Oxidative stress reprograms benign prostatic hyperplasia microenvironments: insights from integrative multi-omics and machine learning

Oxidative stress (OS) is increasingly implicated in benign prostatic hyperplasia (BPH), yet the underlying cellular programs remain unclear. We integrated multi-omics and machine learning to identify OS-associated biomarkers and to characterize OS-linked stromal states, with targeted experimental validation. Two bulk transcriptomic datasets (12 BPH, 16 controls) were integrated to identify DEGs and OS-associated DEGs, followed by WGCNA, enrichment analyses, and four machine-learning algorithms to prioritize hub genes and build a diagnostic nomogram. Consensus clustering defined molecular subtypes. Candidate compounds were screened in silico. Single-cell RNA-seq (124,616 cells) and spatial transcriptomics were analyzed for OS scoring, trajectories, and inferred intercellular communication. Experimentally, WPMY-1 prostatic stromal cells were exposed to LPS to induce OS; intracellular ROS (flow cytometry), ACOX2 expression (RT-qPCR/western blot), proliferation (EdU), and apoptosis (Annexin V) were assessed under ACOX2 overexpression or shRNA knockdown. ACOX2 expression in human prostate tissues was evaluated by immunohistochemistry. We identified 499 DEGs and 26 OS-DEGs. Machine learning converged on three upregulated hub genes: ACOX2, CTSB, and SERPINF1, which with diagnostic AUCs of 0.880 (0.753-1.000), 0.828 (0.663–0.993), and 0.854 (0.711–0.997); however, given the modest sample size, these findings should be interpreted as hypothesis-generating. Two BPH subtypes were identified (immune-infiltrated vs. non-immune). Single-cell analyses showed elevated OS scores across cell types, highest in fibroblasts; ACOX2-high fibroblasts were expanded in BPH and exhibited trajectory-associated increases in ACOX2 with enriched inferred signaling (including TNFSF12-TNFRSF12A). Spatial transcriptomics revealed regional OS heterogeneity and spatial association of hub-gene expression with OS-enriched areas. In vitro, LPS increased ROS and upregulated ACOX2, ACOX2 overexpression increased proliferation and reduced apoptosis, whereas knockdown showed opposite effects and attenuated LPS-associated proliferative phenotypes. IHC showed stronger ACOX2 staining in hyperplastic vs. normal prostate tissues. These results nominate ACOX2, CTSB, and SERPINF1 as candidate OS-associated markers in BPH and support a hypothesis-generating multi-omics signature that warrants validation in larger independent human cohorts. Experimental perturbation and tissue staining provide validation for an ACOX2-associated activation-like phenotype under OS, motivating biomarker-guided stratification and OS-targeted therapeutic exploration.