EGF Receptor Signaling in Time and Space in Tumor Cells

肿瘤细胞中 EGF 受体信号传导的时间和空间

• 批准号: 7579326
• 负责人: ALEXANDER D SORKIN
• 金额: $ 31.87万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-07 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7579326
• 关键词: Address; Behavior; Binding; Biochemical; Cancer cell line; Carcinoma; Cell Line; Cell surface; Cells; Chimeric Proteins; Classification; Complement; Complex; Computer Simulation; Data; Development; Diagnostic; Diffusion; Endocytosis; Endosomes; Epidermal Growth Factor; Epidermal Growth Factor Receptor; Family; Family member; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Goals; Growth Factor; Human; Life; Ligand Binding; Ligands; Location; Lysosomes; MAP Kinase Gene; MAPK Signaling Pathway Pathway; Malignant Epithelial Cell; Mediating; Methods; Microscopy; Mitogen Activated Protein Kinase 1; Mitogen-Activated Protein Kinases; Modeling; Molecular; Oncogenic; Outcome; Output; Pathway interactions; Pattern; Physiological; Play; Population; Process; Prognostic Marker; Proteins; RNA Interference; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Regulation; Role; Route; Scaffolding Protein; Signal Pathway; Signal Transduction; Simulate; Small Interfering RNA; Sorting - Cell Movement; Squamous cell carcinoma; Study models; System; Techniques; Testing; Time; base; cancer cell; cell type; chromophore; computerized data processing; fluorescence imaging; inhibitor/antagonist; insight; knock-down; neoplastic cell; novel; overexpression; prognostic; protein complex; protein protein interaction; public health relevance; receptor; reconstitution; research study; response; scaffold; small molecule; spatiotemporal; therapeutic target; tool; trafficking; tumor progression; vector

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.

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的发展和发展中起着重要作用。阐明家族原型成员的生理调节，表皮生长因子（EGF）受体（EGFR/ERBB1）和其他ERBB，是了解引起其致癌激活的机制的关键。生长因子与EGFR的结合导致启动无数信号转导过程。有丝分裂原激活的蛋白激酶1/2（MAPK/ERK1/2）的激活是由EGFR触发的主要信号通路，它决定了EGFR信号传导的最终结果。最近的研究表明，EGFR信号传导不受细胞表面的信号传导的限制，而是该途径的所有成分在细胞内移动，并且可能与各种细胞内隔室中的和功能相关联。活化的EGFR迅速内化为内体，并将其分类为溶酶体以降解。信号传导和内吞机器之间有许多双向串扰。有人提出，尽管这个问题仍然引起争议，但内吞作用是MAPK激活所必需的。 该建议的总体目标是重建癌细胞中时空的信号传导过程，并将新的实验数据整合到EGFR-MAPK途径动力学的计算模型中。首先，MAPK信号通路的成分将用各种荧光蛋白（XFPS）标记，并在人类癌细胞的生理水平上稳定表达，其中相应的内源性蛋白被载体介导的RNA介导的RNA干扰击倒。然后，将使用定量荧光显微镜在这些新细胞系中分析参与ERK1/2激活的XFP标记蛋白的动态定位和运输模式。其次，将使用RNA干扰，活细胞荧光成像，多共晶荧光荧光共振能量转移显微镜和生化技术，这是MAPK激活过程中多蛋白复合物形成的机制以及这些复合物的定位。第三，对内吞作用和内体在EGFR信号在MAPK调节中的作用的系统分析将在表达各种eGFR水平的人类癌细胞系中进行。使用描述EGFR-MAPK途径组成部分的空间和时间重叠的实验数据，它们的扩散率参数，运输途径和蛋白质 - 蛋白质相互作用途径，一种新的计算计算模型，将EGFR-MAPK信号的空间和时间组织用于不同的EGFR-MAPK信号，以实验验证，EG的浓度，EGFREFR的浓度，EGFR的效果，EGFR的效果，EGFR的效果，EGFR的效果，EGFR的效果，EGFR的效果，EGFR的效果，EGFR的效果，系统对MAPK信号的强度和持续时间的扰动。 公共卫生相关性：EGF受体已成为人类癌的主要预后和诊断标记和重要的治疗靶标。但是，使用EGF受体作为标记和治疗靶标的好处是高度限制。这里提出的研究将增强我们对癌细胞中EGF受体调节的理解，并应有助于制定降低调节EGF受体活性和癌细胞中类似受体的新策略，并可能揭示新的潜在预后标记和治疗靶标。