Abstract 1899: Endocytosis of EGFR and resistance to gefitinib in NSCLC cells with wild-type EGFR

Abstract

Abstract Gefitinib is an oral active, selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) and results in clinical benefit in NSCLC patients with EGFR mutations, but objective clinical responses were hardly seen in the majority of NSCLC patients with wild-type EGFR (wtEGFR). However, approximately 10-20% patients with wtEGFR remain responsive to EGFR TKIs, implying that the existence of unexplored mechanisms contribute to susceptibility to gefitinib. Therefore, in this study, we aimed to identify potential mechanism which can predict the response of gefitinib in NSCLC with wtEGFR. Six NSCLC cells (H358, Calu-3, H1703, Calu-1, H441 and H522) with wtEGFR were selected and tested for sensitivity to gefitinib. MTT and cell counting assay revealed that H358 and Calu-3 cells were more sensitive to gefitinib than H1703, Calu-1, H441 and H522 cells. In addition, light microscopy and wound healing assay showed cell morphology change and suppression of motility by gefitinib treatment in the gefitinib-sensitive cells, but not in the gefitinib-resistant cells. Next, changes in phsosphorylation of EGFR and downstream pathway after gefitinib treatment were examined by Western blot assay in the cells with different gefitinib sensitivity. Interestingly, gefitinib inhibited EGF-induced phosphorylation of EGFR and downstream mediators (AKT and ERK) in both gefitinib-sensitive and -resistant cells. EGFR phosphorylation array confirmed decreased activation of multiple phophorylation sites of EGFR in gefitinib-resistant cells, implying that inhibition of tyrosine kinase activity might not a determinant of gefitinib sensitivity in NSCLC with wtEGFR. Since intracellular activity of EGFR has been reported to be important in cell proliferation, we examined whether intracellular distribution of EGFR after gefitinib treatment had different patterns in between gefitinib-sensitive and -resistant cells. Immunofluorescence microscopy assay revealed that EGFR was internalized from cell surface to intracellular region and then formed dot complex after EGF stimulation in both gefitinib-senstive and -resistant cells. Gefitinib treatment inhibited the EGF-induced internalization of EGFR in gefitinib-sensitive cells, but not in gefitinib-resistant cells. Further flowcytometric analysis using fluorescence labeled EGF and EGFR confirmed internalization of EGFR after genfitinib treatment in gefitinib-sensitive cells. Moreover, internalization inhibitors (dynasore and dynole 34.2) significantly decreased cell viability of gefitinib-resistant cells when combined with gefitinib. In conclusion, our results suggest that endocytosis of EGFR might be one of the contributing factors of gefitinib sensitivity in NSCLC with wtEGFR. (This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare and family Affairs, Republic of Korea (A111218-11-GM04)). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1899. doi:1538-7445.AM2012-1899