AR and c-Myc promote ligand-independent prostate cancer cell growth.

<p>A) LNCaP, abl and 22RV1 cells were transfected with 50 nM of non-targeted control (NTC) or <i>AR</i> RNAi oligonucleotides. Cells were switched to charcoal-stripped serum on the day of transfection. Cell growth was determined 5 days later for LNCaP and 6 days later for abl and CRPC 22RV1 with the trypan blue exclusion method. B) Immunoblot for AR expression. The lower bands in the AR immunoblot in 22RV1 cells reflect the presence of an AR transcript variant <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063563#pone.0063563-Guo1" target="_blank">[7]</a>. B) LNCaP, abl and 22RV1 cells were transfected with 50 nM of NTC or <i>c-Myc</i> RNAi oligonucleotides. Cells were switched to charcoal-stripped serum on the day of transfection. Cell growth was determined 5 days later for LNCaP cells and 6 days later for abl and 22RV1 with the trypan blue exclusion method. Immunoblot for c-Myc protein expression. C) LNCaP cells with stable overexpression of empty vector (EV) or c-Myc were generated. These cells were transfected with 50 nM of non-targeted control (NTC) or AR siRNA oligonucleotides. Cell growth was determined 6 days later with the trypan blue exclusion method. Immunoblot for AR and c-Myc protein expression. The higher bands on the c-Myc immunoblot in the c-Myc-overexpressing cells represent the ectopically-expressed c-Myc. *denotes p<0.05 compared to NTC.</p

<i>c-Myc</i> expression is not activated by androgen ligands.

<p>LNCaP, abl, and 22RV1 cells were grown in charcoal-stripped serum for 72 hours and then treated with 10 nM R1881 (or ethanol vehicle) for 4 hours. A) Chromatin immunoprecipitation was performed to determine the enrichment of AR and histone H3 acetylation (AcH3) at the <i>c-Myc</i> and <i>KLK3</i> enhancer elements. B) QRT-PCR was performed to determine the mRNA levels of <i>KLK3</i> and <i>c-Myc</i> relative to actin. C) Immunoblotting was performed to determine the protein levels of AR, c-Myc, and actin. *denotes p<0.05 compared to vehicle.</p

Androgen Receptor Promotes Ligand-Independent Prostate Cancer Progression through c-Myc Upregulation

<div><p>The androgen receptor (AR) is the principal therapeutic target in prostate cancer. For the past 70 years, androgen deprivation therapy (ADT) has been the major therapeutic focus. However, some patients do not benefit, and those tumors that do initially respond to ADT eventually progress. One recently described mechanism of such an effect is growth and survival-promoting effects of the AR that are exerted independently of the AR ligands, testosterone and dihydrotestosterone. However, specific ligand-independent AR target genes that account for this effect were not well characterized. We show here that <i>c-Myc,</i> which is a key mediator of ligand-independent prostate cancer growth, is a key ligand-independent AR target gene. Using microarray analysis, we found that <i>c-Myc</i> and AR expression levels strongly correlated with each other in tumors from patients with castration-resistant prostate cancer (CRPC) progressing despite ADT. We confirmed that AR directly regulates <i>c-Myc</i> transcription in a ligand-independent manner, that <i>AR</i> and <i>c-Myc</i> suppression reduces ligand-independent prostate cancer cell growth, and that ectopic expression of c-Myc attenuates the anti-growth effects of AR suppression. Importantly, treatment with the bromodomain inhibitor JQ1 suppressed c-Myc function and suppressed ligand-independent prostate cancer cell survival. Our results define a new link between two critical proteins in prostate cancer – AR and c-Myc – and demonstrate the potential of AR and c-Myc-directed therapies to improve prostate cancer control.</p></div

<i>AR</i> suppression recapitulates the effect of <i>c-Myc</i> suppression on <i>c-Myc</i> target gene expression.

<p>LNCaP and abl cells were transfected with 50 nM of non-targeted control (NTC) and either A) <i>AR</i> or B) <i>c-Myc</i> RNAi oligonucleotides. Cells were switched to charcoal-stripped serum on the day of transfection and harvested 96 hours later. QRT-PCR was performed to determine the levels of the indicated <i>c-Myc</i> target genes relative to <i>actin</i>. *denotes p<0.05 compared to NTC.</p

AR promotes ligand-independent expression of <i>c-Myc</i>.

