MET Oncogene Controls Invasive Growth by Coupling with NMDA Receptor

SIMPLE SUMMARY: The MET oncogene, encoding the tyrosine kinase receptor for a hepatocyte growth factor (HGF), plays a key role in the onset and progression of aggressive forms of breast cancer. Recently, it was found that the glutamate receptor, which has a well-known role in the nervous system, is expressed in many types of tumors outside the nervous system and contributes to metastatic behavior in breast cancer cells. Here, we highlight that MET protein physically interacts with glutamate receptors in two highly metastatic breast cancer cell lines. HGF, which creates a supportive proinvasive microenvironment for the tumor cells, stabilizes this interaction. Pharmacological inhibition of glutamate receptors blunts the migration and invasion elicited by HGF, suggesting drug repurposing of glutamate receptor antagonists for anticancer therapy. ABSTRACT: The N-methyl-D-aspartate receptor (NMDAR) is a glutamate-gated ion channel involved in excitatory synaptic transmission. Outside the nervous system, the NMDAR is expressed in a variety of tissues and in cancers, notably in the highly invasive and metastatic triple-negative breast carcinoma. MET encodes the tyrosine kinase receptor for HGF and is a master regulator gene for “invasive growth”. In silico analysis shows that high expression of the NMDAR2B subunit is a negative prognostic factor in human invasive breast carcinoma. Here, we show that in triple-negative breast cancer cell lines NMDAR2B and MET proteins are coexpressed. HGF stimulation of these cells is followed by autophosphorylation of the MET kinase and phosphorylation of the NMDAR2B subunit at tyrosines 1252 and 1474. MET and phosphorylated NMDAR2B are physically associated, as demonstrated by co-immunoprecipitation, confocal immunofluorescence, and proximity ligation assays. Notably, pharmacological inhibition of NMDAR by MK801 and ifenprodil blunts the biological response to HGF. These results demonstrate the existence of a MET-NMDAR crosstalk driving the invasive program, paving the way for a new combinatorial therapy

HGF/Met axis has anti-apoptotic and anti-autophagic function in hypoxic cardiac injury

Ischaemic heart disease is the main cause of death in western countries. Cardiac tissue is primarily damaged by cardiomyocyte cell death triggered by low oxygen supply to the heart (hypoxia). The current therapeutic approach is coronary angioplastic intervention or thrombolytic treatments to resume blood flow in the ischaemic heart. Unfortunately, reperfusion itself causes a burst of ROS production responsible for cardiomyocyte death and myocardial dysfunction. Indeed, the majority of patients surviving to acute myocardial infarction undergoes progressive heart failure, with 50% mortality at five years from diagnosis. Apoptosis of cardiomyocytes is dangerous both during ischaemia and reperfusion. In line with this concept, we have shown that treatment of H9c2 cardiomyoblasts with cobalt chloride (CoCl2), a chemical mimetic of hypoxia, induces caspase-dependent apoptosis. Unexpectedly, we found that 3-methyladenine, an inhibitor of autophagy initiation, partially prevents CoCl2-mediated cell death, indicating that also autophagy contributes to cardiomyoblast death. Consistently, we found an increase in the autophagic flux in dying cells. Mechanistically, we have shown that CoCl2 upregulates Redd1, Bnip3 and phospho-AMPK proteins and causes inhibition of mTOR, the main negative regulator of autophagy.  In light of these observations, it is important to discover new therapeutic tools displaying a dual prosurvival mechanism. To this aim, we have analyzed the cardioprotective action of HGF/Met axis in hypoxic injury. To activate Met signaling we have used either the HGF ligand or two different monoclonal antibodies (mAbs) directed against the extracellular moiety of Met receptor. Owing a divalent structure, the two mAbs can dimerize and activate Met receptor, thus displaying agonist activity. Hypoxic injury was fully prevented by either HGF or Met agonist mAbs through both anti-apoptotic and anti-autophagic functions. By pharmacological inhibition we showed that activation of mTOR is the protective signaling downstream to Met, being involved in the anti-autophagic effect. In conclusion, HGF or Met agonist mAbs promote cell survival by negative dual regulation of apoptotic and autophagic cell death and represent promising new therapeutic tools to manage cardiac diseases

Ligand-regulated binding of FAP68 to the hepatocyte growth factor receptor.

