P2.14-13 Elevated Expression of Eukaryotic Translation Initiation Factor 3 Subunit C Contributes to EGFR-TKI Resistance in Lung Adenocarcinoma

Abstract

The treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as erlotinib, in patients with EGFR-mutant lung adenocarcinoma causes tumor regression and improved prognosis. However, drug resistance and tumor relapse eventually occur, and the molecular mechanisms remain to be fully revealed. In this study, we elucidated the novel mechanism of EGFR-TKI resistance and clarified the clinical significance using erlotinib-resistant lung adenocarcinoma cell lines and patient specimens. The erlotinib-resistant cell line, PC9/ER was obtained from PC9 cell line by exposing to increasing concentrations of erlotinib for 6 months. The sensitivity of erlotinib was evaluated by the MTT assay. In order to identify erlotinib-resistance related factors, we performed a proteomics analysis, followed by western blotting. To evaluate the roles of the candidate factor, we performed gene knockdown analysis using siRNAs. The autophagy activity was assessed by the amount of LC3B-II protein or visualization of autophagosomes using LC3B-RFP expression vector. Immunohistochemical staining was performed on tumor biopsies obtained from lung adenocarcinoma patients with activating mutation in EGFR, and we examined the correlation between the staining intensity and clinical EGFR-TKI resistance. Initially, we performed the sequencing analysis of EGFR exons 18 to 21, which cover the tyrosine kinase domain, and found that there was no difference between PC9 and PC9/ER cells. The proteomics analysis revealed ribosomal and translation-related proteins were abundantly identified in PC9/ER cells, compared with PC9 cells. Among them, we found that eukaryotic translation initiation factor 3 subunit C (eIF3c) was overexpressed in PC9/ER cells. The knockdown of eIF3c expression with siRNA improved the sensitivity of EGFR-TKI in PC9/ER cells. Additionally, we observed that LC3B-II increased in PC9/ER cells, but decreased by the knockdown of eIF3c. Consistently, the overexpression of eIF3c upregulated the number of autophagosomes. Moreover, the inhibition of autophagy with chloroquine attenuated erlotinib-resistance in PC9/ER cells. Finally, the frequency of eIF3c-positive samples was higher in biopsy specimens from the patients’ refractory to EGFR-TKI therapy than in those from the patients prior to the treatment with EGFR-TKI. Furthermore, patients with eIF3c-positive tumors had shorter progression-free survival in EGFR-TKI treatment than eIF3c-negative patients. The increased eIF3c expression conferred the resistance to EGFR-TKI in lung adenocarcinoma cells via enhancement of autophagy. Moreover, the patients with increased eIF3c expression in biopsy samples showed poor prognosis. The inhibition of eIF3c could be a new therapeutic strategy for overcoming EGFR-TKI resistance in lung adenocarcinoma patients.