Mitochondrial Oxidative Stress Alters the Phosphoproteome and Reverses Epithelial–Mesenchymal Transition in Human Breast Cancer Cells via the β‑Catenin/c-Myc Axis

Mitochondrial redox status plays a critical role in cancer progression, yet the effects of mitochondrial oxidative stress on the epithelial–mesenchymal transition (EMT), a key step in metastasis, remain elusive. We have investigated the phosphoproteomic landscape of breast cancer cells exposed to mitochondrial oxidative stress induced by mitochondria-targeted curcumin (mitocurcumin (MC)) and explored its potential as a druggable target. Mitocurcumin led to altered cell morphology, reduced migration, and shift to a cobblestone-like epithelial morphology, indicating EMT reversal. Label-free mass spectrometry-based global phosphoproteomic analysis revealed upregulation of phosphoproteins involved in DNA damage-related processes, transcription termination, and induction of oxidative stress, while downregulated phosphoproteins were linked to translation and amino acid metabolism. Processes related to EMT reversal, such as cytoskeleton organization, cell–cell adhesion, focal adhesion assembly and cell–cell junction organization/assembly, and establishment/maintenance of epithelial cell apical–basal polarity, were enriched. The RAC2/RAC3 GTPase cycle, important for cell migration, was downregulated, along with the Wnt/β-catenin signaling pathway. A decrease in the levels of mesenchymal markers, β-catenin, and its target gene, c-Myc, confirmed the suppression of the mesenchymal phenotype. In conclusion, mitochondrial oxidative stress inhibits the migratory capacity of breast cancer cells by targeting the β-catenin/c-Myc axis, indicating its potential as a novel druggable target to prevent cancer metastasis.