Mechanistic validation of SCF E3 ligase as a cancer and radiosensitizing target

SCF E3 连接酶作为癌症和放射增敏靶点的机制验证

• 批准号: 8054560
• 负责人: YI SUN
• 金额: $ 31.41万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-11 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8054560
• 关键词: Acridine Orange; Adverse effects; Antineoplastic Agents; Antithymoglobulin; Apoptosis; Autophagocytosis; Biochemical; Biological; Biological Process; Bortezomib; Cell Cycle Progression; Cell Death; Clinic; Cullin Proteins; Cycloheximide; DNA Damage; DNA Double Strand Break; DNA biosynthesis; DNA-Binding Proteins; Data; Development; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; FDA approved; Family; Future; Genes; Genomic Instability; Goals; Human; IRS1 gene; Immunoblotting; Immunoprecipitation; In Vitro; Ligase; Light; MAP1 Microtubule-Associated Protein; Malignant Neoplasms; Mantle Cell Lymphoma; Mediating; Multiple Myeloma; Normal Cell; Pathway interactions; Pharmaceutical Preparations; Proteasome Inhibitor; Protein Degradation Inhibition; Proteins; RBX1 gene; Radiation; Radiation Induced DNA Damage; Radiation-Sensitizing Agents; Radiosensitization; Refractory; Relapse; Signal Transduction; Skp1-Cullin-F-Box Proteins; Small Interfering RNA; Staining method; Stains; Testing; Toxic effect; Ubiquitination; Validation; Velcade; Work; analog; cancer cell; cancer therapy; cell killing; genetic regulatory protein; human FRAP1 protein; human ORC1L protein; human RBX1 protein; in vivo; inhibitor/antagonist; innovation; insulin receptor substrate 1 protein; interest; mTOR protein; neoplastic cell; neural precursor cell; novel; radiation effect; response; senescence; tumor; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Targeted cancer therapy relies on a thorough validation of cancer targets. Our long-range goal is to discover a novel class of anticancer drugs that selectively target one type of E3 ubiquitin ligase, which is activated in human cancer. Selective targeting one ubiquitin ligase pathway will reduce normal cell toxicity associated with overall inhibition of protein degradation, as seen in Velcade (also known as Bortezomib or PS-341), the first (and only) class of general proteasome inhibitor, approved by FDA for the treatment of relapsed/refractory multiple myeloma and mantle cell lymphoma. To this end, we have focused on SCF (Skp1-Cullin-F-box proteins) E3 ubiquitin ligases, also known as CRLs (Cullin-RING ubiquitin ligases). SCF E3 ligases, the largest E3 ligase family consisting of Skp1, cullins, F-box proteins, and a RING protein, ROC or RBX, promotes the ubiquitination of a subset of key regulatory proteins for targeted degradation, thus governing important biological processes, including cell cycle progression, signal transduction and DNA replication. While cullin-ROC constitutes the core ligase component, cullin needs to be neddylated via Nedd8-Activating Enzyme (NAE) for its enzymatic activity. Our strong preliminary data showed that ROC1 is over-expressed in a number of human cancers, and inactivation of SCF E3 ubiquitin ligase activity via ROC1 siRNA silencing or by an NAE inhibitor, MLN4924, triggers DNA double strand breaks (DSB) and the DNA damage response (DDR), and induces tumor cell killing via autophagy, senescence and apoptosis. Inhibition of SCF E3 ligase also enhances radiation-induced DDR, leading to radiosensitization. The objective of this proposed study is to elucidate the underlying mechanisms by which inactivation of ROC1-SCF E3 ligase triggers these biochemical and biological changes, leading to various types of cell death, and to validate ROC1-SCF E3 ligase as an anticancer and radiosensitizing target. Our central hypothesis is that inactivation of ROC1-SCF E3 ubiquitin ligase causes accumulation of several key substrates, which triggers DSB and DDR and induces cell death via autophagy, senescence and apoptosis in a sequential or parallel order. These triggered cell killing mechanisms could also enhance radiation effects, leading to radiosensitization of cancer cells. Three specific aims are proposed 1) to determine how inactivation of ROC1-SCF E3 ligase triggers DNA damage and DDR, and induces cell death via different mechanisms; 2) to determine how inactivation of ROC1-SCF E3 ligase blocks mTOR to induce autophagy; and 3) to validate ROC1-SCF E3 ligase as a novel radiosensitizing target. IMPACT: This work is highly innovative and of significant impact with translational value by validating SCF E3 ligase as an anticancer target and by paving the ground for future development of MLN4924 or its analogues as a novel class of radiosensitizers. PUBLIC HEALTH RELEVANCE: Proteasome inhibitor, Velcade is used in the clinic for the treatment of relapsed/refractory multiple myeloma. The drug is in general toxic to normal cells due to its overall inhibition of protein degradation. One way to reduce toxic side-effect is to inhibit a particular E3 ligase that is activated in cancer cells. This study will validate SCF E3 ubiquitin ligase as an anticancer and radiosensitizing target and MLN4924, a newly discovered SCF E3 ligase inhibitor, as a tumor-selective and radiosensitizing agent for cancer therapy.

