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Hepatocyte growth factor/MET in cancer progression and biomarker discovery

Signaling driven by hepatocyte growth factor (HGF) and MET receptor facilitates conspicuous biological responses such as epithelial cell migration, 3‐D morphogenesis, and survival. The dynamic migration and promotion of cell survival induced by MET activation are bases for invasion–metastasis and resistance, respectively, against targeted drugs in cancers. Recent studies indicated that MET in tumor‐derived exosomes facilitates metastatic niche formation and metastasis in malignant melanoma. In lung cancer, gene amplification‐induced MET activation and ligand‐dependent MET activation in an autocrine/paracrine manner are causes for resistance to epidermal growth factor receptor tyrosine kinase inhibitors and anaplastic lymphoma kinase inhibitors. Hepatocyte growth factor secreted in the tumor microenvironment contributes to the innate and acquired resistance to RAF inhibitors. Changes in serum/plasma HGF, soluble MET (sMET), and phospho‐MET have been confirmed to be associated with disease progression, metastasis, therapy response, and survival. Higher serum/plasma HGF levels are associated with therapy resistance and/or metastasis, while lower HGF levels are associated with progression‐free survival and overall survival after treatment with targeted drugs in lung cancer, gastric cancer, colon cancer, and malignant melanoma. Urinary sMET levels in patients with bladder cancer are higher than those in patients without bladder cancer and associated with disease progression. Some of the multi‐kinase inhibitors that target MET have received regulatory approval, whereas none of the selective HGF‐MET inhibitors have shown efficacy in phase III clinical trials. Validation of the HGF‐MET pathway as a critical driver in cancer development/progression and utilization of appropriate biomarkers are key to development and approval of HGF‐MET inhibitors for clinical use.

Signaling driven by hepatocyte growth factor (HGF) and MET receptor facilitates conspicuous biological responses such as epithelial cell migration, 3-D morphogenesis, and survival. The dynamic migration and promotion of cell survival induced by MET activation are bases for invasion-metastasis and resistance, respectively, against targeted drugs in cancers. Recent studies indicated that MET in tumor-derived exosomes facilitates metastatic niche formation and metastasis in malignant melanoma. In lung cancer, gene amplification-induced MET activation and ligand-dependent MET activation in an autocrine/paracrine manner are causes for resistance to epidermal growth factor receptor tyrosine kinase inhibitors and anaplastic lymphoma kinase inhibitors. Hepatocyte growth factor secreted in the tumor microenvironment contributes to the innate and acquired resistance to RAF inhibitors. Changes in serum/plasma HGF, soluble MET (sMET), and phospho-MET have been confirmed to be associated with disease progression, metastasis, therapy response, and survival. Higher serum/plasma HGF levels are associated with therapy resistance and/or metastasis, while lower HGF levels are associated with progression-free survival and overall survival after treatment with targeted drugs in lung cancer, gastric cancer, colon cancer, and malignant melanoma. Urinary sMET levels in patients with bladder cancer are higher than those in patients without bladder cancer and associated with disease progression. Some of the multi-kinase inhibitors that target MET have received regulatory approval, whereas none of the selective HGF-MET inhibitors have shown efficacy in phase III clinical trials. Validation of the HGF-MET pathway as a critical driver in cancer development/progression and utilization of appropriate biomarkers are key to development and approval of HGF-MET inhibitors for clinical use.
T he MET oncogene was first isolated on the basis of its transforming activity, caused by a fusion of genes composed of the translocated promoter region (TPR) locus on chromosome 1 and MET sequence on chromosome 7 (TPR-MET). (1) Isolation of the full-length MET proto-oncogene sequence revealed that it encoded a transmembrane receptor tyrosine kinase (TK). (2) MET was thereafter identified as the receptor for hepatocyte growth factor (HGF). (3) Hepatocyte growth factor was identified and cloned as a mitogenic protein for hepatocytes, (4,5) while subsequent studies indicated that it was the same as scatter factor, an epithelial cell motility factor derived from fibroblasts and mesenchymal cells. (6)(7)(8) Conspicuous responses that are driven by the HGF-MET receptor pathway are dynamic 3-D morphogenesis and survival of cells. The induction of epithelial branching tubulogenesis in a 3-D collagen matrix by HGF had particular impact, because HGF was the first bioactive molecule to induce epithelial tubulogenesis. (9) Impairment in the hepatic progenitor cell survival and the migration of myogenic precursor cells seen in MET knockout mice indicate potent actions of HGF in dynamic migration and promotion of cell survival. (10) It was easy to speculate that the dynamic migration induced by HGF could also contribute critically to the biological basis of invasion and metastasis in tumor tissues. Meanwhile, involvement of the HGF-MET pathway in acquisition of a resistant phenotype against molecular targeted drugs was elucidated. (11,12) The potent action of HGF to promote cell survival is a prevalent biological basis for drug resistance in cancers.
Both HGF and MET are targets in anticancer drug discovery. (13) More than 10 different HGF-MET inhibitors entered into clinical trials, many of which were completed with unsatisfactory results. Recently, previously overlooked mutations in MET, resulting in deletions in the cytoplasmic juxtamembrane (JM) domain, have been found to be potential oncoprotein in non-small-cell lung cancer (NSCLC). Clinical studies have indicated favorable responses to MET inhibitors in patients with this variant MET. (14,15) We describe here recent progress in HGF-MET research on tumor biology and biomarker discovery.

