Inhibition of mitochondrial genome-encoded mitomiR-3 contributes to ZEB1 mediated GPX4 downregulation and pro-ferroptotic lipid metabolism to induce ferroptosis in breast cancer cells

Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, represents a unique vulnerability in cancer cells. However, current ferroptosis-inducing therapies face clinical limitations due to poor cancer cell specificity, systemic toxicity, and off-target effects. Therefore, a deeper understanding of molecular regulators of ferroptosis sensitivity is critical for developing targeted therapies. The metabolic plasticity of cancer cells determines their sensitivity to ferroptosis. While mitochondrial dysfunction contributes to metabolic reprogramming in cancer, its role in modulating ferroptosis remains poorly characterized. Previously, studies have identified that mitochondrial genome also encodes several non-coding RNAs. We identified 13 novel mitochondrial genome-encoded miRNAs (mitomiRs) that are aberrantly overexpressed in triple-negative breast cancer (TNBC) cell lines and patient tumors. We observed higher levels of mitomiRs in basal-like triple-negative breast cancer (TNBC) cells compared to mesenchymal stem-like TNBC cells. Strikingly, 11 of these mitomiRs directly target the 3 ' UTR of ZEB1, a master regulator of epithelial-to-mesenchymal transition (EMT). Using mitomiR-3 mimic, inhibitor and sponges, we demonstrated its role as a key regulator of ZEB1 expression in TNBC cells. Inhibition of mitomiR-3 via sponge construct in basal-like TNBC, MDA-MB-468 cells, promoted ZEB1 upregulation and induced a mesenchymal phenotype. Further, mitomiR-3 inhibition in TNBC cells contributed to reduced cancer cell proliferation, migration, and invasion. Mechanistically, mitomiR-3 inhibition in TNBC cells promote metabolic reprogramming toward pro-ferroptotic pathways, including iron accumulation, increased polyunsaturated fatty acid (PUFA) metabolites, and lipid peroxidation, contributing to ferroptotic cell death via ZEB1-mediated downregulation of GPX4, a critical ferroptosis defense enzyme. We observed that mitomiR-3 inhibition significantly suppressed tumor growth in vivo. Our identified mitomiR-3 has low expression in normal breast cells, minimizing potential off-target toxicity, making them a promising target for pro-ferroptotic cancer therapy. Our study reveals a novel link between mitochondrial miRNAs and ferroptosis sensitivity in TNBC paving a way for miRNA-based therapeutics.