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）的生理调控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。受体激活还会导致溶酶体中EGFR的快速内化和降解，导致受体下调。这种内吞运输决定了细胞中活性受体的数量，因此也决定了信号传导的强度和持续时间。内吞作用还通过确定信号过程的定位和时间动态来协调EGFR信号网络。重要的是，EGFR内吞作用在肿瘤细胞中经常失调。然而，EGFR内吞作用的机制以及内吞作用和信号传导的作用仍然知之甚少。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内吞作用内化步骤的复杂性，这是由多个冗余机制介导的。其中一种机制涉及一种新的翻译后乙酰化修饰EGFR。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证明受损的EGFR内化对致瘤信号的意想不到的抑制作用。基于这些新数据和新技术的可用性，如肿瘤的高分辨率活体成像、活培养细胞的超快速共聚焦成像和定量质谱，我们建议定义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传导作用。本课题的具体目标是：1)明确体外和体内小鼠头颈癌异种移植模型中癌细胞生理条件下EGFR内吞作用机制，包括EGFR乙酰化的机制和作用；2)在小鼠异种移植瘤模型和转基因小鼠肺癌诱导模型中检测抑制EGFR内吞和降解对肿瘤生长的影响；3)明确EGFR泛素化和去泛素化的调控机制，研究EGFR特异性去泛素化酶作为EGFR依赖性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.