c-MET

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INTRODUCTION:

MET (mesenchymal-epithelial transition factor) is a proto-oncogene that encodes a protein MET, also known asc-Met or hepatocyte growth factor receptor (HGFR). MET is a membrane receptor that is essential for embryonic development and wound healing. Hepatocyte growth factor (HGF) is the only known ligand of the MET receptor. MET is normally expressed by cells of epithelial origin, while expression of HGF is restricted to cells of mesenchymal origin. Upon HGF stimulation, MET induces several biological responses that collectively give rise to a program known as invasive growth. Abnormal MET activation in cancer correlates with poor prognosis, where aberrantly active MET triggers tumor growth, formation of new blood vessels (angiogenesis) that supply the tumor with nutrients, and cancer spread to other organs (metastasis). MET is deregulated in many types of human malignancies, including cancers of kidney, liver, stomach, breast, and brain. Normally, only stem cells andprogenitor 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.

The proto-oncogene MET product is the hepatocyte growth factor receptor and encodes 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 PROTEIN STRUCTURE

schematic structure of MET protein
Schematic structure of MET protein

MET is a receptor tyrosine kinase (RTK) 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 Portion:

  • Region of homology to semaphorins (Sema domain), which includes the full α-chain and the N-terminal part of the β-chain;
  • Cysteine-rich MET-related sequence (MRS domain);
  • Glycine-proline-rich repeats (G-P repeats);
  • Four immunoglobuline-like structures (Ig domains), a typical protein-protein interaction region.

Intracellular Portion:

  • Juxtamembrane segment that contains:
    • a serine residue (Ser 985), which inhibits the receptor kinase activity upon phosphorylation
    • a tyrosine (Tyr 1003), which is responsible for MET polyubiquitination, endocytosis, and degradation upon interaction with the ubiquitin ligase CBL. This is broadly considered as a negative regulator for tyrosine kinase activity of the receptor.
  • 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 (Tyr 1349 and Tyr 1356), which are inserted into themultisubstrate docking site, capable of recruiting downstream adapter proteins with Src homology-2 (SH2) domains. The two tyrosines of the docking site have been reported to be necessary and sufficient for the signal transduction both in vitroand in vivo

MET SIGNALLING PATHWAYS AND SPECIFICITY

MET activation by its ligand HGF induces MET kinase catalytic activity, which triggers transphosphorylation of the tyrosines Tyr 1234 and Tyr 1235. These two tyrosines engage various signal transducers, thus initiating a whole spectrum of biological activities driven by MET, collectively knows as the invasive growth program. The transducers interact with the intracellular multisubstrate docking site of MET either directly, such as GRB2 and the p85 regulatory subunit of phosphatidylinositol-3 kinase (PI3K) , or indirectly through the scaffolding protein Gab1 . 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, 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.

MET engagement activates multiple signal transduction pathways :


  • PI3K(Phosphoinositol -3-Kinase) pathway :

Activation of MET receptor leads to the recruitment of the Gab-1 scaffolding protein as mentioned above. In this pathway, the pH domain of the Gab-1 protein binds to the p85 regulatory subunit of the PI3K molecule.PI3K in turn is responsible for the activation of the Akt genes.In humans, there are three genes in the “Akt family”:Akt1, Akt 2 and Akt3. These genes code for enzymes that are members of the serine/threonine-specific protein kinase family. Akt1 is involved in cellular survival pathways, by inhibiting apoptotic processes. Akt1 is also able to induce protein synthesis pathways, and is therefore a key signaling protein in the cellular pathways that lead to skeletal muscle hypertrophy, and general tissue growth. Since it can block apoptosis, and thereby promote cell survival, Akt1 has been implicated as a major factor in many types of cancer. Akt (now also called Akt1) was originally identified as the oncogene in the transforming retrovirus. Akt could promote growth factor-mediated cell survival both directly and indirectly. BAD is a pro-apoptotic protein of the Bcl-2 family. Akt phosphorylates BAD on Ser136 (BAD phosphorylation by Akt), which makes BAD dissociate from the Bcl-2/Bcl-X complex and lose the pro-apoptotic function (BAD interaction with Bcl-2). Akt could also activate NF-κB via regulating IκB kinase (IKK), thus result in transcription of pro-survival genes (regulation of NF-kB).
Akt1 has also been implicated in angiogenesis and tumor development. Deficiency of Akt1 in mice although inhibited physiological angiogenesis, it enhanced pathological angiogenesis and tumor growth associated with matrix abnormalities in skin and blood vessels.
The PI3K pathways have also been implicated in the activation of paxillin, Focal Adhesion Kinases (FAKs) and Intergrins and thereby contributing to cell motility.

