NSUN2–tRNAVal−CAC-axis-regulated codon-biased translation drives triple-negative breast cancer glycolysis and progression

Epitranscriptomic data indicate that aberrant tRNA modifications in malignant diseases can promote tumor growth by facilitating oncogene translation. NSUN2, a 5-methylcytosine (m5C) methyltransferase of tRNA, is elevated in an array of solid cancers, including triple-negative breast cancer (TNBC). However, it remains unclear how NSUN2 drives aggressive behavior and if NSUN2 could be an effective therapeutic target for TNBC. Functional experiments, including RNA interference, lentivirus transduction, and in vivo xenograft models, were conducted to evaluate the role of NSUN2 in TNBC cell proliferation, metastasis, and chemoresistance. Ribosome sequencing (Ribo-seq), tRNA m5C bisulfite sequencing, and codon usage bias analysis were employed to explore the translational mechanisms underlying NSUN2-mediated tRNA modifications. Glycolysis assays and molecular docking were used to investigate metabolic reprogramming and protein interactions. NSUN2 was significantly upregulated in TNBC and correlated with poor patient prognosis. Mechanistically, NSUN2 mediates m5C modification of tRNAVal−CAC, enhancing the codon-frequency-dependent translation of key glycolysis-related genes, including ALDH3A2, ALDH7A1, HK1, and PFKM. Depletion of NSUN2 disrupted tRNAVal−CAC m5C modification, impairing the translation of these metabolic enzymes and suppressing glycolysis, which ultimately inhibited TNBC cell proliferation, migration, and invasion both in vitro and in vivo. Furthermore, NSUN2 overexpression conferred resistance to docetaxel, while its inhibition sensitized TNBC cells to docetaxel treatment. Clinically, elevated expression levels of NSUN2 and glycolysis-related genes were observed in docetaxel-resistant TNBC tissues, further supporting the role of NSUN2 in chemoresistance. This study identifies NSUN2 as a critical regulator of TNBC progression through tRNAVal−CAC m5C modification and codon-biased translation of glycolysis-related mRNAs. Our findings reveal a novel NSUN2–tRNAVal−CAC axis that orchestrates metabolic reprogramming and translational control in TNBC, offering a promising prognostic biomarker and therapeutic target.