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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：
8625717
负责人：
YI SUN
金额：
$ 31.3万
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/8625717
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 public health relevance 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.

描述(由申请人提供)：靶向癌症治疗依赖于对癌症靶点的彻底验证。我们的长期目标是发现一类新型抗癌药物，选择性地针对一种类型的E3泛素连接酶，该酶在人类癌症中被激活。选择性靶向一条泛素连接酶途径将减少与全面抑制蛋白质降解相关的正常细胞毒性，正如VELCADE(也称为Bortezomib或PS-341)所见，VELCADE是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的第一类(也是唯一类)通用蛋白酶体抑制剂。为此，我们重点研究了SCF(Skp1-cullin-F-box Proteins)E3泛素连接酶，也称为CRLS(Cullin-Ring Ubiquitin Ligase)。SCF E3连接酶是最大的E3连接酶家族，由Skp1、Cullins、F-box蛋白和一个环蛋白ROC或RBX组成，促进靶向降解的一组关键调控蛋白的泛素化，从而调控重要的生物学过程，包括细胞周期进程、信号转导和DNA复制。虽然cullin-ROC是连接酶的核心成分，但cullin需要通过Nedd8激活酶(NAE)才能发挥其酶活性。我们强大的初步数据显示，ROC1在许多人类癌症中过表达，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、衰老和凋亡诱导肿瘤细胞死亡。抑制SCF E3连接酶也会增强辐射诱导的DDR，从而导致放射增敏。本研究的目的是阐明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的靶点。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，并依次或平行地通过自噬、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，导致癌细胞的辐射增敏。我们提出了三个具体的目标：1)确定ROC1-SCF E3连接酶失活如何通过不同的机制触发DNA损伤和DDR，并诱导细胞死亡；2)确定ROC1-SCF E3连接酶失活如何阻断mTOR诱导自噬；3)验证ROC1-SCF E3连接酶作为一个新的放射增敏靶点的有效性。影响：这项工作具有很高的创新性和显著的翻译价值，验证了SCF E3连接酶作为抗癌靶点的有效性，并为MLN4924或其类似物作为一类新型放射增敏剂的未来开发奠定了基础。