C-Met
c-Met, also called tyrosine-protein kinase Met or hepatocyte growth factor receptor, is a protein that in humans is encoded by the MET gene. The protein possesses tyrosine kinase activity. The primary single chain precursor protein is post-translationally cleaved to produce the alpha and beta subunits, which are disulfide linked to form the mature receptor.
MET is a single pass tyrosine kinase receptor essential for embryonic development, organogenesis and wound healing. Hepatocyte growth factor/Scatter Factor and its splicing isoform are the only known ligands of the MET receptor. MET is normally expressed by cells of epithelial origin, while expression of HGF/SF is restricted to cells of mesenchymal origin. When HGF/SF binds its cognate receptor MET it induces its dimerization through a not yet completely understood mechanism leading to its activation.
Abnormal MET activation in cancer correlates with poor prognosis, where aberrantly active MET triggers tumor growth, formation of new blood vessels that supply the tumor with nutrients, and cancer spread to other organs. MET is deregulated in many types of human malignancies, including cancers of kidney, liver, stomach, breast, and brain. Normally, only stem cells and progenitor cells express MET, which allows these cells to grow invasively in order to generate new tissues in an embryo or regenerate damaged tissues in an adult. However, cancer stem cells are thought to hijack the ability of normal stem cells to express MET, and thus become the cause of cancer persistence and spread to other sites in the body. Both the overexpression of Met/HGFR, as well as its autocrine activation by co-expression of its hepatocyte growth factor ligand, have been implicated in oncogenesis.
Various mutations in the MET gene are associated with papillary renal carcinoma.
Gene
MET proto-oncogene has a total length of 125,982 bp, and it is located in the 7q31 locus of chromosome 7. MET is transcribed into a 6,641 bp mature mRNA, which is then translated into a 1,390 amino-acid MET protein.Protein
MET is a receptor tyrosine kinase that is produced as a single-chain precursor. The precursor is proteolytically cleaved at a furin site to yield a highly glycosylated extracellular α-subunit and a transmembrane β-subunit, which are linked together by a disulfide bridge.Extracellular
- Region of homology to semaphorins, which includes the full α-chain and the N-terminal part of the β-chain
- Cysteine-rich MET-related sequence
- Glycine-proline-rich repeats
- Four immunoglobulin-like structures, a typical protein-protein interaction region.
Intracellular
- a serine residue, which inhibits the receptor kinase activity upon phosphorylation
- a tyrosine, which is responsible for MET polyubiquitination, endocytosis, and degradation upon interaction with the ubiquitin ligase CBL
- Tyrosine kinase domain, which mediates MET biological activity. Following MET activation, transphosphorylation occurs on Tyr 1234 and Tyr 1235
- C-terminal region contains two crucial tyrosines, which are inserted into the multisubstrate docking site, capable of recruiting downstream adapter proteins with Src homology-2 domains. The two tyrosines of the docking site have been reported to be necessary and sufficient for the signal transduction both in vitro.
MET signaling pathway
Tyr 1349 and Tyr 1356 of the multisubstrate docking site are both involved in the interaction with GAB1, SRC, and SHC, while only Tyr 1356 is involved in the recruitment of GRB2, phospholipase C γ, p85, and SHP2.
GAB1 is a key coordinator of the cellular responses to MET and binds the MET intracellular region with high avidity, but low affinity. Upon interaction with MET, GAB1 becomes phosphorylated on several tyrosine residues which, in turn, recruit a number of signalling effectors, including PI3K, SHP2, and PLC-γ. GAB1 phosphorylation by MET results in a sustained signal that mediates most of the downstream signaling pathways.
Activation of signal transduction
MET engagement activates multiple signal transduction pathways:- The RAS pathway mediates HGF-induced scattering and proliferation signals, which lead to branching morphogenesis. Of note, HGF, differently from most mitogens, induces sustained RAS activation, and thus prolonged MAPK activity.
- The PI3K pathway is activated in two ways: PI3K can be either downstream of RAS, or it can be recruited directly through the multifunctional docking site. Activation of the PI3K pathway is currently associated with cell motility through remodeling of adhesion to the extracellular matrix as well as localized recruitment of transducers involved in cytoskeletal reorganization, such as RAC1 and PAK. PI3K activation also triggers a signal due to activation of the AKT pathway.
- The STAT pathway, together with the sustained MAPK activation, is necessary for the HGF-induced branching morphogenesis. MET activates the STAT3 transcription factor directly, through an SH2 domain.
- The beta-catenin pathway, a key component of the Wnt signaling pathway, translocates into the nucleus following MET activation and participates in transcriptional regulation of numerous genes.
- The Notch pathway, through transcriptional activation of Delta ligand.
