Purpose

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.

Conclusion

Altogether, these results illustrated the essential role of CAD in GBM and revealed a novel therapeutic strategy for CAD-positive and CHIP-negative cancer.

中文翻译：

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CHIP促进CAD泛素化和降解，抑制胶质母细胞瘤细胞的增殖和集落形成

目的

癌细胞的特点是不受控制的增殖，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（氨甲酰磷酸合成酶 2、天冬氨酸转氨甲酰酶和二氢乳清酶）是一种三功能酶，可启动嘧啶从头合成，这种合成在癌细胞中通常会增强，以保留用于细胞分裂的嘧啶库。代表大多数脑癌的神经胶质瘤高度依赖于嘧啶等核苷酸来维持细胞的异常生长和增殖。此前有报道称 CAD 在神经胶质瘤中失调，但其潜在机制仍不清楚。

方法

通过免疫印迹测量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 表达用于特异性过表达或敲低 CAD 和 CHIP。使用细胞计数、集落形成、细胞凋亡和细胞周期测定来评估 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检测来检查 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

在本研究中，我们发现胶质瘤中CAD的表达上调，与胶质瘤患者的肿瘤分级和生存呈正相关。CAD的敲除强烈抑制了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。从机制上讲，我们首先确定 CAD 是通过 K29 连接的多聚泛素化修饰的，该多聚泛素化是由 E3 泛素连接酶 CHIP 介导的。通过与 CAD 相互作用并使 CAD 泛素化，CHIP 增强了其蛋白酶体和溶酶体降解，这解释了 CHIP 在 GBM 细胞中的抗增殖作用。为了维持 CAD 的表达，CHIP 显着下调，这与 GBM 患者的不良预后和生存相关。值得注意的是，低水平的 CHIP 和高水平的 CAD 总体预测 GBM 患者的生存期较短。

结论

总而言之，这些结果说明了 CAD 在 GBM 中的重要作用，并揭示了针对 CAD 阳性和 CHIP 阴性癌症的新治疗策略。