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Clinical Studies

Variability in the Androgen Response of Prostate Epithelium to 5α-Reductase Inhibition: Implications for Prostate Cancer Chemoprevention

Elahe A. Mostaghel, Linda Geng, Ilona Holcomb, Ilsa M. Coleman, Jared Lucas, Lawrence D. True and Peter S. Nelson
DOI: 10.1158/0008-5472.CAN-09-2509 Published February 2010

Abstract

Inhibitors of 5α-reductase (SRD5A) that lower intraprostatic levels of dihydrotestosterone (DHT) reduce the overall incidence of prostate cancer (PCa), but there is significant variation in chemopreventive activity between individual men. In seeking molecular alterations that might underlie this variation, we compared gene expression patterns in patients with localized PCa who were randomized to prostatectomy alone versus treatment with two different doses of the SRD5A inhibitor dutasteride. Prostatic levels of DHT were decreased by >90% in both dutasteride-treated patient groups versus the untreated patient group. Despite significant and uniform suppression of tissue DHT, unsupervised clustering based on prostatic gene expression did not discriminate these groups. However, subjects could be resolved into distinct cohorts characterized by high or low expression of genes regulated by the androgen receptor (AR), based solely on AR transcript expression. The higher-dose dutasteride treatment group was found to include significantly fewer cancers with TMPRSS2-ERG genetic fusions. Dutasteride treatment was associated with highly variable alterations in benign epithelial gene expression. Segregating subjects based on expression of AR and androgen-regulated genes revealed that patients are differentially sensitive to SRD5A inhibition. Our findings suggest that AR levels may predict the chemopreventive efficacy of SRD5A inhibitors. Cancer Res; 70(4); 1286–95

Keywords
  • prostate cancer
  • chemoprevention
  • 5α-reductase
  • androgen receptor
  • dutasteride

Introduction

Androgens and the androgen receptor (AR) regulate prostate development and carcinogenesis (1, 2). Within the prostate, testosterone is converted to the more potent androgen, dihydrotestosterone (DHT), by 5α-reductase (SRD5A) types 1 and 2, and possibly type 3 (3, 4). The importance of SRD5A in mediating prostate carcinogenesis was tested in the Prostate Cancer Prevention Trial (PCPT), where the SRD5A2 inhibitor finasteride decreased prostate cancer (PCa) incidence by 25% (18% versus 24% in placebo-treated patients; ref. 5). This striking benefit was tempered by an increase in high-grade cancers, subsequently attributed to differential effects of finasteride on prostate volume and a possible selective inhibition of low-grade cancers (6). Using the dual SRD5A inhibitor dutasteride, the REduction of DUtasteride of Prostate Cancer Events (REDUCE) study reported a similar 23% reduction in PCa incidence (low-grade cancers in 13.3% versus 18.1% of placebo-treated patients; P < 0.0001), with no difference in high-grade cancers (7).

Mechanisms by which SRD5A inhibition decreases PCa incidence have not been determined but likely reflect decreased AR axis activity caused by reduced tissue DHT, potentially resulting in regression of extant cancers or suppression of de novo tumorigenesis. Importantly, 16% to 18% of subjects in both PCPT and REDUCE were diagnosed with PCa (of low- and high-grade histologies) despite undergoing SRD5A inhibition. Factors responsible for the variable outcome of SRD5A inhibition in reducing PCa incidence among different individuals are unknown.

We sought to identify dutasteride-related molecular alterations underlying the chemopreventive activity of SRD5A inhibition and to identify the effect of SRD5A inhibition on the prostatic AR axis. We evaluated prostate tissues from a randomized study of 81 men with clinically localized PCa undergoing surgery alone or after 4-month dutasteride treatment (8). We quantitated gene expression changes in microdissected prostate epithelium and evaluated gene activity in relation to intraprostatic testosterone and DHT concentrations.

