For instance, compound 8 was a potent and highly selective ATP competitive c-Met inhibitor. Moreover, compound 8 showed good oral bioavailability and good half life and moderate plasma clearance and volume distribution. In addition, Compound 8 also demonstrated effective tumor inhibition. However, it increased heart rate, cardiac output, and induced myocardial degeneration in mice and thus was terminated as a preclinical candidate. Also, adverse effects such as hypertension, decreased appetite, ascites and pyrexia were found in a phase I/II multicenter study of the single-agent foretinib. Additionally, ascites, anemia, abdominal pain, and neutropenia were observed in a phase III study of tivantinib. The reason for these aforementioned side effects may be correlated with the physiological function of HGF/c-Met in many organs, including cytoprotective, regenerative, and reduction of apoptosis after injury. Thus, c-Met inhibitors may block the physiological function of HGF/c-Met and induce side effects. Moreover, the adverse effect of c-Met inhibitors may be due to the inhibition of non-c-Met targets when using non-selective c-Met inhibitors.

29To solve the side effects mentioned above, several therapy strategies have been suggested. Among them, specifically targeting the downstream mediators of c-Met involved in tumor progression is a promising method, including the Grb2 SH2 domain, Src, MAPK, STAT3, Shp2, and Fak. Nevertheless, there are still problems, especially because these signal pathways are shared with other RTKs, which may cause other unpredictable reactions. Therefore, this requires the identification of more specific and suitable downstream targets.

30Recently endosomal processing has been demonstrated to play a pivotal role in the progression of HCC. Receptor endocytosis is crucial for signal transduction, either clathrin-dependent or –independent. HGF binding to c-Met induces the activation of downstream signal mediators, including ERK, c-Jun N-terminal kinase (JNK) and AKT. Both JNK and ERK mediate HCC cell migration by phosphorylating paxillin at serine residues, which is called an HGF-induced focal adhesion signaling molecule. Protein kinases C ε (PKCε) and golgi-localized γ-ear-containing ARF binding protein 3 (GGA3) regulate HGF-induced c-Met endocytosis to direct fluctuating JNK and paxillin signaling pathways which involve HCC cell migration. Importantly, endocytosis blockers, such as dynasore, could prevent the HGF-induced HCC cell migration and invasion by inhibiting critical endosomal components. Thus, critical endosomal components may be promising targets in HGF/c-Met signaling pathways for HCC treatment. In addition, suggested that hydrogen peroxide-inducible clone-5 (Hic-5) may be crucial for c-Met signaling pathways and HCC metastasis because it mediates HGF-induced reactive oxygen species (ROS)-JNK-signaling pathways in HCC and may be also a specific and safe target for treating HCC patients.

31In recent years, more and more studies have found that natural compounds can inhibit the progression of liver cancer. For example, deguelin can suppress tumor angiogenesis on vascular endothelial cells by decreasing autocrine VEGF and repressing HGF-induced c-Met signaling pathways, thereby inhibiting HCC progression. Also, Cinobufacini, a well-known traditional Chinese medicine extracted from toad skins and venom glands, has a therapeutic effect in HCC. A study has found that Cinobufacini could suppress HepG2 cell invasion and metastasis through the inhibition of the c-Met/ERK induced EMT. Moreover, madecassoside (MAD), isolated from Centella asiatica could repress the activation of the HGF-induced c-Met-PKC-ERK1/2-Cyclooxygenase-2 (COX-2)-Prostaglandin E2 (PGE2) cascade to inhibit HCC cell proliferation and invasion. In conclusion, natural compounds and herbals may be potential therapeutic targets for HCC.

32C-Met inhibitor therapy has failed to result in satisfactory outcomes in phase III clinical trials for HCC. Therefore, it is urgent to understand mechanisms and find new strategies such as effective combination therapies. Several studies have shown resistance in c-Met inhibitors due to various mechanisms. First, c-Met inhibitors only affect high-expression c-Met patients with HCC. Thus, enrolled low-expression c-Met patients with HCC could lead to the occurrence of resistance in c-Met inhibitors. Second, as mentioned above, c-Met inhibitors which target the interaction between HGF and c-Met may lose efficacy owing to cell attachment, gene amplification of c-Met, DCP binding to c-Met, gene mutation in the c-Met activation loop and crosstalk with other membrane receptors. Third, inhibition of c-Met signaling pathways triggers the EGFR pathway as a compensatory survival pathway. Fourth, phosphorylation status of FGFR determines different sensitivities of HCC cells to c-Met inhibitors. Fifth, suggested that c-Met inhibitors up-regulate the expression of PD-L1 in HCC cells by suppressing GSK3B-mediated PD-L1 degradation and induce T-cell suppression and tumor evasion of the immune response. Finally, when inhibiting the activation of HGF-induced c-Met, HCC cells can sustain survival through Y1234/1235-dephosphorylated c-Met induced autophagy.

33New therapeutic strategies have been developed against the mechanism of c-Met inhibitors resistance described above. Among them, combined c-Met inhibitors and other pathway inhibitors is a promising treatment. For example, combined inhibition of both c-Met and EGFR pathways could repress the tumor growth of HCC. Additionally, targeting both c-Met and FGFR pathways provides superior suppression of HCC progression. Also, combination of c-Met inhibitor and anti-PD1 treatment represses HCC growth and improves mouse survival. Moreover, targeting c-Met and autophagy could overcome resistance in HCC.

