Hepatocyte Growth Factor Receptor (HGFR) C-Met Pathway

HGFR or Hepatocyte growth factor receptor is an RTK or receptor tyrosine kinase coded for by the Met oncogene. When activated by it’s ligand, the hepatocyte growth factor, it plays an important role in both normal cellular functions as well as oncogenesis. Understanding the intricacies of its structure and pathways is imperative to correctly and effectively identify its oncogenic mechanism as well suitable therapeutic targets.

Role of HGFR:

Unlike several other receptors which are cell specific, HGFR is expressed on a variety of functionally different cells. Hence its activation leads to a cascade of complex and varied biological responses. Depending on the type of cell, its results might include anything from growth, transformation, cell motility and tissue regeneration to epithelial to mesenchymal transition (EMT), to angiogenesis, invasion, and metastasis. It also plays an important role in morphogenesis and growth of multiple embronic tissues, including those of the nervous system. In cancer HGFR has been found to cause cell proliferation, cell survival, invasion, cell motility, metastasis and angiogenesis.

Structure of HGFR:

HGFR is derived from a single chain precursor through proteolytic cleavage. The mature receptor has two distinct subunits, the smaller α-subunit which forms the extracellular region and the comparatively larger β-subunit which forms the transmembrane region. These two regions are linked through a disulphide bond.

The structure includes:

lthe Sema domain (semaphorin domain) -forms the amino terminus, it plays a role in ligand binding and receptor dimerization

la PSI domain (named so as it is found within plexins, semaphorins, and integrins) - forms a bridge between Sema and IPT doamians and is responsible for correct orientation of ligand with respective binding site

lfour IPT repeats- also Ig domains (found within immunoglobulins, plexins, and transcription factors)

la juxtamembrane domain

la tyrosine kinase domain

la carboxy-terminal tail region

The ligand HGF, like its receptor, jas two subunits. The 69kDa alpha and the 34kDa beta subunit. A member of the plasminogen-related growth factor family, it is derived from a 728 AA linear chain precursor. It has a six domain structure and binds to the sema domain with a 2:2 stoichiometric ratio. For this the seven β sheets present in sema bind to the HGF-β chain active site region.

The HGF/HGFR activation pathway:

HGFR as a receptor tyrosine kinase, transduces signal from the matrix into the cytoplasm. HGF also known as scatter factor, binds to the receptor and causes heterodimerization. This in turn activates the kinase domain of the receptor, resulting inphosphorylation of residues Tyr 1234 and 1235 present in the intracellular catalytic region, becoming fully activated. Once activated , it then interacts with downstream signalling molecules like the phosphatidylinositol 3-kinase subunit PIK3R1, PLCG1, src-homology 2, growth factor receptor binding protein 2 (GRB2),

Activation of c-Met pathway 1

signal transducer and activator of transcription (STAT3) or the adapter GRB-associated bindiing protein (GAB1) through its docking site. Recruitment and activation of these effectors by Met leads to the activation of several signaling pathways including the RAS-ERK, PI3 kinase-AKT, PLCϒ-PKC and β-cat/Wnt pathway.

The RAS-ERK activation is associated with the morphogenetic effects while PI3K/AKT coordinates prosurvival effects.

Role of HGFR in cancer:

Over the years autocrine HGFR activation has been associated with several forms of metastatic tumours. Nonautocrine mechanisms have also been known to cause cancerous growth but these cases are much more rare. Several epithelial and mesenchymal cells show marked overexpression of Met gene.

In osteoblasts, higher levels of HGFR and continuous activation have been shown to cause transformation of normal cells to osteosarcoma cells (in-vitro) and osteosarcoma like diseases (in-vivo).

Oversensitization of these receptors in hepatocytes induces hepatocellular carcinoma in transgenic mice.

In case of lung cancer, overexpression, amplification as well as mutation of HGFR have been noted which results in higher pathological stage tumor..

HGFR was also found to be overexpressed in 20 out of 52 squamous cell carcinomas, 34 of 47 adenocarcinomas, and in all 11 non-small cell lung cancer (NSCLC) cell lines that have been studied till date. In NSCLC the HGFR may be over-expressed as much as 2-10 times while presence of HGF may rise 10 to 100 folds.

