Stabilizing nuclear p27kip1 as a therapeutic target for endometrial cancer

描述(申请人提供)：由于细胞周期调控的丧失是癌症发生的标志，识别旨在恢复生长控制的新治疗靶点是癌症治疗的重要方法。子宫内膜癌(ECA)是美国最常见的妇科恶性肿瘤，2013年估计有49,470例新病例和超过8,190例死亡。这项建议的主要目标是解决雌激素(E2)诱导的I型Eca(85%的ECAs)的发病机制，以及一种新的潜在治疗方法，包括抑制肿瘤抑制因子p27kip1(P27)的降解，p27kip1(P27)是一种关键的细胞周期蛋白，可抑制细胞增殖。我们报道，在子宫内膜上皮(EECS)和Eca细胞中，E2通过其特异性的E3连接酶SCF-Skp2/Cks1诱导泛素介导的核p27降解，从而促进细胞增殖。相反，孕酮(PG；一种治疗低度Eca的药物)和转化生长因子-B一样，可以增加细胞核中的p27，并通过阻止p27的降解来抑制细胞增殖。因此，我们提供了令人信服的证据，证明p27是子宫内膜生长调节和子宫内膜癌发生的关键靶点。我们已经发现了新的Skp2/Cks1小分子抑制物(Skp2E3Lis)，它只增加细胞核p27，这是至关重要的，因为细胞质p27介导了迁移/转移。此外，Skp2E3Lis在Eca细胞和小鼠EECS中抑制了E2诱导的核p27的降解和体外增殖。重要的是，Skp2E3LIs有可能成为一种重大的治疗进展，而不是目前通用的蛋白酶体抑制剂，后者不分青红皂白地阻止包括癌基因在内的蛋白质降解。而Skp2/Cks1导致p27降解，而E3连接酶APC/CDH1针对Cks1/Skp2进行破坏，使p27保持不变。有趣的是，我们发现E2降低了CDH1，从而增加了p27降解的Skp2/Cks1，而PG和转化生长因子-B则增加了CDH1，从而增加了核p27。因此，p27的降解可以通过增加CDH1或阻断Skp2来抑制。因此，我们将检验两个主要假设：1.CDH1-Skp2/Cks1-p27轴在泛素蛋白酶体系统(UPS)的细胞周期失调中起重要作用；2.Skp2E3Lis对ECA的治疗具有重要的翻译价值。我们建议：1.使用E_2、PG和转化生长因子β作为分子开关来研究CDH1是如何影响p27水平的。2.表明p27和CDH1在人活检组织中的共同定位是PG治疗阳性结果的生物标志物。{3.对Skp2-Cks1-Skp2E3LI复合体进行共结晶/核磁共振结构分析，对当前的先导化合物进行化学优化，以确定p27在体外和体内ECA中的作用。}4.通过单细胞成像显微镜结合细胞周期分析，量化Skp2E3Lis对p27 Skp2和Cks1在细胞内[核]运输的影响。5.在小鼠模型中检测Skp2E3Lis对人Eca肿瘤生长的阻断作用。由于核p27的降解发生在许多癌症中，我们的研究将通过提供对UPS在癌症中的作用以及Skp2E3LI作为一种新的癌症治疗方法的机械见解来影响癌症生物学领域。

DESCRIPTION (provided by applicant): As loss of cell cycle regulation is a hallmark of carcinogenesis, identifying novel therapeutic targets aimed at restoring growth control is an important approach to cancer therapy. Endometrial cancer (ECA) is the most common gynecologic malignancy in the US with 49,470 new cases and over 8,190 deaths estimated for 2013. The major objectives of this proposal address mechanisms involved in the pathogenesis of estrogen (E2)- induced Type I ECA (85% of ECAs) and a new potential therapy for this disease that involves inhibiting degradation of the tumor suppressor, p27kip1 (p27), a key cell cycle protein that arrests cell proliferation. We reported that E2 induces ubiquitin-mediated degradation of nuclear p27 by its specific E3 ligase, SCF- Skp2/Cks1, in endometrial epithelial (EECs) and ECA cells with increased cell proliferation. Conversely, progesterone (Pg; a therapy for low grade ECA) as well as TGF-ß, increase p27 in the nucleus and inhibit cell proliferation by preventing p27 degradation. Therefore, we provide compelling evidence that p27 is a key target for growth regulation in the endometrium and in endometrial carcinogenesis. We have identified novel small molecule inhibitors of Skp2/Cks1 (Skp2E3LIs) that only increase nuclear p27, which is critical since cytoplasmic p27 mediated migration/metastasis. Furthermore, Skp2E3LIs block both E2-induced degradation of nuclear p27 and proliferation in vitro in ECA cells and {in vivo in mouse EECs}. Importantly, Skp2E3LIs have the potential to be a major therapeutic advancement over current general proteasome inhibitors that indiscriminately block protein degradation including oncogenes. Whereas Skp2/Cks1 causes p27 degradation, the E3 ligase APC/Cdh1 targets Cks1/Skp2 for destruction leaving p27 intact. Interestingly, we show that E2 decreases Cdh1and thereby increases Skp2/Cks1 for p27 degradation whereas Pg and TGF-ß increase Cdh1 to increase nuclear p27. Therefore, p27 degradation can be inhibited both, by increasing Cdh1 or by blocking Skp2. As such, we will test two main hypotheses: 1. that the Cdh1-Skp2/Cks1-p27axis is important in cell cycle dysregulation by the ubiquitin proteasome system (UPS); 2. that Skp2E3LIs have significant translational value for the treatment of ECA. We propose to: 1. Use E2, Pg, and TGF-ß as molecular switches to learn how Cdh1 is controlled to affect p27 levels. 2. Show that co-localization of p27 and Cdh1 in human biopsy tissue is a biomarker for positive outcomes of Pg therapy. {3. Perform co-crystallization/NMR structural analysis of Skp2-Cks1-Skp2E3LI complexes for chemically optimizing current lead compounds to define the role of p27 in ECA in vitro and in vivo.} 4. Quantify the effects of Skp2E3LIs on intracellular [nuclear] trafficking of p27 Skp2, and Cks1 together with cell cycle analysis by single cell imaging microscopy. 5. Test Skp2E3LIs for their efficacy in blocking the growth of human ECA tumors in mouse models. As the degradation of nuclear p27 occurs in numerous cancers, our studies should impact the cancer biology field by providing mechanistic insights into the role of the UPS in cancer and the use of Skp2E3LIs as a novel approach to cancer therapy.