34Recently, a growing body of evidence suggests that aberrant activation of HGF/c-Met signaling is associated with resistance of target therapies, including sorafenib. Sorafenib is a standard therapy for advanced HCC, thus the resistance to sorafenib is a major concerning problem. have found that in HCC patients on long term sorafenib treatment, the upregulation of HGF induces autocrine activation of HGF/c-Met signaling pathways, increasing the invasion and migration abilities of HCC cells and leading to resistance to sorafenib. Moreover, a recent study has demonstrated that tumor associated M2 macrophages secret HGF in a feed-forward manner, leading the resistance to sorafenib. Also, a new study found that HGF activates phosphorylated (P)-ERK/Snail/EMT, P-STAT3/Snail/EMT and AKT/ERK1/2-EGR1 signaling pathways to induce the resistance to sorafenib. According to the study of Chen, IncRNA NEAT1 induces sorafenib resistance of HCC patients through repressing miR-335 expression and activation of c-Met-Akt signaling pathway. Thus, the concentration of HGF in serum may be a potential predictive marker for sorafenib efficacy and the combination of HGF and c-Met inhibitors and sorefenib could improve the efficacy of the first line systemic treatment. For instance, regorafenib plays a crucial role of reversing HGF-induced sorafenib resistance through the inhibition of the EMT. Angiopoietin-like protein (ANGPTL1) not only inhibits sorafenib resistance, but also inhibits cancer stemness and tumor growth of HCC cells via suppressing the EMT through the Met-AKT/ERK-EGR-1-Slug signaling cascade.

35In conclusion, although the tivantinib phase III trial failed and the reason is not clear, the role of c-Met inhibitors in treating HCC can not be denied. C-Met inhibitors are still mainstream of research and deserve more research on its cause of failure and new clinical trials. miRNAs, natural compound and herbal medicines are emerging treatments of HCC which can inhibit multiple pathways including c-Met signaling pathway. But it also means that there may be other side effects. The advantage of c-met downstream pathway inhibitors is that they are highly targeted and have little side effects, but this requires us to further understand the key targets of HCC. The drug resistance may be due to the heterogeneity of liver cancer cells and combination therapy may be a good solution.

36The HGF/c-Met axis has an important role in cellular behaviors such as cell proliferation, migration, survival, migration, morphogenesis, and the EMT. Moreover, it is also essential for liver formation, growth, regeneration and protection and angiogenesis during embryonic development and in adulthood after injury. Especially in chronic liver diseases, inflammation decreases hepatocytes and increases the need for c-Met activity to promote hepatocyte proliferation, regeneration and suppress inflammation. Nevertheless, the aberrant activation of c-Met and downstream signaling pathways through overexpression of HGF or c-Met, gene amplification, mutational activation of c-Met, down-regulation of Met-targeted miRNA, binding to other ligands, autocrine signaling or abnormally high HGF levels initiates and drives tumorigenesis and promotes tumor growth, invasion, metastasis, and angiogenesis in HCC. In the progression of liver cancer, c-Met is regulated by various factors such as miRNAs and SOCS1. Furthermore, c-Met cooperates with other signaling pathways such as MUCI or β-catenin in promoting tumorigenesis. Therefore, the treatment of liver cancer with c-Met as a target is a potential and promising therapy strategy. So far, six c-Met inhibitors have entered clinical trials and have been shown to inhibit tumor growth and invasion. Moreover, selective c-Met inhibitors are superior to non-selective c-Met inhibitors in the treatment of liver cancer due to their low toxicity. However, the failure of the tivantinib phase III trial suggests that we need to further study the causes of failure and the feasibility of c-Met inhibitors in treating HCC.

37In addition, we should also consider the relationship between c-Met inhibitors and liver disease, although there is no clinical evidence that c-Met inhibitors worsen liver function. In chronic liver diseases, c-Met expression is increased to promote hepatocyte proliferation and inhibit inflammation. C-Met inhibitors suppress this positive regulation and may accelerate advanced liver disease. Meanwhile, liver diseases affect the drug pharmacokinetics, pharmacodynamics, reduce enzyme activity, impair hepatic clearance of drugs and even change the interplay between drugs. These effects may alter the dose of the drug needed to reach the desired blood concentration and induce novel toxicities. Thus, we should consider whether patients with Child-Pugh B or C disease can tolerate the dose established in clinical trials because in the past, most clinical trials were conducted in patients with Child-Pugh A disease.

38Besides c-Met inhibitors, many new therapeutic strategies have been developed, such as the use of miRNAs to regulate HGF/c-Met signaling pathways to inhibit liver cancer progression, targeting endocytosis and more downstream molecules, Hic-5 as a therapeutic strategy to reduce side effects of c-Met inhibitors, as well as herbal treatments. c-Met is also involved in the resistance mechanism of sorafenib, which can be solved by the combination of c-Met inhibitor and sorafenib. For the self-resistance of c-Met inhibitors, the combination of c-Met inhibitors and other inhibitors can be used to solve this problem, such as FGFR and EGFT inhibitors, autophagy inhibitor and anti-PD1 treatment. Whether HGF/c-Met can be used as independent diagnostic and prognostic markers still requires further research, but HGF/c-Met in combination with other diagnostic and prognostic markers is valuable. In general, the mechanism of the HGF/c-Met pathway involvement in liver cancer requires more research, and c-Met inhibitors are a potential and promising therapeutic strategy in patients with HCC.

39ZR and ZY proposed the study and the guarantors. HW, BR, and JL performed the research and wrote the first draft. All authors contributed to the interpretation of the study and to further drafts.

40The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.