Stabilizing nuclear p27kip1 as a therapeutic target for endometrial cancer

描述（由申请人提供）：由于细胞周期调节的丧失是癌症发生的标志，确定旨在恢复生长控制的新治疗靶点是癌症治疗的重要途径。子宫内膜癌（ECA）是美国最常见的妇科恶性肿瘤，2013年估计有49470例新病例和超过8190例死亡。该提案的主要目标是研究雌激素（E2）诱导的I型ECA（85%的ECA）的发病机制，以及一种新的潜在治疗方法，该方法涉及抑制肿瘤抑制因子p27kip1 （p27）的降解，p27是一种关键的细胞周期蛋白，可阻止细胞增殖。我们报道了E2通过其特异性E3连接酶SCF- Skp2/Cks1在子宫内膜上皮（EECs）和ECA细胞中诱导泛素介导的核p27降解，并增加细胞增殖。相反，黄体酮（Pg，治疗低级别ECA的一种药物）和TGF-ß可以增加细胞核中的p27，并通过阻止p27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变的关键靶点。我们已经发现了Skp2/Cks1的新型小分子抑制剂（Skp2E3LIs），它只增加核p27，这是至关重要的，因为细胞质p27介导迁移/转移。此外，Skp2E3LIs可阻断e2诱导的体外ECA细胞和小鼠eec细胞中核p27的降解和增殖。重要的是，Skp2E3LIs有可能成为目前一般蛋白酶体抑制剂的主要治疗进展，这些蛋白酶体抑制剂不加区别地阻断蛋白质降解，包括癌基因。虽然Skp2/Cks1导致p27降解，但E3连接酶APC/Cdh1靶向Cks1/Skp2破坏p27，使p27保持完整。有趣的是，我们发现E2降低Cdh1从而增加Skp2/Cks1以降解p27，而Pg和TGF-ß增加Cdh1以增加核p27。因此，p27的降解可以通过增加Cdh1或阻断Skp2来抑制。因此，我们将测试两个主要假设：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的治疗具有重要的转化价值。我们建议：1。利用E2、Pg和TGF-ß作为分子开关，了解如何控制Cdh1影响p27水平。2. 表明p27和Cdh1在人活检组织中的共定位是Pg治疗阳性结果的生物标志物。{3。对Skp2-Cks1-Skp2E3LI配合物进行共结晶/核磁共振结构分析，以化学优化当前的先导化合物，以确定p27在体外和体内ECA中的作用。} 4。量化Skp2E3LIs对p27 Skp2和Cks1细胞内[核]运输的影响，并通过单细胞成像显微镜分析细胞周期。5. 在小鼠模型中检测Skp2E3LIs对人ECA肿瘤生长的抑制作用。由于核p27的降解发生在许多癌症中，我们的研究应该通过提供UPS在癌症中的作用的机制见解以及使用Skp2E3LIs作为癌症治疗的新方法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.