AKT-EZH2-NFKB轴促进胶质瘤恶性生长的分子机制及临床意义

Aberrant DNA methylation of tumors, regulated by multiple factors including DNA methyltransferases, histone modifications and nucleosome structure (Cell Reports 2012), plays a very important role in the initiation and development of malignant glioma. The PI3K-AKT pathway, one of the core signaling pathways in glioma, may be activated by epigenetic silencing of PTEN or miR-149. It is, however, not clear what is the downstream of PI3K-AKT pathway when PTEN or miR-149 is inactivated via promoter hypermethylation. In malignant glioma, the applicants of this proposal found that DNA methylation of the PTEN or miR-149 gene induced phosphorylation of EZH2 protein in an AKT-dependant manner, and that the phosphorylated EZH2 physically interacted with RelA: the key subunit of the nuclear factor-κB (NFKB) molecule. These results indicate that EZH2 might link PI3K-AKT and NFKB pathways, which can induce the formation of an AKT-EZH2-NFKB axis in this type of tumor. Based on these findings, the applicants will first investigate whether there exists the AKT-EZH2-NFKB axis in malignant glioma through completion of multiple experiments such as immunoprecipitation and signaling blockade.Furthermore,the applicants will test whether the axis is essential for the malignant growth of primary tumor cells in vitro and in xenografting nude mice. Lastly, the applicants will exam the relationship between activation of the AKT-EZH2-NFKB axis and pathological grading of tumors or prognosis of patients through clinical analysis. Therefore, elucidation of the activation of the AKT-EZH2-NFKB axis initiated by epigenetic selincing of PTEN or miR-149 may provide a novel and effective strategy for the treatment of malignant glioma.

肿瘤DNA异常甲基化受甲基化酶、组氨酸和核小体等多因素调控(Cell Reports 2012),它是促进恶性胶质瘤发生发展的重要原因。PTEN或miR-149的甲基化失活可激活胶质瘤的关键信号通路PI3K-AKT，但其下游途径则有待深入探索。在恶性胶质瘤中，申请人的前期研究发现PTEN或miR-149甲基化能使EZH2发生AKT依赖性磷酸化；磷酸化的EZH2则进一步与NFKB分子结合。这高度提示EZH2可连接PI3K-AKT和NFKB通路，从而形成AKT-EZH2-NFKB轴。因此，申请人将用免疫沉淀和信号阻断等方法证明该轴在恶性胶质瘤中是否存在；并在原代细胞和荷瘤裸鼠中观察该轴对肿瘤生长的影响；再结合临床资料，分析EZH2磷酸化与肿瘤分级和患者预后的关系。由此可明确PTEN或miR-149的甲基化，是否经AKT-EZH2-NFKB轴而促进胶质瘤的恶性生长，从而为其治疗提供有效的新策略。

恶性胶质瘤是中枢神经系统最常见的恶性肿瘤，生长快且无法完全切除，五年生存率极低。因此，急需对该病的分子机制进行深入研究，以求发现更为有效的治疗策略。肿瘤 DNA 异常甲基化受甲基化酶、组氨酸和核小体等多因素调控，它是促进胶质瘤恶性进展的重要原因。我们发现在胶质瘤中抑癌基因PTEN和miR-149因发生启动子甲基化而失活，它们的失活激活了肿瘤中的重要信号通路PI3K-AKT途径。在胶质瘤中，原癌基因EZH2可作为AKT的磷酸化底物而被激活，激活后的EZH2分子可与NF-κB的RELA分子形成复合物而共同作用下游分子如IL-6，提示在治疗中应针对NF-κB和EZH2联合用药可以达到更好的疗效。与此同时，我们还证明NF-κB能够通过转录激活促进EZH2的表达，进而引起组蛋白H3的K27发生三重甲基化（H3K27me3），最终导致肿瘤发生表观遗传学方式的改变。阻断信号通路后进行裸鼠成瘤，验证了动物体内EZH2和NF-κB激活促进肿瘤发生发展。由此可见，抑癌基因PTEN或miR-149甲基化失活，可经AKT-EZH2-NF-κB轴促进胶质瘤的恶性进展。于此同时，我们通过单细胞测序分析发现，长链非编码RNA的异质性也可能是导致肿瘤异质性从而难以治愈的重要原因。通过我们的研究，可以加深对胶质瘤分子机制的进一步理解；同时我们的结果也提示AKT-EZH2-NF-κB轴可以作为临床治疗的潜在靶标。我们的研究为胶质瘤的有效治疗尤其是联合用药提供了新的思路。项目资助发表SCI共3篇，总影响因子超过20；在研期间培养硕士及博士9人，在读3人，毕业6人。项目投入经费70万，累计支出68.7641万，各项支出基本与预算相符。剩余经费1.2359万元，将用于本项目研究后续支出。