We have used the yeast two-hybrid system to identify proteins that interact with the intracellular portion of the hepatocyte growth factor (HGF) receptor (Met). We isolated a human cDNA encoding a novel protein of 68 kDa, which we termed FAP68. This protein is homologous to a previously described FK506-binding protein-associated protein, FAP48, which derives from an alternative spliced form of the same cDNA, lacking an 85-nucleotide exon and leading to an early stop codon. Here we show that epithelial cells, in which the HGF receptor is naturally expressed, contain FAP68 and not FAP48 proteins. FAP68 binding to Met requires the last 30 amino acids of the C-terminal tail, which are unique to the HGF receptor. Indeed, FAP68 does not interact with related tyrosine kinases of the Met and insulin receptor families. FAP68 interacts specifically with the inactive form of HGF receptor, such as a kinase-defective receptor or a dephosphorylated wild type receptor. In vivo, endogenous FAP68 can be coimmunoprecipitated with the HGF receptor in the absence of stimuli and not upon HGF stimulation. Thus, FAP68 represents a novel type of effector that interacts with the inactive HGF receptor and is released upon receptor phosphorylation. Free FAP68 exerts a specific stimulatory activity toward the downstream target p70 S6 protein kinase (p70S6K). Significantly, nonphosphorylated HGF receptor prevents FAP68 from stimulating p70S6K. These data suggest a role for FAP68 in coupling HGF receptor signaling to the p70S6K pathway

Generation of a truncated hepatocyte growth factor receptor in the endoplasmic reticulum.

The hepatocyte growth factor (HGF) receptor (p190MET) is a tyrosine kinase composed of two disulfide-linked chains, alpha of 50 kDa and beta of 145 kDa. We have previously described an isoform (p140MET) containing a beta chain of 85 kDa, lacking the cytoplasmic kinase domain. The two receptor variants originate by post-translational processing of a common single-chain precursor of 170 kDa (Pr170). In the endoplasmic reticulum a fraction of Pr170 is cleaved at the cytosolic side generating an intermediate product of 120 kDa (Pr120). This molecule 1) is already detectable after 15 min of pulse labeling, 2) contains high mannose-branched oligosaccharides, and 3) accumulates upon treatments inhibiting the export from the endoplasmic reticulum. A second cleavage, occurring after 30 min of chase in the trans-Golgi network, converts the single-chain precursors Pr170 and Pr120 into the mature heterodimers p190MET and p140MET. This process is inhibited by brefeldin A treatment. Conditions leading to Pr170 accumulation in the endoplasmic reticulum, such as receptor overexpression, induce kinase activation and overproduction of Pr120. Conversely, cells expressing a kinase-defective HGF receptor lack the truncated isoform. The proteolytic cleavage of the cytoplasmic domain may thus represent a safety mechanism aimed at preventing ligand-independent intracellular activation of the HGF receptor kinase

A New Transgenic Mouse Model of Heart Failure and Cardiac Cachexia Raised by Sustained Activation of Met Tyrosine Kinase in the Heart

Among other diseases characterized by the onset of cachexia, congestive heart failure takes a place of relevance, considering the high prevalence of this pathology in most European countries and in the United States, and is undergoing a rapid increase in developing countries. Actually, only few models of cardiac cachexia exist. Difficulties in the recruitment and follow-up of clinical trials implicate that new reproducible and well-characterized animal models are pivotal in developing therapeutic strategies for cachexia. We generated a new model of cardiac cachexia: a transgenic mouse expressing Tpr-Met receptor, the activated form of c-Met receptor of hepatocyte growth factor, specifically in the heart. We showed that the cardiac-specific induction of Tpr-Met raises a cardiac hypertrophic remodelling, which progresses into concentric hypertrophy with concomitant increase in Gdf15 mRNA levels. Hypertrophy progresses to congestive heart failure with preserved ejection fraction, characterized by reduced body weight gain and food intake and skeletal muscle wasting. Prevention trial by suppressing Tpr-Met showed that loss of body weight could be prevented. Skeletal muscle wasting was also associated with altered gene expression profiling. We propose transgenic Tpr-Met mice as a new model of cardiac cachexia, which will constitute a powerful tool to understand such complex pathology and test new drugs/approaches at the preclinical level