Construction of a novel cell-based assay for the evaluation of anti-EGFR drug efficacy against EGFR mutation

Hirotaka Hoshi, Gen Hiyama, Kosuke Ishikawa, Kiyoshi Inageda, Jiro Fujimoto, Ai Wakamatsu, Takushi Togashi, Yoshifumi Kawamura, Nobuhiko Takahashi, Arisa Higa, Naoki Goshima, Kentaro Senba, Shinya Watanabe, Motoki Takagi

    Research output: Contribution to journalArticle

    5 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)66-76
    Number of pages11
    JournalOncology Reports
    Volume37
    Issue number1
    DOIs
    Publication statusPublished - 2017 Jan 1

    Fingerprint

    Epidermal Growth Factor Receptor
    Mutation
    Pharmaceutical Preparations
    Drug Resistance
    Exons
    Cell Line
    Neoplasms

    Keywords

    • EGFR mutation
    • EGFR tyrosine kinase inhibitor
    • Epidermal growth factor receptor
    • Lung cancer
    • Molecular target drug
    • Non-small cell lung cancer
    • T790M

    ASJC Scopus subject areas

    • Oncology
    • Cancer Research

    Cite this

    Hoshi, H., Hiyama, G., Ishikawa, K., Inageda, K., Fujimoto, J., Wakamatsu, A., ... Takagi, M. (2017). Construction of a novel cell-based assay for the evaluation of anti-EGFR drug efficacy against EGFR mutation. Oncology Reports, 37(1), 66-76. https://doi.org/10.3892/or.2016.5227

    Construction of a novel cell-based assay for the evaluation of anti-EGFR drug efficacy against EGFR mutation. / Hoshi, Hirotaka; Hiyama, Gen; Ishikawa, Kosuke; Inageda, Kiyoshi; Fujimoto, Jiro; Wakamatsu, Ai; Togashi, Takushi; Kawamura, Yoshifumi; Takahashi, Nobuhiko; Higa, Arisa; Goshima, Naoki; Senba, Kentaro; Watanabe, Shinya; Takagi, Motoki.

    In: Oncology Reports, Vol. 37, No. 1, 01.01.2017, p. 66-76.

    Research output: Contribution to journalArticle

    Hoshi, H, Hiyama, G, Ishikawa, K, Inageda, K, Fujimoto, J, Wakamatsu, A, Togashi, T, Kawamura, Y, Takahashi, N, Higa, A, Goshima, N, Senba, K, Watanabe, S & Takagi, M 2017, 'Construction of a novel cell-based assay for the evaluation of anti-EGFR drug efficacy against EGFR mutation', Oncology Reports, vol. 37, no. 1, pp. 66-76. https://doi.org/10.3892/or.2016.5227
    Hoshi, Hirotaka ; Hiyama, Gen ; Ishikawa, Kosuke ; Inageda, Kiyoshi ; Fujimoto, Jiro ; Wakamatsu, Ai ; Togashi, Takushi ; Kawamura, Yoshifumi ; Takahashi, Nobuhiko ; Higa, Arisa ; Goshima, Naoki ; Senba, Kentaro ; Watanabe, Shinya ; Takagi, Motoki. / Construction of a novel cell-based assay for the evaluation of anti-EGFR drug efficacy against EGFR mutation. In: Oncology Reports. 2017 ; Vol. 37, No. 1. pp. 66-76.
    @article{800f3cbf5f824c2b93e9b1bd36e39cb5,
    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.",
    keywords = "EGFR mutation, EGFR tyrosine kinase inhibitor, Epidermal growth factor receptor, Lung cancer, Molecular target drug, Non-small cell lung cancer, T790M",
    author = "Hirotaka Hoshi and Gen Hiyama and Kosuke Ishikawa and Kiyoshi Inageda and Jiro Fujimoto and Ai Wakamatsu and Takushi Togashi and Yoshifumi Kawamura and Nobuhiko Takahashi and Arisa Higa and Naoki Goshima and Kentaro Senba and Shinya Watanabe and Motoki Takagi",
    year = "2017",
    month = "1",
    day = "1",
    doi = "10.3892/or.2016.5227",
    language = "English",
    volume = "37",
    pages = "66--76",
    journal = "Oncology Reports",
    issn = "1021-335X",
    publisher = "Spandidos Publications",
    number = "1",

    }

    TY - JOUR

    T1 - Construction of a novel cell-based assay for the evaluation of anti-EGFR drug efficacy against EGFR mutation

    AU - Hoshi, Hirotaka

    AU - Hiyama, Gen

    AU - Ishikawa, Kosuke

    AU - Inageda, Kiyoshi

    AU - Fujimoto, Jiro

    AU - Wakamatsu, Ai

    AU - Togashi, Takushi

    AU - Kawamura, Yoshifumi

    AU - Takahashi, Nobuhiko

    AU - Higa, Arisa

    AU - Goshima, Naoki

    AU - Senba, Kentaro

    AU - Watanabe, Shinya

    AU - Takagi, Motoki

    PY - 2017/1/1

    Y1 - 2017/1/1

    N2 - 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.

    AB - 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.

    KW - EGFR mutation

    KW - EGFR tyrosine kinase inhibitor

    KW - Epidermal growth factor receptor

    KW - Lung cancer

    KW - Molecular target drug

    KW - Non-small cell lung cancer

    KW - T790M

    UR - http://www.scopus.com/inward/record.url?scp=85006507869&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=85006507869&partnerID=8YFLogxK

    U2 - 10.3892/or.2016.5227

    DO - 10.3892/or.2016.5227

    M3 - Article

    VL - 37

    SP - 66

    EP - 76

    JO - Oncology Reports

    JF - Oncology Reports

    SN - 1021-335X

    IS - 1

    ER -