  • MAPK (Mitogen Activated Phosphokinase ) Pathway.

The MAPK pathway is possibly the most important pathway which can be activated by the MET activation. This pathway is responsible for various processes of vast importance such as cell motility, cell migration, wound healing, cell proliferation, and also a negative role in metastasis(when the receptor is overexpressed).
The Activation of this pathways can occur either by activation of the Grb-2(Growth Receptor Binding Protein-2) which will consecutively activate the SOS (Sons of Sevenless).SOS then activates the Ras protein (an oncogenic protein that is found in 30% of all cancers) which activates its own pathway(details of which will be discussed later).Ras activates Raf which later activates the ERK/MAPK enzymes which finally will activate the Ets-1 transcription factor. This transcription factor is essential in altering the gene expression of vital components of the cell cycle such as the Cdk6 (Cyclin Dependant Kinase-6) , the p27 and the pRB which help in the regulation of the cell cycle and on overexpression can lead to formation of tumourogenesis.
The exact mechanism of the  effect the MAPK pathway has on the above substrates is unclear. This is as p27 and pRB are tumour suppressor proteins which basically inhibit the replication of damaged DNA and the role of the Ets-1 transcription factors in normal development is unclear, but in tumours, it is evident that the overexpression of MET has some effect on the deactivation of these 2 proteins. The pRB protein plays a checkpoint between the G1 and S phase of the cell cycle, it binds to transcription factors of the E2F family and deactivates them. While p27 plays a checkpoint between the G0 and the G1 phase.
The activation of the MAPK pathway by MET receptor activation also plays an important role in the process of wound healing. MAPK is known to activate 3 crucial substrates of the wound healing process which are Fibronectin, UPA (Urokinase Plasminogen Activator) and MMP’s (Matrix Metalloproteases). Fibronectin is a high-molecular weight extracellular matrix glycoprotein that binds to membrane-spanning receptor proteins called integrins. In addition to integrins, fibronectin also binds extracellular matrix components such as collagen, fibrin and heparan sulfate proteoglycans. Fibronectin plays a crucial role in wound healing. Along with fibrin, plasma fibronectin is deposited at the site of injury, forming a blood clot that stops bleeding and protects the underlying tissue. As repair of the injured tissue continues, fibroblasts and macrophages begin to remodel the area, degrading the proteins that form the provisional blood clot matrix and replacing them with a matrix that more resembles the normal, surrounding tissue. Fibroblasts secrete proteases, including matrix metalloproteinases(also activated by MET activation), that digest the plasma fibronectin, and then the fibroblasts secrete cellular fibronectin and assemble it into an insoluble matrix. Fragmentation of fibronectin by proteases has been suggested to promote wound contraction, a critical step in wound healing. Fragmenting fibronectin further exposes its V-region, which contains the site for α4β1 integrin binding. These fragments of fibronectin are believed to enhance α4β1 integrins-expressing cell binding, allowing them to adhere to and forcefully contract the surrounding matrix. Fibronectin is necessary for embryogenesis, and inactivating the gene for fibronectin results in early embryonic lethality. Fibronectin is important for guiding cell attachment and migration during embryonic development. In mammalian development, the absence of fibronectin leads to defects in mesodermal, neural tube, and vascular development. Similarly, the absence of a normal fibronectin matrix in developing amphibians causes defects in mesodermal patterning and inhibits gastrulation. Fibronectin is also found in normal human saliva, which helps prevent colonization of the oral cavity and pharynx by potentially pathogenic bacteria.
The MAPK pathway is also responsible for the activation of the UPA which is responsible for cleaving HGF from its precursor molecule. It is suggested that the activation of UPA by MET shows a positive feedback mechanism.
There is also certain evidence that shows that the MAPK pathway activates FAK (Focal Adhesion Kinase) , Paxillin and Integrins. Integrin plays a role in the attachment of cells to other cells, and also plays a role in the attachment of a cell to the material part of a tissue that is not part of any cell (the extracellular matrix). Besides the attachment role, integrin also plays a role in signal transduction, a process by which a cell transforms one kind of signal or stimulus into another. It is more common for cells to make new receptors on their surfaces, or remove them if they need to alter their ability to respond to the environment.The integrins are unusual membrane proteins because the signals they convert travel in both outside-in: transducing information from the ECM to the cell, and inside-out: “revealing” the status of the cell to the extracellular world. This allows cells to make rapid and flexible responses. Integrins couple the ECM outside a cell to the cytoskeleton (in particular the microfilaments) inside the cell. Which ligand in the ECM the integrin can bind to is mainly decided by which α and β subunits the integrin is made of. Among the ligands of integrins are fibronectin, collagen, and laminin. The connection between the cell and the ECM may help the cell to endure pulling forces without being ripped out of the ECM. Cell attachment to the ECM is a basic requirement to build a multicellular organism. Integrins are not simply hooks, but give the cell critical signals about the nature of its surroundings. Together with signals arising from receptors for soluble growth factors like VEGF, HGF and many others, they enforce a cellular decision on what biological action to take, be it attachment, movement, death, or differentiation. Thus integrins lie at the heart, both literally and figuratively, of many cellular biological processes. The attachment of the cell takes place through formation of cell adhesion complexes, which consist of integrins and many cytoplasmic proteins which include talin, vinculin, paxillin and alpha-actinin. These act by regulating kinases like FAK (focal adhesion kinase) to phosphorylate substrates such as p130CAS thereby recruiting signaling adaptors such as Crk. These adhesion complexes attach to the actin cytoskeleton. The integrins thus serve to link across the plasma membrane two networks: the extracellular ECM and the intracellular actin filamentous system. One of the most important functions of surface integrins is their role in cell migration. Cells adhere to a substrate through their integrins. During movement, the cell makes new attachments to the substrate at its front and concurrently releases those at its rear. When released from the substrate, integrin molecules are taken back into the cell by endocytosis; they are transported through the cell to its front by the endocytic cycle where they are added back to the surface. In this way they are cycled for reuse, enabling the cell to make fresh attachments at its leading front.

