title: Silencing of SNX1 by siRNA stimulates the ligand-induced endocytosis of EGFR and increases EGFR phosphorylation in gefitinib-resistant human lung cancer cell lines.
abstract: Gefitinib is known to suppress the activation of EGFR signaling, which is required for cell survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. We previously demonstrated that the gefitinib-sensitive NSCLC cell line PC9 shows efficient ligand-induced endocytosis of phosphorylated EGFR (pEGFR). In contrast, the gefitinib-resistant NSCLC cell lines QG56 and A549 showed internalized pEGFR accumulation in the aggregated early endosomes, and this was associated with SNX1, a protein that interacts with and enhances the degradation of EGFR upon EGF stimulation. In the present study, to investigate the role of SNX1 on EGF-stimulated EGFR/pEGFR endocytosis via the endocytic pathway, we examined the effect of depletion of SNX1 expression by siRNA in human NSCLC cell lines. Using immunofluorescence, we demonstrated that transfection of SNX1 siRNA into gefitinib-resistant NSCLC cells resulted in the disappearance of a large amounts of SNX1 staining. In addition, upon 15 min of EGF stimulation, we observed an efficient EGFR phosphorylation and a rapid endocytic delivery of pEGFR from early endosomes to late endosomes. Further, western blot analysis revealed that silencing of SNX1 expression by siRNA in the gefitinib-resistant cells leads to an accelerated degradation of EGFR along with a dramatic increase in the amounts of pEGFR after EGF stimulation. Based on these findings, we suggest that SNX1 is involved in the negative regulation of ligand-induced EGFR phosphorylation and mediates EGFR/pEGFR trafficking out of early endosomes for targeting to late endosomes/lysosomes via the early/late endocytic pathway in human lung cancer cells.
authors.affiliation: Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan. ynkio443@gmail.com
authors.forename: Yukio
authors.initials: Y
authors.lastname: Nishimura
authors.forename: Soichi
authors.initials: S
authors.lastname: Takiguchi
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Yoshioka
authors.forename: Yusaku
authors.initials: Y
authors.lastname: Nakabeppu
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Itoh
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D066246:ErbB Receptors / Q000096:biosynthesis / Q000235:genetics*; D000077156:Gefitinib; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011799:Quinazolines / Q000008:administration & dosage*; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D058305:Sorting Nexins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D011799:Quinazolines; D034741:RNA, Small Interfering; C513978:SNX1 protein, human; D058305:Sorting Nexins; C512478:EGFR protein, human; D066246:ErbB Receptors; D000077156:Gefitinib