Hypoxia Remodels the Extracellular Vesicle Proteome to Drive Metastasis in Breast Cancer via Metabolic–Epigenetic Coupling

This study investigates how hypoxia remodels the extracellular vesicle (EV) proteome to promote metastasis in breast cancer (BC) cells. EVs from hypoxic MCF-7 and MDA-MB-231 cells were characterized and shown to enhance epithelial-mesenchymal transition (EMT), migration, invasion, and clonogenicity in recipient cells. Quantitative proteomics identified over 1250 EV proteins, with 78 commonly regulated by hypoxia across both cell lines. Pathway analysis revealed hypoxia-induced EV enrichment of ribosomal, chromatin remodeling, mitochondrial, and one-carbon metabolism proteins, alongside depletion of immune-modulatory factors. Interestingly, key one-carbon metabolism enzymes (SMS, CAD, and AHCYL1) were consistently upregulated in hypoxic EVs shed by both the cell lines. Notably, AHCYL1, a regulator of the methylation cycle enzyme AHCY, is significantly upregulated under hypoxic conditions. Our findings demonstrate that hypoxic EVs promote an increase in histone H3K9 trimethylation levels in recipient cells. This epigenetic shift downregulated epithelial and metastasis-suppressor genes (CDH1, EpCAM, and DKK1) while sustaining expression of EMT transcription factors (ZEB1 and SNAIL), thereby stabilizing EMT and enhancing invasiveness. Collectively, we describe a hypoxia-driven EV proteome that links metabolic reprogramming to epigenetic enforcement of metastatic traits in BC.