基于高通量组学方法研究Aurora-A对肿瘤干细胞干性的调控机制

Cancer stem cells (CSCs) are an underlying cause of tumor recurrence and metastasis. Uncovering the stemness regulating mechanisms will advance the discovery of CSC-targeting drugs. Our preliminary studies indicated that high-expressed aurora A kinase (AurA) promoted self-renewal of CSCs. However, little is known about the stemness mechanism regulated by AurA. Lately, we have developed an integrated proteomics for the determination of specificity and substrates of kinase by taking advantage of proteome-derived peptide libraries and quantitative proteomics (JPR, 2013, 12: 3813). Here we will systematically screen of the stemness-related substrates of AurA with the above high-throughput proteomics for disclosing the internal stemness-regulating mechanisms for cancer stem cells. Firstly, we will construct a database of AurA substrates and verify the key substrates related to CSCs stemness. Secondly, we will investigate the stemness-regulating mechanism for CSCs mediated by AurA. Further we will verify the function of AurA and its substrates in conditional knock out breast cells and NOD/SCID mice and evaluate the self-renewal ability and tumorigenic capacity of CSCs. In summary, it is the first time to study the function of AurA in CSC self-renewal pathways with the integrated proteomics platform, and this project will shed some light on designing the CSC-target drugs.

肿瘤干细胞是临床肿瘤复发转移的根源，揭示其干性调控过程中关键靶点分子及调控机制有助于抗肿瘤药物的开发。申请人预实验发现Aurora激酶A(AurA)是肿瘤干细胞干性调控的关键分子，并建立了基于蛋白质衍生化肽段库和定量蛋白质组学技术相结合的组学新方法，为发现激酶底物这一可干预的靶点提供了快速、高通量的筛选平台 (JPR,2013,12:3813)。据此，本课题拟针对肿瘤干细胞体系优化已建立的高通量组学方法，筛选AurA底物并探究其对肿瘤干细胞干性的调控机制。构建肿瘤干细胞中AurA的潜在底物库，验证与干性相关的底物；阐明AurA及其底物调控干性的内在分子机制，并在条件敲除乳腺癌细胞和免疫缺陷小鼠模型上验证靶向AurA及其底物对肿瘤干细胞自我更新能力、成瘤能力等生物学特性的影响。本课题首次应用高通量组学筛选方法研究肿瘤干细胞中AurA底物及其干性调控机制，为研发靶向治疗药物提供新的思路。

肿瘤干细胞被认为是肿瘤耐药、复发、转移的根源，揭示其干性调控过程中关键靶点分子及调控机制有助于抗肿瘤药物的开发。作为原癌基因，Aurora A（AurA）激酶高表达可以促进肿瘤干细胞的自我更新，然而目前机制仍不清楚。本课题利用蛋白质组学平台，通过筛选AurA下游底物进而研究其对肿瘤干细胞的干性调控机制。本课题首先建立了蛋白层次上基于微球固载的酶底物筛选方法（Sci Rep, 2016），并进一步优化该方法用于激酶底物筛选，共鉴定到291个潜在AurA激酶底物蛋白上364个磷酸化位点。其中多个底物如PUM2，NUMA1等已经被报道，而且AurA特异性作用模体也与报道的一致，说明我们的方法是可靠的。该数据库是目前最大的数据库，为后续机制研究奠定基础。在机制研究方面，我们发现高表达AurA能够通过磷酸化FOXP1下调FBXL7启动子活性及蛋白表达，抑制survivin降解，增强肿瘤对DNA损伤药物的耐药性（Oncogenesis, 2017）。结合课题组在AurA激酶研究的相关成果，我们系统阐述了近几年AurA在肿瘤中的关键作用及其抑制剂在临床上的应用（Med Res Rev，2016，IF=8.763）。本课题首次利用蛋白质组学平台筛选与干性有关的AurA底物，探讨其对肿瘤细胞自我更新的作用，该研究为肿瘤干细胞靶向药物的开发提供理论支撑。