P01.09.A TARGETING NAT1 INHIBITS GLIOMA PROGRESSION BY MODULATING OXIDATIVE PHOSPHORYLATION AND REPROGRAMMING THE TUMOR IMMUNE MICROENVIRONMENTNT

Abstract BACKGROUND Glioma is the most common malignant brain tumor, characterized by complex metabolic features and an immunosuppressive microenvironment, leading to poor prognosis. N-acetyltransferase 1 (NAT1) plays a key role in various cancers, but its mechanism in glioma remains unclear. METHODS We analyzed NAT1 expression in glioma and normal brain tissues using TCGA and CGGA databases and validated the results via immunohistochemical staining of 85 glioma samples. Survival analyses were performed in public datasets and our cohort. NAT1 knockdown and over-expression models were established in glioma cell lines (U87, U251) via lentiviral transfection, validated by RT-qPCR and Western blotting. Cell proliferation, migration, and invasion assays assessed the impact of NAT1 expression. Transcriptomic and proteomic sequencing compared NAT1-knockdown and wild-type U87 cells to identify pathway alterations. Oxidative phosphorylation parameters (ROS, mitochondrial membrane potential, ATP content, etc.) were measured. We also co-cultured glioma cells with macrophages (THP-1, HMC3) to evaluate the impact of NAT1 on macrophage polarization. Finally, subcutaneous xenograft models in nude mice, with or without macrophage depletion using clodronate liposomes, were used to assess tumorigenicity in vivo. RESULTS NAT1 expression was significantly elevated in glioma tissues and increased with WHO grade in both public datasets and our cohort. High NAT1 expression correlated with shorter overall survival. Functionally, NAT1 knockdown suppressed glioma cell proliferation, migration, and invasion, while over-expression had opposite effects, suggesting NAT1 as a therapeutic target. Transcriptomic analysis identified 546 differentially expressed genes enriched in cytokine-cytokine receptor interaction, IL-17 signaling pathways, etc. Proteomic analysis identified 784 differentially expressed proteins, mainly enriched in oxidative phosphorylation, ROS generation, etc. NAT1 knockdown increased ROS levels and reduced mitochondrial membrane potential, without affecting mitochondrial quantity or ATP levels, indicating disrupted oxidative phosphorylation. In the co-culture system, NAT1 knockdown upregulated M1-associated cytokines (IL1A, IL1B, TNFSF14) and downregulated M2-associated cytokines (TGFB1, IL10, CCL2). Flow cytometry analysis confirmed increased M1 macrophage polarization. In vivo, NAT1 knockdown suppressed tumor growth in nude mice, an effect partially reversed by macrophage depletion, highlighting the role of macrophages in NAT1-mediated glioma suppression. CONCLUSION NAT1 is closely associated with poor prognosis in glioma. NAT1 knockdown inhibits oxidative phosphorylation in tumor cells and promotes M1 macrophage polarization, jointly suppressing glioma malignancy. Targeting NAT1 offers a potential therapeutic strategy.