Abstract
Triple-negative breast cancer (TNBC) is a subtype characterized by the absence of common BC receptors and is closely associated with a hypoxic tumor microenvironment. However, the mechanisms through which TNBCs adapt to hypoxia remain elusive. This study revealed elevated ENO1 levels in various BC datasets and revealed ENO1 protein lactylation in BC samples through 4D label-free lactylation quantitative proteomics analysis. The results indicated that lactylation increases ENO1 protein stability and enzyme activity, which promotes glycolysis. Notably, as lactate levels increased, a positive feedback loop was established, further promoting lactylation of ENO1. This positive feedback mechanism enables TNBC cells to adapt more efficiently to hypoxia and enhances their malignant behaviors. Lactylation prevented the lysosomal degradation of ENO1. In this study, the characteristics of ENO1, an RNA-binding protein, were assessed to determine how to interfere with its lactylation; specifically, an RNA ligand that can be specifically bound by ENO1 was identified. The RNA ligand was found to be linked to the Lamp2a protein in adipose stem cells (ADSCs) after stable transfection with Lamp2a-TAT and TRA-ligand plasmids. ADSCs seeded in a polyglycolic acid scaffold secreted exosomes containing the Lamp2a-linked ligand. This RNA ligand binds to ENO1 after it enters TNBC cells and further induces the lysosomal degradation of ENO1 by the Lamp2a protein. Consequently, glycolysis, which is associated with malignant cell behaviors, is inhibited. Overall, this study elucidated the role of ENO1 lactylation-mediated glycolysis in TNBC adaptation to hypoxia and provides a strategy for targeting ENO1.