Fluorescence resonance energy transfer based quantitative analysis of feedforward and feedback loops in epidermal growth factor receptor signaling and the sensitivity to molecular targeting drugs
Fluorescence resonance energy transfer based quantitative analysis of feedforward and feedback loops in epidermal growth factor receptor signaling and the sensitivity to molecular targeting drugs
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DOI：
10.1111/febs.12852
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发表时间：
2014-07-01
期刊：
影响因子：
5.4
通讯作者：
Aoki, Kazuhiro
中科院分区：
文献类型：
--
作者：
Fujita, Yoshihisa;Komatsu, Naoki;Aoki, Kazuhiro
Ras-ERK 和 PI3K-mTOR 通路在多种恶性肿瘤中过度激活。 Ras-ERK 和 PI3K-mTOR 通路之间的前馈 (FF) 和反馈 (FB) 调节被认为可以减弱针对这些通路的药物的敏感性，并赋予肿瘤对治疗的耐药性。然而，由于对此类调控的分析需要高时间分辨率的测量和扰动，因此 FF 和 FB 调控在分子靶向药物的内在耐药性中所起的定量作用仍不清楚。为了解决这个问题，我们通过 Forster/荧光共振能量转移 (FRET) 成像量化了表皮生长因子受体 (EGFR) 信号通路的 FF 和 FB 调节。通过 FRET 成像测量 EGF 诱导的 EGFR、Ras、细胞外信号调节激酶和 S6K 的激活（无论有或没有抑制剂），并通过半自动图像处理进行分析。根据我们之前的研究确定的成像数据集和动力学参数，我们确定了连贯的 FF 调节和两个负 FB 调节所发挥的作用，其中之一以前未被认识到。系统分析揭示了这些 FF 和 FB 调节如何影响 EGF 刺激后细胞外信号调节激酶活性的时间动态。此外，模拟模型预测了单独或联合使用的分子靶向药物对 BRaf 或 KRas 突变癌细胞系的反应，表明整合 FF 和 FB 调节的定量模型的有效性。
The Ras-ERK and PI3K-mTOR pathways are hyperactivated in various malignant tumors. Feedforward (FF) and feedback (FB) regulations between the Ras-ERK and the PI3K-mTOR pathways have been suggested to attenuate sensitivity to drugs targeting these pathways and confer tumor resistance to therapies. However, because analyses of such regulations require measurements and perturbations with high temporal resolution, the quantitative roles played by FF and FB regulations in the intrinsic resistance to molecular targeting drugs still remain unclear. To address this issue, we quantified FF and FB regulations of the epidermal growth factor receptor (EGFR) signaling pathway by Forster/fluorescence resonance energy transfer (FRET) imaging. EGF-induced activation of EGFR, Ras, extracellular-signal-regulated kinase and S6K with or without inhibitors was measured by FRET imaging, and analyzed by semi-automatic image processing. Based on the imaging data set and kinetic parameters determined by our previous studies, we identified the roles played by a coherent FF regulation and two negative FB regulations, one of which was not recognized previously. The systems analyses revealed how these FF and FB regulations shape the temporal dynamics of extracellular-signal-regulated kinase activity upon EGF stimulation. Furthermore, the simulation model predicts the response of molecular targeting drugs applied solely or in combination with each other to BRaf- or KRas-mutated cancer cell lines, indicating the validity of a quantitative model integrating FF and FB regulations.