Pathogenesis of Cancer - Role of EGF Receptor Endocytosis

描述（由申请人提供）：表皮生长因子（EGF）受体（EGFR）对许多人上皮癌的发展和进展很重要，与不良预后相关，并已成为多种此类癌症的主要预后标志物和治疗靶点。因此，阐明EGFR的生理调节是理解导致其致癌活化和损害EGFR治疗潜力的机制的关键。生长因子与EGFR的结合引发了一系列复杂的信号转导过程。受体活化也通过网格蛋白包被的小凹和溶酶体中配体-受体复合物的降解引起快速内吞作用。因此，内吞运输决定了细胞中活性EGFR的数量，从而决定了信号传导过程的强度和持续时间。内吞作用还在EGFR信号转导的时空调控中发挥作用，可能控制信号转导过程的动力学和特定模式。然而，EGFR内吞作用的机制及其与正常细胞和癌细胞中的信号传导过程的交互作用仍然知之甚少。该提案的主要目标是描述EGFR内吞作用的机制，并确定内吞作用在癌症发展和进展中的作用。在该项目的前一个资助周期中，我们已经确定了控制网格蛋白介导的EGFR内化的生理途径的关键蛋白质。我们的研究还证明了EGFR信号复合物在内体中的定位以及内吞作用对信号传导的重要性。我们已经开发了新的定量分析方法的EGFR复合物在活细胞和一种新的方法的EGFR内吞作用的功能分析，使用高效的RNA干扰。这些重要的进展使得现在可以开发一个全面的模型，EGFR内吞作用是如何在癌细胞中调节，以及内吞过程如何调节癌发生。在这个项目中，我们专注于分析头颈癌（HNC），其中EGFR经常过表达，并被认为是一个重要的治疗靶点。具体目的是：1）明确EGFR通过网格蛋白包被的小凹内吞的分子机制。2）测试具有受损内吞运输的新EGFR突变体是否在体外HNC细胞中产生改变的信号传导模式并影响体内HNC异种移植物的致瘤特性;第三章测试调节EGFR内吞作用的蛋白质和可能的其他致癌受体酪氨酸激酶的表达改变是否影响体外EGFR信号传导，HNC异种移植物在体内的致瘤特性。该提案目标的成功完成将为设计治疗和预防EGFR表达癌症的策略开辟新的途径。

DESCRIPTION (provided by applicant): Epidermal growth factor (EGF) receptor (EGFR) is important for the development and progression of many human epithelial cancers, associated with poor prognosis and has become the major prognostic marker and therapeutic target in a variety of these cancers. Elucidation of the physiological regulation of EGFR is, therefore, a key to understanding of the mechanisms causing its oncogenic activation and impairing the potential of EGFR therapeutics. Growth factor binding to the EGFR triggers a complicated array of signal transduction processes. Receptor activation also causes rapid endocytosis via clathrin-coated pits and degradation of the ligand-receptor complexes in lysosomes. Thus, endocytic trafficking determines the number of active EGFRs in the cell and, therefore, the intensity and duration of signaling processes. Endocytosis also plays role in spatial and temporal regulation of EGFR signaling, which may control the dynamics and the specific pattern of signaling processes. However, the mechanisms of EGFR endocytosis and its cross-talks with signaling processes in normal and cancer cells remain poorly understood. The main goal of this proposal is to delineate the mechanisms of EGFR endocytosis, and to define the role of endocytosis in the development and progression of cancer. During the previous funding cycle of this project we have identified key proteins that control the physiological pathway of clathrin-mediated internalization of EGFR. Our studies also demonstrated localization of EGFR signaling complexes in endosomes and the importance of endocytosis for signaling. We have developed novel quantitative methods of analysis of EGFR complexes in living cells and a novel methodology of the functional analysis of EGFR endocytosis using highly efficient RNA interference. These important advances make it now feasible to develop a comprehensive model of how EGFR endocytosis is regulated in cancer cells and how endocytic processes regulate carcinogenesis. In this project we focus on the analysis of head-and-neck cancer (HNC) where EGFR is frequently overexpressed and considered to be an important therapeutic target. The specific aims are: 1) Define the molecular mechanisms of EGFR endocytosis through clathrin coated pits. This aim will, in particular, define the role of ubiquitylation in EGFR endocytosis using novel methods of mass-spectrometry; 2) Test whether new EGFR mutants with impaired endocytic trafficking produce altered patterns of signaling in HNC cells in vitro and affect tumorigenic properties of HNC xenografts in vivo; 3) Test whether altered expression of proteins regulating endocytosis of EGFR and possibly other oncogenic receptor tyrosine kinases affect EGFR signaling in vitro and tumorigenic properties of HNC xenografts in vivo. The successful completion of the aims of the proposal will open new avenues to design of therapeutic and preventive strategies in treatment of EGFR expressing cancers.