Androgen Regulated Genes in Human Prostate Xenografts in Mice: Relation to BPH and Prostate Cancer

Benign prostatic hyperplasia (BPH) and prostate carcinoma (CaP) are linked to aging and the presence of androgens, suggesting that androgen regulated genes play a major role in these common diseases. Androgen regulation of prostate growth and development depends on the presence of intact epithelial-stromal interactions. Further, the prostatic stroma is implicated in BPH. This suggests that epithelial cell lines are inadequate to identify androgen regulated genes that could contribute to BPH and CaP and which could serve as potential clinical biomarkers. In this study, we used a human prostate xenograft model to define a profile of genes regulated in vivo by androgens, with an emphasis on identifying candidate biomarkers. Benign transition zone (TZ) human prostate tissue from radical prostatectomies was grafted to the sub-renal capsule site of intact or castrated male immunodeficient mice, followed by the removal or addition of androgens, respectively. Microarray analysis of RNA from these tissues was used to identify genes that were; 1) highly expressed in prostate, 2) had significant expression changes in response to androgens, and, 3) encode extracellular proteins. A total of 95 genes meeting these criteria were selected for analysis and validation of expression in patient prostate tissues using quantitative real-time PCR. Expression levels of these genes were measured in pooled RNAs from human prostate tissues with varying severity of BPH pathologic changes and CaP of varying Gleason score. A number of androgen regulated genes were identified. Additionally, a subset of these genes were over-expressed in RNA from clinical BPH tissues, and the levels of many were found to correlate with disease status. Our results demonstrate the feasibility, and some of the problems, of using a mouse xenograft model to characterize the androgen regulated expression profiles of intact human prostate tissues

Real-time qRT-PCR analysis in RNA pools derived from BPH and prostate cancer.

<p>Comparison between gene status in BPH and prostate cancer. BPH categories are mild, moderate (Mod), and severe (Sev) and prostate cancer (CaP) pools are derived from both moderately and poorly differentiated tissues. Average expression levels <1-fold were defined as “low”, levels >6-fold were defined as “high”, and levels between 1-fold and 6-fold were defined as “moderate”. Expression levels are relative to a control pool derived from TZ samples from patients with no appreciable TZ expansion. Genes that appear twice were measured using two distinct primer sets. BPH/CaP is the ratio of the average levels in the BPH pools divided by the average levels in the prostate cancer pools.</p

Selection criteria for candidate biomarker targets.

<p>Potential candidates from our microarray results (androgen-regulated ≥1.5-fold or <i>P</i>≤0.05) were combined with systematically selected genes using overlapping bioinformatics criteria, employing WebGestalt. T14 = testosterone, day 14 and C14 = castrate, day 14.</p

Genes induced in response to androgen supplementation, day 0 vs. day 14.

<p>Genes induced in response to androgen supplementation, day 0 vs. day 14.</p

Immunohistochemical staining by disease severity category.

<p>Percentage staining was scored on a scale of 0 to 4, where 0 = no staining, 1 = less than 25%, 2 = 25% to 50%, 3 = 50% to 75%, and 4 = 75% to 100%. The intensity of staining was scored on a scale of 1 to 3, where 1 = mild, 2 = moderate, and 3 = marked. For a sample yielding no staining, the intensity score was 0. When both stromal and epithelial staining was present, scoring was done separately. For the samples for which two measurements were available, the average score was used to represent the sample. To calculate a protein expression index, the percentage score was summed with the intensity score; the maximum score was 7. The horizontal gray line represents within-group median.</p

Genes induced in response to androgen withdrawal, day 0 vs. day 14.

<p>Genes induced in response to androgen withdrawal, day 0 vs. day 14.</p

Percentages of immunostaining scores from duplicated samples separated by more than 1.

<p>Tissue sections were scored on a scale of 0–7 for combined extent and intensity. The ratios listed are the number of duplicate sections with scores differing by more than 1, divided by the total number of sections scored. Intensity is scored only on the samples for which percentage score is greater than 0.</p

Immunohistochemical staining for FGF2, SMOC1, and TIMP2 in BPH samples with different degrees of severity.

<p>Tissue sections are ∼0.6 mm in diameter. FGF2 and SMOC1 staining was observed in both stromal and epithelial cells while TIMP2 staining was present predominantly in stromal cells.</p

Experimental scheme for addition or withdrawal of testosterone in human prostate xenografts.

<p>TZ tissues from six patients were xenografted beneath the renal capsules of castrated male SCID mice (10 mice per patient). Five mice from each group were then given sub-cutaneous implants containing 25 mg of testosterone. After allowing the xenographs to establish for one month, the implants were removed from the testosterone supplemented mice, and 25 mg testosterone pellets were implanted in mice that had not received testosterone. Control mice were sacrificed at the time of androgen addition or removal, and the remaining mice from each group were sacrificed at 1, 3, 7, and 14 days.</p