Rb1 loss defines distinct migration histories in metastatic prostate cancer subtypes

Rb1 loss is a hallmark of lethal castration-resistant prostate cancer (CRPC), yet its role in shaping metastatic dissemination patterns remains poorly understood. Using our EvoCaP somatically engineered mouse model (SEMM), we generated Pten / Trp53 / Rb1 -deficient ( 2PR ) mice and compared them to Pten / Trp53 -deficient ( 2P ) controls. Loss of Rb1 dramatically increased metastatic burden, particularly to visceral organs, and promoted neuroendocrine (NE) differentiation. Lineage tracing revealed that 2PR tumors establish metastatic hub organs that seed cascade dissemination to multiple secondary sites. Histologically, 2PR tumors displayed distinct NE and mesenchymal-like (ML) compartments, with NE regions showing elevated beta-catenin and EZH2 expression, markers of aggressive castration-resistant prostate cancers including: CRPC-WNT (Wnt signaling driven) and CRPC-NE (EZH2-high) subtypes. Organoids derived from 2PR tumors confirmed these transcriptional changes and showed enhanced sensitivity to EZH2 and WNT inhibitors. These findings reveal how Rb1 loss drives lineage plasticity and metastatic evolution in prostate cancer, identifying therapeutic vulnerabilities in this lethal disease subtype.