Abstract 4755: Clathrin mediated endocytosis as a potential therapeutic strategy for overcoming refractoriness of EGFR TKI

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) have significantly improved on clinical outcomes in many non-small cell lung cancer (NSCLC) patients. However, therapeutic efficacy of EGFR TKIs is limited on patients who have gefitinib-sensitizing EGFR gene mutations (Exon 19del, L858R). Although, not as sensitive as patients who have these mutations, some patients who have wild type EGFR responded to EGFR TKIs. Therefore, we hypothesized that there are underlying mechanisms that cause most of wild type EGFR NSCLC patients to be refractory. In this study, we demonstrated that intractability of EGFR TKIs in wild type EGFR NSCLC was closely related to clathrin mediated endocytosis (CME), which offered a novel strategy for overcoming EGFR TKI resistance. At first, we classified wild type EGFR NSCLC cell lines into gefitinib sensitive cells and refractory cells according to the IC50 values of gefitinib. Then, we performed immunocytochemistry (ICC) and immunoprecipitation (IP) to examine EGFR levels in early endosome with gefitinib treatment in gefitinib sensitive and refractory cell lines. As a result, EGFR endocytosis proceeded in gefitinib refractory cell lines (SNU1327, H1703) despite of gefitinib treatment, but not in sensitive cell lines (H358, Calu-3). EGFR is known to be internalized through CME and non clathrin mediated pathway (NCE). Then, we treated CME inhibitor, Phenylarsine oxide (PAO), or NCE inhibitor, Filipin III, in gefitinib sensitive and refractory cell lines to vefity if there is any difference in dependency of two main endocytosis pathway. As the ICC and IP data shown, blocking NCE did not inhibit EGFR internalization but blocking CME inhibited EGFR endocytosis in gefitinib refractory cell lines. Interestingly, it was the opposite in the gefitinib sensitive cell lines. Furthermore, we observed that cell survival decreased significantly during CME inhibition and combination treatment with gefitinib in refractory cell lines, but not in sensitive cell lines. To enlighten the elusive pathway, we examined the ratio of EGFR recycling and degradation. As a result, we observed EGFR degradation during CME or NCE proceeded conditions in gefitinib sensitive cell lines. However, in gefitinib refractory cell lines, EGFR protein was accumulated in CME dependent manner. These results implied that CME led EGFR recycling and sustained gefitinib refractory cell lines. For further studies, we are confirming signal molecules activated t