Tsg101 - a Modulator of ErbB2 Signaling in Breast Cancer

描述（由申请人提供）：我们的长期目标是阐明生长调节信号网络如何调节正常和肿瘤性乳腺上皮细胞的增殖和存活。我们目前的研究集中在肿瘤易感基因101（Tsg 101）编码的癌蛋白的生物学和生化功能。Tsg 101是转运所需的内体分选复合物（ESCRT）的关键成员。该途径对于泛素化细胞表面受体的转运、分选和溶酶体降解至关重要，特别是诸如EGFR和ErbB 2（Her 2/neu），它们是与乳腺肿瘤发生和其他人类恶性肿瘤有关的重要受体酪氨酸激酶。我们目前研究的一个主要目标是研究Tsg 101如何参与和修饰活性ErbB信号复合物。特别是，我们感兴趣的Tsg 101的内在PTAP氨基酸基序的功能，以调节Tsg 101的活性和其对正常和肿瘤性乳腺上皮细胞中致癌Her 2/neu的下调的影响。我们的中心假设是，改变Tsg 101的表达或修改其活性，通过抑制其自我调节PTAP结构域将影响Her 2/neu介导的乳腺肿瘤的发生和发展。在本提案的第一个具体目标中，我们将确定改变野生型Tsg 101的表达水平如何改变培养中小鼠和人乳腺癌细胞的生长特性以及体内肿瘤进展。我们还将研究Tsg 101缺陷是否影响曲妥珠单抗耐药的人乳腺癌细胞的存活。第二个具体目标集中在Tsg 101的活化机制，其介导Tsg 101蛋白及其货物（即ErbB 2）的加速泛素化和降解。我们将研究组成型活性Tsg 101与泛素连接酶Tal以及ESCRT复合物的已知成员的相互作用。此外，我们将使用蛋白质组学的方法来确定新的调节Tsg 101的激活。在第三个具体目标中，我们将解决激活Tsg 101是否会导致体外和体内乳腺癌细胞中致癌ErbB 2的更有效下调。我们建议开发先进的基因工程模型，通过改变Tsg 101的活性来预测可能的Her 2/neu介导的乳腺癌靶向治疗的结果。使用这些工具，我们可能能够预示这种治疗策略的疗效之前，药物变得可用。公共卫生相关性：浸润性乳腺癌的一个重要子集表现出Her 2/neu基因扩增，预后不良。该提案的目的是阐明如何改变Tsg 101的表达或修改其活性将影响Her 2/neu介导的乳腺癌的发病和进展。本申请中概述的遗传和分子研究将有助于在药物开发和临床前试验测试之前预测Tsg 101作为治疗靶点的潜在价值。

DESCRIPTION (provided by applicant): Our long-term objective is to elucidate how growth-regulatory signaling networks regulate the multiplication and survival of normal and neoplastic mammary epithelial cells. Our current studies focus on biological and biochemical functions of the oncoprotein encoded by the Tumor Susceptibility Gene 101 (Tsg101). Tsg101 is a key member of the endosomal sorting complex required for transport (ESCRT). This pathway is crucial for the transport, sorting, and lysosomal degradation of ubiquitinated cell surface receptors, in particular such as the EGFR and ErbB2 (Her2/neu) that are important receptor tyrosine kinases implicated in breast tumorigenesis and other human malignancies. A primary objective of our current research is to examine how Tsg101 engages with and modifies active ErbB-signaling complexes. In particular, we are interested in the function of Tsg101's intrinsic PTAP amino acid motif to regulate the activity of Tsg101 and its effect on the downregulation of oncogenic Her2/neu in normal and neoplastic mammary epithelial cells. Our central hypothesis is that altering the expression of Tsg101 or modifying its activity through inhibition of its self-regulatory PTAP domain will affect the onset and progression of Her2/neu-mediated mammary tumorigenesis. In the first specific aim of this proposal, we will determine how altering the expression level of wildtype Tsg101 modifies the growth properties of mouse and human mammary cancer cells in culture as well as tumor progression in vivo. We will also examine whether Tsg101 deficiency affects the survival of Trastuzumab-resistant human breast cancer cells. The second specific aim focuses on the mechanisms of the activation of Tsg101 that mediate an accelerated ubiquitination and degradation of the Tsg101 protein and its cargo (i.e. ErbB2). We will study the interaction of constitutively active Tsg101 with the ubiquitin ligase Tal as well as known members of the ESCRT complex. Additionally, we will use a proteomics approach to identify novel regulators for the activation of Tsg101. In the third specific aim, we will address whether activating Tsg101 will result in a more efficient downregulation of oncogenic ErbB2 in mammary cancer cells in vitro and in vivo. We propose the development of advanced genetically engineered models to predict the outcome of a possible targeted therapy against Her2/neu-mediated breast cancer by modifying the activity of Tsg101. Using these tools we might be able to herald the efficacy of this therapeutic strategy before pharmaceutical agents become available. PUBLIC HEALTH RELEVANCE: A significant subset of invasive breast cancers exhibit Her2/neu gene amplification with an unfavorable prognosis. The objective of this proposal is to elucidate how altering the expression of Tsg101 or modifying its activity will affect the onset and progression of Her2/neu-mediated breast cancer. The genetic and molecular studies outlined in this application will help to predict the potential value of Tsg101 as a therapeutic target before a drug will be developed and tested in preclinical trials.