title: Evidence that depletion of the sorting nexin 1 by siRNA promotes HGF-induced MET endocytosis and MET phosphorylation in a gefitinib-resistant human lung cancer cell line.
abstract: The receptor tyrosine kinase MET and its ligand HGF are known 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, exhibited negative regulation of EGFR trafficking out of early to late endosomes in gefitinib-resistant NSCLC cell lines. To investigate the role of SNX1 on HGF-stimulated MET endocytosis and its downregulation via the early/late endocytic pathway, we examined the effect of depletion of SNX1 expression by siRNA in NSCLC cells. Using immunofluorescence, we found that the silencing of SNX1 by siRNA caused a dramatic change in the intracellular distribution of plasma membrane-associated MET and that the resultant MET staining was spread throughout the cytoplasm, and it co-localized well with the endocytosed Texas red-labeled transferrin in the siRNA-SNX1-transfected cells. We also 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. Furthermore, large amounts of phosphorylated MET that had accumulated in late endosomes were seen even after 60 min of HGF-stimulation in the presence of bafilomycin A1, indicating that degradation of phosphorylated MET proceeds in a late endosome/lysosome pathway. Western blot analysis revealed that depletion of SNX1 by siRNA induced a maximal and dramatic increase in phosphorylated MET at 60 min, followed by an accelerated degradation of phosphorylated MET after HGF stimulation in the cells. Taken together, we suggest that SNX1 plays a suppressive role in the regulation of HGF-stimulated MET/phosphorylated MET endocytosis and downregulation via the early/late endocytic pathway in the gefitinib-resistant NSCLC cells.
authors.affiliation: Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Nishimura
authors.affiliation: Institute for Clinical Research, National Kyushu Cancer Center, Fukuoka 811-1395, Japan.
authors.forename: Soichi
authors.initials: S
authors.lastname: Takiguchi
authors.affiliation: Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo 101-0062, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Ito
authors.affiliation: Department of Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511, Japan.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Itoh
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D004705:Endocytosis / Q000187:drug effects*; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D000077156:Gefitinib; D020868:Gene Silencing; D017228:Hepatocyte Growth Factor / Q000494:pharmacology*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics / Q000378:metabolism*; D011799:Quinazolines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D058305:Sorting Nexins / Q000502:physiology*; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D011799:Quinazolines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C513978:SNX1 protein, human; D058305:Sorting Nexins; D017228:Hepatocyte Growth Factor; C491743:MET protein, human; D019859:Proto-Oncogene Proteins c-met; D000077156:Gefitinib