Negative regulation by proteasomal degradation of receptor tyrosine kinase ErbB3

描述(申请人提供)：这个项目的主要目标是描述细胞生长的调节系统，以便为癌症治疗和药物开发提供基础。这一目标很好地反映了国家癌症研究所的使命，因为该项目的解决将提供细胞系统的基本知识，作为一种手段，以获得对癌症新治疗方法的开发的洞察，从而减轻疾病对国家的负担。这个项目的重点将是生长因子受体酪氨酸激酶(RTK)，它的失调有助于实体肿瘤的启动、生长和发展，导致大量和各种癌症类型(1-3)。对这些癌症的治疗通常旨在阻止或延缓RTK的功能，例如ErbB2，一种在25%的乳腺癌中过度表达的RTK，受体活性被曲妥珠单抗(Herceptin)抑制，曲妥珠单抗是一种针对ErbB2的人源化单抗(4)。然而，近年来发现，ErbB3的过度表达有助于ErbB2阳性乳腺肿瘤的生长和发展，并导致治疗耐药，这是乳腺癌治疗的主要障碍[5]，因此需要描述限制正常(未转化)乳腺上皮细胞中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)的乳腺上皮细胞使用强大的转录后负调控机制来限制ErbB3水平，而肿瘤细胞则失活这些机制以促进其生长和恶性(6，7)。RTK的转录后负调控通常通过溶酶体介导的从细胞表面内化的受体的降解来发生(8，9)。然而，我们的实验室最近发现，细胞通过降解内质网(ER)上新合成的受体来抑制ErbB3的水平；外部受体通过泛素化作用靶向蛋白酶体(10)。本项目将探索ErbB3在内质网中的这种新型调控。这一控制机制的机制将被阐明，包括参与ErbB3的靶向、泛素化和反向移位到胞浆中以进行26S蛋白酶体依赖的降解的蛋白质组分。该项目旨在利用生物化学、细胞和分子生物学技术，包括哺乳动物组织培养、免疫沉淀、蛋白质印迹、显微镜和分子克隆。该项目将通过定期出席癌症研究、生物化学和分子生物学中与上文概述的主题和方法相关的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制定对研究的技术和理论方面的进展至关重要的观点和战略。

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.