Proteomic analysis of patient tissue reveals PSA protein in the stroma of benign prostatic hyperplasia

Abstract

Background: Benign prostatic hyperplasia (BPH) is an age-related disease frequently associated with lower urinary tract symptoms (LUTS) that involves hyperplasia of both epithelial and stromal cells. Stromal fibrosis is a distinctive feature of BPH, but the exact mechanisms underlying this phenomenon are poorly understood.

Methods: In the current study, proteomics analyses were utilized to identify proteins altered in the BPH stromal compartment from patients with symptomatic BPH. Stromal cells were isolated from histological nodules of BPH by laser capture microdissection (LCM) and subjected to liquid chromatography/mass spectrometry.

Results: Proteins identified included several stromal-specific proteins involved in extracellular matrix (ECM) remodeling, focal adhesion, and cellular junctions. Additionally, the proteomics array identified the presence of luminal epithelial secretory protein PSA. Immunostaining, ELISA, and in situ hybridization analyses of BPH tissues verified the presence of PSA protein but absence of PSA mRNA in the stromal compartment. E-cadherin was down-regulated in BPH epithelial cells compared to normal adjacent tissues, suggesting that alteration of cellular junctions could contribute to the presence of luminal epithelial secreted proteins PSA and KLK2 in the stromal compartment.

Conclusions: The above findings suggest that the presence of secreted proteins PSA and KLK2 from prostate luminal epithelial cells in BPH stroma is a hallmark of BPH nodules, which could in part be due to alterations in cellular junction proteins and/or increased epithelial barrier permeability. Elucidating the cause and consequence of these secreted proteins in the stromal compartment of BPH may lead to new understanding of BPH pathogenesis as well as approaches to prevent and/or treat this common disease.

Keywords: BPH; PSA; stroma.

Figure 1. Laser capture microdissection of BPH and normal adjacent stroma

Areas of prostate stroma (S) were isolated away from epithelial (E) areas in human prostate BPH and normal adjacent paraffin embedded tissues. Areas captured are outlined in green (center panel, Before LCM) and shown after microdissection (right panel, After LCM).

Figure 2. Detection of PSA protein in the stroma of nodular BPH

A. Immunostaining of serial sections of normal human prostate and treatment naïve BPH with PSA and stromal marker alpha smooth muscle actin (αSMA). Data are representative of 11 out of 11 (100%) patient specimens examined. B. Stromal PSA immunostaining score from representative BPH patient specimens (n=20). Pathology and scores were determined by a board certified genitourinary pathologist (AVP). C. ELISA for stromal PSA protein performed on LCM dissected stroma of BPH and normal adjacent tissue (n=3). Significance was determined by Student’s t-test (*** p, 0.0001, * p, 0.05). D. Stromal KLK2 immunostaining in BPH node. Inset – Arrow denotes edge of BPH node coincides with KLK2 staining in BPH stroma but not normal adjacent stroma.

Figure 3. PSA mRNA expression in nodular BPH

In situ hybridization of PSA mRNA expression (in red) was confined to the luminal secretory epithelial cells in the prostate and was not apparent in the stromal (S) compartment (see DAPI nuclear staining in blue). Left panel is brightfield image depicting PSA in red; center panel is fluorescent image depicting PSA in red; right panel is DAPI nuclear staining in blue.

Figure 4. PSA protein expression in prostate cancer

Immunostaining of serial sections of human prostate cancer, stromal hyperplasia, (negative for PSA n = 0/8) and basal cell hyperplasia (no PSA expression seen in the stromal areas within n= 0/5).

Figure 5. Expression of E-cadherin in BPH and the rat model of prostatic inflammation

A. Immunostaining of normal human prostate (N) and treatment naïve BPH (B) with E-cadherin. Data are representative of 11 out of 11 (100%) patient specimens examined. B. E-cadherin immunostaining in rat ventral prostate stimulated with saline (top panel) or formalin (bottom panel). Red arrows indicate areas of inflammation. Data are representative of at least 6 animals per group.

Figure 6. Schematic of stromal PSA and ECM remodeling in BPH

Increased epithelial barrier permeability due to loss of cellular junctions in BPH could allow the infiltration of PSA into the stromal compartment.