动点微管相关蛋白SKAP磷酸化在食管癌发生发展中的分子调控机制及临床意义探究

Loss or gain of whole chromosome, the form of chromosome instability commonly associated with cancers is thought to arise from aberrant chromosome segregation during cell division. As a highly lethal disease, the development of esophageal cancer is a complicated process involving various factors, multiple stages and multiple gene variation. However, the underlying molecular mechanisms remain undetermined. Previously, we have shown that protein kinase GSK-3β which is highly expressed in esophageal cancer can phosphorylate kinetochore protein SKAP at Ser232/237 and Thr294. We reason that phosphorylated SKAP by GSK-3β ensures is important to mitotic spindle dynamics and chromosome segregation cooperated with Kif2b. Our pre-experiments show that phosphorylated SKAP may also be involved in its kinetchore localization and constitute a dynamic link between spindle microtubule plus-ends and kinetchore cooperated with CLASP-1. We speculate that SKAP, which is phosphorylated by GSK-3β, may play a role in the development of esophageal cancer by participating in the regulation of mitosis. Taken together, in this proposal, we plan to further explore the spatiotemporal dynamic regulation mechanism and biological function of phosphorylated SKAP and synergistic protein Kif2b and CLASP in the cell cycle of esophageal cancer, and the clinical significance in the development of esophageal cancer.

染色体不稳定性或异常分离被广泛认为是癌症发生的标志性事件。食管癌作为一种高致死疾病，其发生发展是一个涉及多因素、多阶段、多基因变异累及相互作用的复杂过程，但具体分子调控机制并不清楚。我们前期工作证实在诸多肿瘤组织中高突变的动点蛋白SKAP被在食管癌中高表达的蛋白激酶GSK-3β磷酸化，位点为其Ser232/237位和Thr294位。磷酸化的SKAP与Kif2b协同参与有丝分裂纺锤体稳定性调控和染色体分离，预实验表明CLASP-1可能协同磷酸化SKAP参与调控其动点定位及动点微管连接。我们推测，被GSK-3β磷酸化的SKAP可能参与食管癌细胞有丝分裂调控，从而在食管癌发生发展中发挥作用。本课题将以磷酸化SKAP为切入点，探究其与协同作用蛋白Kif2b、CLASP-1在食管癌细胞周期中的时空动态性调节机制和生物学功能以及在食管癌发生发展中的临床意义，为治疗食管癌的新医疗手段奠定坚实的理论基础。

食管癌作为一种高致死疾病，其发生发展是一个涉及多因素、多阶段、多基因变异累及相互作用的复杂过程，但具体分子调控机制并不清楚。本研究瞄准在恶性肿瘤中发挥重要作用的蛋白激酶GSK-3β、以及在细胞有丝分裂过程中发挥重要功能的蛋白质SKAP，基于现有的各种平台基础，利用分子生物学、细胞生物学、生物化学、化学生物学及基础医学等手段，从临床组织、细胞、分子以及动物等多个层次，全面、系统地进行了研究。利用原位杂交及生物信息技术检测食管组织及邻近癌旁组织表达差异及表达水平之间的关系；并利用蛋白印迹及qPCR技术对表达差异进行核实和验证。通过在食管癌细胞系中敲低GSK3β或SKAP，观察其对食管癌细胞周期、侵袭、迁移能力等的影响，了解两者在食管癌细胞中的表达相关性及SKAP磷酸化对食管癌进展的影响。进一步地，我们在食管癌细胞系EC109中筛选到了SKAP-WT及模拟磷酸化T294D和非磷酸化T294A突变体的稳转细胞株，进一步分析了被GSK3β磷酸化的SKAP对食管癌细胞的周期、侵袭、迁移和EMT进程的影响。本研究所获得的科研成果为寻找食管癌的靶点药物提供分子实验基础，同时为食管癌的临床诊断和治疗提供新的思路。