Interplay between Cdh1/APC and Rad17 in DNA damage checkpoints and carcinogenesis

描述（由申请人提供）：DNA损伤检查点调节的失败可使细胞易发生癌变。泛素-蛋白体系统（UPS）被认为在调节DNA损伤和DNA修复反应中起重要作用。在试图进一步定义这一作用时，我们观察到调节机制的一个关键组成部分Rad17是一种紫外线诱导的泛素依赖的快速周转蛋白。我们观察到，在检查点应答完成后，Cdh1/APC（泛素蛋白连接酶）对Rad17的时间降解参与了检查点信号的终止，并且通过不可降解的Rad17突变体的功能干扰对Rad17的稳定减弱了检查点终止和dna损伤细胞的去除。我们进一步观察到，蛋白水解受损和持续升高的Rad17是恶性黑素瘤的一个显著特征，并且这种持续水平与从正常黑素细胞到增加程度的不典型增生，最终到黑色素瘤本身的转变相似。此外，稳定的Rad17阻碍了基因组完整性，从而诱导人类原代黑素细胞的恶性转化。我们提出的研究目的是阐明rad17介导的检查点失调的细节，并检查其对黑色素瘤形成过程的影响。我们试图评估Rad17的持久性在某些癌症中可能发挥的致病作用的程度。基于这些证据，我们假设检查点调节的失败是黑色素瘤和其他癌症发展的重要促进因素。我们的具体目标如下：(1)确定调控Cdh1的上游机制，并进一步研究Cdh1/APC与Rad17之间的相互作用如何终止检查点信号传导；(2)研究Rad17在DNA损伤反应中受Cdh1/APC调控的机制；(3)利用已建立的黑色素瘤小鼠模型和3D皮肤重建模型，确定Rad17蛋白水解失调对癌变的影响。通过Rad17蛋白水解调控了解基因组完整性的生物学机制，并阐明Rad17蛋白水解在黑色素瘤发展中的潜在作用，将是未来研究受损DNA损伤信号在肿瘤发生中的作用的重要前提，也是确定治疗干预的潜在靶点的重要前提。

DESCRIPTION (provided by applicant): Failure of DNA damage checkpoint regulation can predispose a cell to become cancerous. The ubiquitin-proteosome system (UPS) is thought to play an important role in regulating responses to DNA damage and DNA repair. In attempting to further define this role, we have observed that a critical component of the regulatory machinery, Rad17, is a UV-induced and ubiquitin-dependent fast-turnover protein. We observed that temporal degradation of Rad17 by Cdh1/APC (ubiquitin-protein ligase) is involved in terminating checkpoint signaling after the completion of the checkpoint response, and that stabilization of Rad17 through functional interference by a non-degradable Rad17 mutant attenuates checkpoint termination and the removal of DNA-damaged cells. We have further observed that impaired proteolysis and persistently elevated Rad17 is a conspicuous feature of malignant melanomas, and that the level of persistence parallels the transition from normal melanocyte through an increasing degree of dysplasia and ultimately to melanoma itself. In addition, stabilized Rad17 impedes genomic integrity, which induces malignant transformation for human primary melanocytes. Our objective in the proposed study is to elucidate details underlying Rad17-mediated checkpoint dysregulation, and examine its impact to the process of melanomagenesis. We seek to assess the extent to which Rad17 persistence may play a pathogenetic role in some cancers. Based on the evidence, we hypothesize that failure of checkpoint regulation is an important facilitator of the development of melanomas and possibly other cancers as well. Our Specific Aims are the following: (1) To determine the upstream mechanism that regulates Cdh1 and further examine how the interplay between Cdh1/APC and Rad17 terminates checkpoint signaling; (2) to study how Rad17 is regulated by Cdh1/APC during the DNA damage response; and (3) to determine the impact of dysregulated Rad17 proteolysis on carcinogenesis using an established melanoma mouse model and 3D skin reconstruction model. Understanding the biological mechanisms involved in genomic integrity via Rad17 proteolytic regulation and elucidating a potential role for Rad17 proteolysis in melanoma development will be an important antecedent to future studies on the role of impaired DNA damage signaling in oncogenesis, as well as for efforts to identify potential targets for therapeutic intervention.