The role of erlotinib-induced autophagy in non-small cell lung carcinoma

Abstract

Background and Aim of Study Non-Small Cell Lung Cancer (NSCLC) is one of the leading causes of cancer deaths. Tyrosine Kinase Inhibitor (TKI) like erlotinib is commonly used as a treatment regimen since Epidermal Growth Factor Receptor (EGFR) is frequently deregulated in NSCLC. Autophagy is a regulated cellular catabolic process in response to deprivation of nutrients or growth factors. This study aims to investigate whether autophagy confers acquired resistance in NSCLC to erlotinib treatment in NSCLC. Methods Two NSCLC cell lines (HCC827 and HCC4006) with EGFR mutations (exon 19 deletions) were selected. Proliferation (MTT) assay and annexin-V binding assay were performed to determine cell proliferation and cell death upon erlotinib treatment. Autophagy induction was monitored by ATG-5/ATG12 conjugation, p62 degradation and conversion of LC3I to LC3II using Western blot. Increase in Acidic Vesicular Organelle (AVO) formation was determined by acridine orange staining. Autophagy inhibitor (chloroquine) and RNA interference were used to study the effect of erlotinib-induced autophagy. Results MTT confi rmed that both cell lines were sensitive to erlotinib (IC50 < 0.5 μM). Erlotinib treatment increased LC3II expression, ATG-5/ATG12 conjugation, formation of AVO and p62 degradation, compatible with induction of autophagy. Combination of 10 μM chloroquine (autophagy inhibitor) with 0.2 μM erlotinib increased apoptotic events compared to erlotinib or chloroquine alone (HCC827: 52.3 ± 2.8% vs 21.2 ± 4.2% vs 13.0 ± 2.5%, p < 0.01; HCC4006: 49.3 ± 4.8% vs 9.4 ± 2.4% vs 9.0 ± 1.8%, p < 0.01; chloroquine + erlotinib vs erlotinib vs chloroquine respectively). Atg5 and beclin 1 knockdown with siRNA resulted in signifi cant autophagy inhibition. Apoptotic cell death in erlotinib combined with ATG-silencing (51.8 ± 1.9%) or beclin-silencing (53.4 ± 2.0%) was increased comparing with erlotinib alone (22.8 ± 3.8%) (p < 0.01), ATG-silencing alone (7.3 ± 4.4%, p = 8.71576E-05) or beclin-silencing alone (10.3 ± 4.6%, p < 0.01) in HCC827 cells. Similarly, in HCC4006, apoptotic cell death was signifi cantly enhanced in erlotinib combined with ATG-silencing (53.1 ± 3.4%) or beclin-silencing (61.9 ± 5.1%), comparing with erlotinib alone (27.4 ± 8.6%, p < 0.01), ATG-silencing alone (6 ± 2.7%, p < 0.01) or beclinsilencing alone (11.5 ± 4.3%, p < 0.01). Conclusions Erlotinib can induce both apoptosis and autophagy in EGFR mutated (exon 19 del) NSCLC cell lines. Inhibition of autophagy with chloroquine or siRNA can enhance erlotinib-induced cell death. Autophagy may serve as a protective mechanism for NSCLC upon treatment with erlotinib.