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

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, acetylCoA 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.