Genomic analysis of benign prostatic hyperplasia implicates cellular re-landscaping in disease pathogenesis

Abstract

Benign prostatic hyperplasia (BPH) is the most common cause of lower urinary tract symptoms in men. Current treatments target prostate physiology rather than BPH pathophysiology and are only partially effective. Here, we applied next-generation sequencing to gain new insight into BPH. By RNAseq, we uncovered transcriptional heterogeneity among BPH cases, where a 65-gene BPH stromal signature correlated with symptom severity. Stromal signaling molecules BMP5 and CXCL13 were enriched in BPH while estrogen regulated pathways were depleted. Notably, BMP5 addition to cultured prostatic myofibroblasts altered their expression profile towards a BPH profile that included the BPH stromal signature. RNAseq also suggested an altered cellular milieu in BPH, which we verified by immunohistochemistry and single-cell RNAseq. In particular, BPH tissues exhibited enrichment of myofibroblast subsets, whilst depletion of neuroendocrine cells and an estrogen receptor (ESR1)-positive fibroblast cell type residing near epithelium. By whole-exome sequencing, we uncovered somatic single-nucleotide variants (SNVs) in BPH, of uncertain pathogenic significance but indicative of clonal cell expansions. Thus, genomic characterization of BPH has identified a clinically-relevant stromal signature and new candidate disease pathways (including a likely role for BMP5 signaling), and reveals BPH to be not merely a hyperplasia, but rather a fundamental re-landscaping of cell types.

Keywords: Cell Biology; Expression profiling; Molecular pathology; Oncology; Prostate cancer.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. BPH transcriptional landscape and clinically relevant stromal signature.

(A) Heatmap of unsupervised clustering of normal prostate, BPH, and BPH stromal nodules (samples color coded) across the 2548 most variably expressed genes. Transcript levels (median-centered log2 reads per kilobase million values) are indicated by color key. Select gene features (clusters) are annotated based on the expression of characteristic marker genes, including a stroma/myofibroblast feature (enframed on the heatmap) enriched in BPH over normal prostate and heterogeneous among BPH cases. Laser capture microdissection (LCM) panel indicates genes more highly expressed in laser microdissected BPH epithelium (orange) or BPH stroma (purple). The graph illustrates correlation of gene expression features (moving average 51 genes) with BPH IPSS and Bother Score. Note: peak correlations reside within the core stromal feature. The data set corresponding to the heatmap is available as Supplemental Table 2. (B) High expression of a 65-gene stromal signature is associated with elevated IPSS (strong trend) and Bother Score. Mean (red) and SD (blue) shown; P values generated from 2-tailed Student’s t test. The 65 genes correspond to the core of the stromal gene feature (indicated in A), with high expression defined as the top 50%. (C) A similarly derived 57-gene AR/Secretory signature showed no association with IPSS or Bother Score.

Figure 2. Genes differentially expressed in BPH versus normal prostate.

(A) Heatmap of genes with significant differential expression in BPH versus normal prostate. Samples are clustered (see color key) and genes ordered by SAM score (t statistic value). (B) Volcano plot (Q value vs. log2 fold change) annotated with topmost genes differentially expressed in BPH. (C) Technical validation of RNA-Seq results by qRT-PCR, confirming elevated expression of BMP5 and CXCL13 in BPH. Five BPH-normal pairs assayed once with technical quadruplicates. Graphed are mean (±1 SD) relative expression levels, normalized to GAPDH and compared against a reference sample (normal prostate sample 8791). Each data point represents 1 of up to 16 pairwise ratios (calculated from the quadruplicate test and reference values). Below, heatmap depiction of corresponding RNA-Seq (median-centered log2 reads per kilobase million) values.

Figure 3. BMP5 addition to cultured prostate cells supports role in BPH disease process.

(A) Addition of BMP5 (250 ng/ml) to RWPE-1 prostate epithelial and WPMY-1 myofibroblast cells activates the canonical SMAD pathway, demonstrated by 15- to 20-fold increased phospho-SMAD1/5/9 on Western blot. (B) BMP5 addition (concentrations indicated) to RWPE-1 cells increases cell numbers. Mean (red) and SD (blue) shown. Multiplicity-adjusted P values generated from 1-way ANOVA with post hoc comparison to no BMP5 control (Dunnett’s test); *P < 0.05. Data are representative of 3 independent experiments, each done with 2 samples assayed per concentration. (C) Addition of BMP5 (250 ng/ml) to RWPE-1 cells leads to dispersed cell growth (with fewer cell clusters) and to increased Transwell migration. Mean and SD shown; P value generated from 2-tailed Student’s t test. Data are representative of 2 independent experiments, each done with 3 samples assayed per condition. (D and E) BMP5 addition to RWPE-1 and WPMY-1 cells generates a transcriptional response significantly enriched for BPH (vs. normal prostate) overexpressed genes (left), and with WPMY-1 cells for BPH stromal signature genes (center), but not AR/secretory signature genes (right). Gene set enrichment analysis (GSEA) enrichment score P values are indicated.

Figure 4. IHC on prostate TMA identifies altered ESR1+ cell subset in BPH.

(A–D) Hematoxylin and eosin stains of representative normal prostate and BPH-matched pair. Original magnifications, ×200 and ×800 (insets). (E–H) CHGA immunostaining (marker of neuroendocrine cells) shown for same case; note depletion of neuroendocrine cells in BPH. (I) CHGA IHC scores across all TMA cases (n = 22 normal and n = 21 BPH). Mean (red) and SD (blue) shown; P values generated from 2-tailed Student’s t test. (J–M) ESR1 immunostaining shown for same case; note depletion of periglandular ESR1+ fibroblast-like cells in BPH. (N) ESR1 IHC scores across all TMA cases (n = 21 normal and n = 20 BPH).

Figure 5. scRNA-Seq reveals altered cell subsets in BPH.

(A) Two-dimensional projection (t-SNE plot) of single-cell transcriptomes stratifies prostate tissue cells (dots) into distinct clusters, identifiable by characteristic expression of marker genes, including (B) KRT13 (basal epithelium) (red intensity scales to maximum log2 expression), (C) KLK3 (secretory epithelium), and (D) DCN (fibroblasts). Additional cell type markers are shown in Supplemental Figure 6. (E) Normal prostate tissue cell subset, illustrating expression of (F) CHGA (neuroendocrine cells), (G) BMP5, and (H) CXCL13. (I) BPH tissue cell subset, illustrating expression of (J) CHGA, (K) BMP5, and (L) CXCL13. Note relative depletion in BPH tissue of CHGA-expressing neuroendocrine cells and enrichment of BMP5- or CXCL13-expressing myofibroblasts. Insets magnify select cell clusters. Inset magnification ×1.6.

Figure 6. WES reveals somatic SNVs in BPH nodules.

(A) Integrated Genome Viewer (IGV) display of mapped reads from WES of BPH and matched normal DNA within the IL1B gene. Red reads correspond to the positive DNA strand, and blue reads correspond to the negative DNA strand; only a subset of mapped reads (totals indicated) shown. Note, the IL1B SNV in BPH leads to a frameshift with early translational termination. (B) Similar IGV display depicts a nonsynonymous SNV in the TTN gene in a different BPH case. (C) Sanger sequencing validation of IL1B SNV; red asterisk marks doublet peak indicative of SNV. (D) Sanger sequencing validation of TTN SNV; green asterisk marks doublet peak indicative of SNV. See Supplemental Table 10 for additional SNVs validated by deep amplicon resequencing.