Background
Prostate cancer (PCa) strongly affects the male population, and is classified as the most commonly diagnosed cancer and a leading cause of cancer death in North American men [
1]. The current prognostic tools, such as pre-operative prostate specific antigen (PSA) levels, histological Gleason grading of biopsy specimens and clinical TNM (tumor, node, metastasis) staging seem unable to accurately risk stratify individual PCa patients at early stages of the disease. Given the wide range of clinical outcomes and the heterogeneity of the disease, the main challenge facing physicians remains to distinguish latent from clinically significant tumors. There is thus a clear need for better prognostic markers.
Androgens are required for maintaining the homeostasis of the normal prostate epithelium. Their effect is mediated via the androgen receptors (AR), a member of the nuclear superfamily of steroid receptor, acting as a transcription factor in prostate cell nuclei. PCa cells have retained the ability to proliferate upon stimulation with androgens, resulting in tumor growth [
2]. Thus, PCa patients that experience a recurrence following localized treatment are subjected to androgen deprivation therapy. Although most patients respond well initially to androgen deprivation therapy, almost all of them will eventually experience resistance to treatment and disease progression [
3]. Therapeutic options for castrate resistant PCa (CRPC) are limited to chemotherapy regimens that show a modest survival benefit [
4]. There is currently no curative treatment for metastatic PCa. Understanding the molecules and the pathways involved in mediating resistance is thus needed for a better clinical management of the disease.
The phosphatidylinositol 3-kinase (PI3K)/AKT signal transduction pathway contributes to cancer growth and survival, and is activated in a broad range of human malignancies including PCa [
5]. The phosphatase and tensin homologue deleted on chromosome 10 (
PTEN) is a tumor suppressor gene on 10q23.3 locus that acts by negatively regulating the PI3K/AKT pathway [
6]. In animal models,
PTEN was shown to be haploinsufficient in tumor suppression [
7].
PTEN genomic deletion has been detected in human tissues representing all stages of PCa development and progression including High Grade Prostatic Intraepithelial Neoplasia (HGPIN), primary PCa and at higher frequency in metastatic PCa and CRPC [
8‐
15]. Using Fluorescent
in situ hybridization (FISH),
PTEN deletion status of primary PCa has been associated with poor outcome [
14]. Previous studies in human PCa cell lines and mice models have suggested that inactivation of
PTEN and PI3K/AKT activation can modulate AR activity and contribute to CRPC [
16‐
18]. These observations provided further rationale to examine PTEN and AR in human prostate tissues.
In this study, we surveyed PCa samples for genomic DNA copy number alterations (CNAs) of the PTEN gene using Fluorescent in situ hybridization (FISH) and AR expression by immunohistochemistry (IHC). An existing PCa microarray dataset of DNA CNAs by array comparative genomic hybridization (CGH) and corresponding gene expression profiling were used to validate these findings.
Methods
Ethics statement
This study was conducted with the written consent of the participants and approved by the Research Ethics Board of the McGill University Health Centre (study BMD-10-115).
Tissue samples
Formalin fixed paraffin embedded (FFPE) blocks (n = 43) of primary tumors and adjacent benign tissues from radical prostatectomy were retrieved from the Department of Pathology. Duplicate tissue cores (1mm diameter) were assembled into tissue microarrays (TMAs). Haematoxylin and eosin (H&E)-stained TMA sections were reviewed to determine the presence of representative areas of the original samples. The clinicopathologic features of the cohort are summarized in Table
1. Recurrence-free interval was defined as the time between date of surgery and the date of first PSA increase >0.2ng/ml or the date of last follow-up without PSA increase (censored). Kaplan-Meier survival analysis (log-rank test) was performed using WinStat (R. Fitch Software).
