Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism

Background: Cancer Stem Cells (CSCs) drive recurrence and therapeutic resistance in triple-negative breast Cancer (TNBC), a highly aggressive breast Cancer subtype. Intratumoral hypoxia, a common feature of solid tumors, promotes CSCs enrichment, yet the mechanisms sustaining CSCs stemness remain poorly understood. Hypoxia-induced Reactive Oxygen Species can oxidatively activate ataxia telangiectasia mutated (ATM) kinase (oxidized ATM, p-ATM) independently of DNA breaks.

Aim: To investigate the role of hypoxia-induced oxidized ATM in sustaining TNBC-CSC stemness through c-Myc-mediated regulation of one-carbon metabolism.

Methods: Hs578T and MDA-MB-231 TNBC cells were cultured under normoxia or hypoxia. CSC stemness was assessed by mammosphere assays and flow cytometry. ATM activity was assessed by pharmacological inhibition (Ku60019) and short hairpin RNA knockdown. c-Myc binding to serine hydroxymethyltransferase 2 (SHMT2) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) promoters was analyzed by dual-luciferase reporter assays and chromatin immunoprecipitation. NADPH/NADP+ ratios were quantified, and metabolic reprogramming was profiled by liquid chromatography-tandem mass spectrometry metabolomics.

Results: Hypoxia significantly increased mammosphere formation in both Hs578T and MDA-MB-231 cells, as reflected by higher numbers of mammospheres (Hs578T: 214 ± 18; MDA-MB-231: 198 ± 16; both P < 0.01) and larger mean diameters (P < 0.01). Hypoxia also elevated CD44+/CD24- cell proportions and stemness gene expression (P < 0.01). Oxidized ATM was activated under hypoxia without γH2AX induction, confirming DNA damage independence. ATM inhibition reduced mammosphere growth and suppressed c-Myc, SHMT2, and MTHFD2. Luciferase and chromatin immunoprecipitation assays confirmed direct c-Myc binding to SHMT2 and MTHFD2 promoters, while mutation of the binding sites abolished promoter activity. NADPH/NADP+ ratios were significantly elevated under hypoxia but reduced following ATM inhibition (P < 0.05). Metabolomics revealed enrichment of serine/glycine one-carbon pathways.

Conclusion: Hypoxia-induced oxidized ATM maintains TNBC-CSC stemness by promoting c-Myc-dependent upregulation of MTHFD2 and SHMT2, linking hypoxia, redox signaling, and one-carbon metabolism. These findings suggest a potential therapeutic axis that could be exploited for TNBC treatment.

Cancer stem cells; Hypoxia; Methylenetetrahydrofolate dehydrogenase 2; One-carbon metabolism; Oxidized ataxia telangiectasia mutated; Serine hydroxymethyltransferase 2; Triple-negative breast cancer.