癌基因B-RAFV600E导致两极纺锤体结构及定位异常的分子机制

The oncogenic B-RAFV600E mutant, drives constitutive activation of the extracellular signal-regulated kinase (ERK) pathway, is implicated in tumorigenesis. In addition to its established roles in promoting cell cycle entry and progression through the G1/S transition, we identify a novel role for the oncogenic B-RAF in driving chromosome instability in melanocytic cells. B-RAFV600E causes aberrant bipolar spindle and its position defect, multipolar spindles, and chromosome missegregation, leading to aneuploidy. Previously, we reported that continuously activated B-RAFV600E signaling may be a possible mechanism for the deregulation of Mps1 stability and kinase activity, and that persistent phosphorylation of Mps1 through B-RAFV600E signaling is a key event in resulting in centrosome amplification, multipolar spindle formation, and chromosome instability that may contribute to tumorigenesis. However, how oncogenic B-RAFV600E causes aberrant bipolar spindles and defect spindle position remains poorly understood. This project will elucidate the molecular mechanism how this occurs by LS-MS analysis, immunofluorescence, live-cell imaging, and RNAi in different retrovirus infected inducible cell lines, which would be important for understanding the mechanism through which B-RAFV600E exerts its oncogenic effects during the tumor development.

BRAFV600E是非常重要的癌基因，通过RAS/RAF/ERK通路调控细胞生长、分化、凋亡等生物学事件，与肿瘤发生发展密切相关。申请者前期研究首次揭示了癌基因BRAFV600E在有丝分裂期的新作用：该基因导致肿瘤细胞两极纺锤体结构及定位异常、多级纺锤体出现、染色体错误分离等有丝分裂期异常现象；阐述了BRAFV600E通过磷酸化修饰纺锤体检测点激酶Mps1而增强其蛋白稳定性、激酶活性，导致中心体过度复制、多级纺锤体出现，进而在肿瘤细胞染色体不稳定性中发挥重要作用的分子机理。但是，关于BRAFV600E基因如何导致两极纺锤体结构及定位异常的分子机制尚不清楚。本项目拟利用可诱导逆转录病毒系统，采用质谱分析、免疫荧光染色、活细胞工作站和RNAi等技术，探讨BRAFV600E基因导致两极纺锤体结构及定位异常的作用途径，为全面理解该基因在肿瘤发生发展中的作用及分子机制、设计抗肿瘤治疗方案提供理论基础

前期研究表明癌基因B-RAFV600E可以导致两极纺锤体结构/定位异常及多极纺锤体出现，最终引起染色体不稳定性及非整倍体肿瘤细胞。然而，关于该基因如何导致两极纺锤体结构及定位异常的分子机制并不清楚。本项目利用串联质谱分析、免疫荧光染色、活细胞监控和基因敲除等技术，发现了癌基因B-RAFV600E通过磷酸化修饰MAP4-T380、S787位点影响纺锤体微管组装的动态平衡及纺锤体定位，导致纺锤体结构出现异常，两极纺锤体出现xy rotation、xy displacement、z rotation等异常现象。同时，我们还发现BRafV600E导致Nudel、Dynein表达水平降低，Nudel与Lamin B相互作用减弱，微管聚集出现障碍。本研究阐明了癌基因BRafV600E导致两极纺锤体结构及定位异常的分子机理，为全面理解癌基因BRafV600E在肿瘤发生发展中的作用及其分子机制、设计抗肿瘤治疗方案提供理论依据。