F box Proteins and Deregulated Proteolysis in Cancer

描述：（由申请人提供）许多细胞调节剂的蛋白水解是 由称为SCF的泛素连接酶控制，因为它们由三个 主要亚基：Skp 1、cull和许多E-box蛋白之一（Fbps）。基板 SCF的特异性由不同的Fbp亚基决定， 底物识别因子我们已经证明了细胞周期 抑制剂p27通过泛素途径通过F-box蛋白降解 Skp 2.重要的是，p27的不稳定，我们和其他人已经记录了 在人类淋巴瘤和上皮癌中， 攻击性。我们对遍在蛋白途径的兴趣将我们引向 26个人类F-box蛋白家族的鉴定， 这个家族的一个成员，称为13-Trcp，调节细胞的稳定性。 原癌基因B-连环蛋白。由于它们在调节细胞中的功能， 根据我们的初步结果，我们假设， F-box蛋白Skp 2和B-Trcp的失调可能参与了 人类癌症的起源。在目标1中，我们将进行免疫组织化学 研究Skp 2和B-Trcp在从两个肿瘤组织中获得的肿瘤样品中的表达， 不同的肿瘤库我们将集中研究乳腺癌，淋巴瘤 和结肠直肠癌。我们收集的肿瘤样本有一致的正常 组织和长期随访。此外，这两个肿瘤银行还拥有 包含临床病理特征信息的广泛数据库， 标准分子标记。使用单变量和多变量分析，我们将 确定Skp 2和B-Trcp作为人类新的预后标志物的价值， 癌的重要的是，我们将调查水平升高的可能原因， 这两种F-box蛋白质，并进行体外测定，以测量其 活动在目标2中，我们将产生和表征转基因小鼠 过量表达Skp 2、B-Trcp或相应的显性失活突变体。使用 通过这种方法，我们将能够研究强制表达的影响， 这些Fbp在乳腺和淋巴器官。组织病理 在转基因动物模型中预期的改变将模仿那些人类肿瘤 其中Skp 2和B-Trcp过表达，这将帮助我们确定致癌基因 在人类肿瘤中发生的分子异常的潜在途径。 最后，在目标3中，我们将测试Skp 2的抑制是否导致 在癌细胞中的细胞抑制或细胞毒性作用。这将通过使用 基于我们的结构设计的特异性膜渗透肽 和生化信息。

DESCRIPTION: (provided by applicant) Proteolysis of many cellular regulators is controlled by ubiquitin ligases called SCFs since they are composed of three major subunits: Skp1, cull and one of many E-box proteins (Fbps). The substrate specificity of SCFs is determined by distinct Fbp subunits that act as substrate recognition factors. We have demonstrated that the cell cycle inhibitor p27 is degraded by the ubiquitin-pathway through the F-box protein Skp2. Importantly, destabilization of p27, which we and others have documented in human lymphomas and in epithelial cancers, correlates with tumor aggressivity. Our interest in the ubiquitin pathway lead us to the identification of a family of 26 human F-box proteins and the discovery that one member of this family, called 13-Trcp, regulates the stability of the proto-oncogene B-catenin. Because of their functions in regulating cell proliferation and given our preliminary results, we hypothesize that deregulation of the F-box proteins Skp2 and B-Trcp may participate in the genesis of human cancers. In Aim 1 we will carry out immunohistochemical studies on Skp2 and B-Trcp expression in cancer samples obtained from two different tumor banks. We will concentrate our study on breast cancer, lymphoma and colorectal cancer. Our collection of tumor samples has concordant normal tissue and long-term follow-ups. In addition, these two tumor banks have extensive databases containing information on clinico-pathological features and standard molecular markers. Using univariate and multivariate analyses, we will determine the value of Skp2 and B-Trcp as novel prognostic markers for human cancers. Importantly, we will investigate possible causes for increased levels of these two F-box proteins and perform in vitro assays to measure their activities. In Aim 2 we will then generate and characterize transgenic mice overexpressing Skp2, B-Trcp or corresponding dominant-negative mutants. Using this approach we will be able to investigate the effects of enforced expression of these Fbps in the mammary gland and lymphoid organs. Histopathological alterations expected in transgenic animal models will mimic those human tumors in which Skp2 and B-Trcp are overexpressed and will help us to define oncogenic pathways underlying the molecular abnormalities occurring in human tumors. Finally, in Aim 3 we will test whether the inhibition of Skp2 results in a cytostatic or cytotoxic effect in cancer cells. This will be achieved using specific membrane permeable peptides designed on the basis of our structural and biochemical information.