Mutations in Met:

Usually, the activity of HGF induced tyrosine kinases are strictly regulated. But through gain of function mutations they may sometimes develop malignant properties. These oncogenic pathways can be stimulated by amplification and/or mutation of met gene, autocrine production of ligands, or non-ligand kinase activation. In some cases mutations can affect inhibitory controls (loss of function) hence allowing the receptor to be either active or hyperresponsive to stimuli. Mutations can also alter the extent to which activation occurs or can even prolong the duration of the signals by inhibiting protein degradation, hence disregulating the pathway.

Since the initial discovery of missense MET mutations in hereditary papillary renal carcinoma, activating MET mutations have been identified in a diverse range of human cancers.

Cytogenic location of Met gene 1

In general hypersensitization of HGFR/HGF pathway is usually governed by two major mechanisms: mutations in the extracellular domain or mutations in the cytoplasmic domain.

The extracellular region of HGFR, i.e the Sema domain, is responsible for ligand binding and dimerization. Hence mutation in this region can greatly affect the working of the receptor. While these mutations have not been intricately studied, they are thought to cause structural and conformational changes in the ligan-binding site. Recent studies have also uncovered germ line mutations in this region.

There have also been identified a cluster of ethnicity specific mutations in this domain, for example N375S which has been exclusively identified in Caucasians or Asian lung cancer patients. Other mutations of this region include E168D, L299F, S323G, and N375S. These MET mutations of the extracellular region affect the binding to HGF, and the N375S mutation, specifically, have been shown to provide resistance to small-molecule inhibition.

Mutations in the juxtamembrane domain have also been shown to cause widespead oncogenic activities though their mechanism is not fully understood. JM mutations found in human cancers may accelerate tumor formation rather than being fully transformed by themselves. For example HGFR-T992I mutationis not known to cause full actuvation of kinase activity in HGFR. However presence of this mutation results in accelerated growth of tumors as compared to normal cells. This mutation is seen in hereditary papillary renal cell carcinoma as well as breast cancer. Besides this, R970C and T992A germline mutations were also identified in 126 patients with adenocarcinomas. In some cases of human melanoma phosphorylation was also observed at the Y1003 activation site. The mutation of this site, which is generally involved with regulation of HGFR, results in transformation of the receptor is fibroblasts and epithilial cells.

Studies suggest that mutations in JM doamin may contribute to enhanced tumorigenicity, cell migration, and phosphorylation of HGFR protein in this disease

Mutations in the kinase domain are the most widely studied mutations of the

Met gene. Kinase domain mutations induce constitutive receptor activation and these mutationally activated Met can be ligand-dependent or ligand-independent.

majority of tyrosine kinase domain activating mutations in MET have been described in sporadic papillary renal carcinomas and hereditary papillary renal cell carcinomas.

These mutations lead to result in an increase in HGFR kinase activity and hence result in increased motility and metastasis.

Other forms of activation:

In addition to mis-sense mutations, HGFR can also be activated by amplification. For instance,10–20% of human gastric carcinomas have Met gene amplification.

HGFR expression can also be driven by low oxygen levels which can then increase HGF-dependent invasion.

In colorectal cancer, upregulation of HGFR is controlled by Wnt signaling.

Regulatory molecules also play an important role in promoting or inhibiting gene transformation.

Therapeutic targets:

Recent researches have been focusing on developing targetted drugs to deal with HGRF pathway and related cancers.

lOne of the most widely used therapeutic target are the tyrosine kinase domains with Tyrosine Kinase Inhibitors (TKI). These have been shown to have significant success in breast cancer (trastuzumab), melanoma (vemurafenib), and lung cancer (erlotinib).

Another effective measure is to inhibit binding of HGFR and HGF using neutralizing antibodies and biological factors.

lHGFR small-molecule inhibitors have been utilized as single agents as well as in combination regimens.

l c-MET kinase-dependent signaling may also be blocked through relevant signal transducers or downstream signaling components hence disrupting the normal functioning of the pathway.

Several small molecule inhibitors are currently used or are being developed for use. Examples of these include:

vCrizotinib (a dual c-MET) and ALK (anaplastic lymphoma kinase) inhibitor -for treating ALK-positive NSCLC.

Cabozantinib and Tivantinib - both multikinase competitive inhibitors that targets c-MET

vOnartuzumab - a humanized monovalent monoclonal antibody directed against c-MET