Hoshi Hirotaka, Hiyama Gen, Ishikawa Kosuke, Inageda Kiyoshi, Fujimoto Jiro, Wakamatsu Ai, Togashi Takushi, Kawamura Yoshifumi, Takahashi Nobuhiko, Higa Arisa, Goshima Naoki, Semba Kentaro, Watanabe Shinya, Takagi Motoki
Medical-Industrial Translational Research Center, Fukushima Medical University, Fukushima, Fukushima 960-1295, Japan.
Japan Biological Informatics Consortium (JBIC), Koto-ku, Tokyo 135-0064, Japan.
Oncol Rep. 2017 Jan;37(1):66-76. doi: 10.3892/or.2016.5227. Epub 2016 Nov 7.
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.
在患有各种癌症的患者肿瘤中，尤其是非小细胞肺癌中，已观察到表皮生长因子受体（EGFR）过表达和EGFR介导的信号通路失调。因此，已经开发了几种抗EGFR药物用于癌症治疗。对于已知具有EGFR激活突变（EGFR外显子19框内缺失和外显子21 L858R替代）的患者，使用EGFR酪氨酸激酶抑制剂（EGFR TKI；吉非替尼、厄洛替尼或阿法替尼）治疗是标准的一线治疗方法。然而，这些TKI的临床疗效最终受到获得性耐药的限制，例如通过守门人T790残基（T790M）的突变。为了克服这种获得性耐药并开发新型抗EGFR药物，需要一种基于细胞的EGFR TKI耐药突变体选择性抑制剂检测系统。我们构建了一种新型的基于细胞的检测方法，用于评估EGFR TKI对EGFR突变的疗效。为此，我们建立了依赖表皮生长因子（EGF）增殖的非致瘤性永生化乳腺上皮细胞（MCF 10A细胞），这些细胞稳定过表达突变型EGFR。我们发现，与表达野生型EGFR的细胞相比，表达含有T790M突变的EGFR的细胞对吉非替尼、厄洛替尼和阿法替尼表现出更高的耐药性。相反，表达L858R突变体的细胞表现出更高的TKI敏感性。T790M选择性抑制剂（奥希替尼和罗西替尼）对表达T790M突变体的细胞的作用明显高于吉非替尼和厄洛替尼。最后，与携带EGFR引入突变的市售同基因MCF 10A细胞系相比，我们的EGFR突变体过表达细胞由于EGFR突变体过表达细胞中EGFR磷酸化水平高于同基因细胞系，因此根据潜在突变对EGFR TKIs表现出明显更高的反应性。总之，我们成功开发了一种新型的基于细胞的检测方法，用于评估抗EGFR药物对EGFR突变的疗效。
