目的:癌细胞的特征是不受控制的增殖,这需要高水平的核苷酸,这些核苷酸是DNA合成和复制的基础。CAD(氨基甲酰磷酸合成酶2,天冬氨酸转碳淀粉酶和二氢乳清酶)是启动从头嘧啶合成的三功能酶,通常在癌细胞中增强,以保留嘧啶库进行细胞分裂。胶质瘤,代表大多数脑癌,高度依赖嘧啶等核苷酸来维持细胞的异常生长和增殖。以前报道CAD在神经胶质瘤中失调,但潜在的机制仍不清楚。
方法:用免疫印迹法测定正常脑细胞和三种胶质母细胞瘤(GBM)细胞系中CAD和CHIP(Hsc70-相互作用蛋白的羧基末端)蛋白的表达。慢病毒介导的靶蛋白或shRNA的表达用于特异性过表达或敲除CAD和CHIP。细胞计数,菌落形成,细胞凋亡和细胞周期测定用于评估CAD和CHIP在GBM细胞增殖和存活中的作用。使用免疫共沉淀和泛素化测定法来检查CHIP与CAD的相互作用以及CAD的泛素化。通过分析GlioVis数据库获得CAD和CHIP表达与GBM患者生存的相关性。
结果:在这项研究中,我们发现CAD在胶质瘤中的表达上调,与胶质瘤患者的肿瘤分级和生存率呈正相关。敲除CAD强烈抑制GBM细胞的细胞增殖和集落形成,表明CAD在GBM发病机制中的重要作用。机械上,我们首先发现CAD被K29连接的聚泛素化修饰,由E3泛素连接酶CHIP介导。通过与CAD交互和泛化,CHIP增强其蛋白酶体和溶酶体降解,这说明了CHIP在GBM细胞中的抗增殖作用。为了维持CAD的表达,芯片大幅下调,这与GBM患者的不良预后和生存有关。值得注意的是,低水平的CHIP和高水平的CAD预测GBM患者的短生存期。
结论:总而言之,这些结果说明了CAD在GBM中的重要作用,并揭示了CAD阳性和CHIP阴性癌症的新治疗策略.
OBJECTIVE: Cancer cells are characterized as the uncontrolled proliferation, which demands high levels of nucleotides that are building blocks for DNA synthesis and replication. CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase and
dihydroorotase) is a trifunctional enzyme that initiates the de novo pyrimidine synthesis, which is normally enhanced in cancer cells to preserve the pyrimidine pool for cell division. Glioma, representing most brain cancer, is highly addicted to nucleotides like pyrimidine to sustain the abnormal growth and proliferation of cells. CAD is previously reported to be dysregulated in glioma, but the underlying mechanism remains unclear.
METHODS: The expression of CAD and CHIP (carboxyl terminus of Hsc70-interacting protein) protein in normal brain cells and three glioblastoma (GBM) cell lines were measured by immunoblots. Lentiviruses-mediated expression of target proteins or shRNAs were used to specifically overexpress or knock down CAD and CHIP. Cell counting, colony formation, apoptosis and cell cycle assays were used to assess the roles of CAD and CHIP in GBM cell proliferation and survival. Co-immunoprecipitation and ubiquitination assays were used to examine the interaction of CHIP with CAD and the ubiquitination of CAD. The correlation of CAD and CHIP expression with GBM patients\' survival was obtained by analyzing the GlioVis database.
RESULTS: In this study, we showed that the expression of CAD was upregulated in glioma, which was positively correlated with the tumor grade and survival of glioma patients. Knockdown of CAD robustly inhibited the cell proliferation and colony formation of GBM cells, indicating the essential role of CAD in the pathogenesis of GBM. Mechanistically, we firstly identified that CAD was modified by the K29-linked polyubiquitination, which was mediated by the E3 ubiquitin ligase CHIP. By interacting with and ubiquitinating CAD, CHIP enhanced its proteasomal and lysosomal degradation, which accounted for the anti-proliferative role of CHIP in GBM cells. To sustain the expression of CAD, CHIP is significantly downregulated, which is correlated with the poor prognosis and survival of GBM patients. Notably, the low level of CHIP and high level of CAD overall predict the short survival of GBM patients.
CONCLUSIONS: Altogether, these results illustrated the essential role of CAD in GBM and revealed a novel therapeutic strategy for CAD-positive and CHIP-negative cancer.