Abstract
The proto-oncoprotein MET is a receptor tyrosine kinase that plays a key role in cancer cell growth and invasion. We have used fluorescence-tagged antibodies to activate MET in live serum-starved glioblastoma cells and monitor the fate of antibody-bound MET receptor in single cell-based assays. We found that the antibodies induced rapid and transient formation of highly polarized MET clusters on the plasma membrane and promoted the activation of MET, resembling the initial effects of binding to its ligand, HGF. However, the antibody-induced clustering and activation of MET led to the rapid removal of the receptor from cell surface and altered its intracellular processing, resulted in rapid degradation of the receptor. Consequently, while cells pre-treated with HGF remain competent to respond to further HGF stimulation, cells pre-treated with antibodies are refractory to further HGF stimulation due to antibody-mediated MET depletion. Removal of MET by sustained treatment of antibodies blocked cancer cell migration and invasion. Our studies reveal a novel mechanism to alter the recycling process of MET in glioblastoma cancer cells by promoting the receptor degradation through a proteasome-sensitive and lysosome-dependent pathway through the ligand-independent activation of MET using anti-MET antibodies.
Highlights
To targeted therapies against VEGFR in GBM11,12 and inhibitors of the EGFR in lung cancers[13,14]
To examine how MET is activated on the plasma membrane, we examined the distribution of the MET protein in serum-starved human U373-MG glioblastoma cells, which express relatively high levels of MET protein[23]
We found that addition of the APCtagged mouse monoclonal anti-MET antibody to the live serum-starved cells detected MET proteins in a highly “clustered” pattern on the plasma membrane in the live and serum-starved cells, while the control APC-tagged mouse IgG did not produce any fluorescence signals in these cells (Fig. 1A, top panels)
Summary
To targeted therapies against VEGFR (vascular endothelial growth factor receptor) in GBM11,12 and inhibitors of the EGFR in lung cancers[13,14]. Since the high level expression of MET is correlated with poor prognosis of various cancers, MET serves as an excellent target for cancer therapy Various approaches, such as the development of small molecular chemical inhibitors or specific monoclonal antibodies, have been explored to inhibit the RTK activity of MET or to block the interaction between the MET receptor and the ligand, HGF, in a wide array of cancers[15,16]. SAIT301, which does not activate the RTK activity of MET, was shown to cause the downregulation of the MET protein after an extended treatment[21] It appears that LY2875358 and SAIT301 employ different cellular processes to down-regulate MET receptors, a direct comparison of these two antibodies is lacking. We have examined the cellular response to the ligand-independent activation of MET receptor on the plasma membrane by antibody-mediated MET receptor clustering
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