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c-MET
by Tanay Surkund
Guide: Mrs. Poonam Advani
INDEX
1. INTRODUCTION
Page 3
2. MET PROTEIN STRUCTURE Page 4
3. MET SIGNALLING PATHWAYS AND
SPECIFICITY.
Page 6
4. DYSREGULATION IN MET TYROSINE KINASE
RECEPTOR IN FORMATION OF INVASIVE
GROWTH TUMOURS.
Page 17
5. CANCER THERAPIES TARGETTING HGF/MET.
Page 22
6. References
Page 27
INTRODUCTION:
MET (mesenchymal-epithelial transition factor) is a protooncogene that encodes a protein MET, also known as c-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 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.
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
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 αsubunit and a transmembrane β-subunit, which are linked
together by a disulfide bridge.
Extracellular Portion:
1. Region of homology to semaphorins (Sema domain),
which includes the full α-chain and the N-terminal part of
the β-chain;
2. Cysteine-rich MET-related sequence (MRS domain);
3. Glycine-proline-rich repeats (G-P repeats);
4. Four immunoglobuline-like structures (Ig domains), a
typical protein-protein interaction region.
Intracellular Portion:
1. Juxtamembrane segment that contains:
o a serine residue (Ser 985), which inhibits the
receptor kinase activity upon phosphorylation
o 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.
2. Tyrosine kinase domain, which mediates MET biological
activity. Following MET activation, transphosphorylation
occurs on Tyr 1234 and Tyr 1235;
3. C-terminal region contains two crucial tyrosines (Tyr
1349 and Tyr 1356), which are inserted into the
multisubstrate 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 :
1. PI3K(Phosphoinositol -3-Kinase) pathway :
Activation of MET receptor leads to the recruitment of the Gab1 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 factormediated 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 proapoptotic 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.
2. 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 insideout: "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.
3. 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 cytoskeletoncontaining eukaryotic cells. Two of its subunits, the ActinRelated 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, NWASP, 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 preexisting filaments. Thus, the Arp2/3 complex binds to preexisting 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 crosslinked, 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 type1 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 actincontaining cytoskeletal filaments. In epithelial cells, E-cadherincontaining cell-to-cell junctions are often adjacent to actincontaining 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 HGFdependent 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 singlechaininactive 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.
4) 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
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.
1. 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.
2. 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 MET amplifications
are highly sensitive to treatment with PHA-665752 .
3. 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 carcinoma, gastric cancer,
and head and neck cancer
4. SGX523 (SGX Pharmaceuticals) specifically inhibits MET
at low nanomolar concentrations.
5. 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 HGFdependent MET activation.
1. 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.
2. 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).
3. 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 HGFdependent 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
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.
1. 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.
2. 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
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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