Negative regulation by proteasomal degradation of receptor tyrosine kinase ErbB3

描述（由申请人提供）：该项目的主要目标是描述细胞生长的调控系统，以便为癌症治疗和药物开发提供基础。这个目标很好地反映了NCI的使命，因为这个项目的解决方案将提供细胞系统的基础知识，作为一种手段，获得对癌症新治疗方法发展的洞察力，从而减轻国家的疾病负担。这个项目的重点将是生长因子受体酪氨酸激酶（RTK），其失调有助于大量和各种癌症类型的实体瘤的发生、生长和进展（1-3）。这些癌症的治疗通常被设计为阻止或延缓RTK的功能，如ErbB2，在25%的乳腺癌中RTK过表达，其中受体活性被曲妥珠单抗（赫赛汀）限制，一种ErbB2特异性的人源化单克隆抗体(4)。然而，近年来发现，ErbB3（同一家族的RTK）的过表达有助于erbb2阳性乳腺肿瘤的生长和进展，以及治疗耐药，这是乳腺癌治疗的主要障碍(5)，因此需要描述正常（未转化）乳腺上皮细胞中限制ErbB3水平的调节机制。我们实验室的研究强烈表明，正常（未转化）乳腺上皮细胞具有有效的转录后负调控机制，限制ErbB3水平，而肿瘤细胞则使这些机制失活，以促进其生长和恶性（6,7）。RTKs的转录后负调控通常通过溶酶体介导的受体降解发生，这些受体已经从细胞表面内化（8,9）。然而，我们的实验室最近发现，细胞通过降解内质网（ER）新合成的受体来抑制ErbB3水平；外受体通过泛素化作用靶向蛋白酶体（10）。本项目将探索ErbB3在内质网的这种新型调控。这一控制机制的机制将被阐明，包括参与ErbB3靶向、泛素化和逆转录到胞质中进行26S蛋白酶体依赖性降解的蛋白质成分。本项目拟使用生物化学、细胞和分子生物学技术，包括哺乳动物组织培养、免疫沉淀、western blotting、显微镜和分子克隆。该项目将通过定期出席与上述主题和方法相关的癌症研究、生物化学和分子生物学主题的科学报告来补充。此外，与该项目有关的课程将有助于发展对研究的技术和理论方面的进展很重要的观点和战略。

DESCRIPTION (provided by applicant): The primary objective of this project is to describe the regulatory systems of cellular growth in order to provide a foundation for cancer therapeutics and drug development. This goal well reflects the mission of the NCI as the resolution of this project would provide fundamental knowledge of a cellular system as a means to gain insight to the development of novel treatments for cancer and thereby reducing the burdens of disease on the state. The focus of this project will be on growth factor receptor tyrosine kinases (RTK) whose dysregulation contributes to solid tumor initiation, growth, and progression in an immense number and variety of cancer types (1-3). Treatments for these cancers are often designed to stem or retard the functionality of RTKs as in the case of ErbB2, an RTK overexpressed in 25% of breast cancers, where receptor activity is curtailed with trastuzumab (Herceptin), a humanized monoclonal antibody specific to ErbB2 (4). However, in recent years it has emerged that the overexpression of ErbB3, an RTK of the same family, contributes to the growth and progression of ErbB2-positive breast tumors, as well as to therapeutic resistance, a major barrier in breast cancer treatment (5), thus leading to a need of describing the regulatory mechanisms that restrict ErbB3 levels in normal (non-transformed) mammary epithelial cells. Studies in our lab strongly suggest that normal (non-transformed) mammary epithelial cells employ potent post-transcriptional negative regulatory mechanisms that restrict ErbB3 levels, while tumor cells inactivate these mechanisms to promote their growth and malignancy (6,7). Post-transcriptional negative regulation of RTKs often occurs through the lysosome-mediated degradation of receptors that have been internalized from the cell surface (8,9). However, our lab has recently discovered that cells suppress ErbB3 levels via the degradation of newly synthesized receptors at the endoplasmic reticulum (ER); extraneous receptors are targeted to the proteasome via their ubiquitination (10). This project will explore this novel type of regulatin of ErbB3 at the endoplasmic reticulum. Mechanisms of this control mechanism will be elucidated including the protein components that are involved in the targeting, ubiquitination, and retrotranslocation of ErbB3 to the cytosol for 26S proteasome-dependent degradation. This project is intended to be carried out using techniques in biochemistry, cell, and molecular biology including mammalian tissue culture, immunoprecipitation, western blotting, microscopy, and molecular cloning. The project will be supplemented by regular attendance of scientific presentations on topics in cancer research, biochemistry, and molecular biology that would be at all relevant to the subject matter and approaches outlined above. Furthermore, courses related to the project would aid in the development of perspectives and strategies important to the progression of the technical and theoretical facets of the research.