Structures and Regulation of HGF-MET
The mature form of MET is composed of a 50-kDa b-chain and 145-kDa a-chain (Fig. 1a). The extracellular region is composed of SEMA, plexin-semaphorin-integrin (PSI), and immunoglobulin-like fold-plexin-transcription factor (IPT) 1-IPT4 domains. The intracellular region contains JM and TK domains. The binding of HGF to MET induces MET clustering and phosphorylation of Y1234 and Y1235, followed by phosphorylation of Y1349 and Y1356 in the carboxyl terminal region, to which adaptor molecules associate and transmit signals downstream. (7,8,13) Hepatocyte growth factor is secreted as a single-chain precursor (pro-HGF) and extracellular processing into a two-chain mature HGF is coupled to the activation of HGF (Fig. 1b). Hepatocyte growth factor-activator and matriptase are the main proteases responsible for the processing of HGF. (16) Hepatocyte growth factor binds to MET through two interfaces: the NK1 (N-terminal and first kringle domains) binds with high affinity whereas the b-chain binds with low affinity. The structure of the complex between the b-chain of HGF and the SEMA-PSI domains of MET were revealed by crystallographic analysis (Fig. 1c). (17) The activation of MET receptor by bivalent MET-binding macrocyclic peptides indicate that stable dimerization of MET with ligands of appropriate length provides a fundamental structural basis for activation of MET. (18) The JM domain, which is composed of 47 highly conserved amino acids, contains two protein phosphorylation sites and acts as a negative regulator in terms of MET-dependent signal transduction. One is Y1003 phosphorylation and the other is S985 phosphorylation. The CBL ubiquitin ligase binds phosphorylated Y1003, and this CBL binding results in MET ubiquitination, endocytosis, and degradation. (19) The CBL-mediated degradation of activated MET provides a mechanism that either attenuates or terminates MET-mediated signaling. Ser985 is phosphorylated by protein kinase-C and is dephosphorylated by protein phosphatase-2A. (20) When MET-S985 is phosphorylated, HGF-induced MET activation and subsequent biological responses are suppressed. (20)

Metastasis and Tumor Microenvironment
A definitive role of stromal fibroblasts in invasion of cancer cells into 3-D collagen was first noted using human oral squamous cell carcinoma cells, (21) and subsequent study indicated neutralization of HGF inhibited 3-D invasion induced by stromal fibroblasts. Independently, induction of invasiveness into collagen by HGF/scatter factor was noted during characterization of scatter factor. (6) These early studies showed the importance of HGF as a fibroblast-derived factor that facilitates the aggressive invasion of cancer cells.
The metastatic tumor microenvironment (premetastatic/metastatic niche) emerged as an important player in metastatic colonization and growth. A variety of stromal cells, such as macrophages, inflammatory cells, endothelial cells, and cancerassociated fibroblasts contribute to the formation of the metastatic microenvironment. (22) Growth factors play promoting roles in forming the metastatic microenvironment. Hepatocyte growth factor functions as a stromal cell-derived factor that strongly influences cancer cell invasiveness in the tumor microenvironment. (22) Selective inhibition of the HGF-MET pathway suppressed metastasis in experimental models. (7,8,13) A recent topic in cancer metastasis is the involvement of exosomes in metastasis. (23) MET in exosomes promotes metastatic microenvironment formation in metastatic melanoma (Fig. 2). (23) The exosomes from highly metastatic mouse and human melanoma cells contained high levels of MET, and exosomes in circulation localized to sites of metastatic tissues and increased vascular permeability, which promotes the migration of tumor cells. The exosomes also increased activated MET in bone marrow-derived cells, thereby being reprogrammed to a proangiogenic phenotype, and the bone marrow-derived cells mobilized to lungs where they could aid angiogenesis, invasion, and metastasis. Administration of exosomes that contained high levels of MET facilitated metastasis of melanoma cells with lower metastatic ability. (24)

