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Comparative Study
. 2015 Jun 30;6(18):16135-50.
doi: 10.18632/oncotarget.3873.

Stromal androgen receptor regulates the composition of the microenvironment to influence prostate cancer outcome

Damien A Leach  1 , Eleanor F Need  1 , Roxanne Toivanen  2 , Andrew P Trotta  1 , Helen M Palethorpe  1 , David J Tamblyn  3 , Tina Kopsaftis  3 , Georgina M England  4 , Eric Smith  1 , Paul A Drew  1   5 , Carole B Pinnock  3 , Peng Lee  6 , Jeff Holst  7   8 , Gail P Risbridger  2 , Samarth Chopra  3   9 , Donald B DeFranco  10 , Renea A Taylor  2   11 , Grant Buchanan  1
Affiliations
Comparative Study

Stromal androgen receptor regulates the composition of the microenvironment to influence prostate cancer outcome

Damien A Leach et al. Oncotarget. .

Erratum in

Abstract

Androgen receptor (AR) signaling in stromal cells is important in prostate cancer, yet the mechanisms underpinning stromal AR contribution to disease development and progression remain unclear. Using patient-matched benign and malignant prostate samples, we show a significant association between low AR levels in cancer associated stroma and increased prostate cancer-related death at one, three and five years post-diganosis, and in tissue recombination models with primary prostate cancer cells that low stromal AR decreases castration-induced apoptosis. AR-regulation was found to be different in primary human fibroblasts isolated from adjacent to cancerous and non-cancerous prostate epithelia, and to represent altered activation of myofibroblast pathways involved in cell cycle, adhesion, migration, and the extracellular matrix (ECM). Without AR signaling, the fibroblast-derived ECM loses the capacity to promote attachment of both myofibroblasts and cancer cells, is less able to prevent cell-matrix disruption, and is less likely to impede cancer cell invasion. AR signaling in prostate cancer stroma appears therefore to alter patient outcome by maintaining an ECM microenvironment inhibitory to cancer cell invasion. This paper provides comprehensive insight into AR signaling in the non-epithelial prostate microenvironment, and a resource from which the prognostic and therapeutic implications of stromal AR levels can be further explored.

Keywords: androgen receptor; extracellular matrix; fibroblasts; prostate cancer; stroma.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors disclose no potential conflicts of interest.