Materials and Methods

Clinical Protocol

We obtained prostate tissue samples from a clinical trial evaluating dutasteride (GlaxoSmithKline) before radical prostatectomy (RP) as described (8). Procedures involving human subjects were approved by Institutional Review Boards of participating institutions; all subjects signed written informed consent. Tissues obtained at prostatectomy were frozen in OCT (Tissue-Tek) and formalin fixed and paraffin embedded. During the original clinical study, resected cancerous prostate tissues were preferentially placed in formalin for paraffin embedding. Histologic analysis of multiple sections from provided frozen tissue blocks revealed malignant prostate glands in <5% of samples; thus, we did not evaluate mRNA levels in malignant prostate epithelium.

Serum and Prostatic Androgen Measurements

Serum and prostate androgen levels were determined using gas chromatography/mass spectroscopy as described (PPD Development; ref. 8). Raw data for individual subjects (previously published in aggregate; ref. 8) were used for present analyses.

Gene Expression Assays

Laser capture microdissection, RNA amplification, and microarray hybridization

We used frozen prostate samples for laser capture microdissection (LCM) of benign tissue (neoplastic tissue was not identified in a sufficient number of frozen samples to allow analysis). We collected 2,000 to 3,000 benign epithelial cells per sample using the Arcturus Veritas LCM System for isolation of total RNA followed by two rounds of linear amplification as described (1, 9). Probe pairs (2 μg amplified RNA from microdissected samples and 30 μg total RNA from a reference prostate cell line RNA pool) were hybridized to custom cDNA microarrays as described (1, 9). Fluorescence images were collected (GenePix 4000B, Axon Instruments) and processed as described (10). We evaluated expression changes using two-sample t tests (Statistical Analysis of Microarray4; ref. 11). False discovery rate (FDR) less than 5% was considered significant. Microarray data can be accessed via the Gene Expression Omnibus database (GSM251831).

Cluster analysis

We performed unsupervised hierarchical average linkage clustering using Cluster 3.0 software5 plotted using TreeView6 version 1.6. We clustered samples using the top 1,000 most variable genes from microarray expression profiling (those with highest interquartile ratio, representing the spread between the 75th and 25th percentile of expression data obtained for each gene) or using a custom list of 90 androgen-regulated genes (see below).

Generation of androgen-regulated gene list for clustering

We included genes in the androgen-regulated set if they were present in at least two of the following sources: NetPath Androgen Receptor Pathway,7 a transcript profiling study of androgen-mediated expression in LNCaP cells by Nelson and colleagues (1), a transcript profiling study of androgen-mediated expression in multiple prostate cell lines by DePrimo and colleagues (≥3-fold change; ref. 12), and results from a whole-genome Agilent microarray comparing LNCaP and VCaP with or without the synthetic androgen R18818 (≥3-fold change). The 90 genes generated by this analysis are presented in Supplementary Fig. S1.

Quantitative reverse transcription-PCR

We validated gene expression changes using quantitative reverse transcription-PCR (qRT-PCR) in triplicate reactions using 5 ng cDNA, 1 μmol/L of each primer pair, and SYBR Green PCR master mix (Applied Biosystems). We normalized mean cycle threshold (Ct) for each gene to a housekeeping gene, RPL13A, in the same sample using the ΔCT method (primer sequences in Supplementary Fig. S2).

Analysis of AR transcript expression in human prostate samples

We used four human prostate microarray data sets to compare AR expression in benign prostate tissue. These consisted of 40 benign prostate samples adjacent to cancer analyzed on spotted 44K human cDNA microarrays by Lapointe and colleagues (13); 50 benign prostate samples adjacent to cancer analyzed on U95Av2 human Affymetrix arrays by Singh and colleagues (14); 23 benign prostate samples from normal donors plus 63 benign prostate samples adjacent to cancer analyzed on Affymetrix U95a, U95b, and U95c chip sets by Yu and colleagues (15); and 11 benign prostate samples on U95Av2 human Affymetrix arrays analyzed by Glinsky and colleagues (16) All studies used bulk tissue. We analyzed two human prostate sample sets by qRT-PCR for AR expression in microdissected benign prostate epithelium: the untreated samples obtained from the dutasteride treatment study reported here and benign prostate epithelium in prostate biopsy cores from 10 untreated individuals.

