Adaptive Evolution of <i>FOXO</i> Family Regulating Lifespan Extension and Cancer Resistance in Long‐Lived Species

The extension of lifespan has evolved independently multiple times in mammals. Long-lived species exhibit markedly lower cancer mortality rates, indicating that they may have evolved enhanced cancer resistance to accommodate their prolonged lifespan. The FOXO protein family is considered to play key roles in modulating lifespan and cancer resistance in model organisms. However, the underlying molecular mechanisms of the FOXO protein family regulating lifespan extension and cancer resistance in long-lived species remain poorly understood. Here, evolutionary analysis of four FOXO genes across 137 mammalian species revealed all the four genes were under overall purifying selection. Nonetheless, we detected modestly elevated ω values in long-lived lineages relative to background groups, and 18 positively selected sites were identified in the four FOXO genes in long-lived mammals. To further test whether these FOXO genes have occurred functional changes, we selected FOXO3 and FOXO4 to perform functional validation. Cell experiments demonstrated that bowhead whale FOXO3 and FOXO4 significantly inhibited HeLa cell proliferation, migration, and invasion. Subcellular localization assays showed that mouse FOXO3 and FOXO4 were mostly distributed in the cytosolic cytoplasm, whereas bowhead whale FOXO3 and FOXO4 were predominantly localized in the nucleus, which may increase transcriptional activity and inhibit tumorigenesis in cetaceans. In particular, bowhead whale FOXO3 was found to upregulate the tumor suppressors PTEN and FASL while downregulating the oncogene BCL6, suggesting that this FOXO3-driven expression pattern may underlie cancer resistance in long-lived mammals. Overall, our findings provide novel insights into the molecular mechanisms underlying the cancer resistance in mammals.