TOPK调节细胞自噬起始信号通路在胶质母细胞瘤发生中的作用研究

Glioblastoma (GBM) is the most common malignant brain tumor and is very poor prognosis. It is very important to explore biomarker as therapeutic target for GBM. TOPK expressed in GBM stem cells promotes glioma initiating cell proliferation and GBM tumorigenesis, but the further mechanism is unknown. Our recent study indicated that: 1)The level of TOPK is related to clinical grading and malignancy of glioma; 2) Knockdown TOPK inhibited Akt-mTOR-autophagy pathway and glioma colony formation; 3) mTOR and ULK1 which are key molecules of autophagy are potential substrates of TOPK by bioinformatics prediction. Therefore, we hypothesize that “phosphorylation of mTOR and ULK1 by TOPK promotes GBM tumorigenesis by inhibiting autophagy initiation”. The following experiments will be performed: 1) Phosphorylation sites of mTOR and ULK1 by TOPK will be identified by in vitro kinase and LC/MS/MS; 2) TOPK phosphorylates mTOR and ULK1 and regulates its function and stability ex vivo; 3) Prepare specific anti-mTOR and anti-ULK1 phsphorylated antibodies; 4) The activity of autophagy, growth and proliferation of stable mutant ULK1 and mTOR cells with high- or low-TOPK would be observed in vivo and ex vivo; 3.Test the expression pattern of TOPK in GBM patients and the possibility of TOPK serving as a biomarker for GBM targeting therapy and prognosis for GBM patients. Reveal of TOPK-mTOR-ULK1-autophagy signaling pathway will help understanding further the role of TOPK in GBM tumorigenesis , and provide more clues to overcome this enigma.

GBM干细胞表达的TOPK引起胶质瘤起始细胞增殖，促进GBM发病，但具体机制不明。申请人发现：TOPK的表达与胶质瘤临床分级及恶性程度正相关；沉默TOPK抑制Akt-mTORC1-自噬信号通路和GBM细胞克隆形成；生物信息学预测mTOR和自噬关键分子ULK1是TOPK潜在底物。本项目拟：1.经预测、突变及体内外激酶实验明确TOPK磷酸化mTOR和ULK1的位点及对其活性及稳定性影响，并制备相应位点特异性磷酸化抗体；2. 抑制、沉默或过表达TOPK，共转mTOR或ULK1磷酸化位点突变质粒，检测GBM细胞自噬活性、增殖能力，探明TOPK磷酸化mTOR和ULK1下调自噬活性促进GBM发生的机制；3.检测TOPK修饰的mTOR和ULK1磷酸化位点突变的GBM细胞在裸鼠的成瘤能力，进一步阐明TOPK-mTOR-ULK1信号通路在GBM发生中的作用。结果为GBM发生机制提供新线索、为防治提供新靶标。

胶质瘤是恶性程度最高的脑肿瘤，手术难以全切，对放化疗易产生耐受。自噬异常是胶质瘤对放化疗产生耐受的原因之一，但其调控的机制不明。研究表明，T-淋巴细胞来源的蛋白激酶（TOPK）在胶质瘤干细胞中特异性表达，与胶质瘤放化疗耐受、复发密不可分，但作用机制尚不清楚。我们研究发现：1）高级别胶质瘤高表达TOPK、P62，低表达LC3-II，且与预后、TMZ耐药密切相关；2）抑制TOPK活性和沉默TOPK表达，抑制肿瘤生长，促进LC3-II表达，抑制P62表达，促进自噬；而过表达TOPK，促进肿瘤生长，抑制LC3-II表达，促进P62表达，抑制自噬；3）ULK1是TOPK底物，且TOPK与ULK1的SPR结构域相互作用；4）TOPK直接磷酸化ULK1的Ser469、Ser495和Ser533三个位点且质谱确认ULK1的S469磷酸化修饰在细胞内存在；5）抑制TOPK，增强ULK1活性和稳定性；而过表达TOPK，减弱ULK1活性和稳定性；6）ULK1的Ser469、Ser495和Ser533三个位点失活突变后，ULK1的活性增加，半衰期延长，并且导致细胞的LC3-II表达上调；7）TOPK抑制剂和TMZ联用，促进高表达TOPK胶质瘤细胞凋亡，抑制细胞增殖和克隆形成能力；用TMZ处理TOPK沉默细胞株，获得相同结果。8）稳定表达Ser469、Ser495和Ser533三位点失活突变ULK1的细胞株，用TMZ处理，细胞凋亡增加，细胞增殖及锚定非依赖生长能力降低。结果表明TOPK通过磷酸化促进ULK1降解进而抑制其活性，进一步抑制自噬起始促进胶质瘤生长和对TMZ耐药。