Drug Resistance
The tumor microenvironment participates not only in cancer metastasis but also resistance to molecular-targeted drugs. Stromal cells influenced the sensitivity to anticancer drugs, and proteomic analysis revealed that stromal cell-derived HGF is a predominant factor that confers resistance to molecular-targeted drugs such as RAF inhibitor. (25) The biochemical basis as to how HGF so potently promotes survival as well as cell motility might relate to the adaptor protein GRB2-associated binding protein 1 (GAB1). The GAB1 protein has a unique recognition structure "MET-binding domain" that mediates its binding to phosphorylated MET. (26) Indeed, phenotypes in MET À/À and GAB1 À/À mice showed extensive similarities. (27) Non-small-cell lung cancer patients developed acquired resistance to epidermal growth factor receptor (EGFR) TK inhibitors (TKIs) within a few years, and 20-25% of the patients showed intrinsic resistance to EGFR-TKIs. As an acquired resistance mechanism, the T790M second mutation in EGFR occurs in approximately half of all patients. (28) As a bypass pathway, MET activation caused by MET gene amplification (11) and HGF-dependent MET activation (12) have been noted as mechanisms by which NSCLC acquires resistance to EGFR-TKIs. MET gene amplification was detected in 5-10% of patients with acquired resistance to EGFR-TKIs, and overexpression of HGF was seen in approximately 61% and 29% of patients with acquired and intrinsic resistance, respectively. (29) After the discovery of EML4-ALK as a driver oncogene in patients with NSCLC, (30) alectinib was developed as a selective anaplastic lymphoma kinase (ALK) TKI. (31) Based on its high objective response rate, long median progression-free survival, and favorable toxicity profile, alectinib has been approved in Japan and the USA. However, patients eventually acquire resistance to alectinib. Among several different mechanisms, alectinib-resistant EML4-ALK-positive NSCLC cells can acquire the ability to express HGF and the ensuing autocrine activation of MET caused by cancer cell-derived HGF confers acquired resistance to alectinib. (32) Collectively, the expression of HGF in cancer cells and/or stromal cells in the tumor microenvironment participates in the resistance to EGFR and ALK TKIs.

MET Mutations
The tight association between MET mutation and cancer development was first reported in hereditary and sporadic forms of papillary renal cell carcinoma. (33) Germline and somatic missense mutations (M1131T, V1188L, L1195V, V1220I, D1228N/H, Y1230C/H, M1250T/I) located in the TK domain of MET are found in papillary renal carcinomas (Fig. 3), and these are likely to be gain-of-function mutations. Missense mutations have been found in childhood hepatocellular carcinoma, head and neck squamous cell carcinoma, ovarian cancer, and small-cell lung cancer. (34) The JM-deleted MET generated by exon 14 skipping (MET-Dexon14) due to intronic mutations was noted in NSCLC cancer tissues and cells. (35) The expression of MET-Dexon14 in cells resulted in the loss of association with the CBL E3 ubiquitin ligase, decreased ubiquitination and prolonged activation of signaling molecules. (35) Considering the notion that MET-Y1003 phosphorylation in the JM domain provides CBL-binding for ubiquitination, MET-Dexon14 variant may have a longer lifespan in terms of protein stability and signaling.
Another mutant variant of MET with deleted extracellular IPT domains was found in approximately 6% of high-grade gliomas. (36) The mutation is caused by intronic mutations and the skipping of exon 7 (encoding a part of IPT1) and exon 8 (encoding a part of IPT2) generates a single pseudo-IPT domain. This MET exon 7-8 skipping variant is mainly Increased MET levels... Fig. 2. Outline of the mechanism for metastasis promoted by the hepatocyte growth factor (HGF)-MET pathway and tumor-derived exosomes in advanced metastatic melanoma. Peinado et al. showed that tumor-derived exosomes from advanced metastatic melanoma contained high levels of MET, and the exosomes induced an increase in the phosphorylated/activated MET in bone marrow-derived cells, thereby resulting in a mobilization of the bone marrow-derived cells to the lungs and lymph nodes, where they initiated metastatic niche formation. (28) Collectively, HGF facilitates local invasion, extravasation, and intravasation, and MET in exosomes facilitates angiogenesis and metastatic niche formation.

Exosomes
present as an unprocessed single chain form and located in the cytoplasm, suggesting an impairment in biosynthetic processing and subsequent translocation to the cell membrane. Missense mutations in MET have been found in a variety of cancers, and the positions of mutational changes are located not only in the intracellular domains, but also extracellular regions (Figs 1C,3A). The significance of these extracellular mutations is unknown.

