干预Skp2以增强胶质瘤细胞治疗敏感性的研究

Glioma is the leading brain tumor in adults and the patient prognosis was limited by treatment resistance greatly. It's been proved that the existence of glioma stem cells and the timely reparation of DNA damages are the main causes of treatment failure. Skp2 is the component of SCF complex, which serves as one of the important E3-ligase in the process of ubiquitination. Our group has demonstrated that Skp2 is involved in the regulation of pool size and self-renewal ability of hematopoietic stem cells, as well as breast cancer stem cells and nasopharyngeal carcinoma stem cell like cells. We also showed that Skp2 ubiquitinate NBS1, the components of MRN complex (Mre11/Rad50/NBS1), to recruit and activate ATM to initiate DNA damage reparation cascade. However, the exact role of Skp2 in the regulation of glioma stem cells and DNA damage reparation in glioma has not been elucidated yet. We propose here that targeting Skp2 could promote radio- and chemotherapy sensitivity through multiple pathways, including suppress function of glioma stem cells and attenuate the reparation of DNA double-strand breaks (DSBs) induced by alkylating reagents and ionizing radiation. We will adopt gene knockdown methods, statins, and small molecules to suppress the level of Skp2, and check pool size and self-renewal ability of glioma stem cell, and the reparation process of DNA DSBs upon radio/chemo treatments. In this way we can evaluate the role of Skp2 in repressing the glioma cell proliferation and subsequently triggering apoptosis. Our project will clarify the novel mechanisms about how Skp2 involved in the glioma radio- and chemotherapy resistance, which will provide a potential target to improve the radio/chemo therapeutic effect through targeting one single target. The fulfillment of our project will definitely provide scientific evidences in improving the prognosis of glioma patients in the near future.

放化疗抗拒是胶质瘤治疗的瓶颈。研究表明，胶质瘤干细胞的存在及DNA损伤的及时修复是胶质瘤治疗失败的重要原因。Skp2是泛素化修饰过程中的关键E3连接酶。我们已证实，Skp2调节造血干细胞及乳腺癌、鼻咽癌干细胞的数量和自我更新能力；Skp2能将MRN复合体的NBS1泛素化，进而募集和活化ATM，促进DNA损伤修复。然而，Skp2在胶质瘤干细胞以及DNA修复中的作用尚未见报导。因此我们推测靶向Skp2能够从多个角度同步增强胶质瘤细胞的放化疗敏感性。本研究将采用si/shRNA及Talen等基因敲除技术，及他汀类药物、小分子抑制剂等抑制Skp2，观察胶质瘤干细胞的数量和自我更新能力，放化疗引起的DNA双联断裂的修复等，从而评价靶向Skp2同步增敏放、化疗的潜力。本项目有望阐明胶质瘤对放化疗抵抗的新机制，为增敏放、化疗提供新的靶点，提供有效抑制剂，为改善胶质瘤患者的预后提供充分的科学依据。

胶质瘤是最常见的原发性中枢神经系统肿瘤，约占所有颅内原发肿瘤的一半。胶质母细胞瘤（glioblastoma, GBM）是成人最常见的原发恶性脑肿瘤，是所有肿瘤中致死性最强的肿瘤之一。GBM多呈浸润生长，增殖迅速，手术往往难以完全切除。因此，手术辅以放、化疗是目前GBM的主要治疗模式，但预后仍较差。Skp2是Skp2-SCF E3连接酶的重要组成分子，通过泛素化修饰途径，调控下游多种底物蛋白的功能与水平。Skp2是肿瘤发生发展中的关键分子，在多种肿瘤组织中呈现高表达，并与患者的不良预后相关，有望成为肿瘤治疗的新靶点。该研究中我们首先分析数据库中SKP2蛋白水平在胶质瘤中的表达情况（GEO, TCGA和GTEx），其次，我们分别进行体内外实验研究Skp2在对胶质瘤发生发展中的作用，并寻找Skp2的小分子抑制剂。研究发现与正常脑组织相比，Skp2的mRNA水平在低级别胶质瘤（LGG）和GBM中均升高，并且在LGG中与患者的预后呈负相关。胶质瘤细胞系中Skp2的表达水平均比正常永生化的星形细胞系的表达水平高；细胞体外生长减慢，克隆形成能力减弱，小鼠体内成瘤能力减弱。敲除Skp2之后，TMZ的细胞杀伤能力增强，IC50降低。动物实验证明敲除Skp2有利于增强肿瘤对TMZ的敏感性。另外，Skp2在胶质瘤干细胞中与胶质瘤干细胞标记分子Sox2和Nestin的表达趋势一致。敲除Skp2之后细胞的球形成能力减弱，细胞衰老增加。洛伐他丁（Lovastatin）是HMG-CoA还原酶的抑制剂，有报道称其可以一直Skp2的活性，但未在胶质瘤中得到证实。洛伐他丁能够降低Skp2的表达，从而上调Skp2下游p21和p27的表达；虽然SZL-P1-41并不影响Skp2的表达，但仍能够上调p21和p27的表达，间接说明SZL-P1-41可以抑制Skp2的功能发挥。体内外实验表明洛伐他丁和SZL-P1-41能够显著增强TMZ的胶质瘤细胞杀伤能力。我们的研究结果充分表明Skp2参与调解胶质瘤细胞的增殖和胶质瘤干细胞的维持，靶向抑制Skp2可以在体内外延缓胶质瘤细胞的增殖，并减弱TMZ的耐药性。小分子抑制剂洛伐他丁和SZL-P1-41能够在体内外促进细胞对TMZ的敏感性，有望成为临床应用的Skp2抑制剂，从而改善胶质瘤患者的生存及预后。