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

描述（由申请人提供）：DNA损伤检查点调控的失败可使细胞易于癌变。泛素-蛋白酶体系统（UPS）被认为在调节DNA损伤和修复反应中起重要作用。在试图进一步确定这一作用，我们已经观察到，一个关键组成部分的监管机制，Rad 17，是一个紫外线诱导和泛素依赖性快速周转蛋白。我们观察到Cdh 1/APC（泛素蛋白连接酶）对Rad 17的暂时降解参与了检查点响应完成后终止检查点信号传导，并且通过不可降解的Rad 17突变体的功能干扰来稳定Rad 17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，受损的蛋白水解和持续升高的Rad 17是恶性黑色素瘤的一个显著特征，并且持续性水平与从正常黑色素细胞到发育不良程度增加并最终到黑色素瘤本身的转变平行。此外，稳定的Rad 17阻碍基因组完整性，这诱导人原代黑素细胞的恶性转化。我们在这项研究中的目的是阐明Rad 17介导的检查点失调的细节，并检查其对黑色素瘤发生过程的影响。我们试图评估Rad 17持久性在某些癌症中可能发挥致病作用的程度。基于这些证据，我们假设检查点调控的失败是黑色素瘤和其他癌症发展的重要促进因素。我们的具体目的是：（1）确定Cdh 1的上游调控机制，并进一步研究Cdh 1/APC和Rad 17之间的相互作用如何终止检查点信号：（2）研究Cdh 1/APC在DNA损伤反应中如何调控Rad 17;以及（3）使用建立的黑色素瘤小鼠模型和3D皮肤重建模型来确定失调的Rad 17蛋白水解对癌发生的影响。了解通过Rad 17蛋白水解调节基因组完整性的生物学机制，阐明Rad 17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，也是确定治疗干预潜在靶点的努力。

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.