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

• 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
• 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

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(申请人提供)：真核细胞已经进化出一个复杂的基因组监测和修复蛋白质网络，以确保DNA复制将以准确和及时的方式发生。这一监测途径被称为S期复制检查点。这种“看守”机制的缺陷会导致基因不稳定，这是人类癌症的一个显著特征。复制检查点监控复制分叉的进度，当分叉停止时，发送延迟S阶段进展的信号，并保持停止的分叉的稳定性，以便在纠正初始错误后可以恢复DNA复制。复制检查点的两个关键组成部分是顶端蛋白激酶ATR及其下游靶蛋白激酶Chk1。ATR或Chk1功能的丧失是致命的，即使在没有外部遗传毒性应激的情况下也是如此，这突显了复制检查点在维持细胞活力方面的重要性。在前期工作中，我们测试了某些抗肿瘤药物，如拓扑异构酶1(Top I)毒药(例如喜树碱(CPT))通过诱导长期、高强度复制应激选择性地杀死癌细胞的假设。我们的研究意外地发现，CPT或其他复制应激诱导剂(如深度缺氧或甲基甲烷磺酸盐)处理会在正常和转化的人类细胞中触发泛素依赖的Chk1降解。Chk1的降解依赖于Skp1-Cullin-F-box(SCF)E3泛素连接酶复合体，严重破坏Chk1的后果是复制叉崩溃和最终细胞死亡。值得注意的是，Chk1降解途径的缺陷使人对CPT的细胞毒作用产生抵抗-这是临床上这类药物的主要问题。因此，这一新的Chk1调节层对于我们理解复制检查点信号以及癌症患者的抗癌耐药机制具有重要的意义。现在，我提议通过追求下列特定目标来阐明应激诱导Chk1破坏的潜在机制和生物学意义：在指导阶段，(1)确定导致Chk1破坏的E3连接酶复合体的F-box蛋白质；在独立阶段，(2)确定Chk1中可能的‘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)在CPT耐药癌细胞中，研究Chk1降解缺陷的分子机制。 相关性：这些研究的结果不仅将促进我们对人类细胞中的遗传毒性应激反应机制的了解，还将为癌症细胞中遗传不稳定和抗癌药物耐药性的原因提供新的见解，这些研究路线对开发针对肿瘤的新型治疗药物具有直接意义。