Targeting ATF4-dependent pro-survival autophagy to synergize glutaminolysis inhibition.
Targeting ATF4-dependent pro-survival autophagy to synergize glutaminolysis inhibition.
复制标题
靶向 ATF4 依赖性促生存自噬以协同抑制谷氨酰胺分解
作者：
Han S;Zhu L;Zhu Y;Meng Y;Li J;Song P;Yousafzai NA;Feng L;Chen M;Wang Y;Jin H;Wang X
由于谷氨酰胺在肿瘤代谢中起着重要作用，因此抑制谷氨酰胺分解已成为理想的抗癌治疗靶点。然而，去甲氨醇分解抑制导致自噬的激活，这损害了其抗肿瘤作用。因此，我们研究了潜在的机制，抑制组胺诱导的促生存自噬。研究方法：对大肠癌（CRC）细胞在去氨酶抑制前后进行高通量测序，以鉴定差异表达的基因。通过基因集富集分析，鉴定了活化转录因子4（ATF 4）途径在氨解抑制细胞中的富集。通过定量实时PCR（qRT-PCR）和蛋白质印迹法评估ATF 4表达。通过蛋白质印迹法评估ATF 4对雷帕霉素（mTOR）调节的机制靶标的功能。荧光素酶报告分析和染色质免疫沉淀用于确认DNA损伤诱导转录本4（DDIT 4）的调节由ATF 4。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 N6-甲基腺苷RNA结合蛋白2（YTHDF 2）和ATF 4之间的关系。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ATF 4对促存活自噬的调节。最后，在氧化偶氮甲烷/葡聚糖硫酸钠小鼠模型中分析了自噬和氨解抑制的协同作用。结果：ATF 4通路在大肠癌细胞中被激活后，抑制氨解。在功能上，ATF 4转录上调DDIT 4以抑制mTOR，这在氨解抑制期间诱导促存活自噬。有趣的是，氨解抑制通过废除N6-甲基腺苷（m6 A）修饰和YTHDF 2介导的RNA衰变促进ATF 4 mRNA表达。最后，抑制ATF 4诱导的自噬增强了去甲氨蝶呤分解抑制的抗肿瘤功效。结论：谷氨酰胺分解抑制以m6 A依赖性方式上调ATF 4表达，以通过mTOR抑制剂DDIT 4的转录激活来激活促存活自噬。靶向ATF 4诱导的自噬是一种协同靶向药物治疗癌症的新策略。
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. Methods: 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. Results: 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. Conclusion: 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.