Table 1
Clinicopathologic parameters of the study subjects
Median age (range, years) | 63 (47–76) |
Median follow-up (months) | 62 |
Median PSA at surgery (ng.ml-1) | 8.7 |
Biochemical recurrence
| 12 (28%) |
Gleason score
| |
≤6 | 13 (30%) |
=7 | 23 (54%) |
≥8 | 7 (16%) |
Pathological stage
| |
≤T2 | 27 (63%) |
≥T3 | 16 (37%) |
Fluorescent in situhybridization (FISH)
Dual-color FISH was carried out on TMA sections using the BAC clone RP11-383D9 (BACPAC Resources Center, Oakland, CA) mapping to the PTEN gene on chromosome 10q23.3 region and the commercially available CEP10 Spectrum Green probe (CEP 10, Abbott Molecular, Abbott Park, IL), which spans the 10p11.1-q11.1 centromeric region. RP11-383D9 DNA was labeled with Spectrum Orange-dUTP (Enzo Life Science, Farmingdale, NY) using the Nick Translation Reagent Kit (Abbott Molecular). The 5 μm TMAs sections were de-paraffinized in 6 changes of xylene before immersion in 95% ethanol. The slides were then placed in 0.2 N HCl solution at room temperature (RT°) for 20 min followed by a 2-hour incubation at 80°C in 10 mM citric acid buffer (pH 6) for pre-treatment. Specimens were digested in 0.1 mg/ml protease I (Abbott Molecular), and then fixed for 10 min in formalin before dehydration in an ethanol series. The two probes and target DNA were co-denatured at 73°C for 6 min and left to hybridize at 37°C O/N using the ThermoBrite system (Abbott Molecular). Post-hybridization washes were performed in 2xSSC and 0.3% NP40/0.4xSSC at 73°C for 2 min and 1 min respectively, followed by a 30 sec incubation at RT° in 2xSSC.
FISH data analysis
In order to evaluate the 10q23.3 copy number, we counted fluorescent signals in 100 non-overlapping interphase nuclei for each sample. 4',6-Diamidino-2-phenylindole (
DAPI III, Abbott Molecular) staining of nuclei with reference to the corresponding H&E-stained tissue identified the areas of adenocarcinoma. Using hybridization in 30 benign control cores, 10q23.3 deletion was defined as ≥15% (mean + 3 standard deviation in non-neoplastic controls as described [
19,
20]) of tumor nuclei containing one or no 10q23.3 locus signal and by the presence of two CEP10 signals. Images were acquired with an Olympus IX-81 inverted microscope at 96X magnification using ImageProPlus 7.0 software (MediaCybernetics, Rockville, MD).
Immunohistochemistry (IHC) staining
Immunostaining of AR on TMAs sections was performed using a mouse anti-AR antibody (N-terminal AR 441, NeoMarker, Fremont, CA) and the Envision detection kit (Dako, Carpinteria, CA). The 5 μm TMAs sections were de-paraffinized in a series of xylene and hydrated in a graded series ethanol solutions. Heat-induced antigen retrieval was performed by immersing the slides in 10 mM citric acid buffer solution (pH 6) and boiling for 30 min using microwave energy. The slides were left in solution to cool down for 30 min at room temperature. Endogenous peroxydase activity was blocked for 5 minutes (Dako). After a 60 min block with 10% normal goat serum in PBS (Dako), the primary antibody (1:50 dilution in Dako antibody diluent) was used for two hours at room temperature. Chromogenic detection was carried out using a peroxidase-conjugated secondary antibody (30 min) and DAB reagents (10 min) provided with the Envision detection kit. Tissue sections were counterstained with Meyer’s Haematoxylin (Thermo Scientific, Waltham, MA).
IHC data analysis
Nuclear staining was assessed by two independent observers using the H-score method described in [
21,
22]. Briefly, H-score was obtained by computing the product of staining intensity (i=0-3) and the proportion of cells with the specific intensity (0–100), in areas of adenocarcinoma as identified with reference to the corresponding H&E-stained tissue. The H-scores were adjusted to give the highest score a value of 100. AR H-scores were compared between
PTEN deleted and non deleted specimens categories with the Mann–Whitney
U-Test (
http://elegans.som.vcu.edu/~leon/stats/utest.html).
Gene set enrichment analysis (GSEA)
Analysis [
23] was performed using GSEA software version 2.07 (Broad Institute, Cambridge, MA) with the previously published gene expression data of 64 prostate tumors by Lapointe et al. [
24] stratified by their
PTEN genomic status as reported in the corresponding array CGH study [
9]. Two androgen-responsive gene sets (R1881-treated LNCaP cells) were tested for enrichment in the gene expression microarray data: a curated set of 82 genes (NELSON_RESPONSE_TO_ANDROGEN_UP, [
25]) from the Molecular Signatures database (MSigDB, C2) and a set of 207 genes reported by DePrimo et al. [
26]. Lapointe et al gene expression study used for GSEA included data for respectively 71 and 204 genes of Nelson et al. and DePrimo et al. androgen-responsive gene sets. A thousand permutations were done and the false discovery rate (FDR) was estimated.
