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. 2022 Apr;256(4):427-441.
doi: 10.1002/path.5857. Epub 2022 Feb 3.

5-Alpha reductase inhibitors induce a prostate luminal to club cell transition in human benign prostatic hyperplasia

Diya B Joseph  1 , Gervaise H Henry  1 , Alicia Malewska  1 , Jeffrey C Reese  2 , Ryan J Mauck  1 , Jeffrey C Gahan  1 , Ryan C Hutchinson  1 , James L Mohler  3 , Claus G Roehrborn  1 , Douglas W Strand  1
Affiliations

5-Alpha reductase inhibitors induce a prostate luminal to club cell transition in human benign prostatic hyperplasia

Diya B Joseph et al. J Pathol. 2022 Apr.

Abstract

Benign prostatic hyperplasia (BPH) is a progressive expansion of peri-urethral prostate tissue common in aging men. Patients with enlarged prostates are treated with 5-alpha reductase inhibitors (5ARIs) to shrink prostate volume by blocking the conversion of testosterone to dihydrotestosterone (DHT). A reduction in DHT levels can elicit atrophy and apoptosis of prostate secretory luminal cells, which results in a favorable clinical response characterized by improved lower urinary tract symptoms. However, the histologic response to 5ARI treatment is often heterogeneous across prostate acini and lower urinary tract symptoms can persist to require surgical intervention. We used two spatial profiling approaches to characterize gene expression changes across histologically normal and atrophied regions in prostates from 5ARI-treated men. Objective transcriptomic profiling using the Visium spatial gene expression platform showed that 5ARI-induced atrophy of prostate luminal cells correlated with reduced androgen receptor signaling and increased expression of urethral club cell genes including LTF, PIGR, OLFM4, SCGB1A1, and SCGB3A1. Prostate luminal cells within atrophied acini adapted to decreased DHT conditions by increasing NF-κB signaling and anti-apoptotic BCL2 expression, which may explain their survival. Using GeoMx digital spatial profiling with a probe set to assess ~18 000 RNA targets, we confirmed that atrophied acini expressing SCGB3A1 displayed higher levels of club cell markers compared with histologically normal acini with NKX3-1 expression. In addition, club-like cells within regions of 5ARI-induced atrophy closely resembled true club cells from the prostatic urethra. A comparison of histologically normal regions from 5ARI-treated men and histologically normal regions from untreated men revealed few transcriptional differences. Taken together, our results describe a heterogeneous response to 5ARI treatment where cells in atrophied acini undergo an adaptation from a prostate secretory luminal to a club cell-like state in response to 5ARI treatment. © 2021 The Pathological Society of Great Britain and Ireland.

Keywords: 5-alpha reductase inhibitor; benign prostatic hyperplasia; club cell; luminal epithelia; spatial transcriptomics.

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Conflict of interest statement

