An m6A-programmed cell death signature predicts prognosis and identifies STK25 as a therapeutic target in colon...

Colon adenocarcinoma (COAD) remains a leading cause of cancer-related mortality worldwide. Although programmed cell death (PCD) and RNA N6-methyladenosine (m6A) modification have each been shown to regulate tumor progression and therapeutic responses, their combined prognostic significance in COAD has not been fully elucidated. The present study aimed to develop an integrated m6A-PCD prognostic model and to identify potential therapeutic targets in COAD. In total, 1,379 genes across 14 PCD-related pathways were systematically analyzed and a 21-gene m6A-PCD signature (MCDI) was constructed following multivariate Cox regression analysis. Single-cell RNA sequencing from publicly available datasets (GSE132465 and GSE205506), together with reverse transcription-quantitative PCR (qPCR) validation using clinical samples, were employed to identify genes with tumor-specific expression patterns. The functional role of serine/threonine kinase 25 (STK25) was further investigated through knockdown experiments, flow cytometry, m6A methylated RNA immunoprecipitation (MeRIP)-qPCR and RIP assays. In addition, small interfering RNAs targeting methyltransferase-like 3 (METTL3) and YTH domain-containing protein 1 (YTHDC1) were used to evaluate the involvement of m6A modification in STK25 mRNA stability and apoptosis regulation. The resulting MCDI signature demonstrated robust and independent prognostic value and effectively predicted differential responses to programmed death-ligand 1 immunotherapy. In total, 5 core genes [microRNA 210, STK25, TGFB2, tripartite motif containing (TRIM)6 and TRIM68] were identified as key prognostic markers. STK25 was specifically upregulated in tumor epithelial cells, and its knockdown significantly promoted apoptosis in COAD cells. Correlation analyses revealed positive associations between STK25 expression and multiple m6A regulators, with METTL3 and YTHDC1 showing the highest targeting credibility. Knockdown of METTL3 or YTHDC1 reduced STK25 mRNA levels. RIP assays confirmed their direct binding to STK25 mRNA, while MeRIP-qPCR demonstrated that METTL3 knockdown decreased the m6A modification level of STK25 mRNA. In conclusion, the reconstructed MCDI was established as a novel m6A-PCD-based prognostic model for patient stratification and the prediction of immunotherapy response in patients with COAD. STK25 was also identified as a potential therapeutic target linking m6A modification to the regulation of apoptosis in COAD.