Glycolytic enolase 1-induced H4K12 lactylation activates quiescent breast cancer stem-like cells in triple-negative breast cancer.

Activation of quiescent or extremely slow-cycling breast cancer stem-like cells (qsBCSCs) contributes to tumor progression, yet the regulatory mechanisms involved in triple-negative breast cancer (TNBC) remain elusive. We developed a dual-reporter system combining NANOG-EGFP and H2B-mCherry pulse-chase labeling to separately identify qsBCSCs and active BCSCs (aBCSCs) in vitro and in vivo. aBCSCs display significant enrichment of glycolysis and lactylation signatures, a finding corroborated by single-cell RNA sequencing (scRNA-seq) of TNBC patient samples. Glycolytic enzyme Enolase 1 (ENO1) expression is positively correlated with elevated histone H4 lysine 12 lactylation (H4K12la) in aBCSCs. Functional studies revealed a positive feedback circuit in which ENO1-increased lactate promotes H4K12la, which in turn activates ENO1 transcription. The ENO1-lactate-H4K12la axis enhances proliferating cell nuclear antigen (PCNA) transcription to activate qsBCSCs. Suppression of ENO1 or H4K12la prevents PCNA upregulation and qsBCSC activation. Importantly, PCNA knockdown alone blocks the activation of qsBCSCs induced by exogenous sodium L-lactate (NALA). In orthotopic breast cancer mouse models, both genetic depletion of ENO1 and pharmacological inhibition of lactate production attenuated tumor growth by blocking qsBCSC activation. Clinically, ENO1 expression was strongly correlated with PCNA expression, with high expression predicting poor OS, RFS, and DMFS. Our results establish the ENO1-H4K12la-PCNA axis as a key metabolic‒epigenetic driver of qsBCSC activation and a therapeutic target in TNBC.