Pathogenesis of cancer: Role of EGF receptor endocytosis

描述(申请人提供)：阐明表皮生长因子受体(EGFR)的生理调节是了解其致癌激活机制的关键。生长因子与EGFR结合触发信号转导过程。受体的激活也会导致溶酶体中EGFR的快速内化和降解，导致受体下调。这种细胞内转运决定了细胞中活跃受体的数量，从而决定了信号的强度和持续时间。内吞作用还通过确定信号过程的局部化和时间动力学来协调EGFR信号网络。重要的是，EGFR内吞作用在肿瘤细胞中经常是失调的。然而，EGFR内吞作用的机制以及内吞作用和信号转导的作用仍不清楚。在这个项目的前一个周期中，我们已经定义了EGFR降解和泛素化的分子机制，泛素化是控制EGFR内吞作用的受体的主要修饰。我们还证明了EGFR内吞的内化步骤的复杂性，这是由多种冗余机制介导的。其中一个机制是通过乙酰化对EGFR进行一种新的翻译后修饰。我们还开发了新的试剂，如降解和内化受损的EGFR突变体，并使用这些新工具来展示受损的EGFR内化对肿瘤发生信号的意外抑制作用。基于新的数据和新技术的可用性，如肿瘤的高分辨率活体成像、活培养细胞的超快共聚焦成像和定量质谱仪，我们建议确定肿瘤细胞系和体内模型中EGFR内化的分子机制，并研究EGFR内吞作用在体内肿瘤模型中的信号转导作用。该建议的具体目的是：1)确定EGFR内吞作用的机制，包括在生理条件下EGFR乙酰化在肿瘤细胞体内和体外的头颈部癌移植模型中的机制和作用；2)在小鼠异种移植模型和转基因小鼠可诱导肺癌模型中检测EGFR被抑制的内吞作用和降解对肿瘤生长的影响；3)确定EGFR泛素化和去泛素化的调节机制，并检测EGFR特异性去泛素化酶作为EGFR依赖肿瘤治疗靶点的潜力。

DESCRIPTION (provided by applicant): Elucidation of the physiological regulation of epidermal growth factor receptor (EGFR) is the key to understanding of the mechanisms causing its oncogenic activation. Growth factor binding to the EGFR triggers the signal transduction process. Receptor activation also causes rapid internalization and degradation of EGFR in lysosomes, leading to receptor down-regulation. This endocytic trafficking determines the number of active receptors in the cell and, therefore, the intensity and duration of signaling. Endocytosis also orchestrates EGFR signaling network by determining the localization and temporal dynamics of signaling processes. Importantly, EGFR endocytosis is frequently dysregulated in tumor cells. However, the mechanisms of EGFR endocytosis and the role of endocytosis and signaling remain to be poorly understood. During the previous cycle of this project we have defined the molecular mechanisms of EGFR degradation and ubiquitination, the major modification of the receptor that controls EGFR endocytosis. We have also demonstrated the complexity of the internalization step of EGFR endocytosis that is mediated by multiple redundant mechanisms. One of these mechanisms involves a novel post-translational modification of EGFR by acetylation. We have also developed new reagents, such as degradation- and internalization-impaired EGFR mutants, and used these new tools to demonstrate unexpected inhibitory effects of impaired EGFR internalization on tumorigenic signaling. Stemming from the new data and based on the availability of new technologies, such as high-resolution intravital imaging of tumors, ultra-fast confocal imaging of living cultured cells and quantitative mass-spectrometry, we propose to define the molecular mechanisms of EGFR internalization in tumor cell lines and in vivo models and examine the role of EGFR endocytosis in signaling in tumor models in vivo. The specific aims of this proposal are: 1) define the mechanisms of EGFR endocytosis, including the mechanisms and role of EGFR acetylation, under physiological conditions in cancer cells in vitro and in vivo in mouse xenograft models of head-and-neck cancer; 2) examine the effects of inhibited endocytosis and degradation of EGFR on tumor growth in mouse xenograft models and transgenic mouse inducible model of lung cancer; 3) define the regulatory mechanisms of EGFR ubiquitination and deubiquitination, and examine the potential of EGFR-specific deubiquitination enzymes as therapeutic targets in EGFR-dependent tumors.