Immunohistochemistry and Fluorescence In situ Hybridization

Tissue microarray and immunohistochemistry

A tissue microarray (TMA) comprising benign and cancer tissue cores from each patient (n = 81) was generously provided by M. Gleave (University of British Columbia, Vancouver, British Columbia). Benign and cancer sites were each sampled thrice (diameter, 0.6 mm), creating a triplet TMA layout (total, 426 cores). Sections (0.5 μm) were mounted on charged slides for staining with TMPRSS2 (previously described; ref. 17) and TFF3 (ab57752, 3 μg/mL; Abcam, Inc.). We individually scored staining in each core on a four-point scale from none (0) to high (3). We compared staining intensity in control and treatment groups by fitting a logistic regression model using generalized estimating equations to account for multiple observations per patient; P values of <0.05 were considered significant.

Fluorescence in situ hybridization

We performed fluorescence in situ hybridization (FISH) analysis for TMPRSS2-ERG chromosomal fusion on the TMA using published probes and methods (18). No cores without carcinoma exhibited a TMPRSS2-ERG fusion. We tabulated fusion status for TMA cores containing cancer blinded to treatment group. We determined significance between treatment groups using a logistic regression model [log(p / (1 − p)) = B0 + B1x1, where p denotes probability of fusion and x1 denotes dutasteride dose] and fit using generalized estimating equations to account for multiple cores as well as different numbers of observations per subject due to loss of cores or absence of cancer.

Results

Serum and prostatic androgen concentrations after dutasteride treatment

To evaluate molecular effects of SRD5A inhibition in vivo, we studied prostate tissues from a clinical trial of dutasteride before RP (8). Eighty-one men aged 45 to 80 years with localized PCa (T1c-T2b), Gleason score of ≤7, and prostate-specific antigen (PSA) of 2.5 to 10 ng/dL were randomized to immediate RP (n = 25) or four months of 0.5 mg dutasteride (n = 26) or 3.5 mg dutasteride (n = 24) orally daily preceding RP.

The proposed mechanism of SRD5A inhibition in PCa chemoprevention is based on an overall reduction in prostate tissue androgens and attendant decrement in AR activity. Accordingly, dutasteride-treated subjects had significantly lower serum and tissue DHT levels than untreated men (Table 1; ref. 8). Compared with untreated samples (3.33 ng/g), prostatic DHT was 93% and 98.8% lower in 0.5 mg (0.23 ng/g) and 3.5 mg (0.04 ng/g) groups, respectively. We observed reciprocal increases in tissue testosterone due to inhibition of SRD5A activity in dutasteride-treated samples. Using a conservative estimate that relative potency of DHT in engaging and maintaining AR activity is twice that of testosterone (19), a “tissue androgen index” (sum of testosterone plus 2× DHT) suggests that overall tissue androgen activity in dutasteride-treated samples is reduced by approximately 40% to 60% (Table 1). Using less conservative DHT to testosterone potency ratios of 5:1 or 10:1 resulted in a 70% to 80% and 80% to 90% difference in estimated tissue androgen activity between untreated and dutasteride-treated samples (Table 1).

View this table:
Table 1.

Serum and tissue androgen levels after dutasteride treatment

Dutasteride induces differential expression of androgen-regulated genes in benign prostate epithelium

To assess effects of SRD5A inhibition on prostate gene expression, we microdissected benign epithelium from untreated and dutasteride-treated cohorts and evaluated transcript expression using cDNA microarrays. Unsupervised hierarchical clustering using all genes did not discriminate treated and untreated samples (data not shown). Clustering based on the 1,000 most variably expressed genes similarly failed to distinguish surgery alone and dutasteride-treated groups (Fig. 1A) and failed to resolve high and low dutasteride cohorts from one another (Fig. 1B). These findings suggest that the overall effects of dutasteride on prostate gene expression were modest and/or heterogeneous and therefore unable to overcome the substantial variability in global transcript expression observed in untreated prostate epithelium.

Figure 1.
Figure 1.

Unsupervised clustering of untreated and dutasteride–treated samples based on prostate epithelial gene expression. Samples from patients undergoing surgery alone (black squares) or treated with 0.5 or 3.5 mg of dutasteride (Dut; white and gray squares) were ordered based on unsupervised clustering using the 1,000 most variably expressed genes. Dendrograms depict relationship of all samples (A) or dutasteride-treated samples (B).

