Persistent PI3K–AKT signaling fortifies cellular defense against oxidative stress and ferroptosis through augmented mitochondrial fitness

Chronic oxidative stress is recognized as a hallmark of cancer and represents a potentially targetable vulnerability in malignant cells. Oncogenic mutations in phosphatidylinositol 3-kinase (PI3K) are frequently observed across diverse malignancies, playing a crucial role in cancer progression. However, the relationship between PI3K–AKT signaling, mitochondrial fitness, and ferroptosis resistance remains poorly understood. We compared the sensitivity of MCF-7 cells (harboring oncogenic PI3K activation) and MDA-MB-231 cells with oxidative stress and ferroptosis inducers using high-content imaging analysis and flow cytometry. RNA-sequencing was performed to identify transcriptomic changes following PI3K inhibition. Mitochondrial fitness was assessed by measuring mitochondrial mass, membrane potential, ATP production, and glutathione levels. Functional validation was conducted through pharmacological manipulation using PI3K–AKT–mTOR pathway inhibitors and AKT activators, as well as genetic approaches involving ectopic expression of oncogenic PIK3CA-E542K in HeLa cells. Cancer cells with constitutive PI3K activation exhibited high resistance to oxidative stress and ferroptosis compared with cells without oncogenic PI3K mutations. Mechanistically, PI3K–AKT signaling orchestrated an augmented mitochondrial gene program, enhancing mitochondrial fitness and antioxidant capacity. Inhibition of the PI3K–AKT–mTOR pathway selectively increased reactive oxygen species levels, compromised mitochondrial fitness, induced mitophagy, and sensitized cells with oncogenic PI3K activation to ferroptosis. Conversely, ectopic expression of oncogenic PIK3CA or pharmacological activation of AKT conferred resistance to oxidative stress and ferroptosis in a mitochondria-dependent manner, as evidenced by the abrogation of protective effects upon mitochondrial uncoupling. Our findings establish a novel link between enhanced mitochondrial fitness and ferroptosis resistance in cancer cells with hyperactive PI3K signaling. These results suggest that combining ferroptosis induction with PI3K inhibition and mitochondrial fitness impairment may offer a promising therapeutic strategy for cancers harboring oncogenic PI3K mutations. This approach provides new insights into potential treatment modalities that exploit the interplay between oncogenic signaling pathways and cellular redox homeostasis in cancer cells.