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

Abstract

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.

Blockade of GCH1/BH4 Axis Activates Ferritinophagy to Mitigate the Resistance of Colorectal Cancer to Erastin-Induced Ferroptosis

“…Additionally, knockdown of GCH1 suppressed FTH1 protein levels ( Figure 1H ). Previous studies demonstrated that nuclear receptor coactivator 4 (NCOA4)-mediated selective autophagic turnover of FTH1 (namely ferritinophagy) is necessary for erastin but unnecessary for RSL3 ( Liu et al, 2020 ; Mancias et al, 2014 ; Gryzik et al, 2021 ). To investigate whether GCH1/BH4 deficiency selectively promotes erastin-induced ferroptosis through activation of ferritinophagy, the NCOA4, FTH1, and LC3B protein levels were assessed.…”

m6A: An Emerging Role in Programmed Cell Death

“…Moreover, METTL3 acts as the target of miR-4443, and regulates the expression of FSP1 to mediate the ferroptosis of non-small cell lung cancer ( Song et al, 2021 ). Autophagy may also represent a targeted pathway by which to regulate the sensitivity of cells to ferroptosis ( Gryzik et al, 2021 ). BECN1 is a key protein that regulates autophagy and promotes ferroptosis by regulating the activity of the cysteine/glutamate antiporter (also referred to as system Xc-) ( Kang et al, 2018 ).…”

Ferroptosis: Redox Imbalance and Hematological Tumorigenesis

“…Nuclear Receptor Coactivator-4 (NCOA4) mediates the degradation of ferritin in an autophagy-dependent pathway where iron is isolated from ferritin and the accumulated free ferrous ions in the cytoplasm promote erastin-induced ferroptosis ( 67 ). Nuclear factor-erythroid 2-related factor 2 (NRF2) regulates the expression of FPN1, FTH, and FTL at the transcriptional level and promotes the stability of the intracellular iron environment ( 68 ).…”