Nogo-A介导氧化还原成瘾信号在恶性胶质瘤发生发展中的作用机制研究

Redox addiction accompanying altered metabolism is one of the predominant phenotypes of malignant glioma, features that could be important in the development of new molecular-targeted drugs. Nogo-A, a myelin-derived neurite outgrowth inhibitor is highly expressed in human glioma samples and related to features of malignancies. Recently, we found that neuronal Nogo-A/amino-Nogo-A can antagonize cellular reactive oxygen species (ROS) generation through interaction with Prdx2. Interestingly, our preliminary data indicate that nucleolar Nogo-A can translocate to cytoplasmic stress granules upon oxidative stress and hypoxia in human U251 glioma cell. We hypothesize that Nogo-A may be a novel mediator of ROS addiction in glioma.This project is designed to investigate the functions of Nogo-A-ROS addiction axis in glioma tumor progression, combined with gene transfection, RNA interference, (co-)immunoprecipitation, protein sulfenylation, reporter gene assay and TAT-mediated protein transduction. We will determine the contribution of Nogo-A to tumor cell proliferation,survival,migration and invasion, with a special focus upon nucleolar-cytoplasmic translocation of Nogo-A due to ROS induction and hypoxia. We will also investigate the underlying molecular mechanisms,mainly focused on Nogo-A regulated ROS homeostastis,ribosomal biogenesis, sulfenylation of some key metabolic remodeling proteins such as PKM2,HIF1A and IDH1/2 in three human glioma cells of LN229,U251 and U87MG,which have very different endogenously expressed Nogo-A. Then we will study the biological roles of Nogo-A and some Cysteine-mutants of PKM2,HIF1A and IDH1/2 in glioma tumor proliferation, invasion and metastasis using a xenograft animal model. Thus, this proposed study will characterize the novel roles and pathways of Nogo-A-ROS addiction in glioma progression. The information gained should be of help in developing new therapeutic approaches to treat malignant glioma.

氧化还原成瘾及其引起的异常的代谢模式是恶性胶质瘤重要的生物学特征之一，是胶质瘤靶向药物设计的新方向。轴突生长抑制蛋白Nogo-A高表达于恶性胶质瘤且与胶质瘤的发生和发展关系密切。我们实验室新发现，神经元内的Nogo-A能够通过与Prdx2互作抑制细胞内的活性氧自由基（ROS）水平的增加。并且，在氧化应激和缺氧条件下时，胶质瘤细胞系U251中核仁定位的Nogo-A转位胞浆。基于该研究基础，本项目拟借助不同Nogo-A表达水平的人胶质瘤细胞系以及小鼠移植瘤模型，联合运用过表达、RNA干扰、免疫沉淀、蛋白氧化修饰、报告基因、蛋白转导等多种技术手段，系统研究Nogo-A-Redox addiction信号调控胶质瘤增殖、存活、迁移和侵袭等生物学功能，揭示该信号影响肿瘤代谢的新机制。该项目对于认识胶质瘤Nogo-A分子的功能和ROS addiction现象以及设计新的分子靶向药物等具有重要的意义。

胶质瘤作为颅内最常见的恶性肿瘤，因其高度的异质性和浸润性生长，预后差、易复发和发生治疗抵抗。尽管目前临床上采取以手术切除辅以放化疗的综合疗法，但是，恶性胶质瘤患者的中位生存期仍在12-15个月。胶质瘤干细胞已被广泛接受且被认为是导致胶质瘤复发和治疗抗性的主要源泉。氧化还原成瘾及其引起的异常的代谢模式是恶性胶质瘤重要的生物学特征之一，是胶质瘤靶向药物设计的新方向。因此，深入研究肿瘤细胞的氧化还原成瘾机制和针对该概念的靶向疗法对克服胶质瘤治疗抗性和改善患者预后具有重要的临床意义。. 本项目中，我们联合运用过表达、RNA干扰、免疫沉淀、报告基因、蛋白转导等多种技术手段，系统研究Nogo-A-Redox addiction信号调控胶质瘤增殖、存活、迁移和侵袭等生物学功能和机制。结果显示，敲减Nogo-A表达后会显著增加肿瘤细胞内的ROS水平，诱导肿瘤细胞和肿瘤干细胞发生恃铁死亡（ferroptosis）。而过表达Nogo-A会抑制细胞的增殖，给予TAT-M9蛋白可以降低细胞内的ROS，且诱导细胞死亡。临床前U87MG细胞小鼠移植瘤模型研究显示TAT-M9可以显著抑制肿瘤生长。研究中，我们还发现Nogo-A的受体信号Lingo1的抑制剂和拮抗肽Lingo1-ecto有非常好的杀灭肿瘤干细胞的作用。Lingo1-ecto的作用是通过整合素受体和EGFR，TrkB等RTK家族成员实现的。并且，我们通过干预细胞内BMI1和RIZ1等ROS的信号通路关键调控分子，可以对肿瘤和肿瘤干细胞的生长实现表观遗传编辑。. 通过过去五年来的系统研究，我们发现Nogo-A 作为细胞ROS调控的“平衡者”角色。这一点与ROS成瘾的作用相匹配。支持我们提出的 “Nogo-A及其信号分子介导胶质瘤细胞氧化还原成瘾和肿瘤发生发展”的科学假设。本项目在《Cancer Res》、《Pharmacology & Therapeutics》、 《Medicinal Research Review》和《Biomaterials》等重要杂志上发表SCI论文23篇，培养研究生3名。. 该项目研究成果对于认识胶质瘤Nogo-A分子的功能和ROS addiction现象打下了坚实基础。我们基于干预肿瘤细胞和肿瘤干细胞中ROS信号的基础和转化研究对于设计新的肿瘤分子靶向药物等具有重要的意义。