CAND1通过STAT3调控胶质瘤干细胞干性机制研究

Glioma stem cells (GSCs) are the origin of glioma occurrence, recurrence, metastasis and drug resistance. Effective removal of GSCs is difficult in glioma therapy. The stemness maintenance of GSCs depends on the deregulation of key molecules, thus to explore the deregulation mechanism of these moleucles might provide new ideas for the glioma therapy. Previous studies suggested that hyperactivation of Signal Transducer and Activator of Transcription 3 (STAT3) played key roles in the stemness maintenance of GSCs. Our studies indicated that downregulation of STAT3 ubiquitin degradation led to significantly higher levels of its expression and phosphorylation in glioma. More importantly, we found CAND1, a noval interaction protein with STAT3, might be involved in regulation of the ubiquitin degradation of STAT3. Previous studies have implied that CAND1 is closely related to the occurrence of cancer. However, whether CAND1 is involved in GSC stemness maintenance has not been reported previously. Our studies showed that CAND1 was downregulated in the GSC and overexpression of CAND1 promoted the ubiquitin degradation of STAT3 and inhibited migration and neurosphere formation of glioma cell line. In this project, we plan to investigate the ubiquitin degradation mechanism of STAT3 regulated by CAND1 through overexpression，downregulation and deletion mutation and further elucidate the roles of CAND1 in the GSC stemness maintenance and the pathogenesis of glioma by in vivo and in vitro experiments. If successful, this study may provide important clues in understanding of the characteristics of glioma and new targets for its treatment.

胶质瘤干细胞（GSC）是胶质瘤发生、复发、转移和耐药的根源，有效清除GSC是胶质瘤治疗的难点。GSC的干性维持依赖于关键分子的失调，研究其失调的机制有望为胶质瘤的治疗提供新思路。研究显示STAT3过激活在GSC的干性维持中起重要作用。我们初步研究提示胶质瘤中STAT3泛素化降解下调引起其含量和磷酸化水平升高，进一步发现了可能调控其泛素化降解的新结合蛋白CAND1。已有研究显示CAND1与肿瘤的发生密切相关，但其是否参与GSC的干性维持尚未见报道。本研究已证实GSC中CAND1下调，其过表达促进STAT3泛素化降解，降低胶质瘤细胞的爬行能力和神经球形成能力。本课题拟通过过表达、下调、缺失突变等方法阐明CAND1调控STAT3泛素化降解的机制；利用体内外实验，阐明CAND1在GSC干性维持和胶质瘤发生发展中的作用机制。如期完成，有望加深对GSC干性和胶质瘤发生发展的认识，为其治疗提供新思路。

胶质瘤干细胞（GSC）是胶质瘤复发和耐药的根源, GSC的干性维持依赖于关键分子的失调，研究其失调机制有望为胶质瘤的治疗提供新思路。STAT3过激活在GSC的干性维持中起重要作用， STAT3泛素化降解下调导致其积累并过激活促进胶质瘤发生发展，但机制尚不清楚。在前期研究的基础上，本课题主要探索了参与STAT3泛素化降解的新调控子CAND1的调控机制。结果：①胶质瘤中CAND1表达下调，而STAT3表达上调，提示其可能参与STAT3的泛素化降解。②CAND1通过与STAT3的SH2结构域相互作用参与STAT3的泛素化降解调控，改变CAND1和STAT3的SH2结构域相互作用则功能丧失。③在胶质瘤细胞系或原代GSC中分别稳定下调和过表达CAND1，结果提示过表达CAND1抑制GSC增殖、肿瘤神经球的形成、提高其对化疗药物替莫唑胺的敏感性、降低干细胞标志物表达，促进细胞凋亡；而下调则结果相反。④过表达CAND1促进TMF/ARA160与Elongin C的结合减少TMF/ARA160的降解，并形成功能性E3识别底物STAT3从而促进STAT3的泛素化降解。⑤建立了立体定位注射模型，结果提示过表达CAND1抑制肿瘤的发生，延长生存期并提高小鼠的生存率，提高GSC分化能力同时降低干细胞标志物的表达。⑥分析了参与STAT3表达调控的LncRNA及其调控机制，揭示了多层次调控的新机制。意义：基本阐明了CAND1调控STAT3泛素化降解的机制及其在胶质瘤发生发展中的作用，为其治疗提供了新的潜在靶点。