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

基本信息

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
项目类别：
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

项目摘要

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）：有针对性的癌症治疗依赖于对癌症靶标的彻底验证。我们的远程目标是发现一种新型的抗癌药物，该药物有选择地靶向一种在人类癌症中激活的E3泛素连接酶。选择性靶向一种泛素连接酶途径将降低与蛋白质降解的总体抑制相关的正常细胞毒性，如Velcade（也称为bortezomib或PS-341），是FDA的一般蛋白酶体抑制剂（唯一的）类别（唯一的）类别的蛋白酶体抑制剂（FDA），FDA用于Relaps/cellaps cells cells cells cells cells and cell and cell and cell and cell and cell and cell and cell and cell and cell and cell and。为此，我们专注于SCF（SKP1-Cullin-F-box蛋白）E3泛素连接酶，也称为CRLS（Cullin-Ring泛素连接酶）。 SCF E3连接酶是由SKP1，Cullins，F-box蛋白和环蛋白（ROC或RBX）组成的最大的E3连接酶家族，促进了靶向降解的关键调节蛋白子集的泛素化，从而促进了重要的生物学过程，包括细胞周期进程，包括信号传递和DNA。虽然Cullin-Roc构成了核心连接酶成分，但需要通过NEDD8激活酶（NAE）对Cullin进行neddydyly，以获得其酶促活性。 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凋亡。 SCF E3连接酶的抑制也增强了辐射诱导的DDR，从而导致放射性化。这项拟议的研究的目的是阐明ROC1-SCF E3连接酶失活的潜在机制触发了这些生化和生物学变化，从而导致各种细胞死亡，并验证ROC1-SCF E3连接酶作为抗癌和射射击靶标。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，这会触发DSB和DDR，并通过序列或平行顺序通过自动噬菌体，衰老和凋亡诱导细胞死亡。这些触发的细胞杀伤机制也可以增强辐射效应，从而导致癌细胞的放射敏化。提出了三个具体目标1）确定ROC1-SCF E3连接酶的失活是如何触发DNA损伤和DDR的三个特定目标，并通过不同的机制诱导细胞死亡； 2）确定ROC1-SCF E3连接酶的失活是如何诱导自噬的； 3）验证ROC1-SCF E3连接酶是一种新型的放射敏感靶标。影响：这项工作是高度创新的，并且通过验证SCF E3连接酶作为抗癌靶标，并通过为MLN4924的未来开发或其类似物作为新型的放射增感器铺平基础，从而具有高度的创新性和显着影响。公共卫生相关性：蛋白酶体抑制剂，Velcade在诊所中用于治疗复发/难治性多发性骨髓瘤。由于其总体抑制蛋白质降解，该药物通常对正常细胞有毒。减少有毒副作用的一种方法是抑制在癌细胞中激活的特定E3连接酶。这项研究将证明SCF E3泛素连接酶是一种抗癌和放射敏化靶标，MLN4924是一种新发现的SCF E3连接酶抑制剂，作为癌症治疗的肿瘤选择性和放射性剂。