Autophagic Degradation of PAX9 Maintain Stemness and Its Reactivation Drives Cellular Senescence in Oral Cancer Stem Cells

Abstract

Paired box 9 (PAX9), a member of the paired box transcription factor family, plays a crucial role in regulating cancer cell growth, proliferation, and differentiation. Its downregulation has been documented in oral cancer and is closely associated with tumor growth and progression. To explore the role of PAX9 in cancer stem cells, we isolated CD44⁺ and CD44⁻ populations from oral cancer cells using magnetic-activated cell sorting (MACS), and validated the separation through flow cytometry and western blot analysis. Notably, PAX9 expression was markedly reduced in CD44⁺ cells compared to CD44⁻ cells, indicating a potential link between PAX9 downregulation and enhanced stemness. Further, PAX9 overexpression in CD44⁺ oral cancer stem cells enhances autophagy and autophagic flux, evidenced by increased tf-LC3 red puncta and LC3-II accumulation after chloroquine treatment. This leads to G₁ cell cycle arrest and suppressed proliferation (EdU and MTT) without triggering apoptosis (no Annexin-V/PI increase). Meanwhile, we further examined the expression of various G1 phase cell cycle arrest markers, including the CDK inhibitors p21, p16, Rb-E2F1, as well as Cyclin D1 and Cyclin E through westernblot. Our data demonstrated that ectopic expression of PAX9 in CD44+ cells significantly increased p21, p16, and Rb levels, while sharply reducing E2F1, Cyclin D1, and Cyclin E in PAX9-overexpressing CD44+ Cal33 and FaDu cells, indicating induction of cellular senescence. Critically, autophagy is required for PAX9’s effects, and knockdown of Beclin-1 or ATG5 reverses growth arrest and G₁ arrest, restoring proliferation. Interestingly, PAX9 itself is degraded via autophagy in CD44⁺ cells, cycloheximide chase shows rapid PAX9 decay, unresolved by proteasome inhibition but stabilized by autophagy inhibitors (CQ, wortmannin, SBI-0206965) and genetic deficiency of autophagy. Co-localization with LC3 and LAMP1, along with cytosolic translocation, confirm autophagic degradation. Moreover, SIRT1 plays a pivotal role, and its inhibition with EX-527 increases PAX9 levels, implying that SIRT1-dependent deacetylation promotes PAX9's autophagic turnover. In conclusion, CD44⁺ CSCs maintain low PAX9 via SIRT1-driven autophagy to support stemness. Overexpressing PAX9 induces autophagy-dependent G₁ arrest. Blocking autophagy or SIRT1 stabilizes PAX9, reversing these effects in CD44⁺ cells.