Discovery of HGF/MET as Biomarkers
Collectively, HGF and sMET in blood, tissues, and/or urine are associated with changes in tumor characteristics and therapeutic responses in several types of tumors, indicating the significance of HGF, sMET, and related molecules as possible biomarkers for evaluation of tumor characteristics and therapeutic responses (Table 1). A substantial number of reports have documented increased circulating levels of HGF in a wide spectrum of cancers, and robust and sensitive immunoassays of soluble HGF protein have become widely available. Inflammatory mediators, including interleukin-1a (IL-1a), IL-1b, tumor necrosis factor-a, and prostaglandin E 2 increase gene expression of HGF in stromal cells. (37) Because these inflammatory mediators are increased in the tumor microenvironment and contribute to a drug-resistant and/or metastatic tumor microenvironment, it is likely that these inflammatory mediators participate in upregulation of HGF in tumors.
MET gene amplification and/or protein overexpression also frequently occur in cancer, which has accelerated investigations into MET gene copy number in tumors or by circulating soluble DNA, as well as MET protein content and phosphorylation (activation) state in tumor samples using a variety of approaches. Technical difficulties associated with the lability of MET and phospho-MET in formalin-fixed, paraffinembedded samples have hindered the development of clinically validated assays for use with archival tumor specimens, but recently reported assays for use with flash-frozen biopsy samples have provided reliable alternatives. (38) Athauda et al. (39) developed two-site electrochemiluminescent immunoassays of MET in flash-frozen samples and sMET ectodomain for plasma, serum, and urine samples, later adapting the assay to detect phospho-MET. (40) Efforts along these lines have identified specific contexts in which HGF/MET signaling contributes to cancer, and for some cancers, may help identify those patients in whom pathway inhibition is likely to have therapeutic benefit.        (13) A complete listing of trials with links to several relevant cancer information sources can be found online (https://ccrod.cancer.gov/confluence/display/CCRHGF/Home). ALK, anaplastic lymphoma kinase; EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; HSP90, heat shock protein 90; IMRT, intensity-modulated radiation therapy; mFOLFOX6, 5-fluorouracil, leucovorin, oxaliplatin; NSCLC, non-small-cell lung cancer; PD, pharmacodynamics; PK, pharmacokinetics; SCLC, smallcell lung cancer.
A recent topic in HGF/MET pathway inhibition is clinical studies in lung cancer patients with MET-Dexon14 alteration. Paik et al. (14) reported that MET-Dexon14 mutation is approximately 4% of lung adenocarcinoma, and three out of four patients with stage IV lung adenocarcinomas harboring MET-Dexon14 mutation had a response to MET TKI. Among 38 028 cancer patients, MET-Dexon14 mutations were found in 221 cases, and MET-Dexon14 mutations are detected most frequently in lung adenocarcinoma (3%), but also frequently in other lung neoplasms (2.3%) and brain glioma (0.4%). (15) In 11 205 lung cancers profiled by comprehensive genomic profiling, 298 (2.7%) carcinomas harbored MET-Dexon14 alterations. (77) Eight patients harboring MET-Dexon14 showed controlled responses, including four cases with partial responses, two cases with complete responses, and two cases with stable disease. (77) Among 1296 Chinese patients with NSCLC, 12 patients (0.9%) had MET-Dexon14 mutation, suggesting a difference in frequency by ethnicity. (78) It is anticipated that ongoing clinical studies will reveal the significance of MET-Dexon14 alteration as a biomarker and therapeutic target for clinical use of HGF-MET inhibitors.

Conclusions
Therapeutic resistance and metastasis are major obstacles to achieving durable clinical responses with molecular-targeted therapies. Signaling pathways driven by HGF and MET participate in invasion, metastasis, and resistance to moleculartargeted drugs. Although selective MET inhibitors have yet shown efficacy in phase II and III clinical trials, ongoing clinical trials have indicated favorable response to MET inhibitors in patients with NSCLC expressing variant MET deleted within the JM domain. Biomarker discovery and the utilization of appropriate biomarkers to validate HGF-MET signaling as a driver in cancer development, metastasis, and drug resistance appears to be key for regulatory approval of HGF-MET inhibitors for clinical use.
Because HGF is biosynthesized as a zymogen-like single chain inactive precursor (capable of MET binding but incapable of MET activation) and the processing to two-chain HGF is coupled to its activation, the measurement and evaluation of HGF activation is also key to understanding the tumor microenvironment that permits tumor metastasis and drug resistance. In the future, elucidation of the 3-D structure(s) of the HGF-MET complex and the MET activation process will provide an opportunity to discover molecular tools applicable to sensitive and specific detection of activation of HGF and MET for diagnosis and evaluation of therapeutics.