Unlocking a TP53 mutation-specific vulnerability: the miR-486-3p/CYB561 axis in bladder cancer

Bladder cancer (BLCA) exhibits profound molecular heterogeneity, with TP53 mutations occurring in approximately 50% of muscle-invasive cases and conferring an aggressive clinical course. However, TP53 mutation-specific miRNA-mRNA regulatory networks and their therapeutic implications remain poorly defined. Multi-omics data from TCGA and six GEO cohorts were integrated to identify TP53 mutation-associated prognostic miRNAs. TargetScan, miRDB, TarBase and correlation analyses were employed to identify downstream mRNA targets. Functional relevance was assessed through in vitro assays in BLCA cell lines. Immunotherapy response was evaluated using the IMvigor210 cohort, and drug sensitivity was predicted via CellMiner and pRRophetic. Gene dependency was analyzed using DepMap. miR-486-3p emerged as a TP53 mutation-specific prognostic biomarker, with lower expression levels independently predicting poor overall survival exclusively in TP53-mutant BLCA. CYB561 was identified as a high-confidence target of miR-486-3p, exhibiting significant negative correlation, and dual-luciferase reporter assay confirmed direct binding of miR-486-3p to the CYB561 3’ UTR. Functional assays further demonstrated that the miR-486-3p/CYB561 axis modulates ferroptosis-related phenotypes, including GPX4 expression and levels of ROS, MDA, and Fe2+. CYB561 overexpression independently predicted adverse survival, particularly in basal/squamous subtypes and tumors with co-mutations in TP53, RB1, and KDM6A. Both overexpression and loss-of-function rescue experiments confirmed CYB561 as a necessary and sufficient downstream mediator of miR-486-3p’s tumor-suppressive effects. High CYB561 expression correlated with reduced cisplatin sensitivity and diminished clinical benefit from anti-PD-L1 immunotherapy. DepMap analysis further identified CYB561 as a context-specific vulnerability in BLCA lineages, with pathway enrichment implicating ferroptosis regulation. miR-486-3p/CYB561 axis represents a TP53 mutation-enriched regulatory circuit with prognostic and therapeutic significance in BLCA, acting at least in part through modulation of ferroptosis. CYB561 emerges as a candidate biomarker for risk stratification and a context-dependent vulnerability, offering potential avenues for targeted intervention in this aggressive disease subset. miR-486-3p is downregulated in BLCA and serves as a TP53 mutation-specific prognostic biomarker, with low expression independently predicting poor survival in this aggressive subset. CYB561 is a direct target of miR-486-3p, validated by dual-luciferase reporter assay, and functions as a downstream effector driving proliferation, invasion, and adverse clinical outcomes. The miR-486-3p/CYB561 axis modulates ferroptosis via GPX4, ROS, MDA, and Fe2+, linking miRNA regulation to iron-dependent cell death in BLCA. High CYB561 expression correlates with cisplatin resistance, reduced anti-PD-L1 immunotherapy benefit, and represents a context-specific vulnerability in TP53-mutant BLCA.