Corresponding Author: Giuseppe Giaccone, Georgetown University, 3970 Reservoir Road NW, Washington, DC.
Mol Cancer Ther. 2013 Nov;12(11):2601-13. doi: 10.1158/1535-7163.MCT-13-0074. Epub 2013 Aug 26.
Developing proteomic biomarkers is valuable for evaluating therapeutic effects of drugs and generating better treatment strategies. However, conventional protein analysis is often challenging due to inadequate sample size of clinical specimens, lack of assay reproducibility, accuracy, and sensitivity. A novel capillary isoelectricfocusing (IEF) immunoassay system (NanoPro) was used to study the dynamic phosphorylation status of signaling molecules in non-small cell lung cancer (NSCLC) cells treated with EGFR tyrosine kinase and MEK inhibitors. NanoPro showed the same dynamic ERK phosphorylation as Western blotting with good assay reproducibility using 1,000 times less protein. The IEF separation in NanoPro system enables multiple protein phosphorylation isoforms to be resolved and detected simultaneously. With NanoPro, we identified a specific on-target mitogen-activated protein/extracellular signal-regulated kinase (MEK) response pattern to MEK inhibitor PD325901, which was not detectable by Western blot analysis. We also revealed a MEK2 signal that may be associated with NSCLC cell sensitivity to the EGF receptor inhibitor erlotinib, and distinguished erlotinib-sensitive cells from intrinsic as well as acquired resistant cells to erlotinib. Moreover, NanoPro could differentiate human ERK1 isoforms from the mouse isoforms based on their isoelectric point differences and showed that erlotinib effectively inhibited ERK phosphorylation in targeted human xenograft cancer cells but not in surrounding mouse stromal cells. With 8 μg of tumor aspirates, we precisely quantified the response of 18 signaling molecules to erlotinib and MEK1 inhibitor treatments in an NSCLC patient. NanoPro's higher sensitivity, better resolution of protein phosphorylation status, and reduced tissue requirement warrant NanoPro's investigation for future drug development and evaluation of drug effects of targeted therapies.
开发蛋白质组学生物标志物对于评估药物的治疗效果和制定更好的治疗策略非常有价值。然而，由于临床标本的样本量不足、检测重现性、准确性和灵敏度差，常规的蛋白质分析往往具有挑战性。一种新型的毛细管等电聚焦（IEF）免疫分析系统（NanoPro）被用于研究 EGFR 酪氨酸激酶和 MEK 抑制剂处理的非小细胞肺癌（NSCLC）细胞中信号分子的动态磷酸化状态。NanoPro 显示与 Western blot 相同的动态 ERK 磷酸化，使用的蛋白量少 1000 倍，具有良好的检测重现性。IEF 分离在 NanoPro 系统中能够同时分离和检测多个蛋白质磷酸化同工型。使用 NanoPro，我们确定了一种针对 MEK 抑制剂 PD325901 的特定的有靶标丝裂原活化蛋白/细胞外信号调节激酶（MEK）反应模式，这在 Western blot 分析中是无法检测到的。我们还揭示了一种 MEK2 信号，它可能与 NSCLC 细胞对表皮生长因子受体抑制剂厄洛替尼的敏感性有关，并将厄洛替尼敏感细胞与内在和获得性的厄洛替尼耐药细胞区分开来。此外，NanoPro 可以根据其等电点的差异将人 ERK1 同工型与小鼠同工型区分开来，并表明厄洛替尼能有效地抑制靶向人异种移植癌细胞中的 ERK 磷酸化，但不能抑制周围的小鼠基质细胞中的 ERK 磷酸化。用 8 μg 的肿瘤抽吸物，我们精确地定量了 18 种信号分子对厄洛替尼和 MEK1 抑制剂治疗的反应，在一位 NSCLC 患者中。NanoPro 的更高灵敏度、更好的蛋白质磷酸化状态分辨率和减少组织需求，保证了 NanoPro 在未来药物开发和靶向治疗药物效果评估中的研究价值。
