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

Abstract

Objective: 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.

Materials and methods: 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.

Results: 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.

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