Role in development
MET mediates a complex program known as invasive growth. Activation of MET triggers mitogenesis, and morphogenesis.During embryonic development, transformation of the flat, two-layer germinal disc into a three-dimensional body depends on transition of some cells from an epithelial phenotype to spindle-shaped cells with motile behaviour, a mesenchymal phenotype. This process is referred to as epithelial-mesenchymal transition. Later in embryonic development, MET is crucial for gastrulation, angiogenesis, myoblast migration, bone remodeling, and nerve sprouting among others. MET is essential for embryogenesis, because MET −/− mice die in utero due to severe defects in placental development. Along with Ectodysplasin A, it has been shown to be involved in the differentiation of anatomical placodes, precursors of scales, feathers and hair follicles in vertebrates. Furthermore, MET is required for such critical processes as liver regeneration and wound healing during adulthood.
HGF/MET axis is also involved in myocardial development. Both HGF and MET receptor mRNAs are co-expressed in cardiomyocytes from E7.5, soon after the heart has been determined, to E9.5. Transcripts for HGF ligand and receptor are first detected before the occurrence of cardiac beating and looping, and persist throughout the looping stage, when heart morphology begins to elaborate. In avian studies, HGF was found in the myocardial layer of the atrioventricular canal, in a developmental stage in which the epithelial to mesenchymal transformation of the endocardial cushion occurs. However, MET is not essential for heart development, since α-MHCMet-KO mice show normal heart development.
Expression
Tissue distribution
MET is normally expressed by epithelial cells. However, MET is also found on endothelial cells, neurons, hepatocytes, hematopoietic cells, melanocytes and neonatal cardiomyocytes. HGF expression is restricted to cells of mesenchymal origin.Transcriptional control
MET transcription is activated by HGF and several growth factors. MET promoter has four putative binding sites for Ets, a family of transcription factors that control several invasive growth genes. ETS1 activates MET transcription in vitro. MET transcription is activated by hypoxia-inducible factor 1, which is activated by low concentration of intracellular oxygen. HIF1 can bind to one of the several hypoxia response elements in the MET promoter. Hypoxia also activates transcription factor AP-1, which is involved in MET transcription.Clinical significance
Role in cancer
MET pathway plays an important role in the development of cancer through:- activation of key oncogenic pathways ;
- angiogenesis ;
- scatter, which often leads to metastasis.
MET amplification has emerged as a potential biomarker of the clear cell tumor subtype.
The amplification of the cell surface receptor MET often drives resistance to anti-EGFR therapies in colorectal cancer.
Role in autism
The SFARIgene database lists MET with an autism score of 2.0, which indicates that it is a strong candidate for playing a role in cases of autism. The database also identifies at least one study that found a role for MET in cases of schizophrenia. The gene was first implicated in autism in a study that identified a polymorphism in the promoter of the MET gene. The polymorphism reduces transcription by 50%. Further, the variant as an autism risk polymorphism has been replicated, and shown to be enriched in children with autism and gastrointestinal disturbances. A rare mutation has been found that appears in two family members, one with autism and the other with a social and communication disorder. The role of the receptor in brain development is distinct from its role in other developmental processes. Activation of the MET receptor regulates synapse formation and can impact the development and function of circuits involved in social and emotional behavior.Role in heart function
In adult mice, MET is required to protect cardiomyocytes by preventing age-related oxidative stress, apoptosis, fibrosis and cardiac dysfunction. Moreover, MET inhibitors, such as Crizotinib or PF-04254644, have been tested by short-term treatments in cellular and preclinical models, and have been shown to induce cardiomyocytes death through ROS production, activation of caspases, metabolism alteration and blockage of ion channels.In the injured heart, HGF/MET axis plays important roles in cardioprotection by promoting pro-survival effects in cardiomyocytes, angiogenesis, inhibition of fibrosis, anti-inflammatory and immunomodulatory signals, and regeneration through activation of cardiac stem cells.
Interaction with tumour suppressor genes
PTEN
is a tumor suppressor gene encoding a protein PTEN, which possesses lipid and protein phosphatase-dependent as well as phosphatase-independent activities. PTEN protein phosphatase is able to interfere with MET signaling by dephosphorylating either PIP3 generated by PI3K, or the p52 isoform of SHC. SHC dephosphorylation inhibits recruitment of the GRB2 adapter to activated MET.VHL
There is evidence of correlation between inactivation of VHL tumor suppressor gene and increased MET signaling in renal cell carcinoma and also in malignant transformations of the heart.Cancer therapies targeting HGF/MET
Since tumor invasion and metastasis are the main cause of death in cancer patients, interfering with MET signaling appears to be a promising therapeutic approach. A comprehensive list of HGF and MET targeted experimental therapeutics for oncology now in human clinical trials can be found .MET kinase inhibitors
Kinase inhibitors are low molecular weight molecules that prevent ATP binding to MET, thus inhibiting receptor transphosphorylation and recruitment of the downstream effectors. The limitations of kinase inhibitors include the facts that they only inhibit kinase-dependent MET activation, and that none of them is fully specific for MET.- K252a is a staurosporine analogue isolated from Nocardiopsis sp. soil fungi, and it is a potent inhibitor of all receptor tyrosine kinases. At nanomolar concentrations, K252a inhibits both the wild type and the mutant MET function.
