Fasting induces ferroptosis by modulating lipid metabolism in breast cancer

Introduction: Cyclic fasting and fasting-mimicking diets (FMDs) showed broad antitumor activity in mice with breast cancer (BC), with promising results in patients (pts) enrolled in conpleted or ongoing clinical trials (NCT03340935; NCT03454282; NCT04248998; NCT05763992 ). However, with the exception of glucose and growth factor modulation, the mechanistic determinants of fasting/FMD antitumor activity are poorly understood. Methods: We combined in vitro and in vivo experiments in BC models with ex vivo analyses of blood and tumor samples collected from BC pts undergoing FMD in the context of clinical trials (NCT03454282; NCT04248998) to investigate the role of lipid metabolism modulation in mediating fasting/FMD antitumor effects. In in vitro experiments, we used six murine and human BC cell lines to study the role of polyunsaturated fatty acids (PUFAs) in affecting cell proliferation (IncucyteS3), survival (propidium iodide), apoptosis (cleaved PARP/caspase 3), lipid peroxidation (malondialdehyde, MDA) and ferroptosis during nutrient starvation. In in vivo experiments, orthotopic mouse BC models (4T1-bearing BALB/c mice, E0771-bearing C57BL/6J mice and MDA-MB-231-bearing NOD-scid IL2rgnull (NSG) mice) were randomized to control conditions (ad libitum diet), intermittent fasting (IF), oral administration of the PUFAs arachidonic acid (AA) or docosaexahenoic acid (DHA), or a combination of IF and PUFAs, with or without carboplatin. We assessed primary tumor growth, lung metastasis formation (through IVIS) and animal survival. Mass Spectrometry analysis was used to quantify plasma and intratumor free fatty acids (FAs), as well as FAs in lipid fractions, in mice and in patients. Results : In both mice and pts (n=112) with BC, fasting/FMD-induced reduction of blood glucose and insulin activated lipolysis in fat tissue, followed by an increase of blood and intratumor AA and DHA. Inhibiting lipolysis through BAY 59-9435 reversed the in vivo antitumor effects of fasting in mice, thus revealing a crucial role of blood FA increase in nutrient starvation antitumor activity. Among several FAs modulated by fasting, AA and DHA accumulate in mitochondrial phospholipids, where they promote radical oxygen species (ROS) formation, lipid peroxidation and ferroptosis. Combining cyclic fasting with AA/DHA administration resulted in cooperative delay of in vivo tumor progression, reduced metastasis formation and prolonged animal survival via ferroptosis activation. These effects were enhanced when chemotherapy was combined with IF plus AA/DHA. Conversely, vitamin E reversed the antitumor effects of nutrient starvation plus AA/DHA. Conclusions: Ferroptosis emerges as a novel determinant of fasting/FMD anticancer activity via PUFA accumulation. Cyclic fasting/FMD plus AA/DHA supplementation is a new, safe and effective antitumor metabolic combination that deserves investigation in phase I/II clinical trials.