Downregulation of PAX1 in OSCC Enhances Stemness and Immunosuppression via IFIT1 and PD-L1 Pathways

Our study investigated how arecoline-induced extracellular vesicle (EV) secretion suppresses PAX1 protein production through DNA hypermethylation and examined whether PAX1 downregulation enhances cancer stemness and immunosuppression in the tumor microenvironment.

EVs were isolated from SAS/TW2.6 cancer cell lines using ultracentrifugation and identified using transmission electron microscopy. PAX1 DNA methylation was tested in an ISO17025-certified lab, with and without EV pretreatment. Stemness and epithelial-mesenchymal transition markers were assessed by western blotting and 3D culture. PAX1, IFIT1, and PD-L1 co-expression were examined through immunofluorescence. Flow cytometry detected various T cells.

Arecoline-induced EVs enhanced PAX1 methylation, suppressing its tumor-suppressive function. Reduced PAX1 mRNA in OSCCs was linked to larger tumors, nodal metastasis, late-stage disease, areca quid chewing, and poor survival. Downregulated PAX1 protein negatively correlated with IFIT1 and PD-L1 expression. Reduced PAX1 promoted stemness via the IFIT1 pathway, increasing PD-L1 secretion and aiding immune evasion. PD-L1 expression correlated with Treg and CD8+ T cell levels in OSCC tissues, and the CD4+/CD8+ T cell ratio was lower in OSCC patients than in controls.

Arecoline-induced EV production, which influences PAX1/IFIT1/PD-L1 function, may serve as a reliable biomarker for targeted therapy in OSCC patients.