However, direct comparison of prostatic gene expression in untreated and dutasteride-treated samples using two-sample t tests did reveal significant dutasteride-related alterations in epithelial gene expression, with 120 genes ≥1.5-fold differentially regulated (FDR <5%; Fig. 2A; Supplementary Fig. S3). Among these, we observed significant alterations in expression of known androgen-regulated genes, including upregulation of IGFBP3 (3.7-fold) and downregulation of TMPRSS2, KLK3 (PSA), KLK2, FKBP5, and KLK4 (1.8-, 1.8-, 2.0-, 2.1-, and 2.6-fold, respectively), confirmed by qRT-PCR (Fig. 2B).

Figure 2.
Figure 2.

Differential gene expression in microdissected prostate epithelium from untreated and dutasteride-treated patients. A, top 60 upregulated or downregulated genes identified in a supervised two-sample t test between untreated (surgery alone) and combined dutasteride-treated samples (≥1.75-fold; FDR, <5%). The full list of 120 genes differentially regulated by ≥1.5-fold is provided in Supplementary Fig. S3. Gray scale represents genes with lower expression as white, no change as gray, and higher expression as black. B, confirmation of microarray data by qRT-PCR of indicated genes. Y axis is fold change in expression (relative to RPL13A). Horizontal bars indicate mean values. Two-sample t tests were used to compare differences in mean cycle thresholds between untreated (Surgery) and dutasteride-treated samples with P values as depicted. Black circles, untreated samples; white circles, 0.5 mg dutasteride; gray circles, 3.5 mg dutasteride.

Variability in the molecular response to SRD5A inhibition distinguishes patients with high and low androgen-responsive programs

A primary measure of the tissue response to dutasteride should be an effect on the cellular androgen axis, as reflected by tissue AR activity. Although expression profiling identified significant dutasteride-related differences in expression of many androgen-regulated genes, substantial heterogeneity among individuals was evident (Fig. 2B). Many dutasteride-treated samples expressed transcripts for androgen-regulated genes within ranges measured in untreated samples and unrelated to treatment dose (Fig. 2B). Moreover, despite significantly lower DHT levels in the 3.5 mg than 0.5 mg cohort [0.04 (±0.02) ng/g versus 0.23 (±0.12) ng/g; P < 0.0001; Table 1], we did not identify differentially regulated genes between the 3.5 mg versus 0.5 mg dutasteride-treated groups (data not shown). These results suggest that AR-mediated transcription reflects the total tissue androgen state and not absolute tissue DHT concentration such that lower tissue DHT in the 3.5 mg group may potentially be attenuated by increases in tissue testosterone. This conclusion is consistent with relatively similar overall estimates of tissue androgen indices in the two dutasteride-treated cohorts (Table 1).

To more specifically interrogate the effect of SRD5A inhibition on the prostatic AR axis, we evaluated dutasteride-treated samples for expression of 90 androgen-regulated genes (Supplementary Fig. S1; refs. 1, 12). Unsupervised hierarchical clustering based on expression of these androgen-regulated genes resolved dutasteride-treated samples into two groups distinguished by markedly different expression levels of genes known to be highly responsive to androgen (FOLH1, NKX3-1, TMPRSS2, KLK2, KLK3, KLK4, and PPAP2A; Fig. 3A), as well as expression of the AR itself. We designated these cohorts ARG-Hi (AR gene activity-high) and ARG-Lo (AR gene activity-low). Unsupervised clustering of the entire sample set (including untreated cases) using the same 90 androgen-regulated genes provided a near-identical segregation of treated samples and placed 7 of 11 untreated samples into the ARG-Hi group (Fig. 3B). Of interest in context of subsequent analyses, three of four untreated samples falling into the ARG-Lo group were among untreated samples with the lowest AR mRNA expression.

Figure 3.
Figure 3.

