Expression of YTHDF1 and YTHDF2 in lung squamous cell carcinoma and their correlation with PD-L1

Lung squamous cell carcinoma (LUSC) is associated with poor prognosis and low 5-year survival rates, and effective targeted therapies remain unavailable in clinical practice. To investigate the expression levels and clinical significance of YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) and YTH N6-methyladenosine RNA-binding protein 2 (YTHDF2) in LUSC tissues, and to preliminarily demonstrate that YTHDF1 promotes the expression of programmed death-ligand 1 (PD-L1) through N6-methyladenosine (m6A) RNA modification.

(I) The Cancer Genome Atlas (TCGA) database was utilized to explore the relationships between the expressions of YTHDF1 and YTHDF2 in LUSC, pathological characteristics, and patient prognosis. (II) Gene set enrichment analysis (GSEA) was used to explore the biological functions and signaling pathways involved in YTHDF1 and YTHDF2. The correlation between YTHDF1 and YTHDF2 expression and 22 immune cells was analyzed using CIBERSORT. (III) Immunohistochemistry is used to detect the expression levels of YTHDF1 and YTHDF2 in 60 LUSC and adjacent tissue samples and their relationship with clinical characteristics, and to evaluate their correlation with PD-L1 expression. (IV) Quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry analysis and other methods are used to detect the expression level of PD-L1 in LUSC cells after overexpression of YTHDF1. (V) Western blot, lactate dehydrogenase (LDH) release assay, T cell-mediated tumor cell-killing assay and other in vitro experiments verify whether YTHDF1 could enhance PD-L1 expression in an m6A-dependent manner in LUSC cells. Registration number for a clinical trial: ChiCTR2600118358.

YTHDF1 and YTHDF2 are elevated in LUSC compared to the adjacent tissues, and their messenger RNA (mRNA) levels correlate with advanced pathological tumor (T), node (N), and metastasis (M) categories and clinical stage. Multivariate Cox regression analysis showed that high YTHDF1 expression and T stage independently predicted a shortened disease-specific survival. GSEA indicated that the gene signatures driven by YTHDF1 and YTHDF2 were enriched for E2F targets and G2M checkpoint pathways. CIBERSORT revealed significant associations with multiple immune-infiltrating cells. Immunohistochemistry confirmed the overexpression of YTHDF1 and YTHDF2 in LUSC specimens, demonstrating a positive correlation between YTHDF2 and PD-L1 expression. YTHDF1 expression is significantly associated with PD-L1 expression. Functional assays showed that YTHDF1 increased PD-L1 levels in an m6A-dependent manner and reduced T-cell cytotoxicity. Bioinformatics analysis showed that YTHDF1 mRNA expression was significantly higher in LUSC tissues than in adjacent non-cancerous tissues (P<0.05), and YTHDF2 mRNA expression was higher in LUSC tissues than in adjacent non-cancerous tissues in paired analysis (P<0.05), but no significant difference was found in the unpaired analysis (P>0.05).

YTHDF1 and YTHDF2 may serve as potential prognostic biomarkers and therapeutic targets, and YTHDF1-mediated m6A modification may open new avenues for LUSC immunotherapy.