NCOA4-mediated ferritinophagy promotes ferroptosis induced by erastin, but not by RSL3 in HeLa cells

Ferroptosis is a regulated cell death characterized by a lethal accumulation of lipid peroxides due to an increase of intracellular iron and a decrease of antioxidant capacity. The reduction of antioxidant activity is obtained by using chemical agents, such as erastin and RSL3, the first one inhibiting the transmembrane cystine-glutamate antiporter causing a cysteine and glutathione depletion and the second one inactivating directly the glutathione peroxidase 4 (GPX4) respectively. The role of iron and its related proteins in supporting the formation of lipid peroxides, is not completely understood hence to try to shed light on it we generated HeLa clones with altered ferritinophagy, the ferritin degradation process, by knocking-out or overexpressing Nuclear Receptor Coactivator 4 (NCOA4), the ferritin autophagic cargo-receptor. NCOA4 deficiency abolished ferritinophagy increasing ferritin level and making the cells more resistant to erastin, but unexpectedly more sensitive to RSL3. Interestingly, we found that erastin promoted ferritinophagy in HeLa cells expressing NCOA4, increasing the free iron, lipid peroxidation and the sensitivity to ferroptosis. In contrast, RSL3 did not modulate ferritinophagy, while NCOA4 overexpression delayed RSL3-induced cell death suggesting that RSL3 mechanism of action is independent of ferritin degradation process. Therefore, the ferritin-iron release in the execution of ferroptosis seems to depend on the inducing compound, its target and downstream pathway of cell death activation. • Erastin induces NCOA4-mediated ferritinophagy for ferroptosis execution • RSL3-mediated ferroptotic cell death does not involve ferritin degradation • NCOA4 expression and/or active ferritinophagy delay RSL3-induced ferroptosis Abbrevations. BH 4 tetrahydrobiopterin CoQ 10 coenzyme Q 10 Cys cysteine Cys-Cys cystine DFO desferoxamine ER erastin FAC ferric ammonium citrate Fer-1 ferrostatin-1 FSP1 ferroptosis suppressor protein 1 FTH ferritin heavy chain FTL ferritin light chain FPN ferroportin GAPDH glyceraldehyde 3-phosphate dehydrogenase GCH1 GTP cyclohydrolase-1 Glu glutamine GPX4 glutathione peroxidase 4 GSH glutathione GSSG oxidized glutathione HO-1 heme oxygenase 1 LC3 (MAP1LC3) microtubule-associated protein 1 light chain 3 LIP labile iron pool LOX5 lipoxygenase 5 NCOA4 nuclear receptor coactivator 4 NRF2 nuclear factor erythroid 2 (NFE2)-related factor 2 NQO1 NAD( P )H quinone dehydrogenase 1 ROS reactive oxygen species RSL3 RAS-selective lethal 3 TfR1 transferrin receptor 1 TxnR thioredoxin reductase 1