<p>LNCaP, abl and 22RV1 cells were transfected with 50 nM of non-targeted control (NTC) or AR RNAi oligonucleotides. Cells were then grown in charcoal-stripped serum for 96 hours. At the end of the treatment, cells were harvested to extract mRNA and protein. A) QRT-PCR was performed to determine the levels of <i>c-Myc</i> relative to <i>actin</i>. B) Immunoblotting was performed to determine the levels of AR, c-Myc and actin. C) Parallel treatments were performed and cells were cross-linked and processed for ChIP to determine AR and histone H3 acetylation (AcH3) enrichment at the <i>c-Myc</i> enhancer. *denotes p<0.05 compared to NTC.</p

AR and c-Myc are critical drivers of ligand-independent mechanisms of prostate cancer progression.

<p>Currently, androgen deprivation therapies that interfere with androgen ligand activation of the AR are primarily used to treat this disease. These therapies suppress AR’s androgen ligand-dependent function and suppress expression of androgen ligand-dependent (AD) AR target genes. However, despite these treatments prostate cancer progression is inevitable. The AR also promotes the expression of androgen ligand-independent pathways such as <i>c-Myc</i>. The <i>c-Myc</i> gene is commonly upregulated in prostate cancer and contributes to androgen ligand-independent prostate cancer progression. This model strongly suggests that AR or c-Myc-directed therapies would complement current androgen deprivation strategies.</p

JQ1 treatment suppresses c-Myc expression and function and reduces ligand-independent prostate cancer cell survival.

<p>LNCaP, abl, and 22RV1 cells were grown in charcoal-stripped serum and treated with vehicle, 50 nM, 250 nM or 500 nM JQ1 every 24 hours for 72 hours. A) Immunoblotting was performed to determine the protein levels of c-Myc. B) QRT-PCR was performed to determine the mRNA level of <i>c-Myc</i> and c-Myc targets genes <i>KIF11, CDKN1A, TPX2</i>, and <i>AURKB</i> relative to <i>actin</i>. *denotes p<0.05 compared to vehicle. C) Cell viability was determined at the end of treatment with the trypan blue exclusion method. p<0.01 for the 250 nM and 500 nM doses vs. vehicle in all three cell lines.</p

<i>AR</i> and <i>c-Myc</i> levels are positively correlated in CRPC specimens.

<p>A) Z-scores (to normal prostate specimens) of <i>AR</i> versus <i>c-Myc</i> mRNA expression across 140 human CRPC metastases. The Pearson correlation coefficient, linear regression, and F test for significantly non-zero slope were performed for each pair of genes. B) Fisher’s exact test and odds ratio on the contingency table analyzing the co-occurrence of tumors with <i>AR</i> or <i>c-Myc</i> z-scores greater than 2.</p

Diagnostic and Prognostic Utility of a DNA Hypermethylated Gene Signature in Prostate Cancer

<div><p>We aimed to identify a <b>p</b>rostate cancer DNA <b>hy</b>permethylation <b>m</b>icro<b>a</b>rray signature (denoted as <b>PHYMA</b>) that differentiates prostate cancer from benign prostate hyperplasia (BPH), high from low-grade and lethal from non-lethal cancers. This is a non-randomized retrospective study in 111 local Asian men (87 prostate cancers and 24 BPH) treated from 1995 to 2009 in our institution. Archival prostate epithelia were laser-capture microdissected and genomic DNA extracted and bisulfite-converted. Samples were profiled using Illumina GoldenGate Methylation microarray, with raw data processed by GenomeStudio. A classification model was generated using support vector machine, consisting of a 55-probe DNA methylation signature of 46 genes. The model was independently validated on an internal testing dataset which yielded cancer detection sensitivity and specificity of 95.3% and 100% respectively, with overall accuracy of 96.4%. Second validation on another independent western cohort yielded 89.8% sensitivity and 66.7% specificity, with overall accuracy of 88.7%. A PHYMA score was developed for each sample based on the state of methylation in the PHYMA signature. Increasing PHYMA score was significantly associated with higher Gleason score and Gleason primary grade. Men with higher PHYMA scores have poorer survival on univariate (p = 0.0038, HR = 3.89) and multivariate analyses when controlled for (i) clinical stage (p = 0.055, HR = 2.57), and (ii) clinical stage and Gleason score (p = 0.043, HR = 2.61). We further performed bisulfite genomic sequencing on 2 relatively unknown genes to demonstrate robustness of the assay results. PHYMA is thus a signature with high sensitivity and specificity for discriminating tumors from BPH, and has a potential role in early detection and in predicting survival.</p></div

Association of PHYMA scores with clinical factors.

<p>(a) Gleason score, (b) Gleason primary grade, (c) difference between low and high grade, and (d) overall survival. Tumor samples have higher PHYMA scores compared to BPH and are associated positively with (a) Gleason score, (b) Gleason primary grade, and (c) difference between high and low grades. (d) Kaplan Meier show samples with higher PHYMA have poorer survival.</p