title: Construction of a novel cell-based assay for the evaluation of anti-EGFR drug efficacy against EGFR mutation.
abstract: Epidermal growth factor receptor (EGFR) overexpression and EGFR-mediated signaling pathway dysregulation have been observed in tumors from patients with various cancers, especially non-small cell lung cancer. Thus, several anti-EGFR drugs have been developed for cancer therapy. For patients with known EGFR activating mutations (EGFR exon 19 in-frame deletions and exon 21 L858R substitution), treatment with an EGFR tyrosine kinase inhibitor (EGFR TKI; gefitinib, erlotinib or afatinib) represents standard first-line therapy. However, the clinical efficacy of these TKIs is ultimately limited by the development of acquired drug resistance such as by mutation of the gatekeeper T790 residue (T790M). To overcome this acquired drug resistance and develop novel anti-EGFR drugs, a cell-based assay system for EGFR TKI resistance mutant-selective inhibitors is required. We constructed a novel cell-based assay for the evaluation of EGFR TKI efficacy against EGFR mutation. To this end, we established non-tumorigenic immortalized breast epithelial cells that proliferate dependent on EGF (MCF 10A cells), which stably overexpress mutant EGFR. We found that the cells expressing EGFR containing the T790M mutation showed higher resistance against gefitinib, erlotinib and afatinib compared with cells expressing wild-type EGFR. In contrast, L858R mutant-expressing cells exhibited higher TKI sensitivity. The effect of T790M-selective inhibitors (osimertinib and rociletinib) on T790M mutant-expressing cells was significantly higher than gefitinib and erlotinib. Finally, when compared with commercially available isogenic MCF 10A cell lines carrying introduced mutations in EGFR, our EGFR mutant-overexpressing cells exhibited obviously higher responsiveness to EGFR TKIs depending on the underlying mutations because of the higher levels of EGFR phosphorylation in the EGFR mutant-overexpressing cells than in the isogenic cell lines. In conclusion, we successfully developed a novel cell-based assay for evaluating the efficacy of anti-EGFR drugs against EGFR mutation.
authors.affiliation: Medical-Industrial Translational Research Center, Fukushima Medical University, Fukushima, Fukushima 960-1295, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Hoshi
authors.affiliation: Medical-Industrial Translational Research Center, Fukushima Medical University, Fukushima, Fukushima 960-1295, Japan.
authors.forename: Gen
authors.initials: G
authors.lastname: Hiyama
authors.affiliation: Japan Biological Informatics Consortium (JBIC), Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Ishikawa
authors.affiliation: Medical-Industrial Translational Research Center, Fukushima Medical University, Fukushima, Fukushima 960-1295, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Inageda
authors.affiliation: Japan Biological Informatics Consortium (JBIC), Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Jiro
authors.initials: J
authors.lastname: Fujimoto
authors.affiliation: Japan Biological Informatics Consortium (JBIC), Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Ai
authors.initials: A
authors.lastname: Wakamatsu
authors.affiliation: Japan Biological Informatics Consortium (JBIC), Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Takushi
authors.initials: T
authors.lastname: Togashi
authors.affiliation: Japan Biological Informatics Consortium (JBIC), Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Kawamura
authors.affiliation: Medical-Industrial Translational Research Center, Fukushima Medical University, Fukushima, Fukushima 960-1295, Japan.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Takahashi
authors.affiliation: Medical-Industrial Translational Research Center, Fukushima Medical University, Fukushima, Fukushima 960-1295, Japan.
authors.forename: Arisa
authors.initials: A
authors.lastname: Higa
authors.affiliation: National Institute of Advanced Industrial Science and Technology (AIST), Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Goshima
authors.affiliation: Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Semba
authors.affiliation: Medical-Industrial Translational Research Center, Fukushima Medical University, Fukushima, Fukushima 960-1295, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Medical-Industrial Translational Research Center, Fukushima Medical University, Fukushima, Fukushima 960-1295, Japan.
authors.forename: Motoki
authors.initials: M
authors.lastname: Takagi
mesh_terms: D000077716:Afatinib; D000970:Antineoplastic Agents / Q000302:isolation & purification* / Q000627:therapeutic use; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000235:genetics*; D000069347:Erlotinib Hydrochloride / Q000494:pharmacology; D000077156:Gefitinib; D006801:Humans; D009154:Mutation; D047428:Protein Kinase Inhibitors / Q000302:isolation & purification* / Q000627:therapeutic use; D011799:Quinazolines / Q000494:pharmacology; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D000077716:Afatinib; D000069347:Erlotinib Hydrochloride; C512478:EGFR protein, human; D066246:ErbB Receptors; D000077156:Gefitinib