Roles of the Checkpoint Kinase Chk1 in Cancer Biology and Therapy

检查点激酶 Chk1 在癌症生物学和治疗中的作用

基本信息

批准号：
7826823
负责人：
YOU-WEI ZHANG
金额：
$ 24.9万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-12-01 至 2012-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7826823
关键词：
ATR protein kinase Alkylating Agents Antineoplastic Agents Apical Biological Camptothecin Cancer Biology Cancer Patient Cancer cell line Cell Cycle Cell Death Cell Respiration Cell Survival Cells Checkpoint kinase 1 Chromosome Segregation Clinical Complex DNA DNA Damage DNA biosynthesis Defect Degradation Pathway Development Drug resistance Environment Eukaryotic Cell Evolution F Box Domain F-Box Proteins Genetic Genome Genotoxic Stress Growth Human Hypoxia Lead Lysine Maintenance Malignant Neoplasms Mentors Metabolic stress Methyl Methanesulfonate Mitotic Chromosome Modification Molecular Monitor Mothers Mutagens Pathway interactions Pharmaceutical Preparations Phase Phosphorylation Phosphotransferases Poison Proteins Regulation Replication-Associated Process Research Resistance Role Signal Transduction Stress Testing Therapeutic Therapeutic Agents Therapeutic Index Topoisomerase Ubiquitin Ubiquitination Work antitumor agent assault biological adaptation to stress cancer cell cytotoxic daughter cell insight killings multicatalytic endopeptidase complex neoplastic cell novel novel therapeutics repaired response tumor ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): Eukaryotic cells have evolved an elaborate network of genome surveillance and repair proteins to insure that DNA replication will occur in an accurate and timely fashion. This surveillance pathway is termed the S phase replication checkpoint. Defects in this 'caretaker' machinery lead to genetic instability, a hallmark feature of human cancers. The replication checkpoint monitors the progress of replication forks, and when fork stalls, transmits signals that delay S-phase progression, and maintains the stability of stalled forks so that DNA replication can resume after the initial error is corrected. Two key components of the replication checkpoint are the apical protein kinase, ATR, and its downstream target kinase, Chk1. Loss of ATR or Chk1 function is lethal, even in the absence of extrinsic genotoxic stress, underscoring the importance of the replication checkpoint in the maintenance of cell viability. In preliminary work, we tested the hypothesis that certain anti-tumor agents, such as the topoisomerase 1 (Top I) poisons (e.g., camptothecin (CPT)) selectively kill cancer cells through the induction of protracted, high-intensity replication stress. Our studies unexpectedly revealed that treatment with CPT or other replication stress inducers (e.g., deep hypoxia or methylmethane sulfonate) triggers the ubiquitin-dependent degradation of Chk1 in both normal and transformed human cells. The degradation of Chk1 was dependent on the Skp1-Cullin-F-box (SCF) E3 ubiquitin ligase complex, and the consequences of severe Chk1 destruction were replication fork collapse and ultimate cell death. Remarkably, defects in the Chk1 degradation pathway confer resistance to the cytotoxic effect of CPT - a major problem with this class of drugs in the clinical arena. Thus, this novel layer of Chk1 regulation has important implications for our understanding of replication checkpoint signaling, as well as mechanisms of anticancer resistance in cancer patients. I now propose to elucidate the underlying mechanisms and biological significance of the stress-induced Chk1 destruction through pursuit of the following specific aims: In the mentored-phase, (1) To identify the F-box proteins of the E3 ligase complexes responsible for Chk1 destruction; In the independent phase, (2) To identify the putative 'Degron' region and the lysine residues targeted for ubiquitin modification in Chk1; (3) To investigate the roles of de-phosphorylation in Chk1 degradation; (4) To characterize the molecular mechanisms underlying the defect in Chk1 degradation in CPT-resistant cancer cells. Relevance: The results of these studies will not only advance our understanding of the genotoxic stress response machinery in human cells, but also provide novel insights into the causes of genetic instability and anticancer drug resistance in cancer cells, and these lines of research have direct implications for the development of novel therapeutic agents targeted against tumors.

描述（由申请人提供）：真核细胞已经进化出一个复杂的基因组监视和修复蛋白网络，以确保 DNA 复制以准确和及时的方式发生。该监视途径称为 S 期复制检查点。这种“看护”机制的缺陷会导致遗传不稳定，这是人类癌症的一个标志性特征。复制检查点监视复制叉的进展，当复制叉停滞时，传输延迟S期进展的信号，并维持停滞的叉的稳定性，以便在纠正初始错误后DNA复制可以恢复。复制检查点的两个关键组成部分是顶端蛋白激酶 ATR 及其下游靶激酶 Chk1。即使没有外在基因毒性应激，ATR 或 Chk1 功能的丧失也是致命的，这强调了复制检查点在维持细胞活力中的重要性。在初步工作中，我们测试了这样的假设：某些抗肿瘤药物，例如拓扑异构酶 1 (Top I) 毒物（例如喜树碱 (CPT)），通过诱导持久、高强度的复制应激选择性杀死癌细胞。我们的研究出乎意料地表明，在正常和转化的人类细胞中，用 CPT 或其他复制应激诱导剂（例如深度缺氧或甲基甲磺酸盐）治疗会触发 Chk1 泛素依赖性降解。 Chk1 的降解依赖于 Skp1-Cullin-F-box (SCF) E3 泛素连接酶复合物，Chk1 严重破坏的后果是复制叉崩溃和最终细胞死亡。值得注意的是，Chk1 降解途径的缺陷导致对 CPT 的细胞毒性作用产生抵抗——这是此类药物在临床领域的一个主要问题。因此，Chk1 调控的这一新层对于我们理解复制检查点信号传导以及癌症患者的抗癌耐药机制具有重要意义。我现在建议通过追求以下具体目标来阐明应激诱导的 Chk1 破坏的潜在机制和生物学意义：在指导阶段，（1）识别负责 Chk1 的 E3 连接酶复合物的 F-box 蛋白破坏；在独立阶段，（2）鉴定Chk1中假定的“Degron”区域和针对泛素修饰的赖氨酸残基； (3) 研究去磷酸化在Chk1降解中的作用； (4) 表征 CPT 耐药癌细胞中 Chk1 降解缺陷的分子机制。相关性：这些研究的结果不仅将增进我们对人类细胞中基因毒性应激反应机制的理解，而且还为癌细胞中遗传不稳定性和抗癌药物耐药性的原因提供了新的见解，并且这些研究具有直接意义用于开发针对肿瘤的新型治疗剂。