Cbx4经SUMO化修饰增强HIF-1a转录活性的分子机制与肝癌发生发展

HIF-1 is a key transcriptional factor for cellular response to hypoxia, one of the critical in vivo microenvironmental factors of cancers including hepatocellular carcinoma (HCC). It consists of an oxygen-sensitive HIF-1a (alpha) and constitutively expressed HIF-1beta subunits. Stability and transactivation of HIF-1a are widely regulated by post-translational modifications. Recently we report that the exepression of Cbx4, which acts as a member of polycomb repressive complex 1 and also presents the SUMO E3 ligase activity, is significantly correlated with VEGF, angiogenesis and the overall survival in HCC patients and transplanted HCC mice. Furthermore, we showed that Cbx4 interacts with and enhances sumoylation of HIF-1a, by which Cbx4 increases HIF-1 transactivation, governing the hypoxia-induced angiogenesis in HCC cells and transplanted HCC mice. In this project, we aim to investigate the following concerns: (1) Because Cbx4-enhanced sumoylation of HIF-1a has no effect on the stability of HIF-1a protein but increases its transcriptional activity, while PIASy, another known SUMO E3 ligase，promotes the HIF-1a degradation by inducing its sumoylation. We propose that they sumoylate different lysine sites of HIF-1a protein. Therefore, we attempt to identify the sumoylating sites of PIASy for HIF-1a protein to understand the difference of effects of Cbx4 and PIASy-enhanced HIF-1a sumoylation. （2）Our preliminary data show that Cbx4-enhanced HIF-1a sumoylation block the interaction of HIF-1a and Sirt6, a histone deacetylase which is also shown to act as the corepressor for HIF-1. We propose that sumoylation at some sites of HIF-1a can modulate its interactions with some co-activators and/or co-repressors. Hence, we attempt to use protein-protein interaction-based proteomics to identify sumoylated HIF-1a-interacted proteins to explore how Cbx4-enhanced HIF-1a sumoylation increase the transcriptional activity of HIF-1.（3）Cbx4 also increases the expressions of other HIF-1 targeted genes such as PDK1 and PGK1 besides VEGF, which exert an important role in the Warburg effect of cancers. Therefore, we also attempt to explore the potential role of these regulations of Cbx4 on HIF-1 targeted genes in the pathogenesis of HCC. （4）We also want to identify the crystal structure of Cbx4 and sumoylated Cbx4-HIF-1a complex with and without the interacting proteins identified above to understand the structural basis for HIF-1-Cbx4 interaction and increased transcriptional activity of Cbx4-sumoylated HIF-1a. Based on this, we shall design and discover some compounds to interfere sumoylating activity of Cbx4 and/or Cbx4-HIF-1 interaction, and identify their potential in vitro and in vivo pharmalogical actions against HCC. These works would provide new insights for Cbx4 function, HIF-1a sumoylation and its regulation on its transactivation as well as their roles in the pathogenesis of HCC, and get some new lead compounds for selectively inhibiting HCC.

低氧诱导因子-1 (HIF-1)是调控细胞对低氧反应的重要转录因子。最近，本课题组在《Cancer Cell》报道Cbx4表达与肝癌组织的VEGF表达、微血管密度和病人预后密切相关。进一步的研究显示Cbx4通过SUMO化修饰HIF-1a(alpha)蛋白的391和477位赖氨酸，显著增强HIF-1的转录活性，进而增加VEGF表达，促进新生血管生成和肝癌转移。本课题拟以这些新发现为基础，深入探寻Cbx4通过SUMO化修饰HIF-1a蛋白致其转录活性增加的分子机制，探讨Cbx4对其它HIF-1靶蛋白表达调控及其在肝癌发生发展中的作用，并获得靶向Cbx4的SUMO化修饰活性和(或)抑制Cbx4-HIF-1a相互作用位点的化合物，研究其体内外抗肝癌效应。这些研究工作对于进一步揭示Cbx4功能，HIF-1的SUMO化修饰和转录调控机制及其在肝癌发生发展中的作用提供新的发现，也为肝癌的治疗提供线索。

本课题在课题组已有工作的基础上，进一步明确了Cbx4在肝癌中的作用，其表达水平是肝癌病人的独立预后因素；并且是临床动脉栓塞治疗/动脉栓塞联合化疗（TAE/TACE）治疗的蛋白标志物；通过构建双荧光素酶报告系统运用高通量筛选的方法寻找获得5个能够抑制Cbx4增强HIF-1转录活性的活性化合物，在细胞水平明确了其中3个化合物能够抑制Cbx4的SUMO化修饰活性，2个抑制Cbx4与HIF-1的相互作用。在此基础上，通过一系列的基团取代，获得了活性更优的化合物。同时，通过Click反应，初步发现其中1个化合物作用的靶标蛋白；发现低氧能够调控HIF-1α共抑制蛋白Sirt6的表达并进行了相关的机制研究；发现无糖增强Cbx4的乙酰化修饰并寻找到了发生乙酰化修饰的位点及其乙酰转移酶和去乙酰化酶。Cbx4乙酰化修饰水平的增高能够促进其SUMO E3连接酶活性。通过裸鼠成瘤实验发现，抑制Cbx4乙酰化修饰能够抑制肿瘤的生长。在国际重要学术刊物上发表15篇学术论文。培养10余名新的学科骨干和研究生。