Unsupervised clustering of dutasteride-treated samples based on expression of androgen-regulated genes. Benign prostate epithelial samples from dutasteride patients (white squares, 0.5 mg; gray squares, 3.5 mg) were ordered based on unsupervised hierarchical clustering using 90 androgen-regulated genes. A, dutasteride-treated samples segregated into two groups based on low expression of androgen-regulated genes (ARG-Lo; green shaded portion of heat map) versus higher expression of androgen-regulated genes (ARG-Hi; red shaded). Depicted is a node containing a subset of 90 androgen-regulated genes used to perform clustering. B, unsupervised clustering of entire sample set (including untreated cases, black squares) using the same 90 androgen-regulated genes. C, surgery alone and dutasteride-treated samples were separately ordered based on AR transcript expression level (microarray data; red, higher expression; green, lower expression). Dutasteride treatment and whether each sample was designated as high (ARG-Hi) or low (ARG-Lo) for expression of androgen-regulated genes from the cluster analysis in A is indicated. Crosshatching denotes the one sample with lower AR transcript expression that was assigned to ARG-Hi in the cluster analysis.

Variation in tissue response to dutasteride might simply reflect differences in efficacy of SRD5A inhibition, thus correlating with intraprostatic DHT levels or the calculated androgen index. However, dutasteride was uniformly effective in reducing prostatic DHT, with no DHT levels in treated samples exceeding untreated samples (Supplementary Fig. S4A). Testosterone levels were more variable, as were measures of the estimated androgen index (Supplementary Fig. S4B–E). However, plotting samples by tissue DHT, testosterone, or the calculated androgen index did not correlate with segregation into ARG-Lo or ARG-Hi cohorts (Supplementary Fig. S5). Accordingly, we found no differences in expression of SRD5A1, SRD5A2, or SRD5A3 in ARG-Lo or ARG-Hi groups (Supplementary Fig. S6), nor any difference in SRD5A2 single-nucleotide polymorphism alleles known to modulate SRD5A2 functional activity (including A49T, P48R, V89L, and F194L; data not shown; refs. 20, 21). Thus, we could not attribute observed variation in AR-responsive genes to differences in dutasteride effects on DHT reduction.

SRD5A effects on the AR program are highly correlated with cellular AR expression and weakly associated with tissue androgen levels

To further examine prostatic androgen responses to dutasteride, we identified genes in ARG-Hi and ARG-Lo groups most closely associated with sample segregation. With one exception, simply ordering dutasteride-treated samples based solely on AR mRNA abundance partitioned samples into the same ARG-Hi and ARG-Lo categories (Fig. 3C) identified by clustering of samples with the 90 androgen-regulated genes (Fig. 3A). Using qRT-PCR, we confirmed that segregation of dutasteride-treated samples into ARG-Hi and ARG-Lo groups similarly showed relatively high and low expression of AR, PSA, and TMPRSS2 (Fig. 4A). Moreover, PSA and TMPRSS2 showed a highly significant correlation with AR transcript expression [r2 = 0.45 (P < 0.001) and r2 = 0.52 (P < 0.0001), respectively; Fig. 4B]. Accordingly, known AR-regulated genes predominated among those whose microarray expression profile was most strongly correlated with AR transcript expression (Supplementary Fig. S7).

Figure 4.
Figure 4.

Comparison of androgen-responsive gene status with AR transcript abundance. A, dot plots showing expression of indicated genes by qRT-PCR in surgery alone and dutasteride-treated groups; designation of dutasteride-treated samples into ARG-Hi and ARG-Lo is based on clustering of microarray data in Fig. 3A. Black circles, untreated samples; white circles, 0.5 mg dutasteride; gray circles, 3.5 mg dutasteride. B, linear regression of AR transcript expression with that of PSA (squares; slope = 0.4203) and TMPRSS2 (triangles; slope = 0.5442) by qRT-PCR.

In contrast, we observed a much weaker correlation between androgen-regulated genes and tissue androgen levels. Plotting transcript levels against tissue DHT, testosterone, or androgen index yielded modestly significant correlations between TMPRSS2 and DHT (r2 = 0.21; P = 0.04) and androgen index (r2 = 0.28; P = 0.01), whereas correlations for PSA only trended toward significance (P = 0.06 and 0.08, respectively; Supplementary Fig. S8). In addition, we observed no differences in polyQ and polyG trinucleotide repeats (associated with AR transactivating capacity) in ARG-Lo and ARG-Hi groups (data not shown; refs. 22, 23). These observations suggest that the primary driver of prostatic androgen response in dutasteride-treated tissues is the level of AR itself.

