Abstract
Citrate, a substance being related to de novo fatty acid synthesis and tricarboxylic acid (TCA) cycle, has a pivotal role in cell survival. However, the molecular mechanisms that regulate intracellular citrate in triple-negative breast cancer (TNBC), especially under hypoxic condition, remain poorly understood. Here we find that hypoxia (1% O2) induces DNA damage-independent ATM activation (oxidized ATM) and suppression of oxidized ATM reduces intracellular citrate via decreasing the levels of phosphofructokinase (PFKP) and citrate synthase (CS), two key glucose metabolism-associated enzymes. Mechanistically, PFKP is regulated by HIF1A at the translational level, whereas CS is of posttranscriptional regulation by UBR5-mediated ubiquitination. Interestingly, accumulation of citrate in cytoplasm or exogenous citrate significantly enhances cell migration, invasion, and metastasis of hypoxic TNBC cells in vitro and in mice xenografts. The underlying mechanism mainly involves citrate-stimulated activation of the AKT/ERK/MMP2/9 signaling axis. Our findings unravel a novel function of oxidized ATM in promoting migration, invasion, and metastasis of TNBC.
Highlights
Breast cancer is a major cause of cancer mortality among women worldwide[1]
We reveal that DNA damage-independent ATM activation induces energy metabolism reprogramming (EMR) through HIF1A-mediated transcriptional upregulating of phosphofructokinase (PFKP) and UBR5-mediated ubiquitination degradation of citrate synthase (CS)
MMP2 and MMP9, two well-known proteins related with tumor invasion (Figure S8E–S8G), were decreased in hypoxic BT549 and Hs578T cells transfected with shATM, shPFKP, or shCS compared with their control cells (Fig. 8a), and increased in ATM knockdown BT549 and Hs578T cells under combined treatment with citrate under hypoxia (Fig. 8b)
Summary
Reagents, plasmids, and cell transfection BT549 and Hs578T were cultured in RPMI 1640 medium (Gibco-BRL, Australia) containing 10% fetal bovine serum (FBS) (Gibco-BRL, Australia) at 37 °C in humidified atmosphere containing 5% CO2 with 1% O2 or 21% O2. 105 cells in 2 ml RPMI 1640 per well and transfected with constructor and/or treated with specific reagents according to the designed experiments. All experiments were performed at least three times and the data were normalized by the cell numbers or protein content. After 8 h incubation, the parent cells were treated with or without KU60019 (10 μM) in RPMI 1640 medium with 10% FBS and all cells were continuously incubated for 12 h in hypoxia. After culture under normoxia for 24 h, parent cells were treated with 10 μM of KU60019 and incubated in 1% O2 for around 12 h, and 10 nM of MitoTracker Green FM dye (Invitrogen) were added to all cells and continuously incubated for 1 h at 37 °C before dying.
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