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.