EMAP-II诱导胶质瘤干细胞自噬发挥抗肿瘤作用的分子机制

Recently, we had reported the mechanisms of low-dose EMAP-II on the opening of blood-tumor barrier (BTB) and inhibition of brain glioma growth. We verified that EMAP-II could open the BTB selectively, meanwhile induce apoptosis and autophagy of U251 glioma cells to result in cell death. Our recent research found that EMAP-II inhibited the proliferation, migration and invasion of GSCs, and also decreased the mitochondrial membrane potential (MMP). 3-MA or zVAD-fmk partly blocked the inhibition of GSCs proliferation and decreased the MMP induced by EMAP-II. The synergistic administration of the above two inhibitors might entirely block the EMAP-II-induced process, which suggested EMAP-II-induced death of GSCs was related to apoptosis and autophagy. EMAP-II upregulated the expression of LC3-II,beclin1, Atg2B and Atg7, however, the potential mechanisms of EMAP-II-induced autophagic death are not clarified yet. Building on our previous research and related reports, we proposed the following hypothesis: EMAP-II might inhibit PI3K/Akt/mTOR signaling pathway to induce autophagic death of GSCs. The following three pathways might be involved in regulating the autophagy of GSCs, after the inhibition of PI3K/Akt/mTOR signaling: ① through initiating the autophagy of GSCs directly; ② through inhibiting the function of bcl-2. The inhibition of bcl-2 might upregulate beclin1 to promote the formation of beclin1 complex and autophagosomes in GSCs; ③ through inhibiting the phosphorylation of FOXO1. The inhibition of p-FOXO1 might promote FOXO1 nuclear import and increase Atg2B gene expression. Meanwhile, the nuclear import of FOXO1 might upregulate Atg7 by inhibiting miR-372. The increased Atg2B and Atg7 might promote the autophagosome formation via different pathways and furtherly cause the autophagic death of GSCs. To date, there has been no report about the GSCs autophagy initiated by EMAP-II-induced inhibition of PI3K/Akt/mTOR signaling, and about autophagosome formation increased by EMAP-II-induced upregulation of beclin1, Atg2B and Atg7 to cause the autophagic death of GSCs. To verify the above hypothesis, based on the clarification of EMAP-II-induced autophagic death of GSCs, this project proposal is to investigate whether EMAP-II promote the autophagic death of GSCs directly via inhibiting the PI3K/Akt/mTOR signaling. Then, this project will attempt to verify whether EMAP-II promote the upregulation of beclin1 and formation of beclin1 complex by inhibiting bcl-2, and to analyze the molecular mechanisms of EMAP-II-induced autophagosome formation. Finally, we will investigate the potential mechanisms of FOXO1 on the transcriptional regulation of Atg2B. Meanwhile, the regulatory effects of FOXO1 on miR-372, and the regulation of miR-372 on Atg7 expression and function will also be investigated to make clear how EMAP-II promotes the autophagosome formation. The project will provide a new strategy to enhance the therapeutic efficacy for glioma.

前期研究发现EMAP-II在抑制人脑胶质瘤干细胞(GSCs)增殖、迁移和侵袭的同时能促进LC3-I向LC3-II转变，上调beclin1、Atg2B和Atg7的基因和蛋白表达，3-MA减弱了EMAP-II引起的GSCs死亡。通过系列前期工作推测，EMAP-II可能通过抑制PI3K/Akt/mTOR信号通路，调控上述自噬相关基因的表达，促进GSCs自噬性死亡。本项目拟先研究EMAP-II是否通过抑制PI3K/Akt/mTOR信号通路直接促进GSCs自噬性死亡；明确EMAP-II是否通过抑制bcl-2，上调beclin1表达，诱导自噬体形成的机制；最后研究EMAP-II作用下FOXO1对Atg2B的转录调节效果及机制，阐明miR-372对Atg7表达和功能的调控，明确EMAP-II促进自噬体形成的分子机制。本项目能明确EMAP-II促进GSCs自噬性死亡的机制，为脑胶质瘤的治疗提供新途径。

本项目通过系列研究在国内外率先发现并报道了EMAP-II诱导胶质瘤肝细胞(GSCs)自噬性死亡的作用效果与分子机制。本项目研究结果显示：(1) EMAP-II能够以剂量依赖方式显著抑制GSCs细胞的细胞活力、降低线粒体膜电位、诱导GSCs细胞S期阻滞，自噬抑制剂3-MA能够阻断EMAP-II的上述作用，而凋亡抑制剂Z-VAD则无影响，证明EMAP-II通过自噬途径抑制GSCs细胞的细胞活力。(2) 研究发现EMAP-II作用能诱导自噬体形成，增加LC3-II/LC3-I的比值，降低p62/SQSTM1的表达，诱导GSCs细胞自噬性死亡；并且证明EMAP-II能够通过抑制PI3K/AKT/mTOR信号通路直接诱导GSCs细胞自噬性死亡。(3) EMAP-II能够通过抑制PI3K/AKT/mTOR信号通路，下调bcl-2的表达，上调beclin-1的表达，并且增加beclin1与Vps34和beclin1与UVRAG的结合能力，进而诱导诱导GSCs细胞自噬性死亡。(4) EMAP-II能够通过抑制PI3K和AKT的表达，降低细胞浆中FOXO1的磷酸化水平，诱导FOXO1进入细胞核。FOXO1进入细胞核后，一方面通过与Atg2B启动子的‘‘TATTTT’’ DNA序列靶向结合，转录抑制Atg2B的表达，诱导GSCs自噬性死亡；另一方面通过显著降低miR-372的表达，上调Atg7的表达(Atg7是miR-372的靶基因，miR-372能够靶向转录抑制Atg7的表达)，诱导GSCs细胞自噬性死亡。(5) 在裸鼠脑胶质瘤模型上，证明了EMAP-II能够抑制PI3K/AKT/mTOR信号通路，上调细胞核中FOXO1的表达，下调miR-372的表达，这与细胞水平的实验结果相一致。(6) 在裸鼠脑胶质瘤模型上，证明EMAP-II分别与PI3K、Akt、mTOR的特异性抑制剂、以及Ad-FOXO1和/或LNA anti-miR-372联合应用，能够显著抑制裸鼠脑胶质瘤的生长，增强化疗药物卡铂的治疗效果。Ad-FOXO1和LNA anti-miR-372单独以及联合应用具有潜在的临床应用价值。本项目的研究结果不仅明确了EMAP-II能够通过PI3K/Akt/mTOR通路直接或通过自噬相关因子Atg7和Atg2B诱导GSCs自噬死亡的分子机制，而且为脑胶质瘤的治疗提供了新途径，为切实