Comprehensive multi-omics analysis reveals a robust transcriptomic signature for biochemical recurrence prediction and identifies SPAG5 as a targetable oncogene with small-molecule compounds in prostate cancer

Biochemical recurrence (BCR) after radical prostatectomy indicates occult progression in prostate cancer (PCa), but reliable prognostic tools and targeted therapies for high-risk patients remain lacking. We integrated TCGA and GEO transcriptomic and clinical data. BCR-associated genes were identified using DESeq2/limma, and a prognostic model was constructed using Cox/LASSO regression. Model performance was assessed by Kaplan-Meier, ROC, calibration, and nomogram. GSEA and CIBERSORT explored pathways and immune infiltration. Focusing on SPAG5, we characterized its expression using multi-omics datasets (bulk transcriptome, DNA methylation, spatial and single-cell transcriptomes, and somatic mutations), and performed in vitro (CCK-8, colony formation, wound healing, Transwell, cell cycle) and in vivo (mouse xenograft, including shRNA-resistant SPAG5 rescue) functional assays. Virtual screening identified small-molecule compounds targeting SPAG5, and their binding affinity, thermal stabilization, and degradation mechanisms were validated by cellular thermal shift assay (CETSA), isothermal dose-response CETSA, and proteasome/autophagy inhibition experiments. A four-gene signature (SPAG5, LPL, ASPN, THBS2) stratified patients into high- and low-risk BCR groups, with robust accuracy in training and validation cohorts. High-risk patients showed enrichment of oncogenic pathways and elevated M2 macrophage infiltration. SPAG5 had the highest risk coefficient. Multi-omics analysis linked SPAG5 promoter hypomethylation to overexpression, and spatial and single-cell analyses showed enrichment in malignant regions and tumor-associated cell populations. Somatic mutation analysis linked SPAG5 overexpression to TP53 mutations. Consistently, SPAG5 was significantly overexpressed in PCa tissues and cells. In vitro, SPAG5 knockdown suppressed proliferation, migration, and invasion, and induced G1 arrest, whereas overexpression had opposite effects. In vivo, SPAG5 knockdown attenuated tumor growth, partially rescued by shRNA-resistant SPAG5. Two compounds, (-)-Hydroxycitric acid lactone and 2,4,6-Trihydroxybenzoic_acid, directly bound and thermally stabilized SPAG5 and promoted its proteasomal degradation, reducing PCa cell viability with little effect on SPAG5-low RWPE-1 cells. The rescue group restored compound sensitivity with a significantly lower IC50 than sh-SPAG5 cells. This four-gene signature provides a robust and clinically applicable framework for BCR risk stratification in PCa. Furthermore, SPAG5 is identified as a functionally relevant oncogenic driver and a potential therapeutic target, and the identified small-molecule compounds represent potential hit compounds that warrant further optimization for targeted therapy in recurrent PCa.