EGF Receptor Signaling in Time and Space in Tumor Cells

描述（由申请人提供）：ErbB家族的受体酪氨酸激酶在各种类型的人类癌症的发生和发展中起重要作用。阐明该家族的原型成员表皮生长因子（EGF）受体（EGFR/ErbB 1）和其他ErbB的生理调节是理解其致癌激活机制的关键。生长因子与EGFR的结合导致无数信号转导过程的启动。丝裂原活化蛋白激酶1/2（mitogen-activated protein kinase 1/2，MAPK/ERK 1/2）的激活是EGFR激活的主要信号通路，决定了EGFR信号通路的最终结局。最近的研究表明，EGFR信号传导不受细胞表面信号传导的限制，而是该途径的所有组分在细胞内移动，并可能与各种细胞内区室相关并在其中发挥作用。活化的EGFR被迅速内化到内体中，并被分选到溶酶体中进行降解。在信号传导和内吞机制之间存在许多双向的交叉对话。已经提出内吞作用是MAPK激活所必需的，尽管这个问题仍然存在争议。 该提案的总体目标是重建癌细胞中空间和时间的信号传导过程，并将新的实验数据整合到EGFR-MAPK通路动力学的计算模型中。首先，MAPK信号通路的组分将被各种荧光蛋白（XFP）标记，并在人癌细胞中以生理水平稳定表达，其中相应的内源性蛋白已通过载体介导的RNA干扰被敲低。然后将使用定量荧光显微镜在这些新细胞系中分析参与ERK 1/2激活的XFP标记蛋白的动态定位和运输模式。第二，结合RNA干扰、活细胞荧光成像、多发色团荧光共振能量转移显微镜和生物化学技术，研究MAPK激活过程中多蛋白复合物的形成机制及其定位。第三，将在一系列表达不同水平EGFR的人癌细胞系中对内吞作用和内体在调节EGFR信号传导至MAPK中的作用进行系统分析。使用描述EGFR-MAPK途径的组分的空间和时间重叠、它们的扩散速率参数、运输途径和蛋白质-蛋白质相互作用的实验数据，将产生EGFR-MAPK信号传导的空间和时间组织的新计算模型，实验验证并用于预测不同EGFR表达水平的影响，EGFR配体的浓度和系统的各种扰动对MAPK信号传导的强度和持续时间的影响。 公共卫生相关性：EGF受体已成为人类肿瘤的主要预后和诊断标志物以及重要的治疗靶点。然而，使用EGF受体作为标记物和治疗靶点的益处目前非常有限。本文提出的研究将增强我们对癌细胞中EGF受体调节的理解，并有助于开发下调癌细胞中EGF受体和类似受体活性的新策略，还可能揭示新的潜在预后标志物和治疗靶点。

DESCRIPTION (provided by applicant): Receptor tyrosine kinases of the ErbB family play important roles in the development and progression of various types of human carcinoma. Elucidation of the physiological regulation of a prototypical member of the family, epidermal growth factor (EGF) receptor (EGFR/ErbB1), and other ErbBs, is the key to understanding the mechanisms causing their oncogenic activation. Growth factor binding to the EGFR results in initiation of a myriad of signal transduction processes. Activation of the mitogen-activated protein kinase 1/2 (MAPK/ERK1/2) is the major signaling pathway triggered by EGFR, which determines the ultimate outcome of the EGFR signaling. Recent studies revealed that EGFR signaling is not limited by signaling at the cell surface, but, rather, all components of the pathway move within the cell and may associate with and function in various intracellular compartments. The activated EGFR is rapidly internalized into endosomes and sorted to lysosomes for degradation. There are numerous bi-directional cross-talks between signaling and endocytic machineries. It has been proposed that endocytosis is necessary for MAPK activation, although this issue remains controversial. The overall goal of this proposal is to reconstitute the signaling process in space and time in cancer cells and to integrate the new experimental data into a computational model of the dynamics of the EGFR-MAPK pathway. Firstly, the components of the MAPK signaling pathway will be tagged with various fluorescent proteins (XFPs) and stably expressed at physiological levels in human carcinoma cells, in which the corresponding endogenous protein had been knocked-down by vector-mediated RNA interference. The patterns of the dynamic localization and trafficking of XFP-tagged proteins involved in ERK1/2 activation will then be analyzed in these new cell lines using quantitative fluorescence microscopy. Secondly, using a combination of RNA interference, live-cell fluorescence imaging, multi-chromophore fluorescence resonance energy transfer microscopy and biochemical techniques, the mechanisms of the formation of the multi-protein complexes during MAPK activation and the localization of these complexes will be investigated. Thirdly, a systematic analysis of the role of endocytosis and endosomes in the regulation of EGFR signaling to MAPK will be performed in a range of human cancer cell lines expressing various levels of EGFR. Using the experimental data describing the spatial and temporal overlap of the components of the EGFR-MAPK pathway, their diffusion rate parameters, routes of trafficking and protein-protein interactions, a new computational model of spatial and temporal organization of EGFR-MAPK signaling will be generated, experimentally validated and used to predict the effects of different EGFR expression levels, concentrations of EGFR ligands and various perturbations of the system on the intensity and duration of MAPK signaling. PUBLIC HEALTH RELEVANCE: The EGF receptor has become the major prognostic and diagnostic marker and an important therapeutic target in human carcinoma. However, the benefits from using the EGF receptor as a marker and a therapeutic target are currently highly limited. The studies proposed here will enhance our understanding of the regulation of EGF receptors in cancer cells and should help in developing new strategies of down regulating the activity of EGF receptors and similar receptors in cancer cells and may also reveal new potential prognostic markers and therapeutic targets.