Although AR activity (measured by epithelial expression of androgen-regulated genes) correlated closely with AR transcript levels, it is unknown whether variable AR expression in dutasteride-treated samples reflects steady-state AR status or if a subset of subjects responds to decreased androgen levels caused by SRD5A inhibition by modulating AR transcript synthesis or stability. As this study did not include pretreatment and posttreatment samples, we cannot directly address this possibility. However, the wide variation in AR transcript levels in untreated subjects suggests that “intrinsic” AR levels could account for the range of AR expression observed in dutasteride-treated samples. To confirm that AR levels normally exhibit substantial variability, we examined AR transcript abundance in four published microarray data sets (13–16) and in microdissected benign epithelium from untreated prostatectomy (n = 12) and needle biopsy (n = 10) specimens. Consistent with the range of AR expression observed in the present study, AR abundance in untreated prostate samples from published data sets ranged 16-fold (Supplementary Fig. S9).

As SRD5A inhibition has been associated with histopathologic measures of epithelial cell atrophy in some (but not all) studies (6, 8, 24–26), we examined whether differences in AR transcript expression between ARG-Lo and ARG-Hi cohorts might reflect a differential induction of luminal cell atrophy, with relative loss of AR-expressing luminal cells but continued presence of AR-negative basal cells in the ARG-Lo cohort. We found no consistent difference in expression of four basal (TP63, CD44, KRT5, and KRT7) or four luminal cell markers (KRT8, KRT14, KRT17, and KRT18) between ARG-Lo and ARG-Hi cohorts (by qRT-PCR; data not shown), nor did we observe histopathologic differences in relative proportion of epithelial glands showing microcystic atrophy (data not shown).

SRD5A inhibition affects TMPRSS2 expression in cancerous epithelium

Although androgens and the AR are clearly associated with PCa development and progression, determining specific androgen-regulated genes directly contributing to carcinogenesis has proven elusive. To assess potential functional importance of dutasteride-associated transcriptional changes, we evaluated protein expression of trefoil factor 3 (TFF3; downregulated 2.1-fold by dutasteride; Fig. 2A) and TMPRSS2, epithelial genes implicated in PCa genesis and/or progression (17, 18, 27, 28). As previously observed, TFF3 exhibited a spectrum of expression with more intense staining in cancer than benign epithelium (27). Expression was generally low in control samples and further decreased by dutasteride (Fig. 5A and C), with no effect of dutasteride in cancer tissue. TMPRSS2 also stained more intensely in cancer than benign epithelium, with dutasteride reducing staining intensity in both benign and cancer tissues (Fig. 5B and C).

Figure 5.
Figure 5.

Analysis of TFF3 and TMPRSS2 expression in neoplastic prostate epithelium. TFF3 and TMPRSS2 expression was evaluated using a TMA comprising triplicate benign and cancer cores per patient. Staining was scored on a four-point scale from none (0) to high (3). The distribution of cores in each treatment group displaying no (0), low (1), moderate (2), or high (3) intensity staining is shown for TFF3 (A) and TMPRSS2 (B). Staining intensity in control and treatment groups was compared using a logistic regression model to account for multiple observations per patient, with statistically significant changes indicated (*). Decreases in TFF3 staining were significant in the benign tissue (dutasteride 3.5 mg group; P < 0.01) and for TMPRSS2 in the benign (3.5 mg dutasteride group; P = 0.03) and cancer tissue (0.5 mg dutasteride, P = 0.04; 3.5 mg dutasteride; P = 0.02). C, representative images (20×) showing TFF3 and TMPRSS2 staining in benign untreated (surgery alone) and dutasteride-treated cores.

