A network pharmacology-guided multi-omics and spatial single-cell framework nominates WT1 as a spironolactone-linked...

Spironolactone (SPI), a mineralocorticoid receptor antagonist with anti-androgenic activity, has emerged as a candidate for drug repurposing in prostate cancer (PCa). However, the cellular pharmacology governing its impact on the tumor microenvironment and specific molecular targets remains incompletely understood. This study aims to elucidate the regulatory circuitry linking SPI to immune modulation and tumor suppression in the PCa ecosystem.

We employed an integrated framework combining network pharmacology with bulk, single-cell (scRNA-seq), and spatial transcriptomics (ST) to prioritize SPI-associated targets and map their microenvironmental context. The clinical and immunological relevance of the top candidate, Wilms Tumor 1 (WT1), was assessed using TCGA and GEO datasets. Finally, molecular docking and in vitro gain-of-function assays (proliferation, migration, clonogenicity, and apoptosis) in DU145 and PC-3 cell lines were conducted to validate functional mechanisms.

Network pharmacology identified WT1 as a central regulatory node in the SPI-PCa interaction network. In clinical cohorts, WT1 was significantly downregulated in tumor tissues compared to normal prostate; however, preserved high WT1 expression correlated with improved disease-free survival. Crucially, WT1-high tumors exhibited an "immune-hot" phenotype characterized by enhanced T-cell infiltration and antigen presentation pathways. scRNA-seq and ST analyses revealed that WT1 is heterogeneously expressed across malignant and stromal compartments, localizing to specific immune-interacting niches. Molecular docking suggested a potential structural compatibility between SPI and WT1, although direct binding remains to be experimentally confirmed. Functionally, restoring WT1 expression in PCa cells potently suppressed malignant behaviors, inhibiting proliferation and migration while triggering apoptosis.

This study defines a novel SPI-WT1 axis, positioning WT1 as a druggable, immune-correlated biomarker in prostate cancer. By linking SPI pharmacology to WT1-associated microenvironmental features and tumor suppression, our findings provide a mechanistic rationale for repurposing Spironolactone as an immunomodulator to overcome therapeutic resistance in genitourinary oncology.