  • Ras Pathway and consecutive activation of  Rac-1 and CDC42.

On activation of the MET receptor, the Grb-2 substrate through the SOS protein is responsible for the activation of the Ras pathway which is crucial with respect to cell motility. The Ras pathway activates the Rho family of GTPases. In mammals, the Rho family contains 20 members. Almost all research involves the two most common members of the Rho family: Cdc42 and Rac1.These members are responsible for possessing a unique effect on the cytoskeleton. They are responsible for the action of actin filament rearrangement which contributes to the formation of the lamellopodia and filopodia, which are considered the motility engines of the cell during the process of cell migration. The lamellipodium  is a cytoskeletal actin projection on the mobile edge of the cell. It contains a two-dimensional actin mesh; the whole structure pulls the cell across a substrate. Within the lamellipodia are ribs of actin called microspikes, which, when they spread beyond the lamellipodium frontier, are called filopodia . The lamellipodium is born of actin nucleation in the plasma membrane of the cell and is the primary area of actin incorporation or microfilament formation of the cell. They are believed to be the actual motor which pulls the cell forward during the process of cell migration. The tip of the lamellipodium is the site where exocytosis occurs in migrating mammalian cells as part of their clathrin-mediated endocytic cycle. This, together with actin-polymerisation there, helps extend the lamella forward and thus advance the cell’s front. It thus acts as a steering device for cells in the process of chemotaxis. It is also the site from which particles or aggregates attached to the cell surface migrate in a process known as cap formation. Structurally, the plus ends of the microfilaments (localized actin monomers in an ATP-bound form) face the “seeking” edge of the cell, while the minus ends (localized actin monomers in an ADP-bound form) face the lamella behind
This creates treadmilling throughout the lamellipodium, which aids in the retrograde flow of particles throughout
Arp2/3 complexes are present at microfilament-microfilament junctions in lamellipodia, and help create the actin meshwork. Arp 2/3 can only join onto previously existing microfilaments, but once bound it creates a site for the extension of new microfilaments, which creates branching. Arp2/3 complex is a seven-subunit protein that plays a major role in the regulation of the actin cytoskeleton. It is a necessary component of the actin cytoskeleton and is therefore ubiquitous in actin cytoskeleton-containing eukaryotic cells. Two of its subunits, the Actin-Related Proteins ARP2 and ARP3 closely resemble the structure of monomeric actin and serve as nucleation sites for new actin filaments. The complex binds to the sides of existing (“mother”) filaments and initiates growth of a new (“daughter”) filament at a distinctive 70 degree angle from the mother. Branched actin networks are created as a result of this nucleation of new filaments. The regulation of rearrangements of the actin cytoskeleton is important for processes like cell locomotion, phagocytosis, and intracellular motility of lipid vesicles. Many actin-related molecules create a free barbed end for polymerization by uncapping or severing pre-existing filaments The nucleation core activity of Arp2/3 is activated by members of the Wiskott-Aldrich syndrome family protein (WASP, N-WASP, WAVE, and WASH proteins). The V domain of a WASP protein interacts with actin monomers while the CA region associates with the Arp2/3 complex to create a nucleation core. However, de novo nucleation followed by polymerization is not sufficient to form integrated actin networks, since these newly synthesized polymers would not be associated with pre-existing filaments. Thus, the Arp2/3 complex binds to pre-existing filaments so that the new filaments can grow on the old ones and form a functional actin cytoskeleton. Capping proteins limit actin polymerization to the region activated by the Arp2/3 complex, and the elongated filament ends are recapped to prevent depolymerization and thus conserve the actin filament.and using these as nucleation cores. However, the Arp2/3 complex stimulates actin polymerization by creating a new nucleation core.
