METTL3 promotes pancreatic cancer immune escape by m6A modification of lncRNA AFAP1-AS1 to enhance EGR2 stability.

BACKGROUND AND AIMS: Pancreatic cancer (PC) exhibits significant immune evasion. The roles of N6-methyladenosine (m6A) RNA modification and long non-coding RNAs (lncRNAs) in PC immunity are emerging. We aimed to elucidate the function and mechanism of METTL3-mediated m6A modification of lncRNA AFAP1-AS1 in PC progression and immune escape, focusing on its interaction with early growth response 2 (EGR2). METHODS: Expression of METTL3, AFAP1-AS1, and EGR2, along with AFAP1-AS1 m6A levels, were measured in human PC tissues/cell lines via quantitative reverse transcription PCR, Western blot, and methylated RNA immunoprecipitation. Functional assays (proliferation, invasion, CD8(+) T cell co-culture) were performed using knockdown/overexpression strategies in PANC-1 cells. Mechanistic insights were gained through RNA immunoprecipitation, stability assays, chromatin immunoprecipitation (ChIP), and fluorescence in situ hybridization. An in vivo murine subcutaneous tumor model validated key findings, with comprehensive immune microenvironment profiling including regulatory T cells (Tregs) and tumor-associated macrophages (TAMs). RESULTS: METTL3, AFAP1-AS1, and EGR2 were significantly upregulated in PC, where AFAP1-AS1 exhibited increased m6A modification and correlated with poor prognosis. METTL3 directly mediated AFAP1-AS1 m6A modification, enhancing its stability through the m6A readers YTHDF2 and IGF2BP1. Functionally, AFAP1-AS1 promoted PC cell malignancy and immune evasion, characterized by resistance to T cell killing, suppressed T cell function, and increased programmed death-ligand 1 (PD-L1). Mechanistically, AFAP1-AS1 stabilized EGR2 mRNA through direct binding, elevating EGR2 protein expression. ChIP assays confirmed that EGR2 directly binds to the promoters of PD-L1 and Snail. EGR2 knockdown phenocopied AFAP1-AS1 knockdown effects, and EGR2 overexpression rescued the AFAP1-AS1 knockdown phenotype. In vivo, METTL3 depletion inhibited tumor growth and enhanced anti-tumor immunity, characterized by decreased Tregs and M2 macrophages infiltration, effects partially reversed by EGR2 restoration. CONCLUSION: METTL3 enhances AFAP1-AS1 stability via m6A modification recognized by YTHDF2 and IGF2BP1, which subsequently increases EGR2 expression by improving its mRNA stability. EGR2 directly activates transcription of immune checkpoint and epithelial-mesenchymal transition (EMT)-related genes. This METTL3/AFAP1-AS1/EGR2 signaling axis drives PC malignancy and immune resistance, offering potential therapeutic targets for PC treatment.