延胡索酸酶通过影响组蛋白甲基化对基因转录调控的分子机制及其在肺癌发展中的作用

Local metabolic effect of fumarase (FH) regulates histone methylation. However, whether this regulation is implicated in gene transcription remains unclear. Notch signaling has been known to negatively regulate Transforming growth factor β (TGF-β)-induced growth arrest, while the mechanisms underlying this signal crosstalk and their relevance to tumorigenesis remains further investigated. Currently, we have found that TGF-β induces p38-mediated fumarase phosphorylation, which promotes its binding to CSL, the core transcriptional factor under Notch signaling pathway, which further lead to a complex formation of FH /CSL/p53 and the recruitment of FH to p21 promoter. Promoter-associated FH inhibits KDM2A histone demethylase-mediated histone H3 Lys 36 demethylation; subsequently, this antagonizes the inhibitory effect of CSL on p53 and promotes the transcription of p21 and the cell cycle arrest. Meanwhile, FH is found to be largely phosphorylated by PAK4 in NSCLC cells; and this phosphorylation blocks FH /CSL/p53 complex formation. These findings demonstrate the critical role of FH /CSL/p53 signaling pathway in gene transcription and cell growth. in the regulation of cross talk between TGF-β and Notch pathway, and its dyregulation drives tumor development. In the present project, we are going to further investigate the molecular mechanism by which promoter-associated fumarate regulates gene transcription. Furthermore, we will utilize the cancer cell lines and mice tumor model to test the inhibitory effect of PAK4 on TGF-beta-CSL/p53/FH-p21 signaling pathway and the relevant effect on tumor development. Finally, we will set forth to examine the relationship between FH phosphorylation and patient survival. Collectively, here we expect to uncover the functional role of FH in gene transcription and tumorigenesis.

已有研究表明：FH能通过调节组蛋白甲基化介导DNA损伤修复。但FH能否通过影响组蛋白甲基化介导基因转录仍不明了。已知CSL能够抑制p53转录效应；我们前期研究发现：TGF-beta刺激正常细胞导致p38磷酸化FH，促进FH与CSL/p53相互作用，从而推动FH在p21启动子的积累、增强p21启动子区域甲基化、p21转录, 细胞生长抑制。另一方面，肿瘤细胞中PAK4磷酸化FH抑制了FH-CSL相互作用及下游效应。提示TGF-beta-CSL/p53/FH-p21信号通路影响基因转录以及细胞生长。本项目拟研究：FH在启动子局部调节基因转录的分子机制；细胞水平与动物模型中检测PAK4抑制TGF-beta-CSL/p53/FH-p21信号通路对肿瘤生长的影响；在临床肿瘤样本测序或免疫组化分析FH磷酸化状态与病人预后的相关性。本项目将阐明FH调节基因转录的分子机制以及其在肿瘤中的作用。

细胞代谢发生异常重编程是肿瘤发生的重要原因。代谢酶FH在细胞内可利用其酶活性调控组蛋白表观修饰，进而调控基因表达影响细胞生命活动。p53是细胞内的抑癌因子，可介导细胞生长阻滞和细胞凋亡，其功能缺失在多数肿瘤细胞中较常见。据报道，TGF-β信号通路的激活可促使p53发挥抑癌功能。同时经报道，Notch通路的效应分子CSL可抑制p53介导的细胞衰老。基于这些依据，作者开始探索Notch信号通路和TGF-β信号通路的交互作用对p53功能的调控，以及FH在其中所扮演的角色。. 研究人员首先发现在TGF-β的刺激和Notch通路激活下，FH的90位苏氨酸可被p38磷酸化并与CSL-p53形成复合物，从而被招募到p21启动子处；且此过程对TGF-β介导的细胞生长阻滞必不可少。同时，FH酶活突变型与野生型对照实验、外源延胡索酸添加等实验表明，FH催化活性及其在细胞核内产生的延胡索酸可抑制KDM2活性，维持组蛋白H3的36位赖氨酸甲基化水平，这对于TGF-β介导的细胞生长阻滞必不可少。进一步研究发现，在非小细胞肺癌细胞中，FH的46位丝氨酸被PAK4磷酸化后与14-3-3蛋白结合，抑制了FH-CSL-p53复合物的形成，促进肿瘤细胞生长增殖。最后，研究人员在小鼠体内和临床样本层面验证了上述机制。该研究结果为临床非小细胞肺癌的精准诊治提供了新的理论基础。