The Arp2/3 complex simultaneously controls nucleation of actin polymerization and branching of filaments. Moreover, autocatalysis is observed during Arp2/3-mediated actin polymerization. In this process, the newly formed filaments activate other Arp2/3 complexes, facilitating the formation of branched filaments.
Rac and Cdc42 are the two Rho-family GTPases which are normally cytosolic but can also be found in the cell membrane under certain conditions. When Cdc42 is activated, it can interact with Wiskott-Aldrich syndrome protein (WASp) family receptors, in particular N-WASp, which then activates Arp2/3. This stimulates actin branching and increases cell motility. Rac1 induces cortactin to localize to the cell membrane, where it simultaneously binds F-actin and Arp2/3. The result is a structural reorganization of the lamellipodium and ensuing cell motility.
The filopodia (also microspikes) are slender cytoplasmic projections, similar to lamellipodia, which extend from the leading edge of migrating cells. They contain actin filaments cross-linked into bundles by actin-binding proteins, e.g. fimbrin. Filopodia form focal adhesions with the substratum, linking it to the cell surface. A cell migrates along a surface by extending filopodia at the leading edge. The filopodia attach to the substratum further down the migratory pathway, then contraction of stress fibres retracts the rear of the cell to move the cell forwards.
Activation of the Rho family of small Ras-related GTPases and their downstream intermediates results in the construction of actin fibers. Growth factors bind to receptor tyrosine kinases resulting in the polymerization of actin filaments, which cross-linked, make up the supporting cytoskeletal elements of filopodia. Rho activity also results in the activation of the phosphorylation of the ezrin-moesin-radixin group promoting the binding of actin filaments to the filopodia membrane.
To close a wound in vertebrates, growth factors stimulate the formation of filopodia in fibroblasts to direct fibroblast division and close the wound. In developing neurons, filopodia extend from the growth cone at the leading edge. In neurons deprived of filopodia by the removal of actin filaments, growth cone extension continues as normal but direction of growth is disrupted and highly irregular. Another molecule that is often found in polymerizing actin with Arp2/3 is cortactin, which appears to link tyrosine kinase signalling to cytoskeletal reorganization in the lamellipodium and its associated structures. This molecule has now been found to be activated by Rac-1.
This pathway is also responsible for Cadherin rearrangement, without which the motile cells would not be able to deattach themselves from the cell junction. Cadherins are a class of type-1 transmembrane proteins. They play important roles in cell adhesion, ensuring that cells within tissues are bound together. They are dependent on calcium(Ca2+) ions to function, hence their name. Alpha-catenin participates in regulation of actin-containing cytoskeletal filaments. In epithelial cells, E-cadherin-containing cell-to-cell junctions are often adjacent to actin-containing filaments of the cytoskeleton.
E-cadherin is first expressed in the 2-cell stage of mammalian development, and becomes phosphorylated by the 8-cell stage, where it causes compaction. In adult tissues, E-cadherin is expressed in epithelial tissues, where it is constantly regenerated with a 5-hour half-life on the cell surface.
Loss of E-cadherin function or expression has been implicated in cancer progression and metastasis. E-cadherin downregulation decreases the strength of cellular adhesion within a tissue, resulting in an increase in cellular motility.This in turn may allow cancer cells to cross the basement membrane and invade surrounding tissues.