The significant, albeit modest, dutasteride-associated reductions in TMPRSS2 transcript and protein levels are noteworthy in view of the frequent chromosomal rearrangements found in PCa and preneoplastic lesions placing ETS oncogene family members under control of genes with androgen-regulated promoters such as TMPRSS2 (18). To evaluate potential effects of dutasteride on oncogenic events driven by these rearrangements, we assessed TMPRSS2-ERG fusion status using FISH analysis of triplicate cancer cores per patient. Of samples with sufficient cancer for analysis, we identified the TMPRSS2-ERG fusion in 49% of cancer cores from untreated patients (n = 19 evaluable) and in 57% and 25% of the low-dose (n = 18 evaluable) and high-dose (n = 16 evaluable) dutasteride-treated groups, respectively (Supplementary Fig. S10A; the number of positive and negative cores for each case is given in Supplementary Fig. S10B). The frequency of fusion-positive cancer cores was significantly lower in high-dose dutasteride versus untreated samples (P = 0.048). The significance of this observation is uncertain given the relatively short duration of dutasteride treatment before prostatectomy but is consistent with the hypothesis that cancers harboring TMPRSS2-ERG fusions may be sensitive to modulation by dutasteride.

Discussion

PCa prevention remains an attractive approach for reducing morbidity and mortality (29), and a substantial effect might be achieved by decreasing the rate at which relatively indolent foci of malignancy (present in the majority of men with advancing age) progress to clinically detectable disease (30). Increasingly, there is momentum to “treat” these common low-grade cancers with surveillance plans incorporating administration of natural products or pharmaceuticals with minimal side effects and low attendant risk profiles (6, 31). In this context, evaluation of SRD5A inhibitors has a strong rationale centered on a mechanism-based role for androgens and the AR in maintaining physiologic functions of benign and neoplastic prostate epithelium.

The PCPT formally tested the hypothesis that decreasing SRD5A activity would lower PCa incidence. Compared with placebo, finasteride reduced the 7-year period prevalence of PCa by 25% (5). However, 803 of 4,368 (∼18%) finasteride-treated men were diagnosed with PCas spanning low- and high-grade histologies, as were ∼16% of dutasteride-treated men in the REDUCE study (7). Many unexplained questions remain about preventive versus regressive effects of SRD5A inhibition on PCa development and progression. Importantly, it is unknown why SRD5A inhibition was not effective in all individuals nor what factors underlie the variable outcomes observed.

Finasteride and dutasteride substantially reduce intraprostatic DHT levels and have been shown to reduce prostate volume and improve voiding symptoms related to benign prostatic hypertrophy (32–36). However, measurable effects of these agents toward prostate epithelium and vasculature are not consistent. Remarkably, studies including analyses of prostate samples from the PCPT (6) have documented that histologic assessments are not capable of distinguishing tissues treated with SRD5A inhibitors versus placebo. A report by the Proscar Long-Term Efficacy and Safety Study group concluded that no significant histologic differences were discernible in either benign or cancerous prostate tissues when comparing finasteride and placebo (24). Randomized studies of dutasteride treatment have not found reproducible associations with measures of microvessel density, epithelial cell atrophy, proliferation rates, or apoptosis (8, 25). Although these results may partly reflect the substantial normal variability in aging-related androgen decline and attendant tissue effects in untreated individuals, other influences may inhibit or accentuate responses to SRD5A inhibitors, with biological consequences important for the efficacy of this strategy in PCa prevention and treatment.

In this study, we identified several possible mechanisms by which dutasteride could modify the development and progression of PCa. Several gene products with known roles or associations with prostate carcinogenesis were altered by dutasteride therapy: prostate epithelium from treated subjects expressed elevated levels of IGFBP3 transcripts (37) and lower levels of TFF3 (27, 28) and TMPRSS2 (10, 17). Reduced TMPRSS2 expression is particularly relevant in view of recurrent genomic rearrangements involving TMPRSS2 and members of the ETS oncogene family that occur in >50% of all PCas (18). In support of the hypothesis that cancers harboring TMPRSS2-ETS family rearrangements may be driven by AR signaling, we found that the frequency of cancers with TMPRSS2-ERG fusion was reduced in the cohort of patients treated with high-dose dutasteride, although the implication of this observation is limited by the relatively small sample size and short treatment duration.

