NOTCH3 regulates myofibroblastic CAF differentiation via the P62–ROS signaling axis to promote bladder cancer progression

Abstract Cancer-associated fibroblasts (CAFs) are important contributors to malignant progression in bladder cancer (BLCA), yet the mechanisms by which they promote tumor progression remain incompletely understood. In this study, we integrated patient-derived CAFs with bulk and single-cell transcriptomics, spatial transcriptomics, and multi-cohort clinical datasets to systematically define the molecular mechanisms underlying CAF-driven BLCA progression. Integrated analyses of bulk RNA sequencing, single-cell transcriptomics, spatial transcriptomics, immunohistochemistry, and multiplex immunofluorescence of paraffin-embedded tissues, together with PCR, immunoblotting, and immunofluorescence in patient-derived normal fibroblasts and CAFs, consistently demonstrated marked enrichment of NOTCH3 in CAFs in bladder cancer. Silencing of NOTCH3 attenuated extracellular matrix deposition, contractile capacity, intracellular ROS accumulation, and the ability of CAFs to support tumor growth. Mechanistically, TGF-β induced NOTCH3 transcription through SMAD2 binding to the NOTCH3 promoter. In turn, NOTCH3 recruited HSPA8 to promote K48-linked ubiquitination and proteasomal degradation of P62, thereby suppressing the P62–NRF2 antioxidant pathway and sustaining intracellular ROS accumulation. Restoration of P62 expression or pharmacological scavenging of ROS effectively reversed NOTCH3-driven myofibroblastic differentiation and stromal support of tumor growth. In vivo, CAF-specific deletion of NOTCH3 significantly inhibited tumor growth, reduced collagen deposition, and attenuated stromal remodeling in both subcutaneous and orthotopic bladder cancer models. Collectively, these findings identify CAF-intrinsic NOTCH3 as important regulator of myofibroblastic differentiation through integration of upstream TGF-β signaling with redox and proteostatic control, highlighting stromal NOTCH3 as a potential therapeutic target to limit malignant progression in BLCA.