DYSREGULATION IN MET TYROSINE KINASE RECEPTOR IN FORMATION OF INVASIVE GROWTH TUMOURS

Dysregulation of Met activity in cells is thought to be a key event underlying tumour metastasis, and indeed, Met overexpression and hyperactivation are reported to correlate with metastatic ability of the tumor cells.
LIGAND DEPENDANT MECHANIMS OF MET ACTIVATION
Met activation in tumor cells can occur through any of several molecular mechanisms, the simplest of which involve HGF-dependent Met activation, much as occurs in normal cells. In some cases, tumor cells express both HGF and its receptor, setting the stage for an autocrine loop in which secreted HGF binds to Met and causes constitutive activation of Met and its downstream signaling pathways, thus enhancing tumor growth and invasive behavior. Such HGF-Met autocrine loops have been detected in gliomas, osteosarcomas, and mammary, prostate, breast, lung, and other carcinomas; they are often associated withmalignant progression of tumors and correlate with poor prognosis. Interference with either HGF or Met expression can inhibit tumorigenic transformation, angiogenesis, tumor growth, and invasion.
Under physiological conditions HGF is not an autocrine, but rather a paracrine, factor: Mesenchymal cells produce HGF, which acts on epithelial and other cells that express Met. Similarly, Met-positive tumor cells that do not produce HGF may nevertheless respond to HGF produced by stromal cells. However, since HGF is secreted by cells as a single-chaininactive precursor (pro-HGF), which must be activated by proteolytic cleavage, HGF-Met autocrine and paracrine loops depend on a third component — an enzyme capable of processing pro-HGF to produce HGF. A number of serine-like proteases, including urokinase-type plasminogen activator and coagulation factor XII, have such an activity and have beendetected in some tumors. Nevertheless, the mechanism by which pro-HGF is converted to HGF in tumor tissues remains to be established.

