Loss of NUMB drives aggressive bladder cancer via a RHOA/ROCK/YAP signaling axis.
We have discovered a previously uncharacterized tumor suppressor function of Numb in the homeostasis of the normal bladder mucosa. Targeted ablation of Numb in the basal layer of the urothelium drives spontaneous bladder tumorigenesis, driving progression from preneoplastic to preinvasive and overtly invasive tumors, and accelerates tumorigenesis in the presence of oncogenic insults. Using 3D-Matrigel organoid cultures to recapitulate bladder tumorigenesis in vitro, we found that Numb deficiency enhances the proliferative and invasive potential of both mouse and human bladder cancer 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 bladder cancer. 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 bladder cancers, opening avenues for the development of targeted therapies. Furthermore, retrospective cohort studies revealed that a Numb-deficient status correlates with worse overall survival in post-cystectomy muscle-invasive bladder cancer (MIBC) patients and increased risk of progression to MIBC in non-muscle-invasive bladder cancer (NMIBC) patients. The Numb-deficient status was associated with a 27-gene prognostic signature that has the potential to identify high-risk NMIBC patients who are eligible for targeted RhoA/ROCK/YAP therapies. Overall design: Gene expression profiling of NUMB-Low (HT1376, CLS439, 5637) and NUMB-High cell lines (KK47, RT4, RT112); NUMB-High cell lines (KK47, RT4, RT112) silenced for NUMB or scramble; NUMB-High cell lines (KK47, RT4, RT112) silenced for NUMB and treated with verterporfin, ROCKi or vehicle; RT4 cell line silenced for NUMB and then silenced for CYR61 and CTGF or scramble; Mouse Bladder Organoids (MBO) derived from WT and NUMB-KO mice; MBO NUMB-KO organoids treated with verteporfin, ROCKi or vehicle; tumor cells derived from WT and NUMB-KO mice treated with BBN; BBN NUMB-KO tumor cells treated with verteporfin, ROCKi or vehicle; BBN NUMB-KO tumor cells expressing shCYR61+shCTGF or shLuc.