A Role for SNX1 in the Regulation of EGF-Dependent Phosphorylated EGFR Endocytosis Via the Early/Late Endocytic Pathway in a Gefitinib-Sensitive Human Lung Cancer Cells

Yukio      Nishimura; Kiyoko      Yoshioka; Soichi      Takiguchi; Biborka      Bereczky; Yusaku      Nakabeppu; Kazuyuki      Itoh

doi:10.2174/157436211797484047

Abstract

The EGF and its receptor (EGFR) play an important role in the pathogenesis of different tumors. Gefitinib (Iressa) has been shown to suppress the activation of EGFR signaling required for cell survival and proliferation in nonsmall cell lung cancer (NSCLC) cell lines. We recently demonstrated that gefitinib-sensitive cells show efficient endocytosis of EGFR. In contrast, gefitinib-resistant cells showed internalized EGFR accumulation in the aggregated early endosomes that was revealed to be associated with SNX1, originally identified as a protein that interacts with EGFR. These results indicate an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/ lysosomes in the gefitinib-resistant cells. Here, we conducted the present study to investigate the relationship between EGF-stimulated pEGFR endocytosis and SNX1 trafficking via the endocytic pathway in the gefitinib-sensitive and gefitinib-resistant NSCLC cells by employing confocal immunofluorescence microscopy. We demonstrate that EGF stimulates efficient EGFR phosphorylation and the rapid endocytic delivery of pEGFR from the plasma membrane to early endosomes/late endosomes/lysosomes in a gefitinib-sensitive NSCLC cell line. Moreover, we made the novel observation that after EGF stimulation there is a rapid release of SNX1 from early endosomes in gefitinib-sensitive cells along with a concomitant time-dependent increase in nuclear SNX1. In contrast, following EGF-stimulation of gefitinibresistant cells, most SNX1 staining remained associated with aggregated early endosomes in which internalized pEGFR gradually accumulated instead of being trafficked to late endosomes/lysosomes, suggesting that the EGF-dependent nuclear translocation mechanism of SNX1 is significantly perturbed. Moreover, this translocation of SNX1 from early endosomes in gefitinib-sensitive cells was completely abolished by latrunculin B treatment, indicating that the actin cytoskeleton may mediate the EGF-stimulated nuclear translocation of SNX1. Collectively, we postulate that SNX1 is involved in the negative regulation of EGF-dependent EGFR trafficking out of early endosomes to late endosomes/ lysosomes, and suggest that impairment of the nuclear trafficking machinery of SNX1 regulating pEGFR endocytosis might perturb pEGFR release from early endosomes, which subsequently leads to the acquisition of gefitinib-resistance in NSCLC cell lines.

Keywords: orting nexin 1 (SNX1), Phosphorylated EGF receptor (pEGFR), Endosomes/Lysosomes, Non-small cell lung cancer cell lines, SNX1, pEGFR, EGFR, Endocytosis, Gefitinib, Endosomes, Lysosomes, Nucleus, NSCLC