Figures

Figure 1
Figure 1. The expression of stromal AR is related to clinical parameters and outcomes of prostate cancer
A-C. Patient-matched duplicate cores of BPH and cancer were immunostained with anti-AR antibody. Samples were scored by two independent researchers, using a scale of high (3), moderate (2), low (1) intensity or absent (0) immunostaining in the epithelia and stroma and averaged between the duplicate samples and scorers. B. Scores were evaluated in relation to disease state for stroma (St) and epithelia (Ep) and compared using the Wilcoxon Rank-Sum test C. Mean AR score ± SEM for both the cancer stroma (Ca-St) and epithelia (Ca-Ep) was calculated for each Gleason grade.
Figure 2
Figure 2. Loss of myofibroblast AR protects cancerous prostatic epithelia from castration induced apoptosis
Tissue recombination of patient prostate cancer tissues co-grafted with either PShTert-AR or PShTert-ctrl myofibroblasts into immune-deficient host mice. After 8 weeks, host mice were castrated for a further three days. A. Human tissue was identified by dual immunostaining of basal cell marker p63 (brown stain) and epithelial marker CK8/18 (pink stain); cancer foci were p63−CK8/18+ highlighted by white outline. AR levels were assessed in samples immunostained with anti-AR antibody. B. Epithelial proliferation was determined by the percentage of cells immunostained for anti-Ki-67. C. Human cancer tissue grafts from castrated mice was assessed for CK8/18, p63 and AR as described in (A). D. Epithelial cell death was measured through cleaved caspase-3 immunostaining and percent positive cells counted (*, p<0.05, **, p<0.01, ***, p<0.001, Student's T-test).
Figure 3
Figure 3. Cell specificity of AR action may be mediated by interactions of AR with DNA
A. Lysates from C4-2B and PShTert-AR cells treated with or without 10 nM DHT and 10 μM bicalutamide (BIC) were probed for AR and FKBP5. B. Affymetrix 1.0st Gene Array of 10 nM DHT or vehicle control (V.C.) treated PShTert-AR or C4-2B cells, presented as a Venn-diagram of genes with >0.5 log2 fold change in expression between treatments. C-E. Microarray was validated via RT-qPCR of independent samples produced under the same conditions. Data is represented as mean + SEM of triplicate biological replicates (V.C. vs DHT * p<0.05, ** p<0.01, ***p<0.001 Student's T-test). F-H. Chromatin immunoprecipitation (ChIP) was performed on C4-2B and PShTert-AR cells treated with 10 nM DHT or vehicle, and immunoprecipitated with anti-AR N20 or nonspecific IgG antibody. ChIP samples were quantified by RT-qPCR and mean percent input for each binding region in the proximity of (F) FBXO32, (G) PSA and (H) FKBP5 was normalized to a non-specific binding region.
Figure 4
Figure 4. C4-2B and PShTert-AR cells have different proliferative responses to DHT
A-B. Proliferative response of C4-2B and PShTert-AR cells to 10 nM DHT was measured daily via Trypan blue exclusion assays. C,D. The androgen mediated gene and DNA-licensing factor, FBXO32, was silenced via siRNA (C) and the effect on PShTert-AR growth in response to 10 nM DHT was measured via Trypan blue exclusion assay (D). E,F. The effect of conditioned media from PShTert-AR and PShTert-ctrl on C4-2B and PC-3 cells was measured as in A. Data represents the mean number of viable cells in triplicate wells ± SEM. G,H. The presence of DHT in the conditioned media was assessed via transactivation assays performed on C4-2B (G) and PShTert-AR (H) cells. Data presented as mean relative light units (RLU) ± SEM of six independently transfected wells.
Figure 5
Figure 5. DHT has pro-adherent effects on fast and long term adherence of myofibroblast cells
A-C. The quantity of C4-2B, PShTert-ctrl, and PShTert-AR cells, treated with 10 nM DHT or equivalent vehicle (V.C.), remaining after trypsinization over 15 min was measured using crystal violet staining. Presented as mean ± SEM of six technical replicates, and representative of three independent experiments. D,E. Adherence was measured by manually counting the number of 10 nM DHT, V.C. or 10 μM bicalutamide (BIC) treated C4-2B, PShTert-ctrl, and PShTert-AR cells adhering after 30 min. Data is presented as mean ± SEM of four samples and is representative of three independent experiments. (* p<0.05 V.C. vs DHT, # p<0.05 DHT vs DHT+BIC Student's T-test). F,G. PShTert-AR cells transfected with siRNA against Hic-5 or scrambled control were assayed for adherence as described in D but measured over a 2 h period. Data is presented as mean ± SEM of four replicates and representative of three independent experiments. H-I. Hic-5 contribution to androgen-mediated attachment was assayed as described in A-C. For all time course adherence data, significance (p<0.05) was determined by one-way ANOVA.
Figure 6
Figure 6. AR in cancer associated fibroblasts and model of AR action in prostate stroma
A. PC-3 attachment to ECM deposited by PShTert-ctrl and PShTert-AR cells treated with or without 10 nM DHT ± 10 μM BIC was measured as described in Fig. 5D. Data presented as mean adherence per mm2 of four wells ± SEM. (* p<0.05 vehicle control (V.C.) vs DHT, # p<0.05 DHT vs DHT+BIC Student T-test). B. Migration of PC-3 cells over matrices created from V.C. or DHT treated PShTert-AR myofibroblasts was assessed by measuring the area of the cell-free gap over a 15 hour time period and calculated as a percentage of time point 0. Data represents mean ± SEM of three replicates. C-E. PShTert-AR or PShTert-ctrl cells were grown to confluence on a gelatin layer and allowed to deposit a 3D-ECM for 8 d following 10 nM DHT or V.C. treatment before myofibroblast removal. C. Adherence of 5 × 104 C4-2B cells to the 3D-matrices was determined after an hour. Data is presented as mean ± SEM of four replicates and is representative of three independent experiments. D. The effect of the 3D-matrices on epithelial proliferation was determined via Trypan blue exclusion assay. Data is presented as mean ± SEM of four replicates and is representative of three independent experiments. E. Invasion of calcein-labeled C4-2B cells through the myofibroblast 3D-matrices was determined via a modified Boyden chamber technique. Data is presented as mean ± SEM of six samples and is representative of three independent experiments. F. RT-qPCR analysis for expression of selected ECM genes in PShTert-AR cells. Data represents the mean + SEM from triplicate biological replicates. G. ELISA analysis of collagen-1 (COL1) levels in conditioned media from DHT treated PShTert-AR cells. Data is presented as the mean + SEM from six replicates representative of two independent experiments. H. RT-qPCR gene analysis in human patient cancer associated fibroblasts (CAF), BPH associated fibroblasts (BAF), and normal prostatic fibroblasts (NPFs), isolated and treated with either V.C. or 100 nM DHT. Data represents the mean of technical triplicates (± SEM) from N=1 for each cell type (in all panels * p<0.05, ***p<0.001, Student's T-test). I. Model of AR action in prostate myofibroblasts. The AR signaling in myofibroblasts causes increased production of ECM components and inhibition of MMP enzymes. When AR signaling in myofibroblasts is lost, decreased expression of ECM components and enhanced MMP expression create an environment which decreases cancer cell attachment and increases cancer cell invasion.
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References

    1. American Cancer Society I. Cancer Facts & Figures 2012. American Cancer Society; 2012.
    1. Amling CL, Blute ML, Bergstralh EJ, Seay TM, Slezak J, Zincke H. Long-term hazard of progression after radical prostatectomy for clinically localized prostate cancer: continued risk of biochemical failure after 5 years. J Urol. 2000;164:101–105. - PubMed
    1. Freedland SJ, Humphreys EB, Mangold LA, Eisenberger M, Dorey FJ, Walsh PC, Partin AW. Risk of prostate cancer-specific mortality following biochemical recurrence after radical prostatectomy. JAMA. 2005;294:433–439. - PubMed
    1. Saadi A, Shannon NB, Lao-Sirieix P, O'Donovan M, Walker E, Clemons NJ, Hardwick JS, Zhang C, Das M, Save V, Novelli M, Balkwill F, Fitzgerald RC. Stromal genes discriminate preinvasive from invasive disease, predict outcome, and highlight inflammatory pathways in digestive cancers. Proc Natl Acad Sci U S A. 2010;107:2177–2182. - PMC - PubMed
    1. Ma XJ, Dahiya S, Richardson E, Erlander M, Sgroi DC. Gene expression profiling of the tumor microenvironment during breast cancer progression. Breast Cancer Res. 2009;11:R7. - PMC - PubMed

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