NCOA4-driven ferritinophagy and GSH reprogramming underlie maslinic acid-induced ferroptosis and autophagy in breast cancer.

BACKGROUND: Breast cancer is the most prevalent cancer globally, with limited efficacy of traditional therapies and high drug resistance. Maslinic acid (MA) has broad-spectrum anti-tumor activity. This study aimed to investigate whether MA exerts its anti-tumor effects in breast cancer by inducing ferroptosis and autophagy.

METHODS: Breast cancer cell proliferation was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-ethynyl-2'-deoxyuridine (EdU) assays. Autophagy was evaluated by monitoring microtubule-associated protein 1A/1B-light chain 3 (LC3) expression and sequestosome 1 (p62) levels, combined with mRFP-GFP-LC3 plasmid transfection assay. MA-induced ferroptosis was investigated by measuring reactive oxygen species (ROS), glutathione (GSH), glutathione disulfide (GSSG), Glu, and Fe2+ levels. Western blotting and bioinformatics analysis were performed to assess the molecular mechanisms underlying MA-regulated autophagy and ferroptosis.

RESULTS: MA significantly inhibited breast cancer cell proliferation. It activated autophagy in MDA-MB-231 cells autophagy by upregulating tumor protein p53 (p53) expression and inhibiting mammalian target of rapamycin (mTOR) signaling, evidenced by increased levels of beclin 1 (BECN1), unc-51 like autophagy activating kinase 1 (ULK1), autophagy protein 5 (ATG5), and phosphatidylethanolamine-conjugated LC3 (LC3-II), as well as decreased p62 expression, indicating enhanced autophagic flux. Meanwhile, MA induced MDA-MB-231 cell ferroptosis, characterized by elevated ROS, decreased GSH levels, and downregulated expression of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11). Furthermore, MA increased Fe2+ levels and up-regulated nuclear receptor coactivator 4 (NCOA4) expression; this NCOA4 upregulation directly caused ferritin heavy chain 1 (FTH1) downregulation via ferritinophagy, thereby driving ferroptosis. Additionally, co-treatment with the autophagy inhibitor chloroquine (CQ) attenuated MA-induced FTH1 downregulation and rescued MA-induced inhibition of cell viability in MDA-MB-231 cells. Consistently, knockdown of NCOA4 attenuated MA-induced FTH1 degradation and reduced MA-caused Fe2+ accumulation. Bioinformatics analysis identified key hub genes associated with MA-induced autophagy, including tumor protein p53 (TP53), heat shock protein 90 alpha family class a member 1 (HSP90AA1), AKT serine/threonine kinase 1 (AKT1), mTOR, tumor necrosis factor (TNF), interleukin 6 (IL6), epidermal growth factor receptor (EGFR) and BCL2 apoptosis regulator (BCL2), as well as hub genes related to ferroptosis, such as mitogen-activated protein kinase 3 (MAPK3), RELA proto-oncogene (RELA), epidermal growth factor receptor (EGFR), prostaglandin-endoperoxide synthase 2 (PTGS2), SRC proto-oncogene (SRC), interleukin 1 beta (IL1B), interleukin 6 (IL6), and peroxisome proliferator activated receptor gamma (PPARG).

CONCLUSIONS: In MDA-MB-231 cells, MA triggered autophagy through p53 upregulation and mTOR signaling inhibition, and induced ferroptosis by reprogramming GSH metabolism and activating the NCOA4-mediated ferritinophagy pathway, lead to FTH1 degradation.