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Targeting ATF4-dependent pro-survival autophagy to synergize glutaminolysis inhibition
Theranostics ( IF 12.4 ) Pub Date : 2021-7-25 , DOI: 10.7150/thno.60028
Shuting Han 1 , Liyuan Zhu 2 , Yiran Zhu 2 , Yuan Meng 2 , Jiaqiu Li 1 , Ping Song 1 , Neelum Aziz Yousafzai 3 , Lifeng Feng 2 , Miaoqin Chen 2 , Yanmei Wang 1 , Hongchuan Jin 2 , Xian Wang 1
Affiliation  

As glutamine plays a central role in cancer metabolism, inhibition of glutaminolysis has become an ideal anticancer therapeutic target. However, glutaminolysis inhibition leads to activation of autophagy, which compromises its antitumor effect. Hence, we investigated the mechanism underlying glutaminolysis inhibition-induced pro-survival autophagy./nMethods: High-throughput sequencing was performed on colorectal cancer (CRC) cells before and after glutaminolysis inhibition to identify differentially expressed genes. Activating transcription factor 4 (ATF4) pathway enrichment in glutaminolysis inhibited cells was identified through gene set enrichment analysis. ATF4 expression was assessed by quantitative real-time PCR (qRT-PCR) and western blotting. The function of ATF4 on mechanistic target of rapamycin (mTOR) regulation was assessed by western blotting. Luciferase reporter assays and chromatin immunoprecipitation were used to confirm the regulation of DNA damage inducible transcript 4 (DDIT4) by ATF4. mRNA half-life assays, RNA immunoprecipitation, qRT-PCR and western blotting were performed to determine the relationship between FTO alpha-ketoglutarate dependent dioxygenase (FTO), YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), and ATF4. ATF4 regulation of pro-survival autophagy was measured by tandem monomeric red fluorescent protein-green fluorescent protein fluorescence microscopy. Finally, the synergistic effect of autophagy and glutaminolysis inhibition was analyzed in an azoxymethane/dextran sodium sulfate mouse model./nResults: The ATF4 pathway was activated in CRC cells upon glutaminolysis inhibition. Functionally, ATF4 transcriptionally upregulated DDIT4 to suppress mTOR, which induced pro-survival autophagy during glutaminolysis inhibition. Interestingly, glutaminolysis inhibition promoted ATF4 mRNA expression by abrogating N6-methyladenosine (m6A) modification and YTHDF2-mediated RNA decay. Finally, inhibition of ATF4-induced autophagy enhanced the antitumor efficacy of glutaminolysis inhibition./nConclusion: Glutaminolysis inhibition upregulated ATF4 expression in an m6A-dependent manner to activate pro-survival autophagy through transcriptional activation of the mTOR inhibitor DDIT4. Targeting ATF4-induced autophagy is a new strategy to synergize glutaminolysis-targeting therapies for cancer treatment.

中文翻译:

靶向 ATF4 依赖性促生存自噬以协同抑制谷氨酰胺分解

由于谷氨酰胺在癌症代谢中起核心作用,抑制谷氨酰胺分解已成为理想的抗癌治疗靶点。然而,抑制谷氨酰胺分解会导致自噬激活,从而损害其抗肿瘤作用。因此,我们研究了谷氨酰胺分解抑制诱导的促存活自噬的机制。/n方法:在谷氨酰胺分解抑制前后对结直肠癌 (CRC) 细胞进行高通量测序,以鉴定差异表达的基因。通过基因集富集分析确定了谷氨酰胺分解抑制细胞中激活转录因子 4 (ATF4) 途径的富集。通过定量实时 PCR (qRT-PCR) 和蛋白质印迹评估 ATF4 表达。通过蛋白质印迹评估 ATF4 对雷帕霉素 (mTOR) 调控机制靶点的功能。荧光素酶报告基因检测和染色质免疫沉淀用于确认 ATF4 对 DNA 损伤诱导转录物 4 (DDIT4) 的调节。进行 mRNA 半衰期测定、RNA 免疫沉淀、qRT-PCR 和蛋白质印迹以确定 FTO α-酮戊二酸依赖性双加氧酶 (FTO)、YTH N 之间的关系6-甲基腺苷 RNA 结合蛋白 2 (YTHDF2) 和 ATF4。通过串联单体红色荧光蛋白-绿色荧光蛋白荧光显微镜测量促生存自噬的 ATF4 调节。最后,在偶氮甲烷/葡聚糖硫酸钠小鼠模型中分析了自噬和谷氨酰胺分解抑制的协同作用。/n结果:在抑制谷氨酰胺分解后,CRC 细胞中的 ATF4 通路被激活。在功能上,ATF4 转录上调 DDIT4 以抑制 mTOR,这在谷氨酰胺分解抑制期间诱导促生存自噬。有趣的是,谷氨酰胺分解抑制通过消除 N 6 -甲基腺苷 (m 6A) 修饰和 YTHDF2 介导的 RNA 衰变。最后,抑制 ATF4 诱导的自噬增强了谷氨酰胺分解抑制的抗肿瘤功效。/n结论:谷氨酰胺分解抑制以 m 6 A 依赖性方式上调ATF4表达,通过 mTOR 抑制剂 DDIT4 的转录激活激活促生存自噬。靶向 ATF4 诱导的自噬是一种协同谷氨酰胺分解靶向疗法用于癌症治疗的新策略。
更新日期:2021-08-15
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