- SU11274 specifically inhibits MET kinase activity and its subsequent signaling. SU11274 is also an effective inhibitor of the M1268T and H1112Y MET mutants, but not the L1213V and Y1248H mutants. SU11274 has been demonstrated to inhibit HGF-induced motility and invasion of epithelial and carcinoma cells.
- PHA-665752 specifically inhibits MET kinase activity, and it has been demonstrated to represses both HGF-dependent and constitutive MET phosphorylation. Furthermore, some tumors harboring MET amplifications are highly sensitive to treatment with PHA-665752.
- ARQ197 is a promising selective inhibitor of MET, which entered a phase 2 clinical trial in 2008.
- Foretinib targets multiple receptor tyrosine kinases with growth-promoting and angiogenic properties. The primary targets of foretinib are MET, VEGFR2, and KDR. Foretinib has completed a phase 2 clinical trials with indications for papillary renal cell carcinoma, gastric cancer, and head and neck cancer.
- SGX523 specifically inhibits MET at low nanomolar concentrations.
- MP470 is a novel inhibitor of c-KIT, MET, PDGFR, Flt3, and AXL. Phase I clinical trial of MP470 had been announced in 2007.
HGF inhibitors
- NK4 competes with HGF as it binds MET without inducing receptor activation, thus behaving as a full antagonist. NK4 is a molecule bearing the N-terminal hairpin and the four kringle domains of HGF. Moreover, NK4 is structurally similar to angiostatins, which is why it possesses anti-angiogenic activity.
- Neutralizing anti-HGF antibodies were initially tested in combination, and it was shown that at least three antibodies, acting on different HGF epitopes, are necessary to prevent MET tyrosine kinase activation. More recently, it has been demonstrated that fully human monoclonal antibodies can individually bind and neutralize human HGF, leading to regression of tumors in mouse models. Two anti-HGF antibodies are currently available: the humanized AV299, and the fully human AMG102.
- Uncleavable HGF is an engineered form of pro-HGF carrying a single amino-acid substitution, which prevents the maturation of the molecule. Uncleavable HGF is capable of blocking MET-induced biological responses by binding MET with high affinity and displacing mature HGF. Moreover, uncleavable HGF competes with the wild-type endogenous pro-HGF for the catalytic domain of proteases that cleave HGF precursors. Local and systemic expression of uncleavable HGF inhibits tumor growth and, more importantly, prevents metastasis.
Decoy MET
Immunotherapy targeting MET
Drugs used for immunotherapy can act either passively by enhancing the immunologic response to MET-expressing tumor cells, or actively by stimulating immune cells and altering differentiation/growth of tumor cells.Passive immunotherapy
Administering monoclonal antibodies is a form of passive immunotherapy. MAbs facilitate destruction of tumor cells by complement-dependent cytotoxicity and cell-mediated cytotoxicity. In CDC, mAbs bind to specific antigen, leading to activation of the complement cascade, which in turn leads to formation of pores in tumor cells. In ADCC, the Fab domain of a mAb binds to a tumor antigen, and Fc domain binds to Fc receptors present on effector cells, thus forming a bridge between an effector and a target cells. This induces the effector cell activation, leading to phagocytosis of the tumor cell by neutrophils and macrophages. Furthermore, NK cells release cytotoxic molecules, which lyse tumor cells.- DN30 is monoclonal anti-MET antibody that recognizes the extracellular portion of MET. DN30 induces both shedding of the MET ectodomain as well as cleavage of the intracellular domain, which is successively degraded by proteasome machinery. As a consequence, on one side MET is inactivated, and on the other side the shed portion of extracellular MET hampers activation of other MET receptors, acting as a decoy. DN30 inhibits tumour growth and prevents metastasis in animal models.
- OA-5D5 is one-armed monoclonal anti-MET antibody that was demonstrated to inhibit orthotopic pancreatic and glioblastoma tumor growth and to improve survival in tumor xenograft models. OA-5D5 is produced as a recombinant protein in Escherichia coli. It is composed of murine variable domains for the heavy and light chains with human IgG1 constant domains. The antibody blocks HGF binding to MET in a competitive fashion.
Active immunotherapy