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>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

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

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

<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 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

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

Cellular Adhesion Promotes Prostate Cancer Cells Escape from Dormancy

<div><p>Dissemination of prostate cancer (PCa) cells to the bone marrow is an early event in the disease process. In some patients, disseminated tumor cells (DTC) proliferate to form active metastases after a prolonged period of undetectable disease known as tumor dormancy. Identifying mechanisms of PCa dormancy and reactivation remain a challenge partly due to the lack of <i>in vitro</i> models. Here, we characterized <i>in vitro</i> PCa dormancy-reactivation by inducing cells from three patient-derived xenograft (PDX) lines to proliferate through tumor cell contact with each other and with bone marrow stroma. Proliferating PCa cells demonstrated tumor cell-cell contact and integrin clustering by immunofluorescence. Global gene expression analyses on proliferating cells cultured on bone marrow stroma revealed a downregulation of TGFB2 in all of the three proliferating PCa PDX lines when compared to their non-proliferating counterparts. Furthermore, constitutive activation of myosin light chain kinase (MLCK), a downstream effector of integrin-beta1 and TGF-beta2, in non-proliferating cells promoted cell proliferation. This cell proliferation was associated with an upregulation of CDK6 and a downregulation of E2F4. Taken together, our data provide the first clinically relevant <i>in vitro</i> model to support cellular adhesion and downregulation of TGFB2 as a potential mechanism by which PCa cells may escape from dormancy. Targeting the TGF-beta2-associated mechanism could provide novel opportunities to prevent lethal PCa metastasis.</p></div

A Three-Marker FISH Panel Detects More Genetic Aberrations of <i>AR</i>, <i>PTEN</i> and <i>TMPRSS2/ERG</i> in Castration-Resistant or Metastatic Prostate Cancers than in Primary Prostate Tumors

<div><p><i>TMPRSS2</i>/<i>ERG</i> rearrangement, <i>PTEN</i> gene deletion, and androgen receptor (<i>AR</i>) gene amplification have been observed in various stages of human prostate cancer. We hypothesized that using these markers as a combined panel would allow better differentiation between low-risk and high-risk prostate cancer. We analyzed 110 primary prostate cancer samples, 70 metastatic tumor samples from 11 patients, and 27 xenograft tissues derived from 22 advanced prostate cancer patients using fluorescence in situ hybridization (FISH) analysis with probes targeting the <i>TMPRSS2</i>/<i>ERG</i>, <i>PTEN</i>, and <i>AR</i> gene loci. Heterogeneity of the aberrations detected was evaluated. Genetic patterns were also correlated with transcript levels. Among samples with complete data available, the three-marker FISH panel detected chromosomal abnormalities in 53% of primary prostate cancers and 87% of metastatic (Met) or castration-resistant (CRPC) tumors. The number of markers with abnormal FISH result had a different distribution between the two groups (<i>P</i><0.001). At the patient level, Met/CRPC tumors are 4.5 times more likely to show abnormalities than primary cancer patients (<i>P</i><0.05). Heterogeneity among Met/CRPC tumors is mostly inter-patient. Intra-patient heterogeneity is primarily due to differences between the primary prostate tumor and the metastases while multiple metastatic sites show consistent abnormalities. Intra-tumor variability is most prominent with the <i>AR</i> copy number in primary tumors. <i>AR</i> copy number correlated well with the <i>AR</i> mRNA expression (rho = 0.52, <i>P</i><0.001). Especially among <i>TMPRSS2:ERG</i> fusion-positive CRPC tumors, <i>AR</i> mRNA and <i>ERG</i> mRNA levels are strongly correlated (rho = 0.64, <i>P</i><0.001). Overall, the three-marker FISH panel may represent a useful tool for risk stratification of prostate cancer patients.</p></div