No conflicts of interest were declared

Figures

Figure 1.
Figure 1.. Increased transcriptional heterogeneity of prostate epithelial cells in 5ARI-treated men.
(A) Cryosections from untreated and 5ARI-treated prostates were placed on Visium spatial gene expression slides. Transcriptional data from individual capture dots were compared to normal prostate single cell RNA-sequencing dataset Joseph et al 2021 [9] to obtain identities for each capture dot. Cell type probability scores for 11 cell types in the prostate were calculated which together sum to 1. The cell type associated with the highest probability score value was assigned as the identity of the capture dot. Capture dot identities were overlaid on the image of the original section stained with hematoxylin and eosin. Dots identified as prostate luminal and club were overlaid on slide scans of prostates from (B) untreated and (C) 5ARI-treated men. Magnified regions from (B) underlying dots identified as prostate luminal are shown in B’,B”. Magnified regions from (C) underlying dots identified as prostate luminal and club are shown in C’ and C” respectively. Scale bar, 100 μm. Abbreviations: peFib-Peri epithelial fibroblast, iFib-Interstitial fibroblast, SM-Smooth muscle.
Figure 2.
Figure 2.. Spectrum of prostate luminal and club scores in 5ARI-treated men.
Club score values are plotted as a spatial heatmap on prostates from (A) untreated and (B) 5ARI-treated BPH patients. Prostate luminal score values are plotted as a spatial heatmap on (C) untreated and (D) 5ARI-treated BPH samples. Dots identified as prostate luminal and club were subsetted. Prostate luminal and club score values of these dots are plotted against each other. Dots are shaded by (E) cell identity and (F) treatment status.
Figure 3.
Figure 3.. Club like gene expression is associated with morphological atrophy in 5ARI-treated men.
Dots identified as prostate luminal and club were subsetted. (A) Prostate luminal dots were categorized into ‘prostate luminal’ (<0.1 club score) and ‘intermediate luminal’ (>0.1 club score). Club dots were classified into ‘club-like’ (<0.1 prostate luminal score) and ‘intermediate club-like’ (>0.1 prostate luminal score). The four categories were overlaid on images from (B) untreated and (C) 5ARI-treated BPH prostates. Images of magnified regions underlying (D) untreated prostate luminal dots, (E) 5ARI-treated prostate luminal dots, (F) 5ARI-treated intermediate luminal dots, (G) 5ARI-treated intermediate club-like dots and (H) 5ARI-treated club-like dots. Morphometric analysis was performed on acini from all 5 categories. Plots showing (I) acinus area, (J) acinus circularity (values from 0–1, 1 is a perfect circle), (K) epithelial cell heights, (L) lumen area, (M) lumen circularity and (N) cell layer thickness. Scale bar, 100 μm. Asterisks indicate significant p-values (* p<0.05, **<0.01, ***<0.001, ****<0.0001). Abbreviations: Int-Intermediate, PrLE-Prostate luminal epithelium, IntLE-Intermediate luminal epithelium, IntClub-Intermediate club.
Figure 4.
Figure 4.. Progressive increase in club gene expression in situ in prostate luminal cells from 5ARI-treated men.
Violin plot depicting expression of the androgen receptor target genes (A) MSMB, (B) KLK3 and (C) NKX3-1 in prostate luminal, intermediate luminal, intermediate club-like and club-like dots. (D) Violin plot for LTF expression. Immunostaining with antibodies against LTF (in red), KRT5 (in white) and NKX3-1 (in green) in (E) untreated prostates, (F) regular acini and (G) atrophic acini from 5ARI-treated prostates. (H) Violin plot for PIGR expression. Immunostaining with antibodies against PIGR (in red), KRT5 (in white) and NKX3-1 (in green) in (I) untreated prostates, (J) regular acini and (K) atrophic acini from 5ARI-treated prostates. (L) Violin plot for OLFM4 expression. Immunostaining with antibodies against OLFM4 (in red), KRT5 (in white) and NKX3-1 (in green) in (M) untreated prostates, (N) regular acini and (O) atrophic acini from 5ARI-treated prostates. (P) Violin plot for SCGB1A1 expression. Immunostaining with antibodies against SCGB1A1 (in red), KRT5 (in white) and NKX3-1 (in green) in (Q) untreated prostates, (R) regular acini and (S) atrophic acini from 5ARI-treated prostates. (T) Violin plot for SCGB3A1 expression. In situ hybridization with probes against SCGB3A1 (in red) and immunostaining with antibodies against KRT5 (in white) in (U) untreated prostates, (V) regular acini and (W) atrophic acini from 5ARI-treated prostates. DAPI staining is in blue. Scale bars, 100 μm. Abbreviations: PrLE-Prostate luminal epithelium, IntLE-Intermediate luminal epithelium, IntClub-Intermediate club.
Figure 5.
Figure 5.. NF-κB and anti-apoptotic signaling is increased in atrophied regions from 5ARI-treated men
(A) Violin plot depicting NF-κB target gene scores in prostate luminal, intermediate luminal, intermediate club-like and club-like dots. Immunostaining with antibodies against phospho-P65 (in red) and KRT5 (in green) in (B) untreated prostates, (C) regular acini and (D) atrophic acini from 5ARI-treated prostates. (E) Violin plot depicting anti-apoptotic target gene scores in prostate luminal, intermediate luminal, intermediate club-like and club-like dots. Immunostaining with antibodies against BCL2 (in red) and KRT5 (in green) in (F) untreated prostates, (G) regular acini and (H) atrophic acini from 5ARI-treated prostates. (I) Primary prostate epithelial cells treated with TNFα in combination with the NF-κB pathway inhibitor BMS-345541. Results representative of experiments conducted on primary prostate cells derived from at least 2 separate patients. Asterisks represent significant p values (* p<0.05, *** p<0.001). (J) Schematic showing decrease in AR signaling and a reciprocal increase in NF-κB signaling after 5ARI treatment along with a transition from in-folded prostate acini to small atrophied acini. DAPI staining is in blue. Scale bars, 100 μm. Abbreviations: PrLE-Prostate luminal epithelium, IntLE-Intermediate luminal epithelium, IntClub-Intermediate club.
Figure 6.
Figure 6.. Spatial profiling of segmented cell types in prostatic urethra and BPH confirms transcriptional similarity between urethral club cells and club-like atrophy
Paraffin tissue sections labeled with probes to NKX3-1 (in green) and SCGB3A1 (in red) were hybridized with the GeoMx whole transcriptome atlas RNA probe set. (A) Representative images of scanned slides showing ROI selection and cell marker segmentation in prostate sections from (A) untreated and (B) 5ARI-treated BPH patients. Images are representative of n=3 slides/group. Magnified regions show representative ROIs and segmentation of (A’) urethral club cells (SCGB3A1+) and (A”) histologically normal prostate luminal regions (NKX3-1+) from untreated men. Representative ROIs and segmentation of (B’) atrophied club-like regions (SCGB3A1+) and (B”) histologically normal prostate luminal regions (NKX3-1+) from 5ARI-treated men. (C) UMAP plot of 4 segmented categories. An intermediate prostate luminal category from 5ARI-treated patients is circled by dotted lines. (D–I) Images of segmented ROIs labeled on the UMAP plot in panel (C) representing the spectrum of normal to atrophied prostate acini. (J) Differential expression analysis was performed comparing club-like and PrLE categories from 5ARI-treated patients (n=18 ROIs/group from 3 patients). Top 25 genes sorted by log2 fold-change that were up and down in the club-like category (FDR<0.05) were used for unsupervised clustering of all ROIs from 5ARI and untreated patients. Results are depicted in a heatmap of normalized target counts. (K) DEGs in urethral club versus prostate luminal segments from untreated men. (L) DEGs in club-like cells versus prostate luminal segments from 5ARI-treated men. (M) Common DEGs between (K) and (L). (N) DEGs between urethral club segments in untreated men versus club-like segments in 5ARI-treated men. (O) DEGs between prostate luminal segments in untreated versus 5ARI-treated men. (P) Volcano plot of DEGs in urethral club versus prostate luminal segments from untreated men. (Q) Volcano plot of DEGs in prostate luminal cells versus club-like segments from 5ARI-treated men. Syto13 staining for DNA is in blue. Gray scale bar, 2.5 mm. White scale bar, 100 μm. Abbreviations: PrLE-Prostate Luminal, Ur-Urethral.
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