LIGAND INDEPENDENT MECHANISMS OF MET ACTIVATION
1) MET Overexpression
Met can also be activated in an HGF-independent manner in tumors, particularly as a result of Met overexpression, which occurs in almost every case of differentiated papillary carcinomas.Increased Met expression can be mediated by MET gene amplification, by enhanced transcription, or by posttranscriptional mechanisms. Increased expression of Met on the cell surface apparently favors ligand independent activation through spontaneous Met dimerization, but it is not generally sufficient to trigger Met activation. In some cases, even very high expression of Met does not cause constitutive receptor activation. Noncovalently associated, inactive clusters of these receptors have been identified on the cell surface,perhaps explaining the cells’ resistance to transformation,even in the face of high Met levels (12). An additional signal, such as Met transactivation by other membrane receptors, may be required to activate signalling by these receptors. Alternatively, these clustersmay contain suppressor molecules that prevent spontaneous Met activation in normal cells but may be lost or inactivated in tumor cells.

2) Gene Arrangement (TPR-MET)
One well-known oncogenic form of Met, first identified in the chemically transformed human osteosarcoma cell line HOS, is the product of the TPR-MET fusion, which arises through a chromosomal rearrangement. The resulting chimeric gene contains the promoter and the N-terminal sequence of the TPR gene from chromosome 1, fused with the C-terminal sequence of MET, which maps to chromosome 7. The TPR-MET chimeric gene encodes a cytoplasmic protein with molecular weight 65 kDa comprising the TPR leucine zipper domain and the Met kinase domain. This protein is constitutively active as a result of TPR leucine zipper interactions, which allow for Met kinase dimerization, transphosphorylation, and activation, and it is potently oncogenic in vitro and in vivo.
3) Absences of negative regulators
Abnormal processing or the absence of normal negative regulators can also lead to constitutive Met activation and tumorigenesis. The mature Met consists of two subunits, α and β, arising from proteolytic cleavage of the single-chain precursor. As a result of defective posttranslational processing, the precursor fails to be cleaved in the colon carcinoma cell line LoVo; consequently, Met is expressed on the cell surface as a single- chain molecule, which is constitutively tyrosinephosphorylated. In metastatic B16 melanoma cells, on the other hand, cytosolic phosphatases that normally mediate Met dephosphorylation, internalization, and degradation are downregulated, leading to constitutive Met activation.
4) Mutations
A large class of somatic or inherited mutations in the MET gene can lead to active, typically ligand-independent, Met signaling in tumor cells. For instance, a mutant in which the Met cytoplasmic domain is truncated immediately below the trans-membrane domain encodes a constitutively active signalling domain that can transform rodent fibroblasts. A similar, naturally occurring truncation has been detected in malignant human musculoskeletal tumors. This short 85-kDa N-terminally truncated Met is tyrosine-phosphorylated and located in the cytoplasm. The mechanism by which this truncated Met is produced is not known, but its constitutive activation suggestsa role in tumorigenesis. Missense point mutations in MET have been identified in hereditary and sporadic papillary renal carcinomas, childhood hepatocellular carcinomas, gastric carcinomas, and head and neck squamous cell carcinomas. At present, 21 such mutations have been described. All identified Met mutations in the kinase domain increase Met tyrosine kinase activity. Although mutations in the juxtamembrane domain do not trigger ligand-independent Met activation, receptors carrying the P1009I mutation show persistent Met activation in response to HGF. This mutated form of Met demonstrates transforming potential and invasive activity in vitro and in vivo.
5) MET transactivation via other membrane receptors.
Recent investigations have shown that Met kinase activity can be regulated through other receptors by HGF independent mechanisms. Thus, Met can be activated by stimuli that do not directly interact with Met, including adhesive receptors, such as various integrins and CD44, and signal transducing receptors like Ron and the EGF receptor. Integrins are cell surface receptors that mediate cell adhesion to the ECM. Plating of Met expressing cells on ECM, and the consequent ligation of cell surface integrins, can cause ligand-independent Met tyrosine phosphorylation . Interestingly, transgenic mice expressing Met in hepatocytes have activated Met and develop hepatocellular carcinoma, despite the absence of detectable HGF expression, perhaps as a result of cellular adhesion in this tissue.CD44, a cell surface receptor for hyaluronic acid (a major glycosaminoglycan component of the ECM),regulates a number of normal cell functions and has been implicated in tumor progression and metastasis.This receptor can promote Met activation by two mechanisms. First, binding of CD44 to hyaluronic acid causes HGF-independent Met activation, leading to cell growth and migration. Second, a heparan sulphate proteoglycan isoform of CD44 binds HGF and presents it to Met in the form of a multivalent complex inducing a high level of Met activation in comparison with soluble nonbound HGF. Because Ron belongs to the same family of receptor tyrosine kinases as Met and shares many common structural features, it is perhaps not surprising that activated Ron can transphosphorylate Met, and vice versa, as was recently shown. Pre-existing, ligand-independent heterodimers between Met and Ron have been detected on the cell surface (23), indicating that these receptors are colocalized and may be able to transphosphorylate and to activate one another. In addition, some human hepatoma cell lines — but not normal hepatocytes are activated by a TGF-α–EGF receptor autocrine loop, which leads to constitutive, ligand-independent tyrosine phosphorylation of Met.

