Stabilizing nuclear p27kip1 as a therapeutic target for endometrial cancer

稳定核 p27kip1 作为子宫内膜癌的治疗靶点

• 批准号: 9094005
• 负责人: TIMOTHY J CARDOZO
• 金额: $ 7.94万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-13 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9094005
• 关键词: Address; Affect; Atypical Endometrial Hyperplasias; Binding; Biological Availability; Biological Markers; Biopsy; Cancer Biology; Cell Cycle; Cell Cycle Proteins; Cell Cycle Regulation; Cell Nucleus; Cell Proliferation; Cells; Cessation of life; Chemicals; Complex; Crystallization; Cyclin-Dependent Kinase Inhibitor; Data; Development; Disease; Endometrial; Endometrial Carcinoma; Endometrium; Epithelial; Epithelial Cell Proliferation; Epithelial Cells; Estrogens; Event; G1 Arrest; G1 Phase; Generations; Growth; Growth Inhibitors; Gynecologic; Health; Hormonal Carcinogenesis; Hormones; Human; Image Analysis; Immunocompromised Host; In Vitro; Injection of therapeutic agent; Knowledge; Lead; Learning; Left; Libraries; Malignant - descriptor; Malignant Neoplasms; Mediating; Microscopy; Molecular; Molecular Target; Mus; Neoplasm Metastasis; Nuclear; Oncogenes; Outcome; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Progesterone; Proteasome Inhibitor; Proteins; Publishing; Regulation; Reporting; Role; System; Testing; Therapeutic; Therapeutic Agents; Therapeutic Intervention; Therapeutic Uses; Tissues; Toxic effect; Transforming Growth Factor beta; Tumor Suppressor Proteins; Tumor Volume; Ubiquitin; X-Ray Crystallography; Xenograft procedure; biophysical analysis; cancer cell; cancer therapy; carcinogenesis; cellular imaging; chemical group; in vivo; inhibitor/antagonist; insight; knock-down; migration; mouse model; multicatalytic endopeptidase complex; new therapeutic target; novel; novel strategies; novel therapeutic intervention; pharmacophore; prevent; protein degradation; scaffold; small molecule; spatiotemporal; therapeutic target; trafficking; treatment strategy; tumor; ubiquitin ligase; ubiquitin-protein ligase

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.

描述（由申请人提供）：由于细胞周期调节的丧失是致癌的标志，因此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 例新发病例和超过 8,190 例死亡。该提案的主要目标是解决雌激素 (E2) 诱导的 I 型 ECA（占 ECA 的 85%）的发病机制，以及针对该疾病的新的潜在疗法，包括抑制肿瘤抑制因子的降解， p27kip1 (p27)，一种阻止细胞增殖的关键细胞周期蛋白。我们报道，在子宫内膜上皮 (EEC) 和 ECA 细胞中，E2 通过其特异性 E3 连接酶 SCF-Skp2/Cks1 诱导泛素介导的核 p27 降解，并增加细胞增殖。相反，黄体酮（Pg；低级别 ECA 的一种治疗方法）和 TGF-β 会增加细胞核中的 p27，并通过防止 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发生的关键靶标。我们已经鉴定出新型 Skp2/Cks1 小分子抑制剂 (Skp2E3LIs)，它们仅增加核 p27，这至关重要，因为细胞质 p​​27 介导迁移/转移。此外，Skp2E3LIs 可以阻断 E2 诱导的核 p27 降解以及体外 ECA 细胞和{小鼠 EEC 体内}的增殖。重要的是，与目前通用的蛋白酶体抑制剂相比，Skp2E3LI 有可能成为一项重大的治疗进展，后者可以无差别地阻止包括癌基因在内的蛋白质降解。 Skp2/Cks1 会导致 p27 降解，而 E3 连接酶 APC/Cdh1 则以 Cks1/Skp2 为目标进行破坏，从而使 p27 保持完整。有趣的是，我们发现 E2 会减少 Cdh1，从而增加 Skp2/Cks1 以促进 p27 降解，而 Pg 和 TGF-ß 会增加 Cdh1 以增加核 p27。因此，可以通过增加 Cdh1 或阻断 Skp2 来抑制 p27 降解。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) 引起的细胞周期失调中发挥重要作用； 2. Skp2E3LIs对于ECA的治疗具有显着的转化价值。我们建议： 1. 使用 E2、Pg 和 TGF-ß 作为分子开关来了解如何控制 Cdh1 来影响 p27 水平。 2. 表明 p27 和 Cdh1 在人体活检组织中的共定位是 Pg 治疗积极结果的生物标志物。 {3.对 Skp2-Cks1-Skp2E3LI 复合物进行共结晶/NMR 结构分析，以化学方式优化当前的先导化合物，从而定义 p27 在体外和体内 ECA 中的作用。} 4. 通过单细胞成像显微镜进行细胞周期分析，量化 Skp2E3LI 对 p27 Skp2 和 Cks1 细胞内 [核] 运输的影响。 5. 在小鼠模型中测试 Skp2E3LI 阻断人类 ECA 肿瘤生长的功效。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有关 UPS 在癌症中的作用的机制见解以及使用 Skp2E3LI 作为癌症治疗的新方法来影响癌症生物学领域。