NSUN2-mediated m5C hypermethylation of hsa_circ_0004516 promotes breast cancer brain metastasis by activating AKT signaling

Breast cancer brain metastasis (BCBM) remains fatal with elusive mechanisms. Here, we unveil the first circRNA m5C methylation landscape in BCBM through MeRIP-seq (methylated RNA immunoprecipitation next-generation sequencing) identifying 7465 BCBM-specific m5C peaks versus 5929 in primary breast cancer (BC). A total of 48 hypermethylated and 128 hypomethylated m5C sites in BCBM (231-BR) were identified compared to BC. Bioinformatics enrichment analysis revealed hypermethylated circRNAs enriched in ERBB/VEGF signaling pathways. Among 8 validated differentially methylated circRNAs, <i>hsa_circ_0004516</i> was consistently upregulated in BCBM tissues/cells and exhibited NSUN2-dependent m5C modification. Mechanistically, NSUN2-mediated m5C methylation enhanced <i>hsa_circ_0004516</i> stability, evidenced by significantly shortened half-life upon NSUN2 depletion. Crucially, catalytic mutant NSUN2 (C271A/C321A) abolished this effect. Functional assays demonstrated that <i>hsa_circ_0004516</i> knockdown in 231-BR cells suppressed proliferation, migration, and invasion by reducing p-AKT (Ser473) levels. The AKT activator SC79 reversed these phenotypic impairments, definitively linking <i>hsa_circ_0004516</i>-driven metastasis to AKT signaling activation. Our study establishes the NSUN2-m5C-<i>hsa_circ_</i>0004516-AKT axis as a novel therapeutic target and biomarker for BCBM.