Cancer Therapies Targeting HGF/MET
cancer therapies targeting MET and HGF

Strategies to inhibit biological activity of 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.
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 (Fermentek Biotechnology) is a staurosporine analogue isolated from Nocardiopsis sp. soil fungi , and it is a potent inhibitor of all receptor tyrosine kinases (RTKs). At nanomolar concentrations, K252a inhibits both the wild type and the mutant (M1268T) MET function.
  • PHA-665752 (Pfizer) specifically inhibits MET kinase activity, and it has been demonstrated to represses both HGF-dependent and constitutive MET phosphorylation. Furthermore, some tumors harboring METamplifications are highly sensitive to treatment with PHA-665752 .
  • ARQ197 (ArQule) is a promising selective inhibitor of MET, which has entered a phase 2 clinical trial in 2008. (Exelixis) targets multiple receptor tyrosine kinases (RTKs) with growth-promoting and angiogenic properties. The primary targets of XL880 are MET, VEGFR2 and KDR. XL880 has completed a phase 2 clinical trials with indications for papillary renal cell carcinomagastric cancer, and head and neck cancer
  • SGX523 (SGX Pharmaceuticals) specifically inhibits MET at low nanomolar concentrations.
  • MP470 (SuperGen) is a novel inhibitor of c-KIT, MET,  and AXL. Phase I clinical trial of MP470 had been announced in 2007.

HGF Inhibitors
Since HGF is the only known ligand of MET, formation of a HGF:MET complex blocks MET biological activity. For this purpose, truncated HGF, anti-HGF neutralizing antibodies, and an uncleavable form of HGF have been utilized so far. The major limitation of HGF inhibitors is that they block only HGF-dependent MET activation.

  • NK4 competes with HGF as it binds MET without inducing receptor activation, thus behaving as a fullantagonist. 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 (AVEO), and the fully human AMG102 (Amgen).
  • 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
Decoy MET refers to a soluble truncated MET receptor. Decoys are able to inhibit MET activation mediated by both HGF-dependent and independent mechanisms, as decoys prevent both the ligand binding and the MET receptor homodimerization. CGEN241 (Compugen) is a decoy MET that is highly efficient in inhibiting tumor growth and preventing metastasis in animal models.

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

1. Passive Immunotherapy
Administering monoclonal antibodies (mAbs) is a form of passive immunotherapy. MAbs facilitate destruction of tumor cells by complement-dependent cytotoxicity (CDC) and cell-mediated cytotoxicity (ADCC). 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 (phagocytes and NK 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.

2. Active Immunotherapy

Active immunotherapy to MET-expressing tumors can be achieved by administering cytokines, such as interferons(IFNs) and interleukins (IL-2), which triggers non-specific stimulation of numerous immune cells. IFNs have been tested as therapies for many types of cancers and have demonstrated therapeutic benefits. IL-2 has been approved by FDA for the treatment of renal cell carcinoma and metastatic melanoma, which often have deregulated MET activity.

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