Spatial and single-cell transcriptomics reveal a HIF-1α/NF-κB-driven hypoxia-induced senescence axis in BPH...

Benign prostatic hyperplasia (BPH) is an age-related prostate disorder with incompletely defined mechanisms. We integrated laser capture microdissection RNA sequencing, public single-cell RNA sequencing, public spatial transcriptomics, human tissue validation, mouse-model analyses, and in vitro perturbation assays to characterize hypoxia-associated epithelial senescence in BPH. SA-β-gal-positive cells were predominantly epithelial. RNA sequencing of SA-β-gal-positive epithelial cells revealed enrichment of hypoxia/HIF-1, NF-κB, cell-cycle arrest, and senescence-associated programs, with increased HIF1A, senescence-marker, and SASP-associated gene expression. Single-cell and spatial analyses showed coordinated activation of hypoxia-response, NF-κB-related, and senescence-associated programs in luminal epithelial cells or luminal-dominant spots. In 52 human BPH specimens, epithelial HIF-1α staining was modestly but significantly associated with p21, p27, and Rb. In vitro, 1% O₂ exposure or HIF1A overexpression induced senescence-associated phenotypes in BPH-1 and RWPE-1 cells, whereas HIF-1α inhibition or HIF1A knockdown attenuated these effects. NF-κB inhibition or RELA knockdown partially reversed HIF-1α-associated senescence phenotypes. Conditioned media from hypoxia-treated epithelial cells promoted stromal-cell proliferation and SASP-associated cytokine secretion in a partly HIF1A-dependent manner. In a testosterone propionate-induced BPH-like mouse model, HIF-1α or NF-κB inhibition attenuated prostatic hyperplasia, epithelial remodeling, and senescence-marker expression. These findings link hypoxia-associated HIF-1α/NF-κB signaling to epithelial senescence and epithelial-stromal crosstalk in BPH.