Uncoupling FoxO3A mitochondrial and nuclear functions in cancer cells undergoing metabolic stress and chemotherapy.

While aberrant cancer cell growth is frequently associated with altered biochemical metabolism, normal mitochondrialfunctions are usually preserved and necessary for full malignant transformation. The transcription factor FoxO3A is akey determinant of cancer cell homeostasis, playing a dual role in survival/death response to metabolic stress andcancer therapeutics. We recently described a novel mitochondrial arm of the AMPK-FoxO3A axis in normal cells uponnutrient shortage. Here, we show that in metabolically stressed cancer cells, FoxO3A is recruited to the mitochondriathrough activation of MEK/ERK and AMPK, which phosphorylate serine 12 and 30, respectively, on FoxO3A N-terminaldomain. Subsequently, FoxO3A is imported and cleaved to reach mitochondrial DNA, where it activates expression ofthe mitochondrial genome to support mitochondrial metabolism. Using FoxO3A−/−cancer cells generated with theCRISPR/Cas9 genome editing system and reconstituted with FoxO3A mutants being impaired in their nuclear ormitochondrial subcellular localization, we show that mitochondrial FoxO3A promotes survival in response tometabolic stress. In cancer cells treated with chemotherapeutic agents, accumulation of FoxO3A into themitochondria promoted survival in a MEK/ERK-dependent manner, while mitochondrial FoxO3A was required forapoptosis induction by metformin. Elucidation of FoxO3A mitochondrial vs. nuclear functions in cancer cellhomeostasis might help devise novel therapeutic strategies to selectively disable FoxO3A prosurvival activity