天冬酰胺内肽酶通过微囊泡转运驯化基质细胞促进胶质母细胞瘤发展的机制研究

Crosstalk between tumor cells and stromal cells in tumor microenvironment is crucial for tumor progression. In recent years, tumor cell derived microvesicles have been found as one of the key communicators between tumor cells and stromal cells in malignant glioma - glioblastoma (GBM). We previously found a novel protease- asparaginyl endopeptidase (AEP) was highly enriched in serum of patients who suffered from GBM. And the amount of AEP was associated with the poor prognosis of patients. Further animal studies showed the importance of AEP in GBM progression. More interestingly, the circulating AEP in serum was found largely in microvesicles, and these AEP containing microvesicles were captured by stromal cells including astrocytes and endothelial cells. Therefore, in this study we would like to systematically investigate the function and molecular mechanisms of microvesicle’s AEP in modulating stromal cells especially focusing on astrocytes and endothelial cells. The biochemical studies found that engulfed AEP in stromal cells can specifically degraded wild-type P53 protein and eventually promoted the survival and proliferation abilities. Meanwhile, the endocytosed AEP co-localized with integrin on the cellular membrane which helps degradation of extracellular matrix such as fibronectin. Altogether, this study will illustrate the functions and molecular mechanisms of GBM cell derived microvesicle’s AEP in GBM progression and might provide a novel therapeutic target in GBM therapy.

肿瘤细胞对其微环境中间质细胞的驯化是肿瘤发展的关键。近年研究提示，肿瘤细胞分泌的微囊泡是恶性脑胶质瘤-胶质母细胞瘤（GBM）影响间质细胞功能的重要方式之一。我们的前期工作发现一种独特的天冬酰胺内肽酶（AEP）在GBM患者的血清中大量存在，与患者预后密切相关;并证实其是GBM发生发展的重要因子。深入研究发现AEP富集于GBM细胞分泌的微囊泡中；且此微囊泡被微环境中的间质细胞所摄取。故本项目拟进一步研究GBM微环境中的间质细胞摄取富含AEP的微囊泡后的功能变化及其分子机制。预初工作发现，内吞的AEP通过特异酶切失活野生型P53蛋白，促进间质细胞存活和增殖；不仅如此，摄取的AEP和细胞膜表面的整合素结合，降解胞外纤连蛋白，由此促进GBM的侵润。因此，本研究将揭示GBM中肿瘤细胞通过分泌富含AEP的微囊泡使得间质细胞发生恶性功能转变，促进GBM发展的新分子机制，为GBM的治疗提供新的潜在靶标。

揭示胶质母细胞瘤（Glioblastoma，GBM）发生发展的核心通路、发现新治疗靶标一直是神经肿瘤领域的研究难重点。申请者在前期国自然基金-青年项目的研究中发现天冬酰胺内肽酶（AEP）在GBM组织中高表达，且与患者的不良预后密切相关。本课题从底物蛋白P53出发，深入探讨GBM细胞的AEP通过微囊泡转运驯化基质细胞促进GBM发展的新机制。. 研究发现：1、AEP富集于IDH WT-GBM细胞分泌的微囊泡中；且此微囊泡被微环境中的基质细胞所摄取；2、AEP能够特异结合野生型P53，并于其第311位天冬酰胺位点特异性切割以失活P53功能，促进GBM细胞克隆形成、增殖和存活；3、被摄取的微囊泡AEP酶切失活肿瘤微环境中基质细胞包括血管内皮细胞，胶质细胞的野生型P53蛋白，协同促进GBM的恶性进展。. 不仅如此，本项目对AEP上游调控因子进行了新的探索，研究发现：1、假基因LGMNP1、circAEP（circ_0033009）在GBM中表达增高，且与患者预后密切相关；2、LGMNP1、circAEP可促进GBM细胞的增殖、侵袭等生物学行为；3、LGMNP1作为ceRNA分子竞争结合miR-495-3p，解除了该miRNA对下游AEP的抑制作用，促进AEP表达；circAEP能够竞争结合miR-127-3p，上调AEP，促进GBM恶性进展；4、在荷胶质瘤原位动物模型中验证了LGMNP1、circAEP通过miRNAs调控AEP，促进GBM生长。. 本研究重点探讨了蛋白酶AEP促进GBM恶性进展的新机制，为GBM的诊疗提供重要的理论基础和新策略。