Prognostic biomarkers and Hedgehog pathway activation in early prostate cancer neuroendocrine differentiation via spatial profiling.

Prostate cancer (PCa) progression to treatment-related neuroendocrine PCa (t-NEPC) often involves an early neuroendocrine differentiation (NED) phase lacking identified biomarkers. We utilized GeoMx Digital Spatial Profiling and single-cell RNA sequencing on human PCa tissues ranging from hormone-naïve (HNPC) to t-NEPC, with extensive multi-omics validation. Our results established early NED as a distinct, prognostically adverse subtype. We identified and validated 6 NED markers (FMN2, APLP1, SCG2, SCG3, CHGB, and RIMBP2), demonstrating specificity for NE cells and consistent upregulation throughout NEPC progression. Notably, the Hedgehog (Hh) pathway was specifically activated in early NED, manifested by high SHH ligand in neuroendocrine cells, GLI1 upregulation, and intercellular communication mediated via the SHH-Hh axis. Conversely, in late-stage t-NEPC, Hh pathway activity was significantly downregulated, while MYC-driven proliferation pathways predominated. A risk score derived from the 6-gene NED markers effectively stratified patients, correlating with pronounced NE features and shorter progression-free survival in 86 endocrine-treated patients and externally in The Cancer Genome Atlas (TCGA). In conclusion, this study delineates the dynamic molecular landscape of early NED, establishing a clinically promising biomarker panel and highlighting the Hh pathway as a stage-specific therapeutic target.