天冬酰胺内肽酶AEP促进多形性胶质母细胞瘤发生发展的作用及其机制研究

Glioblastoma multiforme (GBM) is the most common brain malignant tumor with poor prognosis. And aberrant P53 and RTK/PI(3)K pathways are identified in most, and perhaps all, GBM tissues through integrated analyses of multi-dimensional genomic data. The applicant had ever found that AEP is vital for tumor invasion and metastasis (Journal of National Cancer Institute, 2014). However, the functions and molecular mechanisms of AEP in GBM progression are unknown. Our preliminary experiments showed that AEP interacted with and specifically cleaved P53 upon EGFR activation, which inhibited expression of its downstream target genes, Bax and P21, resulting in losing of tumor-suppressor function. Meanwhile, we found that AEP was highly expressed in GBM tissues and was associated with poor prognosis. Based on these observations, in this project, we will use in vitro studies and in vivo animal models and human clinical samples to explore a novel mechanism- "AEP regulates P53 inactivation upon EGFR activation", which is important for GBM progression. And we further investigate the potential of AEP as a new target for GBM diagnosis and therapy. These original researches are significant as (1) they elucidate a novel mechanism of EGFR mediated P53 inactivation during GBM development; (2) they establish AEP as a new potential oncogene; (3) they imply AEP may serve as a biomarker for GBM diagnosis and targeted therapy.

多形性胶质母细胞瘤（GBM）是颅内最常见的恶性肿瘤,预后极差；目前研究揭示几乎全部GBM中均存在P53及RTK/PI3K通路的异常。申请人曾发现天冬酰胺内肽酶AEP对肿瘤侵袭与转移非常重要（Journal of National Cancer Institute,2014）。然而，AEP在GBM发展中的作用及其信号通路未有研究。预实验发现EGFR激活上调的AEP结合并特异的降解P53，干扰其下游靶基因Bax，P21等的转录，使之失去肿瘤抑制功能。临床样本检测发现AEP在GBM组织中高表达且与不良预后相关。本课题将在此基础上，利用体外实验和体内动物模型及临床标本，揭示GBM发生发展的新机制-“EGFR激活调控的AEP降解抑癌基因P53”，并探索AEP作为新型GBM诊治靶标的潜能。这些原创性研究将揭示一种全新的EGFR和P53通路间的互作及调控机制，为GBM的诊治提供重要的理论基础和策略。

胶质母细胞瘤（Glioblastoma，GBM）是最常见的恶性脑胶质瘤。GBM因侵润性强，和正常脑组织没有明显界线，手术难以彻底切除并对放化疗均不甚敏感，使得其复发率高，五年生存率仅3%左右，预后极差。美国癌症基因图谱研究（The Cancer Genome Atlas, TCGA）于2008年解析了209例GBM样本，发现在大部分甚至全部GBM中均存在RTK/PI(3)K，P53和Rb信号通路的异常，该研究揭示了GBM发生发展的核心通路，为GBM的治疗提供新的思路和靶点。因此，深入探索这些通路，发现新的分子机制及治疗靶标成为目前的难热点。天冬酰胺内肽酶（Asparaginyl Endopeptidase，AEP），是一种溶酶体蛋白酶，属于半胱氨酸蛋白酶C13家族，具有很高的保守性，且是哺乳动物中唯一已知的天冬酰胺内肽酶。不同于其他蛋白酶，AEP对底物的选择有严格的专一性，切割底物时要求底物分子的P1位点必须为天冬酰胺基团，提示其功能的特异性和重要性。在本项目中，通过我科的胶质瘤组织芯片，发现AEP在正常组织或低级别的胶质瘤中表达低，而在GBM组织中高表达；且与患者不良预后密切相关。不仅如此，脑胶质瘤患者血清和脑脊液中AEP的含量明显高于正常人，且高级别胶质瘤患者血清中AEP的含量又明显高于低级别胶质瘤。进而，在GBM细胞中敲低AEP后，GBM细胞的成瘤能力，生长和侵袭能力均受到影响。深入实验发现，EGF刺激或稳定表达EGFR的GBM细胞株中，EGFR的激活促使AEP的蛋白水平增加。反之，EGFR抑制剂（AG1478）或EGFR的敲低导致AEP的表达和活性均降低；且AEP能够直接结合野生型P53，并于其第311位天冬酰胺（N311）特异切割，从而破坏其正常功能。同时，我们和上海交通大学化学教研室刘坚华老师合作，合成针对AEP的特异小分子化合物。在颅内移植瘤模型中证实，AEP是促进GBM发生发展的重要因子和潜在靶标。综上所述，我们首次发现在p53野生型的GBM细胞中AEP特异切割失活野生型p53的机制，为GBM的诊治提供新的科学依据和潜在靶标。