MECHANISMS OF CELL CYCLE CONTROL DURING MAMMARY GLAND DE

本研究的目的是探讨乳腺发育过程中细胞周期调节的机制及其在乳腺肿瘤发生过程中改变的后果。特别是，我们将启动以小鼠为动物模型系统的研究，以确定最近发现的SCFSKP2复合物（Skp1, Cullins/CDC53， F-box蛋白），一种泛素E3连接酶，在G1/S转变过程中的作用，以及SCFSKP2表达致病性改变的致瘤潜力。我们最初分离出的p45SKP2蛋白在许多转化的肿瘤细胞中被高度诱导。我们的研究表明，SKP2是SCFSKP2泛素E3连接酶的一个组成部分，是进入s期所必需的。我们发现，在G1/S转变过程中，SKP2特异性结合并靶向CDK抑制剂p27Kip1进行泛素依赖性蛋白水解。在静止细胞中，SKP2的表达足以促进p27降解、CDK活化和s期进入。P27是一种主要的细胞周期调节因子，受多种信号过程控制，包括细胞附着和可溶性生长因子。p27主要控制在蛋白质稳定性水平，G1/S转变需要其降解。包括PTEN在内的几种肿瘤抑制因子与p27的调控有关。p27的下调与包括乳腺癌在内的多种人类癌症的恶性肿瘤有关。研究表明p27的缺失与许多年轻乳腺癌患者的低生存率有关。为了研究乳腺组织增殖、分化的调控以及导致乳腺癌的机制，我们建议利用小鼠转基因和基因敲除技术确定SKP2在乳腺发育中的作用，并研究其致瘤潜力。我们的具体目的是：1)分析SKP2、p27、cyclin D等细胞周期调节因子在乳腺发育过程中的表达。2)构建在乳腺组织中特异性表达SKP2的转基因小鼠系，研究其对乳腺增殖、分化及潜在致瘤性的影响。3)在小鼠中构建条件SKP2基因敲除构建，并检测敲除表型对乳腺组织和乳腺发育的影响。这些研究将有助于建立乳腺癌小鼠模型，并揭示正常乳腺发育过程中乳腺增殖和致瘤过程的控制机制及其致病改变的影响。这些研究也为设计新的乳腺癌诊断、预后和治疗策略提供了基础。

The goal of this proposal is to investigate the mechanism of cell cycle regulation during the mammary gland development and the consequence of its alteration during breast tumorigenesis. In particular, we will initiate studies using mouse as an animal model system to determine the roles of recently identified SCF SKP2 complex (Skp1, Cullins/CDC53, F-box proteins), an ubiquitin E3 ligase, during the G1/S transition and the tumorigenic potential of the pathogenic alteration of SCFSKP2 expression. We have originally isolated p45SKP2 as a protein that is highly induced in many transformed tumor cells. Our studies suggest that SKP2 is a component of SCFSKP2 ubiquitin E3 ligase and is required for the S-phase entry. We found that SKP2 specifically binds to and targets the CDK inhibitor p27Kip1 for ubiquitin- dependent proteolysis during the G1/S transition. Expression of SKP2 is sufficient to promote p27 degradation, CDK activation and S-phase entry in quiescent cells. p27 is a major cell cycle regulator that is controlled by a variety of signaling processes including cell attachment and soluble growth factors. p27 is primarily controlled at the level of protein stability and its degradation is required for the G1/S transition. Several tumor suppressors, including PTEN, have been implicated in p27 regulation. The down-regulation of p27 is correlated with malignancy of a wide variety of human cancers, including breast cancers. Studies suggest that loss of p27 is associated with poor survival of many young breast cancer patients. To investigate the regulation of breast tissue proliferation, differentiation, and the mechanisms that cause breast cancer, we propose to determine the role of SKP2 in mammary gland development and to examine its tumorigenic potential using the mouse transgene and knockout techniques. Our specific aims are: 1) to analyze the expression of SKP2, p27, cyclin D and other cell cycle regulators during mammary gland development. 2) To construct a transgenic mouse line to specifically express SKP2 in breast tissues to determine its effect on proliferation, differentiation, and potential tumorigenicity of the mammary gland. 3) To make a conditional SKP2 knockout construct in mice and to examine knockout phenotypes on breast tissue and mammary gland development. These studies should help to establish mouse models for breast cancer research and to reveal novel insights into the mechanism by which proliferation and tumorigenic processes of mammary gland are controlled during normal mammary gland development and the effect of its pathogenic alteration. These studies should also provide a basis to design novel strategies for diagnosis, prognosis, and therapeutic treatment of breast cancers.