The receptor tyrosine kinase MET and its ligand HGF have been reported to be overexpressed in malignant tumor cells, and they have been implicated in gefitinib-resistance in lung cancer cells. We recently found that sorting nexin 1 (SNX1), a protein that interacts with EGFR, showed negative regulation of EGFR trafficking out of early to late endosomes in gefitinib-resistant NSCLC cell lines. To further investigate the role of SNX1 on HGF-stimulated MET endocytosis followed by its down-regulation, we examined the effect of silencing of SNX1 expression by siRNA in NSCLC cells. Western blot analysis demonstrated that depletion of SNX1 protein induced a dramatic increase in phosphorylated MET at 60 min, followed by an accelerated degradation of phosphorylated MET after HGF stimulation in the cells. Furthermore, using immunofluorescence, we showed that the silencing of SNX1 by siRNA caused a substantial change in the intracellular distribution of plasma membrane-localized MET and that the resultant MET staining was spread throughout the cytoplasm, and it co-localized well with the early endosomes in the siRNA-SNX1-transfected cells. Moreover, we found efficient MET phosphorylation and rapid endocytic delivery of phosphorylated MET from early endosomes to late endosomes in the siRNA-SNX1-transfected cells. By contrast, the siRNA-control transfected cells showed inefficient endocytic delivery of phosphorylated MET from early endosomes to late endosomes. Also, in the presence of bafilomycin A1, large amounts of phosphorylated MET that had accumulated in late endosomes were seen even after 60 min of HGF-stimulation, suggesting that degradation of phosphorylated MET proceeds in a late endosome/lysosome pathway. Collectively, we infer that SNX1 plays a negative role in the regulation of HGF-stimulated MET/phosphorylated MET endocytosis and downregulation in the gefitinib-resistant NSCLC cells.
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