Activation of a FOXO3-induced cell cycle arrest regulates ferroptosis

FOXO transcription factors act downstream of PI3K signaling, and FOXO transcriptional activity is inhibited through nuclear exclusion by PKB/AKT-mediated phosphorylation. Many studies have shown FOXO to contribute to organismal homeostasis by mitigating (extra)cellular stress to prevent cell death (reviewed in [1]). Here we show that FOXO3 activation protects cells from ferroptosis, an iron-dependent form of non-apoptotic cell death. In untransformed hTERT-RPE-1 cells, FOXO3 activation reduces ferroptosis in a multilayered manner. First, FOXO3 mediates protection from ferroptosis in part through a p27-induced G1 cell cycle arrest. Second, FOXO3 activation reduces cellular H₂O₂ levels, thereby limiting substrate availability for the Fenton reaction, which fuels hydroxyl radical formation for lipid peroxidation. Third, FOXO3 activation lowers cellular iron content by reducing TFR1 expression, which, combined with the lowering of cellular H₂O₂ levels, likely further reduces the formation of hydroxyl radicals through the Fenton reaction. Finally, FOXO3 activation reduces expression of long-chain-fatty-acid—CoA ligase 4 (ACSL4) and Peroxisomal targeting signal 1 receptor (PEX5), proteins involved in lipid metabolism and protection against ferroptosis. Taken together, we show that FOXO3 activation results in protection from ferroptosis, adding to the repertoire of FOXO-controlled cell protection programs.