title: Evidence for efficient phosphorylation of EGFR and rapid endocytosis of phosphorylated EGFR via the early/late endocytic pathway in a gefitinib-sensitive non-small cell lung cancer cell line.
abstract: Gefitinib (Iressa)-a specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase-has been shown to suppress the activation of EGFR signaling required for cell survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. We recently provided novel evidence that gefitinib-sensitive PC9 cells show normal endocytosis of EGFR: internalized EGF-EGFR complexes were transported to late endosomes/lysosomes 15 min after EGF stimulation, and then degraded within the lysosomes. However, gefitinib-resistant QG56 cells showed internalized EGFR accumulation in early endosomes after 60 min of internalization, instead of its trafficking to lysosomes, indicating an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes. Therefore, we postulate that impairment in some steps of EGF-EGFR trafficking from early endosomes to late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines. To further substantiate the detailed internalization mechanism of gefitinib-sensitive and gefitinib-resistant cells, using confocal immunofluorescence microscopy, we examined the endocytic trafficking of phosphorylated EGFR (pEGFR) in the absence or presence of gefitinib. In PC9 and QG56 cells without EGF stimulation, a large number of pEGFR-positive small vesicular structures not colocalized with late endosomes/lysosomes were spread throughout the cytoplasm, and some pEGFR staining was distributed in the nucleus. This implies a novel intracellular trafficking pathway for pEGFR from cytoplasmic vesicles to the nucleus. Furthermore, an aggregated vesicular structure of early endosomes was observed in the perinuclear region of QG56 cells; it was revealed to be associated with SNX1, originally identified as a protein that interacts with EGFR. Therefore, we confirmed our previous data that an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes occurs in QG56 cells. Furthermore, in PC9 cells, efficient phosphorylation of EGFR and rapid internalization of pEGFR was observed at 3 min after EGF stimulation; these internalized pEGFR-positive vesicles were trafficked to late endosomes at 15 min, indicating rapid trafficking of EGF-pEGFR complexes from early to late endosomes in PC9 cells. Gefitinib treatment strongly reduced the phosphorylation level of EGFR, and subsequent endocytosis of EGFR was significantly suppressed in PC9 cells. In contrast, in QG56 cells, EGFR trafficking via the early endocytic pathway was basically impaired; therefore, gefitinib appeared to slightly suppress the internalization of pEGFR. Collectively, our data provide novel evidence that extensive impairment in pEGFR endocytosis via the early endocytic pathway might confer gefitinib-resistance in QG56 cells.
authors.affiliation: Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. nishimur@bioc.phar.kyushu-u.ac.jp
authors.forename: Yukio
authors.initials: Y
authors.lastname: Nishimura
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Yoshioka
authors.forename: Biborka
authors.initials: B
authors.lastname: Bereczky
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Itoh
mesh_terms: D001692:Biological Transport / Q000187:drug effects; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D004705:Endocytosis / Q000187:drug effects*; D011992:Endosomes / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D000077156:Gefitinib; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D008247:Lysosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010766:Phosphorylation / Q000187:drug effects; D011799:Quinazolines / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D058305:Sorting Nexins; D033921:Vesicular Transport Proteins / Q000378:metabolism
chemical_list: D011799:Quinazolines; C513978:SNX1 protein, human; D058305:Sorting Nexins; D033921:Vesicular Transport Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D000077156:Gefitinib