PAQR4 stabilizes NRF2 to suppress ferroptosis and enhance cisplatin resistance in bladder cancer

The Progestin and AdipoQ Receptor 4 (PAQR4) has been implicated in multiple malignancies, yet its role in bladder cancer (BCa) progression and cisplatin resistance remains poorly understood. Here, we investigated the expression, biological function, and molecular mechanism of PAQR4 in BCa. PAQR4 expression was analyzed in BCa tissues and cell lines using TCGA-BLCA database, western blot, and RT-qPCR. Functional assays (CCK-8, colony formation, transwell, wound healing, EdU) and xenograft models were employed to assess the role of PAQR4 in BCa progression. Mechanistic studies included co-immunoprecipitation, mass spectrometry, ubiquitination assays, dual-luciferase reporter assays, and ferroptosis evaluation (lipid peroxidation, ROS, GSH, Fe²⁺, mitochondrial morphology). Rescue experiments using the NRF2 agonist tBHQ or NRF2 overexpression were performed to confirm pathway dependence. PAQR4 was significantly upregulated in BCa tissues and cell lines, and high PAQR4 expression correlated with poor patient prognosis. PAQR4 knockdown suppressed BCa cell proliferation, migration, invasion, and tumor growth in vivo. Mechanistically, PAQR4 bound to the DGR domain of KEAP1, competitively disrupting KEAP1-NRF2 interaction, thereby inhibiting NRF2 ubiquitination and proteasomal degradation. This led to NRF2 stabilization, enhanced NRF2 nuclear accumulation and transcriptional activity, and upregulation of its downstream targets, including the ferroptosis defense enzymes SLC7A11 and GPX4. Consequently, PAQR4 knockdown sensitized BCa cells to erastin-induced ferroptosis (10 µM, 24 h), as evidenced by increased lipid peroxidation, mitochondrial shrinkage, elevated ROS and Fe²⁺, and reduced GSH—effects specifically reversed by Fer-1. Moreover, PAQR4 knockdown resensitized cisplatin-resistant T24/DDP cells to cisplatin in vitro and in vivo, accompanied by increased γH2AX expression and reduced ERCC1 expression. Rescue experiments confirmed that NRF2 is necessary and sufficient for PAQR4-mediated ferroptosis inhibition and cisplatin resistance. This study identifies PAQR4 as a novel negative regulator of ferroptosis and a critical mediator of cisplatin resistance in BCa via the KEAP1-NRF2 axis, suggesting that targeting PAQR4 may represent a promising therapeutic strategy to suppress tumor progression and overcome chemoresistance.