title: EGFR-independent autophagy induction with gefitinib and enhancement of its cytotoxic effect by targeting autophagy with clarithromycin in non-small cell lung cancer cells.
abstract: Gefitinib (GEF), an inhibitor for EGFR tyrosine kinase, potently induces autophagy in non-small cell lung cancer (NSCLC) cell lines such as PC-9 cells expressing constitutively activated EGFR kinase by EGFR gene mutation as well as A549 and H226 cells with wild-type EGFR. Unexpectedly, GEF-induced autophagy was also observed in non-NSCLC cells such as murine embryonic fibroblasts (MEF) and leukemia cell lines K562 and HL-60 without EGFR expression. Knockout of EGFR gene in A549 cells by CRISPR/Cas9 system still exhibited autophagy induction after treatment with GEF, indicating that the autophagy induction by GEF is not mediated through inhibiting EGFR kinase activity. Combined treatment with GEF and clarithromycin (CAM), a macrolide antibiotic having the effect of inhibiting autophagy flux, enhances the cytotoxic effect in NSCLC cell lines, although treatment with CAM alone exhibits no cytotoxicity. GEF treatment induced up-regulation of endoplasmic reticulum (ER)-stress related genes such as CHOP/GADD153 and GRP78. Knockdown of CHOP in PC-9 cells and Chop-knockout MEF both exhibited less sensitivity to GEF than controls. Addition of CAM in culture medium resulted in further pronounced GEF-induced ER stress loading, while CAM alone exhibited no effect. These data suggest that GEF-induced autophagy functions as cytoprotective and indicates the potential therapeutic possibility of using CAM for GEF therapy. Furthermore, it is suggested that the intracellular signaling for autophagy initiation in response to GEF can be completely dissociated from EGFR, but unknown target molecule(s) of GEF for autophagy induction might exist.
authors: Department of Biochemistry, Tokyo Medical University, Tokyo, Japan.
authors: Shohei
authors: S
authors: Sugita
authors: Department of Biochemistry, Tokyo Medical University, Tokyo, Japan.
authors: Kentaro
authors: K
authors: Ito
authors: Department of Biochemistry, Tokyo Medical University, Tokyo, Japan.
authors: Yutaro
authors: Y
authors: Yamashiro
authors: Department of Biochemistry, Tokyo Medical University, Tokyo, Japan.
authors: Shota
authors: S
authors: Moriya
authors: Department of Biochemistry, Tokyo Medical University, Tokyo, Japan.
authors: Xiao-Fang
authors: XF
authors: Che
authors: Department of Clinical Oncology, Tokyo Medical University, Tokyo, Japan.
authors: Tomohisa
authors: T
authors: Yokoyama
authors: Department of Biochemistry, Tokyo Medical University, Tokyo, Japan.
authors: Masaki
authors: M
authors: Hiramoto
authors: Department of Biochemistry, Tokyo Medical University, Tokyo, Japan. Electronic address: miyazawa@tokyo-med.ac.jp.
authors: Keisuke
authors: K
authors: Miyazawa
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D001343:Autophagy / Q000187:drug effects*; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D017291:Clarithromycin / Q000008:administration & dosage; D004357:Drug Synergism; D000091342:Endoplasmic Reticulum Chaperone BiP; D066246:ErbB Receptors / Q000378:metabolism*; D000077156:Gefitinib; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D011799:Quinazolines / Q000008:administration & dosage; D016896:Treatment Outcome
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D011799:Quinazolines; C512478:EGFR protein, human; D066246:ErbB Receptors; D017291:Clarithromycin; D000077156:Gefitinib
keywords: Autophagy; EGFR; ER-stress; Gefitinib; Macrolide antibiotics; Non-small cell lung cancer