Methyltransferase-like 3/14-mediated m⁶A Silencing of GPx3 Drives Lipophagy Dysfunction and Ferroptosis Resistance in...

Background: Colorectal cancer (CRC) is a major cause of cancer morbidity and mortality worldwide, and its progression is closely associated with redox imbalance. The biological role and upstream regulation of the antioxidant enzyme glutathione peroxidase 3 (GPx3) remain poorly understood in CRC. Methods: This study integrated analyses from clinical CRC tissues, in vitro functional experiments, molecular biology assays, and public The Cancer Genome Atlas datasets. Techniques included gene expression profiling, N ⁶-methyladenosine methylation analysis, mitochondrial reactive oxygen species detection, lipophagy assays, lipid droplet staining, transcriptomic and proteomic analyses, ferroptosis sensitivity testing, and in vivo xenograft and metastasis models to investigate the role and underlying mechanisms of GPx3 in CRC. In addition, lung metastatic burden was assessed using in vivo bioluminescence imaging and high-resolution micro-computed-tomography scanning. Results: GPx3 was substantially down-regulated in CRC tissues and cells, primarily due to N ⁶-methyladenosine modification catalyzed by methyltransferase-like protein 3 (METTL3) and METTL14. Down-regulation of GPx3 expression elevated mitochondrial reactive oxygen species, impaired lipophagy flux, and caused excessive lipid droplet accumulation. Multiomics analyses identified Dickkopf-1 (DKK1) as a downstream target negatively regulated by GPx3 through reactive-oxygen-species-dependent β-catenin signaling. The GPx3-DKK1-solute carrier family 7 member 11 (SLC7A11) axis modulated lipid peroxidation and influenced ferroptosis sensitivity. In vivo xenograft and metastatic models showed that GPx3 suppressed tumor growth and metastasis, and these effects were partially reversed by DKK1. Conclusion: This study demonstrates that METTL3/14-mediated N ⁶-methyladenosine modification is a key upstream mechanism leading to GPx3 down-regulation in CRC. Down-regulation of GPx3 expression promotes malignant progression by increasing mitochondrial oxidative stress, disrupting lipophagy, and cooperating with the DKK1-SLC7A11 pathway to reduce ferroptosis sensitivity. Targeting this regulatory axis may provide promising molecular strategies for CRC diagnosis and therapy.