IDH1 R132突变体通过上调GLS增强谷氨酰胺代谢促进肿瘤的形成

Excessive glutamine metabolism is recognized as a distinctive feature in addition to Warburg effect in cancer. Alterations of oncogenes have been reported to be involved in glutamine metabolism regulation. Mutations at the R132 residue of IDH1 were frequently found in various cancers. To investigate whether these tumor associated mutations play roles in glutamine metabolism regulation, we construct IDH1 R132Q mutant knockin MEFs and found that IDH1 R132 mutant significantly enhanced glutamine metabolism in vitro. Further study showed that GLS protein level was dramatically increased in IDH1 R132 mutant cells. In this project, we will try to clarify the mechanism how GLS is up regulated by using IDH1 R132Q knockin MEFs and HT1080 fibrosarcoma cells which express endogenous IDH1 R132C mutant, and then to investigate whether and how GLS upregulation influences glutamine metabolism and whole cell metabolism. We will also investigate the effect of GLS upregulation on cellular ROS level, proliferation and apoptosis. Moreover,we will perform allograft tumor assays and collect glioma samples from patients to further dissect the mechanism by which IDH1 R132 mutant leads to tumorigenesis in glioma via upregulating GLS and glutamine metabolism.

谷氨酰胺代谢增强是肿瘤细胞除Warburg效应外又一重要的能量代谢方式。很多癌基因编码的蛋白产物参与了谷氨酰胺代谢的调控。IDH1 R132位点的突变与多种肿瘤的发生密切相关，为了探索该突变体是否调节谷氨酰胺代谢，我们构建了IDH1 R132Q基因敲入MEF细胞，并发现该突变体剧烈地增强了谷氨酰胺代谢。进一步研究表明该突变体能够显著上调谷氨酰胺酶（GLS）的蛋白水平。在本项目中，我们拟首先利用IDH1 R132Q MEF及携带IDH1 R132C天然突变的纤维肉瘤HT1080细胞，研究该突变体上调GLS的机理；进而研究GLS上调对谷氨酰胺代谢及整个细胞代谢的综合影响；以及GLS上调对IDH1突变细胞内ROS水平、细胞增殖和凋亡的影响；最后通过裸鼠成瘤模型，并结合对临床胶质瘤标本的研究揭示该突变体通过上调GLS增强谷氨酰胺代谢，导致肿瘤发生的机理。

异柠檬酸脱氢酶1（IDH1）R132位点的突变与多种肿瘤的发生发展密切相关。谷氨酰胺是人体内含量最丰富的自由氨基酸，也是生物合成中碳源和氮源的重要提供者，多数肿瘤细胞的生长和增殖依赖于谷氨酰胺代谢。然而IDH1 R132突变体是否调节了谷氨酰胺代谢还不清楚。在本项目中，我们发现IDH1 R132突变体通过产生2-HG显著上调谷氨酰胺酶（GLS）的表达，促进谷氨酰胺代谢。通过荧光定量PCR和蛋白半衰期测定以及相关过表达和敲减实验，我们发现IDH1 R132突变体通过c-Myc-miR23a/b-GLS信号通路影响GLS的翻译过程。在IDH1 R132突变体细胞中敲低GLS会显著削弱IDH1 R132突变体促进的谷氨酰胺代谢、DNA合成、细胞增殖。最后通过裸鼠成瘤实验和临床胶质瘤样品证明IDH1 R132突变体上调GLS促进了肿瘤的发生发展。综上，本项目的研究揭示了IDH1 R132突变体上调GLS的机理，为肿瘤的治疗提供了新的思路。