Chemical Proteomics Uncovers EPHA2 as a Mechanism of Acquired Resistance to Small Molecule EGFR Kinase Inhibition
Chemical Proteomics Uncovers EPHA2 as a Mechanism of Acquired Resistance to Small Molecule EGFR Kinase Inhibition
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DOI：
10.1021/acs.jproteome.5b00161
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发表时间：
2015-06-01
影响因子：
4.4
通讯作者：
Kuster, Bernhard
中科院分区：
文献类型：
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作者：
Koch, Heiner;Busto, M. Estela Del Castillo;Kuster, Bernhard
酪氨酸激酶抑制剂（TKI）已成为治疗多种癌症的重要治疗选择。吉非替尼是一种表皮生长因子受体 (EGFR) 抑制剂，在临床上用于治疗含有激活 EGFR 突变的非小细胞肺癌 (NSCLC)。然而，尽管初始反应率很高，但许多患者对吉非替尼产生了耐药性。 TKI 耐药的分子机制通常仍不清楚。在这里，我们描述了一种化学蛋白质组学方法，包括激酶亲和纯化（kinobeads）和定量质谱法，用于鉴定癌细胞中激酶抑制剂的耐药机制。我们鉴定了之前描述的 MET 扩增，并发现 EPHA2 在吉非替尼耐药的 HCC827 细胞中过表达超过 10 倍（p < 0.001），表明在产生耐药性中具有潜在作用。 siRNA 介导的 EPHA2 敲低或用多激酶抑制剂达沙替尼处理细胞可恢复对吉非替尼的敏感性。在所有达沙替尼靶标中，EPHA2 表现出最显着的效果 (p < 0.001)。此外，EPHA2 敲低或 ephrin-A1 处理耐药细胞会降低 FAK 磷酸化和细胞迁移。这些发现证实 EPHA2 是一个可行的药物靶点，为药物组合方法提供了合理的基础，并表明化学蛋白质组学广泛适用于发现激酶抑制剂耐药性。
Tyrosine kinase inhibitors (TKIs) have become an important therapeutic option for treating several forms of cancer. Gefitinib, an inhibitor of the epidermal growth factor receptor (EGFR), is in clinical use for treating non-small cell lung cancer (NSCLC) harboring activating EGFR mutations. However, despite high initial response rates, many patients develop resistance to gefitinib. The molecular mechanisms of TKI resistance often remain unclear. Here, we describe a chemical proteomic approach comprising kinase affinity purification (kinobeads) and quantitative mass spectrometry for the identification of kinase inhibitor resistance mechanisms in cancer cells. We identified the previously described amplification of MET and found EPHA2 to be more than 10-fold overexpressed (p < 0.001) in gefitinib-resistant HCC827 cells suggesting a potential role in developing resistance. siRNA-mediated EPHA2 knock-down or treating cells with the multikinase inhibitor dasatinib restored sensitivity to gefitinib. Of all dasatinib targets, EPHA2 exhibited the most drastic effect (p < 0.001). In addition, EPHA2 knockdown or ephrin-A1 treatment of resistant cells decreased FAK phosphorylation and cell migration. These findings confirm EPHA2 as an actionable drug target, provide a rational basis for drug combination approaches, and indicate that chemical proteomics is broadly applicable for the discovery of kinase inhibitor resistance.