Transcriptomic analysis of benign prostatic hyperplasia identifies critical pathways in prostatic overgrowth and 5‐alpha reductase inhibitor resistance

BACKGROUND: The medical therapy of prostatic symptoms (MTOPS) trial randomized men with symptoms of benign prostatic hyperplasia (BPH) and followed response of treatment with a 5α-reductase inhibitor (5ARI), an alpha-adrenergic receptor antagonist (α-blocker), the combination of 5ARI and α-blocker or no medical therapy (none). Medical therapy reduced risk of clinical progression by 66% but the reasons for nonresponse or loss of therapeutic response in some patients remains unresolved. Our previous work showed that prostatic glucocorticoid levels are increased in 5ARI-treated patients and that glucocorticoids can increased branching of prostate epithelia in vitro. To understand the transcriptomic changes associated with 5ARI treatment, we performed bulk RNA sequencing of BPH and control samples from patients who received 5ARI versus those that did not. Deconvolution analysis was performed to estimate cellular composition. Bulk RNA sequencing was also performed on control versus glucocorticoid-treated prostate epithelia in 3D culture to determine underlying transcriptomic changes associated with branching morphogenesis. METHOD: Surgical BPH (S-BPH) tissue was defined as benign prostatic tissue collected from the transition zone (TZ) of patients who failed medical therapy while control tissue termed Incidental BPH (I-BPH) was obtained from the TZ of men undergoing radical prostatectomy for low-volume/grade prostatic adenocarcinoma confined to the peripheral zone. S-BPH patients were divided into four subgroups: men on no medical therapy (none: n = 7), α-blocker alone (n = 10), 5ARI alone (n = 6) or combination therapy (α-blocker and 5ARI: n = 7). Control I-BPH tissue was from men on no medical therapy (none: n = 8) or on α-blocker (n = 6). A human prostatic cell line in 3D culture that buds and branches was used to identify genes involved in early prostatic growth. Snap-frozen prostatic tissue taken at the time of surgery and 3D organoids were used for RNA-seq analysis. Bulk RNAseq data were deconvoluted using CIBERSORTx. Differentially expressed genes (DEG) that were statistically significant among S-BPH, I-BPH, and during budding and branching of organoids were used for pathway analysis. RESULTS: Transcriptomic analysis between S-BPH (n = 30) and I-BPH (n = 14) using a twofold cutoff (p < 0.05) identified 377 DEG (termed BPH377) and a cutoff < 0.05 identified 3377 DEG (termed BPH3377). Within the S-BPH, the subgroups none and α-blocker were compared to patients on 5ARI to reveal 361 DEG (termed 5ARI361) that were significantly changed. Deconvolution analysis of bulk RNA seq data with a human prostate single cell data set demonstrated increased levels of mast cells, NK cells, interstitial fibroblasts, and prostate luminal cells in S-BPH versus I-BPH. Glucocorticoid (GC)-induced budding and branching of benign prostatic cells in 3D culture was compared to control organoids to identify early events in prostatic morphogenesis. GC induced 369 DEG (termed GC359) in 3D culture. STRING analysis divided the large datasets into 20-80 genes centered around a hub. In general, biological processes induced in BPH supported growth and differentiation such as chromatin modification and DNA repair, transcription, cytoskeleton, mitochondrial electron transport, ubiquitination, protein folding, and cholesterol synthesis. Identified signaling pathways were pooled to create a list of DEG that fell into seven hubs/clusters. The hub gene centrality was used to name the network including AP-1, interleukin (IL)-6, NOTCH1 and NOTCH3, NEO1, IL-13, and HDAC/KDM. All hubs showed connections to inflammation, chromatin structure, and development. The same approach was applied to 5ARI361 giving multiple networks, but the EGF and sonic hedgehog (SHH) hub was of particular interest as a developmental pathway. The BPH3377, 5ARI363, and GC359 lists were compared and 67 significantly changed DEG were identified. Common genes to the 3D culture included an IL-6 hub that connected to genes identified in BPH hubs that defined AP1, IL-6, NOTCH, NEO1, IL-13, and HDAC/KDM. CONCLUSIONS: Reduction analysis of BPH and 3D organoid culture uncovered networks previously identified in prostatic development as being reinitiated in BPH. Identification of these pathways provides insight into the failure of medical therapy for BPH and new therapeutic targets for BPH/LUTS.

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