NEK2调控NIK/NF-κB信号转导通路促进胶质瘤干细胞增殖及放疗抵抗的机制研究

Accumulating evidence suggests that glioma stem-like cells (GSCs), which are critical therapeutic targets related to tumor proliferation, infiltration and therapy resistance in glioblastoma (GBM), are a small subset of cells within GBM tumor and are functionally defined by enhanced tumorigenicity and multipotency to generate heterogeneous tumor cells in vivo. In the preliminary experiments, we found that NIMA-related kinase 2 (NEK2) was essential to proliferation and radio-resistance in GSCs. Additionally, Gene Ontology and KEGG analysis indicated that NIK/NF-κB signaling pathway (Nuclear factor-κB-inducing kinase/Nuclear factor-κB signaling pathway) was one of the most correlated activated pathway to NEK2. Moreover, immunoprecipitation results indicated a formation of NEK2/NIK protein complex, however, knock-down of NEK2 reduced phosphorylation level and protein stability of NIK. Based on these findings, we hypothesize that NEK2 promotes kinase activity and enhances protein stability of NIK via phosphorylation of NIK, thus activates NF-κB signaling pathway and promotes proliferation and radio-resistance of GSCs. To this end, we first plan to establish NEK2 knock-down GSCs by using lentivirus infection and investigate the functional role of NEK2 in activation of NIK/NF-κB signaling pathway, as well as the promotion of proliferation and radio-resistance in GSCs by using Real-time PCR, Western blotting and immunofluorescence. Moreover, we plan to identify the novel phosphorylation site of NIK via co-immunoprecipitation followed by mass spectrometry. Furthermore, GST-pull down assay and in vitro kinase phosphorylation assay will be performed to clarify the mechanism of NEK2 kinase activity-dependent stabilization and phosphorylation of NIK, in purpose to clarify the biological function of NEK2/NIK/NF-κB axis in GSCs.

胶质瘤干细胞（GSCs）是胶质母细胞瘤（GBM）中一类具有自我更新和多向分化能力的细胞，对GBM的增殖及放、化疗抵抗具有重要意义。前期研究发现NEK2能够促进GSCs增殖并诱导其产生放疗抵抗性；蛋白免疫沉淀实验证实NEK2能够和NIK形成蛋白复合体，敲低NEK2后NIK的磷酸化程度和蛋白稳定性显著下调。据此，我们提出假设：NEK2可能通过介导NIK特异性位点的磷酸化，激活NIK激酶活性并增强其蛋白稳定性，进而激活NF-κB信号转导通路，最终促进GSCs的增殖并诱导其产生放疗抵抗性。因此，本研究拟利用慢病毒转染构建的NEK2敲低GSCs验证NEK2激活NIK/NF-κB信号转导通路并增强GSCs放疗抵抗性的作用，明确NEK2磷酸化NIK的特异性位点并进一步研究NEK2激酶活性对NIK磷酸化及蛋白稳定性的作用，阐释NEK2参与NIK蛋白修饰及调控NIK/NF-κB信号转导通路的分子生物学机制。

胶质母细胞瘤（Glioblastoma，GBM）是中枢神经系统最具侵袭性的肿瘤之一，具有浸润性生长、易复发、死亡率高等特点，是对人类健康威胁最大的恶性肿瘤之一。目前GBM的标准治疗策略包括最大安全切除、放疗和替莫唑胺（TMZ）化疗，但其治疗效果一直不能取得根本性的提高，大多数GBM患者仍在短时间内出现复发。为深入探索GBM恶性生物学行为调控的分子生物学机制并寻找可能的分子治疗靶点，利用高通量测序和生物信息学分析，我们发现丝氨酸/苏氨酸蛋白激酶NEK2（NIMA Related Kinase 2）的表达在GBM中显著上调并且和GBM患者的预后生存以及肿瘤的恶性程度紧密相关；同时，细胞划痕实验、基质胶侵袭实验、细胞成球实验、克隆形成以及裸鼠原位胶质瘤模型的结果表明，下调GBM细胞中NEK2的表达能够有效抑制其增殖、迁移、侵袭及致瘤能力；反之，外源性过表达NEK2能够增强GBM细胞的恶性生物学行为；通过高通量测序和生物信息学技术对现有数据库进行分析，我们发现NEK2和NF-κB信号通路的激活显著相关；此外，NEK2敲低能够显著抑制GBM细胞的迁移、侵袭及体外致瘤能力，而NF-κB通路激动剂TNF-α处理能够部分逆转NEK2敲低带来的抑制效果，提示NEK2可能通过激活NF-κB通路参与GBM恶性生物学行为的调控；通过分子生物学实验我们发现，NEK2能够特异性磷酸化并增强NIK激酶的稳定性，进而参与IKK的磷酸化调控并通过非经典通路激活NF-κB信号通路及其下游分子靶点，最终增强GBM细胞的恶性生物学行为。综上所述，我们的研究结果证实了NEK2通过磷酸化NIK进而增强NIK蛋白的稳定性，并通过非经典途径激活NF-κB信号通路，进而增强GBM的恶性生物学行为并最终导致肿瘤复发，可作为GBM的预后相关分子标记物和靶向治疗药物的潜在靶点。