以去泛素化酶UCH37为靶点治疗脑胶质瘤恶性增殖的分子机理研究

Malignant glioma is the most common primary brain tumor and is very refractory to treatment with high rate of recurrence and mortality. Lacking of significant progresses in therapeutics, it is of very high priority to elucidate mechanisms underlying gliomagenesis and explore novel therapeutic approaches and/or targets. Insights from our earlier tissue microarray profiling and bioinformatics analysis suggest that the expression of a group of genes including deubiquitinating enzymes UCH37 and USP14, and E3 ubiquitin ligase CHIP positively correlated with the degree of glioma malignancy and may play crucial roles in gliomagenesis and/or disease progression. Importantly, the small molecule inhibitor of UCH37 and USP14 is now under Phase II clinical trial to treat chronic myeloid leukemia. Thus, in the present study, we first wish to utilize both established cell lines and primary glioma cells and carry out functional and mechanistic dissection of UCH37 in gliomagenesis, proliferation, invasion, drug sensitivity and disease relapse. Moreover, working with both in vitro primary cell culture and in vivo exnograft model, we are keen to evaluate the efficacy and the potential usage of UCH37 inhibitor in treating glioma. Positive outcomes shall not only strongly indicate a crucial role for UCH37 in gliomagenesis, but also provide a novel drug target and viable individualized treatment for patients with the related lesions.

恶性胶质瘤是最常见的难治性神经系统恶性肿瘤，有易复发和高死亡率的特性，使现有治疗方式和理念一直缺乏突破性进展，阐明脑胶质瘤发生分子机理，开拓新干预靶点是该领域非常迫切的前沿课题。前期课题组提炼胶质瘤芯片数据和信息分析发现去泛素化酶UCH37和USP14表达与恶性程度密切相关，调控胶质瘤发生发展，同时UCH37和USP14特异性抑制剂金诺芬正开展临床II抗肿瘤试验性治疗。项目拟用慢病毒稳转胶质瘤细胞株、RNA干扰靶蛋白表达siRNA及回复技术、蛋白芯片、免疫共沉淀和与microRNA相关调控等方法研究UCH37参与胶质瘤增殖侵袭、放化疗敏感中上下游调控分子、蛋白信号通路和恶性生物学行为的分子机制；在此基础上，构建裸鼠荷瘤动物模型在体内研究去泛素化酶抑制剂潜在的治疗价值，评价UCH37作为治疗靶点及判断胶质瘤生存期及复发独立因子的可行性，为个体化治疗选择提供分子依据，有重要临床价值。

研究背景：恶性胶质瘤是最常见的难治性神经系统恶性肿瘤，有易复发和高死亡率的特性，使现有治疗方式和理念一直缺乏突破性进展，阐明脑胶质瘤发生分子机理，开拓新干预靶点是该领域非常迫切的前沿课题，研究者前期已通过分析临床数据、免疫组化芯片，发现UCH37在脑胶质瘤中表达上调，其表达高低可判断胶质瘤患者预后。因此，UCH37可作为胶质瘤的预后靶标和潜在治疗靶点，需要进一步在原代细胞中研究其促进肿瘤恶性增殖与侵袭的机制，并探究其成为治疗靶点的潜在可能性。.研究内容：本课题通过在细胞系和原代细胞中调控UCH37的表达，研究其对脑胶质瘤细胞恶性生物学行为影响，初步探究了小分子抑制剂b-AP15在体外的抗肿瘤作用，并通过酵母双杂法，筛选并鉴定去泛素化酶UCH37可能互作的关键蛋白，并对其在关键肿瘤信号通路中的影响进行研究。.研究结果：在胶质瘤细胞系和原代细胞中敲减和敲除UCH37后，胶质瘤细胞的细胞增殖能力减弱，G1期细胞比例明显上升，抑制了细胞迁移和侵袭能力，且小分子抑制剂b-AP15具有体外抑制胶质瘤细胞恶性增殖的能力；通过酵母双杂交，课题组筛选出UCH37可能互作的蛋白23个，并挑选SESN1作为候选蛋白进行免疫共沉淀验证。结果提示UCH37蛋白可与SESN1互作，且SESN1蛋白表达与UCH37正相关，而酶突变失活的UCH37C88A则无法改变SESN1的蛋白表达，提示SESN1的蛋白稳定可能是通过UCH37的去泛素化作用导致的。UCH37的表达可影响细胞迁移与侵袭通路中关键蛋白的变化。敲减UCH37后的细胞侵袭表型可被过表达SESN1回复。.研究结论与意义：去泛素化酶UCH37在胶质瘤中作为癌基因，可促进胶质瘤恶性增殖、迁移与侵袭，且小分子靶向抑制剂b-AP15具有体外抗肿瘤作用，可作为潜在药物靶点；本研究首次筛选并鉴定UCH37互作蛋白SESN1，UCH37可通过稳定SESN1促进胶质瘤恶性迁移与侵袭。