Autophagy regulates the cancer stem cell phenotype of head and neck squamous cell carcinoma through the noncanonical FOXO3/SOX2 axis.

Autophagy is an essential catabolic process that orchestrates cellular homeostasis and plays dual roles in tumor promotion and suppression. However, the mechanism by which autophagy affects the self-renewal of cancer stem cells (CSCs) remains unclear. In this study, we investigated whether autophagy activation contributes to CSC properties of head and neck squamous cell carcinoma (HNSCC). The results showed that the autophagy level and CSC properties of HNSCC cells were elevated in response to several adverse conditions, including treatment with cisplatin, starvation, and hypoxia. Pretreatment with autophagy inhibitors, such as 3-MA and chloroquine, diminished the CSC properties acquired under adverse conditions. In addition, the isolated CSCs were endowed with stronger autophagic activity than non-CSCs, and the CSC properties were dampened when autophagy was inhibited either by 3-MA, chloroquine, or Beclin1 knockdown. Notably, the tumor-initiating activity of CSCs was decreased upon knocking down Beclin1. Further study revealed that FOXO3, a substrate for autophagy, was enriched in the nucleus of cells with lower autophagy levels. Nuclear FOXO3 directly bound to the promoter region of SOX2 and negatively regulated its transcriptional activity. Overexpression of FOXO3 decreased the expression of SOX2 and thereby impaired the CSC phenotype both in vitro and in vivo. Taken together, our findings suggest that the activation of autophagy is essential for the acquisition of CSC properties in adverse conditions and the self-renewal of CSCs. We clarify the role of autophagy in regulating the CSC phenotype and demonstrate that the noncanonical FOXO3/SOX2 axis is the intrinsic regulatory mechanism.