NKAP在DNA损伤保护性应答中的功能及机制研究

In response to diverse genotoxic stresses, cells activate DNA damage checkponit pathways to protect genomic integrity and promote survival of the organism.Defects in the ability to properly respond to and repair DNA damage underlie many forms of cancer.In the past several years, We have devoted to understanding the roles of the DNA damage response(DDR) in genomic stability and cancer, and made some notable findings.We identified sox4, a new DNA damage sensor, which is required for the activation of p53 in response to DNA damage. It promotes cell cycle arrest and apoptosis,and it inhibits tumorigenesis in a p53-dependent manner. (PNAS, 2009).We also identify CUEDC2 as a new player in silencing the inhibitory checkpoint signal.CUEDC2 is highly expressed in many types of tumors and its dysregulation might contribute to tumor development by causing chromosomal instability (Nature Cell Biology, 2011).To identify new genes involved in the DNA damage checkpoints, we performed a siRNA library screen.We found that NF-kappa B activating protein(NKAP) is required for DNA damage repair.DNA damage was observed in NKAP knock down cells, which are associated with defective DNA double - strand break repair. Notably, immunohistochemistry analysis showed NKAP is lost in the majority skin cancer tissues. Our prelimary data suggest that NKAP may be a potential key factor in regulating DDR-associated cancer. In this proposal, we would dynamically observe NKAP interactive proteins with/without DNA damage. With immunoaffinity purification and mass analysis, it is possible to find specific NKAP-bing proteins induced by DNA damage. Applying various cell biological and molecular biological approaches, together with UV induced mice skin cancer models,we will unravel the detailed functions of NKAP in DNA damage response, which will pave a way to better understand the underlying mechanisms underlying DDR associated cancer, and probably lead to finding of new cancer therapy target.

基因组DNA时刻受到各种损伤因素的危害，细胞对DNA损伤反应的异常将导致肿瘤的发生。本室近年来关注该领域，发现了新的DNA损伤效应分子SOX4（PNAS,2009）；细胞分裂纺锤体检查点调控蛋白CUEDC2（NCB, 2011）。近期，在基于RNAi的高通量筛选中，我们发现了一个新的DNA损伤效应蛋白NKAP。沉默其表达导致细胞S期阻滞，DNA损伤增加，IR照射后DNA修复受阻，免疫组化显示NKAP在皮肤癌中显著低表达，提示NKAP在DNA损伤保护性应答以及阻止皮肤癌发生中的重要作用。本项目在前期已取得重要线索基础上，通过免疫亲和纯化和质谱分析鉴定在DNA损伤中与NKAP动态结合的蛋白质，利用干涉、过表达细胞系以及动物模型，结合周期分析、激酶检测等手段，深入阐明NKAP对DNA损伤诱导周期检查点以及损伤修复的调控模式，揭示其功能缺失在DNA损伤导致皮肤癌发生中的作用及分子机制。

细胞内外的各种损失因素（毒物、辐射、细菌病毒等病原体感染、低氧等）都有可能导致细胞有丝分裂的异常和基因组的不稳定。而基因组不稳定和多种疾病的发生密切相关，如衰老、神经系统疾病和肿瘤等。在本项基金的资助下，我们发现了一个在基因组稳定性维护中起到重要作用的蛋白质NKAP，揭示了NKAP在染色体正确排列和分离、维护染色体稳定性、抑制肿瘤发生的重要作用和分子机制。我们发现NKAP在细胞微管与着丝粒的相互作用中发挥着重要作用。NKAP影响了细胞着丝粒上的一个负责细胞染色体排列的重要蛋白CENP-E的着丝粒的定位。课题组进一步详细揭示了NKAP通过SUMO化修饰调控CENP-E定位的分子机制。重要的是NKAP缺失导致细胞染色体分离错误和基因组不稳定性。并且在人软组织肉瘤、胰腺癌、甲状腺癌等人的肿瘤样本中发现了NKAP的缺失，揭示了NKAP功能异常在肿瘤发生中的重要作用。我们的工作发现了一个在维持基因组稳定性中具有重要作用的新抑癌基因。部分研究结果已发表在今年十月的Nature Communications 上。