GM-CSF通过谷氨酰胺代谢促进肾透明细胞癌转移的机制研究

Metastatic Renal Cell Carcinoma usually has a pivotal influence on RCC prognosis. Our preliminary study showed expression level of GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor) was associated with metastasis and overall survival of clear cell renal cell carcinoma (CCRCC). GM-CSF could boost glutamine metabolism via enhancing the expression of GOT2 (Glutamic-oxaloacetic transaminase), which could promote invasion and metastasis of CCRCC. The detailed mechanism that GM-CSF reprograming glutamine metabolism is still not clear. Besides, we found GM-CSF could enhance transcription activity of ATF4. GOT2 promoter has a potential binding site for ATF4 that was analyzed by bioinformatic resources. Therefore, we propose a hypothesis: GM-CSF enhances glutamine metabolism by activating ATF4/GOT2 signal axis, then promotes metastasis of CCRCC. We plan to knock out GM-CSF in CCRCC cell lines by using CRISPR/Cas9 and construct an orthotopic metastasis model of CCRCC to understand the role of GM-CSF in the metastasis progress of CCRCC. We would use dual luciferase-report system and CHIP-qPCR to figure out if ATF4 could bind to GOT2 promoter. Hope this project could provide theoretical basis for GM-CSF as a future therapeutic target or prognosis factor of CCRCC.

肾透明细胞癌（CCRCC）发生转移是影响其预后的关键。申请人前期研究发现，粒细胞-巨噬细胞刺激因子（GM-CSF）与CCRCC转移和预后密切相关，GM-CSF能增强谷氨酸-草酰乙酸转氨酶2（GOT2）表达，促进CCRCC谷氨酰胺代谢，调控其侵袭和转移，但具体机制尚不清晰；同时发现GM-CSF激活转录因子ATF4，增强GOT2表达，而GOT2启动子含有ATF4潜在结合位点。因此，本项目提出假说：GM-CSF激活ATF4/GOT2信号轴，增强谷氨酰胺代谢，促进CCRCC侵袭和转移。我们拟通过敲除和过表达GM-CSF，构建CCRCC原位转移模型，明确GM-CSF在体内外对CCRCC转移的影响；使用双荧光素酶报告基因以及CHIP-qPCR等实验明确ATF4是否在转录水平调控GOT2表达，重编谷氨酰胺代谢，促进肾CCRCC侵袭和转移，以期为GM-CSF作为CCRCC的分子靶点和预后因子提供理论依据。

在缺氧微环境中，肾癌细胞自分泌GM-CSF，促进肾癌细胞侵袭喝转移，进一步在细胞、分子水平探究 GM-CSF 通过激活 ATF4/GOT2/IDH2，调控谷氨酰胺代谢，在缺氧时，GM- CSF不仅通过ATF4增强肾癌细胞糖酵解速率以及谷氨酰胺的代谢速率，为肾癌细胞供能，还保护线粒体功能，帮助肾癌细胞在缺氧环境中存活，同时通过上调PD- L1介导免疫逃逸。