ONECUT1 activates PSAT1 to facilitate PD-L1 expression and mediate immune evasion of lung squamous cell carcinoma.

[BACKGROUND] PD-1/PD-L1, a classic immune checkpoint commonly employed in targeted therapy, has proven to yield only limited benefits for patients with lung squamous cell carcinoma (LUSC). Unraveling the intrinsic mechanisms underlying the progression of LUSC serves as the foundation for discovering more effective treatment strategies.

[METHODS] A study was conducted on the differential expression of PSAT1 and ONECUT1 in LUSC based on data from the TCGA database. Pearson correlation analysis was undertaken to examine the association between PSAT1 expression and CD8 T-cell infiltration or ONECUT1 expression in LUSC. Based on the hTFtarget and JASPAR databases, upstream regulators and potential binding sites for PSAT1 were screened and predicted. An analysis of the prognostic impact of PSAT1 on lung cancer was based on the Kaplan-Meier Plotter website. In qPCR analysis, the mRNA expression of PSAT1 and ONECUT1 was detected. Clinical samples of tumor tissue and adjacent normal tissues were collected, with IHC analysis undertaken on them to determine the PSAT1 protein content, as well as the infiltration level of CD8 T cells in the tumor tissue. Through MTT, cell viability was evaluated. Western blot (WB) was utilized to detect the expression of PD-L1 protein. The ANNEXIN V-FITC experiment was carried out to assess the level of apoptosis in cancer cells. CFSE, ELISA, and Transwell experiments were all employed to assess the activation level and chemotaxis of CD8 T cells. ChIP and dual luciferase assay were carried out to investigate the binding relationship between ONECUT1 and PSAT1.

[RESULTS] In tumors and LUSC lines, PSAT1 exhibited significant overexpression, which was linked with adverse prognosis and inversely correlated with CD8 T cell infiltration. In PSAT1-overexpressing tumor tissues, the infiltration level of CD8 T cells was greatly lower than that in tissues with low PSAT1 expression. Knocking down PSAT1 considerably decreased the viability and expression of PD-L1 protein in LUSC cells, as well as upregulated apoptosis levels and the activation and chemotactic capacity of CD8 T cells in co-culture systems. ONECUT1 was a potential upstream regulator of PSAT1, displaying a positive correlation and possessing potential binding sites within the promoter region of PSAT1. Furthermore, ONECUT1 had remarkable overexpression in LUSC tissues and cells. The ChIP and dual luciferase assays confirmed that ONECUT1 effectively bound to PSAT1 and activated PSAT1 expression. Overexpression of ONECUT1 restored the negative regulation on cell viability and positive activation effect on CD8 T cells caused by PSAT1 knockdown.

[CONCLUSION] ONECUT1 transcriptionally activates PSAT1 to reinforce LUSC immune evasion through upregulation of PD-L1 expression.