低氧下氧化型ATM通过胞内柠檬酸累积促进人乳腺癌细胞的迁移和侵袭

Oxidized ATM Promotes Migration and Invasion of Hypoxic Breast Cancer Cells via Intracellular Citrate Accumulation

该研究主要探讨低氧下共济失调毛细血管扩张突变(ataxia-telangiectasia mutated,ATM)蛋白激酶活化通过糖代谢重塑对人乳腺癌MCF7及MDA-MB-231细胞迁移和侵袭的影响。构建靶向ATM基因的慢病毒载体(shATM)及其阴性对照病毒(shNC),感染人乳腺癌MCF7和MDA-MB-231细胞,建立ATM基因稳定沉默细胞系。实验设阴性对照组(shNC)、ATM基因沉默组(shATM)以及ATM特异性抑制剂KU60019处理组,利用实时荧光定量PCR检测ATM下调效果;Western blot检测常氧及低氧下ATM、p-ATM、γ-H2AX、p-CHK2(T68)的蛋白表达水平,利用伤口划痕愈合及Transwell实验分别检测细胞迁移和侵袭能力的改变,用糖代谢相关试剂盒检测糖代谢过程中相关底物的变化。结果表明,低氧处理12 h时(此时尚未发生DNA损伤),人乳腺癌MCF7和MDA-MB-231细胞p-ATM(即氧化型ATM)表达量显著增加,其迁移和侵袭能力增加。糖代谢改变表现为葡萄糖消耗量、丙酮酸产量、乙酰辅酶A产量明显高于乳酸生成量;进而检测三羧酸循环(tricarboxylic acid cycle,TCA)产物,发现与柠檬酸相比,下游琥珀酸和延胡索酸产生了较大程度的累积。而shATM和KU60019细胞处理组能有效抑制p-ATM的表达量、细胞迁移和侵袭能力、葡萄糖消耗、丙酮酸产量、乙酰辅酶A产量,但对于乳酸产量的抑制作用并不明显;TCA中柠檬酸的产量的降低程度明显大于其下游琥珀酸和延胡索酸的降低量。在低氧下,shATM细胞中补充外源性柠檬酸,肿瘤细胞的迁移侵袭能力均得到一定程度回补。研究结果揭示,低氧情况下,氧化型ATM的表达量明显增加,并通过胞内柠檬酸的累积促进人乳腺癌细胞的迁移和侵袭。

This article was perfromed to investigate the effect of ATM (ataxia-telangiectasia mutated) protein kinase activation on the migration and invasion of human breast cancer cells (MCF7 and MDA-MB-231) via remodeling of glucose metabolism.MCF7 and MDA-MB-231 cells were infected with recombinant lentivirus vector (shATM) and its negative control (shNC) to establish cell lines of stable ATM gene silencing,and treated with KU60019 to downregulate the expression of p-ATM.All the cells were cultured in hypoxia (1% O2) or normoxia (21% O2).The expression of ATM mRNA was detected by quantitative Real-time PCR.The protien levels of ATM,p-ATM and DNA damage markers [γ-H2AX,p-CHK2(T68)] were evaluated by western blotting.The abilities of cell migration and invasion were separately analyzed by wound healing and transwell assay.The substrates related to glucose metabolism were measured according to instruction of reagent kits.Results showed that hypoxia (1% O2 12 h) induced the p-ATM expression without a notable effect of DNA damage.The DNA damage-independent ATM was called oxidized ATM.Moreover,cell migration and invasion abilities of hypoxic MCF7 and MDA-MB-231 significantly increased compared with corresponding normoxic cells.A moderate increased lactate was detected in cells after hypoxia treatment.However,remarkable enhanced glucose consumption,pyruvate production,acetyl-CoA production and lactic acid production were also observed in hypoxia.Furthermore,citrate had a greater accumulation than that of the downstream succinate and fumarate.After KU60019 treatment or ATM knockdown under hypoxia (1% O2 12 h),there were sharply decrease of p-ATM expression,the ability of cell migration and invasion,glucose consumption,pyruvate,acetyl-CoA production and citrate production,and a moderate reduce of lactate production,as well as no significant change of succinate and fumarate.The migration and invasion abilities of shATM cells were rescued by exogenous citrate treatment.The results showed that oxidized ATM significantly increased under the condition of hypoxia (1% O2 12 h),which promoted the migration and invasion of human breast cancer cells via intracellular citrate accumulation.