Yuan-Yuan Li1, Sze-Kwan Lam1, Judith Choi-Wo Mak1, Chun-Yan Zheng1, James Chung-Man Ho2. 1. Division of Respiratory Medicine, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong Special Administrative Region. 2. Division of Respiratory Medicine, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong Special Administrative Region. Electronic address: jhocm@hku.hk.
Abstract
PURPOSE: Erlotinib is a commonly used tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC). Autophagy is a catabolic process in response to stress and deprivation of nutrients. This study aims to investigate whether autophagy confers acquired resistance to erlotinib treatment in NSCLC. METHODS: Four NSCLC cell lines (HCC827, HCC4006, H358 and H1975) with different epidermal growth factor receptor (EGFR) mutation status (exon 19 deletion, exon 19 deletion, wild-type and L858R/T790M respectively) were selected. MTT assay, crystal violet staining and Annexin-V assay were performed to determine cell viability and apoptosis. Autophagic proteins were detected by Western blot. Acidic vesicular organelle (AVO) formation was determined by acridine orange staining. Autophagy inhibitor (chloroquine) and RNA interference were used to demonstrate the biological effect of erlotinib-induced autophagy. RESULTS: In line with EGFR mutation status, it was shown that both HCC827 and HCC4006 cells were sensitive to erlotinib, while H358 and H1975 cell lines were resistant. Erlotinib treatment at clinically relevant concentrations induced autophagy (increased LC3II expression, Atg-5/Atg12 conjugation, formation of AVO and p62 degradation) in sensitive NSCLC cell lines, via p53 nuclear translocation, AMPK activation and mTOR suppression. Addition of chloroquine, as an autophagy inhibitor, enhanced erlotinib sensitivity in sensitive cells. Similarly, silencing of Atg5 or Beclin-1 significantly increased sensitivity to erlotinib in both sensitive cell lines. In contrast, there was no induction of autophagy in resistant H358 and H1975 cell lines upon erlotinib exposure. CONCLUSIONS: Erlotinib can induce both apoptosis and autophagy in sensitive NSCLC cell lines with activating EGFR mutation (exon 19 del). Inhibition of autophagy can further enhance sensitivity to erlotinib in EGFR-mutated NSCLC, suggesting that autophagy may serve as a protective mechanism.
PURPOSE:Erlotinib is a commonly used tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC). Autophagy is a catabolic process in response to stress and deprivation of nutrients. This study aims to investigate whether autophagy confers acquired resistance to erlotinib treatment in NSCLC. METHODS: Four NSCLC cell lines (HCC827, HCC4006, H358 and H1975) with different epidermal growth factor receptor (EGFR) mutation status (exon 19 deletion, exon 19 deletion, wild-type and L858R/T790M respectively) were selected. MTT assay, crystal violet staining and Annexin-V assay were performed to determine cell viability and apoptosis. Autophagic proteins were detected by Western blot. Acidic vesicular organelle (AVO) formation was determined by acridine orange staining. Autophagy inhibitor (chloroquine) and RNA interference were used to demonstrate the biological effect of erlotinib-induced autophagy. RESULTS: In line with EGFR mutation status, it was shown that both HCC827 and HCC4006 cells were sensitive to erlotinib, while H358 and H1975 cell lines were resistant. Erlotinib treatment at clinically relevant concentrations induced autophagy (increased LC3II expression, Atg-5/Atg12 conjugation, formation of AVO and p62 degradation) in sensitive NSCLC cell lines, via p53 nuclear translocation, AMPK activation and mTOR suppression. Addition of chloroquine, as an autophagy inhibitor, enhanced erlotinib sensitivity in sensitive cells. Similarly, silencing of Atg5 or Beclin-1 significantly increased sensitivity to erlotinib in both sensitive cell lines. In contrast, there was no induction of autophagy in resistant H358 and H1975 cell lines upon erlotinib exposure. CONCLUSIONS:Erlotinib can induce both apoptosis and autophagy in sensitive NSCLC cell lines with activating EGFR mutation (exon 19 del). Inhibition of autophagy can further enhance sensitivity to erlotinib in EGFR-mutated NSCLC, suggesting that autophagy may serve as a protective mechanism.
Authors: Aikaterini Lampada; James O'Prey; Gyorgy Szabadkai; Kevin M Ryan; Daniel Hochhauser; Paolo Salomoni Journal: Cell Death Differ Date: 2017-05-05 Impact factor: 15.828
Authors: Sarmistha Talukdar; Anjan K Pradhan; Praveen Bhoopathi; Xue-Ning Shen; Laura A August; Jolene J Windle; Devanand Sarkar; Frank B Furnari; Webster K Cavenee; Swadesh K Das; Luni Emdad; Paul B Fisher Journal: Proc Natl Acad Sci U S A Date: 2018-05-14 Impact factor: 11.205
Authors: Christina H Eng; Zuncai Wang; Diane Tkach; Lourdes Toral-Barza; Savuth Ugwonali; Shanming Liu; Stephanie L Fitzgerald; Elizabeth George; Elizabeth Frias; Nadire Cochran; Rowena De Jesus; Gregory McAllister; Gregory R Hoffman; Kevin Bray; LuAnna Lemon; Judy Lucas; Valeria R Fantin; Robert T Abraham; Leon O Murphy; Beat Nyfeler Journal: Proc Natl Acad Sci U S A Date: 2015-12-17 Impact factor: 11.205