Importantly, the substantial degree of heterogeneity in the molecular response to dutasteride therapy was unanticipated. Despite near-uniform reduction of tissue DHT levels to the lowest limits of assay detection, many dutasteride-treated tissues continued to exhibit robust activity of the AR gene expression program. Moreover, we found only weak associations between AR activity and measures of tissue testosterone, DHT, or a composite metric of androgen concentrations. Notably, the molecular feature most closely associated with status of the AR expression program was the level of AR itself.

AR expression levels have previously been found to contribute to the development of castration-resistant PCa. Using isogenic PCa xenograft models, Chen and colleagues (38) found that a modest increase in AR was the only change consistently associated with development of resistance to antiandrogen therapy. Elevated AR levels converted cancers from androgen sensitive to castration resistant, with cancer growth proceeding by enhanced output of the signal contributed by low residual androgens.

Although oncogenic events such as AR gene amplification or dysregulated kinase signaling may lead to increased AR transcript expression in PCa (39, 40), our studies of benign epithelium shows that a wide range of AR expression normally exists (or can be induced) in prostatic epithelium without a requirement for aberrant genomic or epigenetic events that accompany neoplastic growth. The wild-type AR gene has been shown to function as a self-regulating transcription factor capable of binding to response elements within its coding region, leading to increased mRNA levels (41). Thus, natural variations in levels of coactivator/corepressors or polymorphisms involving genomic regulatory sites in the AR itself may contribute to intrinsic differences in AR regulation.

A potential limitation of our study is that we evaluated the molecular effect of SRD5A inhibition on benign epithelium of men with known PCa. However, a study evaluating chemoprevention in subjects without known cancer is also likely to harbor a certain percentage of men with undiagnosed PCa (as shown by the PCPT). Moreover, emerging data implicate a field cancerization effect underlying multifocal PCa development (42), suggesting that our findings in benign-appearing epithelium of men with cancer are likely to be relevant to the effect of SRD5A inhibition in modulating progression of premalignant lesions to overt cancer.

Variation in the molecular program of AR gene regulation has important implications for the optimal use of SRD5A inhibitors in PCa prevention and treatment. Our data suggest the hypothesis that under conditions of relative androgen depletion, high AR levels in benign prostate epithelium can maintain AR transcriptional network activity, whereas low AR levels cannot compensate for low ligand concentrations. The lack of compensatory ability in some individuals could affect development and/or progression of initiated/preneoplastic prostate lesions (possibly those harboring an androgen-driven TMPRSS2-ERG rearrangement). Although mechanisms responsible for variation in AR transcript expression have not been defined, our data lead to the hypothesis that pretreatment tissue AR levels may predict response to SRD5A-directed therapies, a question that can be tested in an appropriately designed clinical study.

Disclosure of Potential Conflicts of Interest

L.D. True has received grant support from GlaxoSmithKline. P.S. Nelson has consulted and received research support from GlaxoSmithKline.

Acknowledgments

We thank Roger Coleman and Andrew Morgan for expert technical assistance, Farinaz Shokri for performing immunohistochemical stains, Roman Gulati for advice on statistical methods, Alex Moreno for administrative assistance, and Dr. Roger Rittmaster for review and helpful comments. We also thank Bostwick Laboratories for providing tissues, and the patients and treating clinicians for study participation.

Grant Support: American Society of Clinical Oncology Cancer Foundation Young Investigator Award (E.A. Mostaghel), Prostate Cancer Foundation Career Development Award (E.A. Mostaghel), GlaxoSmithKline (L.D. True and P.S. Nelson), and NIH grants 1K23 CA122820 (E.A. Mostaghel) and the Pacific Northwest Prostate Cancer SPORE P50CA97186 (P.S. Nelson).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

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Cancer Research: 70 (4)
February 2010
Volume 70, Issue 4

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Variability in the Androgen Response of Prostate Epithelium to 5α-Reductase Inhibition: Implications for Prostate Cancer Chemoprevention
Elahe A. Mostaghel, Linda Geng, Ilona Holcomb, Ilsa M. Coleman, Jared Lucas, Lawrence D. True and Peter S. Nelson
Cancer Res February 15 2010 (70) (4) 1286-1295; DOI: 10.1158/0008-5472.CAN-09-2509
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