CEBPD作为缺氧调控分子网络关键节点促进胶质瘤抗血管治疗耐药性的机制研究

Anti-angiogenesis therapy was the only type of novel drugs that are approved by FDA for treatment of gliomas in recent years. However, anti-angiogenesis therapy can induce drug resistance and progressed invasion growth of gliomas, which is closely related to hypoxia induced by anti-angiogenesis therapy. Hypoxia regulates a lot of responsive genes which form a complex molecular network. Therefore, it would be important for theoretical research and clinical practice to find key points of this network as therapeutic target. Our bioinformatics analysis suggested that transcriptional activator CEBPD is a critical point for the hypoxia regulated molecular network in glioma stem like cells (GSCs). Our further studies demonstrated that CEBPD was over-expressed in gliomas and high CEBPD expression predicted poor prognosis. The expression levels of CEBPD was positively correlated with that of several angiogenesis factors. In addition, CEBPD positively regulated the proliferation of GSCs. Based on these findings, the current project will focus to explore the roles of CEBPD in the hypoxia regulated network. We would study the underlying mechanisms of CEBPD induced angiogenesis, drug resistance and progressed invasion growth of gliomas through the hypoxia regulated molecular network, under the conditions of hypoxia and/or administration of anti-angiogenesis agent. The project will provide foundations for establishing novel therapeutic strategies against hypoxia by targeting CEBPD, which would strengthen the therapeutic effect of anti-angiogenesis agent and avoid induction of drug resistance.

抗血管发生药物是近年来唯一获得FDA批准用于胶质瘤治疗的新型药物。然而，抗血管治疗会导致胶质瘤耐药性产生及浸润性进展，这与抗血管治疗引发的肿瘤缺氧反应密切相关。缺氧调控的大量分子形成一复杂网络，以此网络关键节点为治疗靶点从而抑制缺氧反应具有重要的理论价值与应用前景。我们通过对胶质瘤干细胞（GSC）缺氧调控分子网络的生物信息学分析，发现转录激活因子CEBPD是此网络关键节点。进一步研究证实，CEBPD在胶质瘤高表达，且表达高者预后差，其表达水平与多个血管发生因子正相关；CEBPD正向调控GSC增殖。基于这些发现，本项目将深入研究CEBPD在GSC缺氧调控分子网络中的作用，阐明在缺氧及使用抗血管药物的情况下，该分子通过缺氧分子网络促进胶质瘤血管发生、耐药性产生、浸润性进展的作用机制，为探索以抗缺氧反应为目标、以CEBPD为靶点，提高胶质瘤抗血管治疗效果、避免耐药性产生的新策略奠定基础。

胶质母细胞瘤（GBM）患者预后差，生存期只有1年左右，尚无理想治疗方法。抗血管发生药物是近年来唯一获得FDA批准用于胶质瘤治疗的新型药物。然而，抗血管治疗会导致胶质瘤耐药性产生及浸润性进展，这与抗血管治疗引发的肿瘤缺氧反应密切相关。缺氧调控的大量分子（HRGs）形成一复杂网络（缺氧调控分子网络，HRGs-MINWs），我们通过对胶质瘤干细胞（GSC）缺氧调控分子网络的生物信息学分析，发现转录激活因子CEBPD是此网络关键节点。本课题研究指出，CEBPD的mRNA及蛋白水平均在胶质瘤高表达，且与胶质瘤的级别正相关，其中表达高者预后差。我们采取慢病毒沉默U87及GSC中CEBPD的表达。生长曲线、细胞克隆、transwell实验及血管发生实验指出，CEBPD沉默后胶质瘤细胞增殖、生长、浸润性、血管发生均受到明显抑制；体内移植瘤实验指出，CEBPD沉默后胶质瘤的体内致瘤性受到了明显的抑制。免疫组化染色指出，在胶质瘤标本中，CEBPD表达高者，其CD34的微血管面积及血管分形维度均明显增加。在缺氧的研究中，CEBPD在缺氧情况下表达上升，且其表达受到缺氧诱导因子HIF1a的调控。基因芯片分析指出，CEBPD调控许多与血管发生密切相关的重要基因，包括：EPM2, ETS2, AKAP12, HAS2, COL4A2，且在胶质瘤标本中CEBPD的表达与这些基因呈正相关。为进一步研究CEBPD对缺氧调控分子网络的影响，我们以GBM标本的基因芯片数据为基础，基于信息学构建了GBM-HRGs-MINWs，并发现该网络具有不同于随机网络及正常脑组织网络的代数拓扑学结构。进一步对CEBPD表达高与低的GBM标本的分析指出，CEBPD的表达可明显影响GBM-HRGs-MINWs的代数拓扑学结构。采取贝伐单抗抗血管治疗胶质瘤体内移植瘤后，GBM细胞可产生耐药性。分析指出，在移植瘤未产生耐药的时候，CEBPD表达略有升高，而在耐药发生后标本中CEBPD显著升高；而在CEBPD沉默后，给予贝伐单抗治疗后GBM移植瘤未产生明显耐药性。综上所述，CEBPD是GBM-HRGs-MINWs的关键分子，其受到缺氧的调控，可促进胶质瘤的增殖、血管发生及体内致瘤性，并参与胶质瘤抗血管治疗耐药，是针对血管发生、抗血管治疗耐药及缺氧的潜在的胶质瘤治疗靶点。