ABSTRACT Bladder cancer (BCa) is one of the most challenging and costly cancers to treat, yet little progress has been made on the development of predictive biomarkers and targeted therapies. Here, we uncover a critical function of Numb as a tumor suppressor in the bladder, identifying loss of Numb expression as a causal alteration in BCa that underlies biological aggressiveness and disease progression. Through retrospective cohort studies, we established that a Numb-deficient tumor status correlates with worse overall survival in post-cystectomy muscle-invasive bladder cancer (MIBC) patients and increased risk of MIBC progression in non-muscle-invasive bladder cancer (NMIBC) patients. The prognostic value of Numb loss can be attributed to its crucial role as a determinant of aggressive bladder tumorigenesis, as demonstrated in mouse and human models. Targeted Numb ablation in the basal layer of the urothelium was alone sufficient to trigger spontaneous bladder tumorigenesis and drive progression from preneoplastic to preinvasive and, ultimately, overtly invasive tumors. Additionally, Numb ablation sensitized the urothelium to other oncogenic insults, accelerating tumor onset and progression. Using 3D-Matrigel organoid cultures to recapitulate bladder tumorigenesis in vitro , we found that Numb loss heightens the proliferative and invasive potential of both mouse and human BCa cells. Integrative transcriptomic and functional analyses revealed that downregulation of the canonical Hippo pathway, resulting in enhanced YAP transcriptional activity, underlies the biological aggressiveness of Numb-deficient BCa. These molecular events are dependent on the activation of RhoA/ROCK signaling subsequent to Numb loss. Thus, a dysfunctional Numb–RhoA/ROCK–Hippo/YAP regulatory network is at play in aggressive Numb-deficient BCa and represents a therapeutic vulnerability. A 27-gene prognostic signature capable of identifying high-risk Numb-deficient patients could provide the basis of a clinical tool to stratify patients for innovative RhoA/ROCK/YAP targeted therapies. One Sentence Summary Numb loss-directed hyperactivation of RhoA/ROCK/YAP underlies aggressive bladder cancer biology.

Loss of the tumor suppressor NUMB drives aggressive bladder cancer through hyperactivation of a RhoA/ROCK/YAP signaling circuitry