Tsg101 - a Modulator of ErbB2 Signaling in Breast Cancer

描述(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控正常和肿瘤乳腺上皮细胞的增殖和存活。我们目前的研究主要集中在肿瘤易感基因101(Tsg101)编码的癌蛋白的生物学和生化功能上。Tsg101是运输所需的内体分选复合体(ESCRT)的关键成员。这一途径对泛素化细胞表面受体的运输、分选和溶酶体降解至关重要，尤其是EGFR和ErbB2(Her2/neu)，它们是与乳腺肿瘤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Tsg101是如何与活性ErbB信号复合体结合并修饰的。特别是，我们感兴趣的是Tsg101的S固有的PTAP氨基酸基序调节Tsg101活性的功能及其在正常和肿瘤乳腺上皮细胞中下调致癌基因Her2/neu的作用。我们的中心假设是，改变Tsg101的表达或通过抑制其自我调节的PTAP结构域来改变其活性将影响Her2/neu介导的乳腺肿瘤的发生和发展。在这项提案的第一个具体目标中，我们将确定改变野生型Tsg101的表达水平如何改变培养中的鼠和人乳腺癌细胞的生长特性以及体内的肿瘤进展。我们还将研究Tsg101缺乏是否会影响曲妥珠单抗耐药的人乳腺癌细胞的存活。第二个特定目的集中于Tsg101激活的机制，它介导了Tsg101蛋白及其货物(即ErbB2)的加速泛素化和降解。我们将研究组成活性Tsg101与泛素连接酶Tal以及ESCRT复合体的已知成员之间的相互作用。此外，我们将使用蛋白质组学方法来确定Tsg101激活的新调节因子。在第三个具体目标中，我们将研究激活Tsg101是否会在体外和体内更有效地下调乳腺癌细胞中致癌的ErbB2。我们建议开发先进的基因工程模型，通过改变Tsg101的活性来预测针对Her2/neu介导的乳腺癌的可能靶向治疗的结果。使用这些工具，我们也许能够在药物制剂可用之前预测这一治疗策略的有效性。公共卫生相关性：浸润性乳腺癌的一个重要亚组表现为Her2/neu基因扩增，预后不良。该建议的目的是阐明改变Tsg101的表达或改变其活性将如何影响Her2/neu介导的乳腺癌的发生和发展。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之前预测Tsg101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.