多聚ADP核糖基化修饰的OVOL2在促进肿瘤细胞形成多倍体和诱导细胞衰老中的机制研究

OVOL2 is a member of a family of zinc finger transcriptional repressors. Previous studies demonstrated that OVOL2 plays a pivotal role in development and cancer invasion and metastasis; however, weather OVOL2 regulates the formation of cancers is still unknown. Our preliminary data of this proposal showed that OVOL2 could be modified by PARylation, which is essential for its binding with target promoter DNA. Moreover, OVOL2 was found to promote Cyclin E protein expression through transcriptionaly repress the expression of Fbw7, an E3 ubiquitin ligase. As such, Cyclin E could induce the formation of plyploidy, and facilitate CDK2 to phosphorylate NPM1, resulting in the disassociation of NPM1 from centrosome and the amplification of centrosome. Consequently, chromosome instability was induced and cell transformation occurred. On the other hand, Cyclin E could induce the expression of P21, which induces the senescence of cells. The choice between cell transformation and cell senescence was controlled by the third function of OVOL2, which transcriptionaly repressed the expression of HEST to inhibit the centrosome clustering. When cells could not effectively pass through mitosis phase, the cells chose senescence. In the present proposal, we will systematically study the mechanism and function of OVOL2 in regulating the formation of polyploidy and induction of senescence, in molecular, cellular, mouse tumor models and clinical tumor sample levels. Our study will reveal a possibility that OVOL2 may act as a novel target for gene therapy of cancers.

OVOL2是含锌指蛋白结构的转录抑制因子，之前的研究表明，OVOL2在组织发育和肿瘤侵袭转移中发挥重要作用，然而在肿瘤发生过程中的作用仍然未知。本课题前期实验发现，OVOL2在细胞内会被PARylation修饰,这种修饰为OVOL2的DNA结合所必须。OVOL2会通过转录抑制泛素连接酶Fbw7使CyclinE蛋白持续表达。一方面，CyclinE引起多倍体形成，并促进CDK2磷酸化NPM1，使NPM1从中心体分离以及诱导中心体复制，最终引起染色体不稳定，促进细胞转化。另一方面，CyclinE引起P21表达，可以促进细胞衰老。二者的选择由OVOL2的第三个作用所控制，即OVOL2通过转录抑制HEST而阻碍中心体成簇，在细胞不能有效分裂时，细胞选择衰老死亡。我们将从分子、细胞、小鼠以及临床标本等几个层面系统研究PARylation修饰的OVOL2在促进肿瘤细胞形成多倍体和诱导细胞衰老中的的机制。

Poly（ADP-核糖基）（PAR化）是一种翻译后修饰，其中聚ADP-核糖（PAR）聚合物通过PAR聚合酶（PARP）共价地加入到蛋白质中。在此，利用蛋白质组学方法，我们确定转录调节因子OVOL2是PARP1的一种新底物，可以在其C2H2锌指结构域内的残基赖氨酸145、赖氨酸176和赖氨酸212处对位酰化。PARylated OVOL2的过度表达改变了细胞形态，并诱导染色体滞后和非整倍体。为了明确OVOL2诱导异常细胞周期和中心体扩增的潜在分子机制，我们发现OVOL2通过增强其稳定性来提高细胞周期蛋白E的蛋白质水平。此外，我们还将周期蛋白E的E3泛素连接酶Skp2确定为PARylated OVOL2的直接靶点。通过芯片分析，Skp2启动子区域的OVOL2结合位点被定位。为了进一步探索其生理效应，我们发现PARylated OVOL2可以诱导细胞死亡。此外，为了研究PARylated OVOL2在体内的功能，我们进一步开发了一种无效小鼠异种移植模型，并生成MMTV-PyVT转基因小鼠，并监测宽型OVOL2和非PARylated OVOL2-3K/a突变体对肿瘤进展的影响。一致地，在空白小鼠异种移植和MMTV-PyVT转基因小鼠中，宽型OVOL2的过度表达分别显示出显著的肿瘤进展减少，进一步表明OVOL2作为体内肿瘤抑制因子的功能受到PARylation的高度调节。综上所述，我们的研究揭示了PARP1诱导的PAR酸化是OVOL2介导的染色体完整性调节和抑制癌细胞生长的关键事件。