描述（由申请人提供）：靶向癌症治疗依赖于对癌症靶标的彻底验证。我们的长期目标是发现一类新型抗癌药物，选择性地靶向一种在人类癌症中被激活的 E3 泛素连接酶。选择性靶向泛素连接酶途径将减少与蛋白质降解总体抑制相关的正常细胞毒性，如 Velcade（也称为 Bortezomib 或 PS-341）中所见，Velcade 是第一类（也是唯一一类）通用蛋白酶体抑制剂，经 FDA 批准用于治疗复发/难治性多发性骨髓瘤和套细胞淋巴瘤。为此，我们重点关注 SCF（Skp1-Cullin-F-box 蛋白）E3 泛素连接酶，也称为 CRL（Cullin-RING 泛素连接酶）。 SCF E3 连接酶是最大的 E3 连接酶家族，由 Skp1、cullins、F-box 蛋白和 RING 蛋白、ROC 或 RBX 组成，可促进关键调节蛋白子集的泛素化以进行靶向降解，从而控制重要的生物过程，包括细胞周期进程、信号转导和 DNA 复制。虽然 cullin-ROC 构成了核心连接酶成分，但 cullin 需要通过 Nedd8 激活酶 (NAE) 进行 neddylated 才能发挥其酶活性。我们强有力的初步数据表明，ROC1 在多种人类癌症中过度表达，通过 ROC1 siRNA 沉默或 NAE 抑制剂 MLN4924 使 SCF E3 泛素连接酶活性失活，触发 DNA 双链断裂 (DSB) 和 DNA 损伤反应 (DDR)，并通过自噬、衰老和细胞凋亡诱导肿瘤细胞杀伤。 细胞凋亡。抑制 SCF E3 连接酶还会增强辐射诱导的 DDR，从而导致放射增敏。这项研究的目的是阐明 ROC1-SCF E3 连接酶失活触发这些生化和生物学变化，导致各种类型的细胞死亡的潜在机制，并验证 ROC1-SCF E3 连接酶作为抗癌和放射增敏靶点。我们的中心假设是，ROC1-SCF E3 泛素连接酶失活会导致几种关键底物的积累，从而触发 DSB 和 DDR，并通过顺序或并行顺序的自噬、衰老和凋亡诱导细胞死亡。这些触发的细胞杀伤机制还可以增强辐射效应，导致癌细胞的放射增敏。提出了三个具体目标：1）确定ROC1-SCF E3连接酶失活如何触发DNA损伤和DDR，并通过不同机制诱导细胞死亡； 2) 确定ROC1-SCF E3连接酶失活如何阻止mTOR诱导自噬； 3) 验证 ROC1-SCF E3 连接酶作为新型放射增敏靶点。影响：这项工作具有高度创新性，具有重大转化价值，验证了 SCF E3 连接酶作为抗癌靶点的作用，并为 MLN4924 或其类似物作为新型放射增敏剂的未来发展奠定了基础。 公共卫生相关性：蛋白酶体抑制剂 Velcade 在临床上用于治疗复发/难治性多发性骨髓瘤。由于该药物全面抑制蛋白质降解，因此通常对正常细胞有毒。减少毒副作用的一种方法是抑制癌细胞中激活的特定 E3 连接酶。这项研究将验证 SCF E3 泛素连接酶作为抗癌和放射增敏靶点，以及新发现的 SCF E3 连接酶抑制剂 MLN4924 作为癌症治疗的肿瘤选择性和放射增敏剂。