Discussion
In this study, we have shown in two independent sets of PCa samples that the PTEN genomic deletion was associated with early disease recurrence and reduced levels of AR expression. In microarray gene expression data, the PTEN deletion was also associated with a down regulation of AR-driven genes.
The frequency of
PTEN deletion in our FISH study (40%) is within the range of previous reports [
8,
10,
12,
14,
15]. Our survival analysis further confirms the association of
PTEN genomic deletion and poor outcome of PCa reported earlier [
14] and its potential use as a prognostic marker. Clinical relevance is also supported by the recent literature detecting
PTEN deletion at high frequency in CRPC samples [
11], in circulating tumor cells [
27] and its association with PCa death [
11,
28]. Further validation in larger cohorts would be critical to compare its predictive value with the current prognostication tools.
The intriguing finding of our study was the reduced levels of AR expression quantified by H-score in tumors harboring a
PTEN deletion. We found a similar association between
PTEN deletion and AR transcript levels in a PCa microarray dataset. The differential expression of AR according to the
PTEN tumor status has not been well documented so far. A pilot IHC study has found a positive correlation between AR and PTEN expression [
29]. In contrast, Sircar et al. reported a positive correlation between
PTEN deletion status and AR expression [
11] in CRPC samples. These results likely reflect two different stages of the disease: CRPC and untreated PCa. The genomic amplification of AR is known to occur in CRPC but rarely in untreated PCa [
30], thereby explaining differences in results.
Previous
in vitro studies in cell lines derived from advanced PCa suggested that PTEN could act as suppressor of AR activity [
31,
32]. It was also reported that the activation of PI3K/AKT pathway can suppress the AR activity in low passage LNCaP and enhance AR activity in high passage, hence suggesting modulation as cells evolve towards less responsive status [
33]. In models representing less advanced disease, re-expression of PTEN in
PTEN null murine cells did not affect AR expression, but upregulated the AR transcriptional activity [
34]. Another group reported that
PTEN null murine prostate cells had a reduced AR protein levels compared to wild-type
PTEN cells and the AR protein levels were partly restored by the PI3K/mTOR inhibitor BEZ235 [
35]. The latter observation would suggest that the activation of PI3K pathway may in part explain the reduced AR levels in
PTEN deleted tumors. A shown by Lin et al., it is also possible that PTEN interacts directly with AR and promotes its degradation [
31]. Underlying mechanisms of how
PTEN deletion in human tumors is associated with lower AR expression and transcriptional activity need to be further explored.
Given their reduced levels of AR expression, the
PTEN deleted tumor cells are expected to be less responsive to androgen ablation treatment. In support of this hypothesis, it was reported that CRPC and early biochemical recurrence were associated with reduced immunoreactivity of PTEN and AR in the PCa samples harvested before treatment initiation [
29]. The addition of an inhibitor of PI3K/mTOR to the standard androgen ablation treatment of advanced PCa may therefore be beneficial to patients with
PTEN deleted tumor.
Some previous studies have found that low levels of AR were associated with PCa recurrence [
36,
37] while others reported the opposite [
38,
39]. In our study, AR levels of expression were not significantly associated with PCa recurrence. The antibody used, IHC technique and scoring methods may explain the differences in the findings. Given the limited number of patients of our study, a detailed analysis of AR and PTEN in a large cohort of patients with follow-up is warranted.
During the course of our study, two groups also showed a reduced expression of androgen regulated genes in human
PTEN deleted PCa by microarray analysis [
34,
35]. In our analysis, the androgen regulated genes enriched in tumor with no deletion of
PTEN include genes expressed in normal prostate luminal epithelium such as KLK3 (PSA), TMPRSS2, and NKX3-1. Of interest, the list includes AZGP1 previously reported as a surrogate marker for subtype-1 tumors, a favourable prognostic subclass of PCa defined by gene expression pattern analysis [
24]. AZGP1 prognostic value was further confirmed by two other studies [
40,
41]. Previous GSEA has also revealed enrichment of androgen-responsive genes in subtype-1 tumors [
42]. Consistant with our findings, the confirmation of intact
PTEN status in subtype-1 tumors from the array CGH data may, at least in part, explain their androgen-regulated gene expression feature and good clinical outcome.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
Conceived and designed the experiments: KC, JE, JL. Performed the experiments: KC, JE. Analyzed the data: KC, JE, FB, JL. Contributed materials/clinical data: AA. Wrote the paper: KC, JE, SC, JL. All authors read and approved the final manuscript.