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Cancer Research 68, 2678, April 15, 2008. doi: 10.1158/0008-5472.CAN-07-6017
© 2008 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Androgen Receptor Overexpression in Prostate Cancer Linked to Pur{alpha} Loss from a Novel Repressor Complex

Longgui G. Wang1, Edward M. Johnson2, Yayoi Kinoshita3, James S. Babb1, Michael T. Buckley1, Leonard F. Liebes1, Jonathan Melamed1, Xiao-Mei Liu1, Ralf Kurek4, Liliana Ossowski3 and Anna C. Ferrari1

1 New York University Cancer Institute, New York, New York; 2 Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, Virginia; 3 Mount Sinai School of Medicine, New York, New York; and 4 Stadtische Kliniken, Offenbach, Germany

Requests for reprints: Anna C. Ferrari, New York University Cancer Institute, New York University School of Medicine, 8th Floor, 160 East 34th Street, New York, NY 10016. Phone: 212-731-5389; Fax: 212-731-5455; E-mail: anna.ferrari{at}nyumc.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Increased androgen receptor (AR) expression and activity are pivotal for androgen-independent (AI) prostate cancer (PC) progression and resistance to androgen-deprivation therapy. We show that a novel transcriptional repressor complex that binds a specific sequence (repressor element) in the AR gene 5'-untranslated region contains Pur{alpha} and hnRNP-K. Pur{alpha} expression, its nuclear localization, and its AR promoter association, as determined by chromatin immunoprecipitation analysis, were found to be significantly diminished in AI-LNCaP cells and in hormone-refractory human PCs. Transfection of AI cells with a plasmid that restored Pur{alpha} expression reduced AR at the transcription and protein levels. Pur{alpha} knockdown in androgen-dependent cells yielded higher AR and reduced p21, a gene previously shown to be under negative control of AR. These changes were linked to increased proliferation in androgen-depleted conditions. Treatment of AI cells with histone deacetylase and DNA methylation inhibitors restored Pur{alpha} protein and binding to the AR repressor element. This correlated with decreased AR mRNA and protein levels and inhibition of cell growth. Pur{alpha} is therefore a key repressor of AR transcription and its loss from the transcriptional repressor complex is a determinant of AR overexpression and AI progression of PC. The success in restoring Pur{alpha} and the repressor complex function by pharmacologic intervention opens a promising new therapeutic approach for advanced PC. [Cancer Res 2008;68(8):2678–88]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Recurrent prostate cancer (PC) remains a therapeutic challenge in part because the mechanisms of progression to androgen independence (AI) and the reasons for the development of resistance to androgen deprivation therapy in hormone-refractory (HR) PC patients are still unknown. A large proportion of patients with high-grade localized cancer (1), metastatic disease (2), and HR-PC (3) show increased expression of the androgen receptor (AR), suggesting that this plays a key role in disease progression. Approximately 30% of cases have AR gene amplification (4) and 10% have mutations (5), but the vast majority of cases have increased AR gene transcription and/or altered receptor protein stability (6). Although PC cells can derive proliferative signals through several pathways, it is apparent that the AR is essential to PC progression in the presence or absence of the androgen ligand (7).

Increased AR expression is also a feature of the emergent AI phenotype in PC models. To mimic the AI phenotype that emerges during androgen deprivation therapy, we chronically deprived an androgen-dependent (AD) cell line (LNCaP) of androgen (8). We found that, similar to some advanced PC xenografts (9, 10), the emergent AI cell line had 2-fold to 4-fold more AR than the parental AD cells (8) resulting from transcriptional up-regulation of the AR. Increased AR was also described in another pair of LNCaP-AD cells and their AI derivatives (9). More recently, a microarray analysis of seven HR/hormone-sensitive isogenic pairs of human PC xenografts showed that among 12,500 gene probe sets tested, only AR mRNA was differentially expressed in all seven cases (10). In the latter study, the HR-PC had more AR protein, and even a modest change in AR level was able to shift the relative abundance of coactivators and corepressors assembled on the promoters of androgen target genes. Moreover, an increase in the AR level of a magnitude similar to that observed in our AI-LNCaP cells caused AR antagonists to function as agonists (10).

Overall, this body of evidence indicates that AR expression level is an important determinant of biological outcomes and provides a compelling reason to study the mechanisms responsible for AR overexpression (10–12). Indeed, although multiple alterations at the gene and protein level have been described in AI PC (13), there is only limited information regarding alterations in the regulation of AR transcription that may account for increased AR mRNA levels (14). We found that a suppressor element (ARS), originally identified in a mouse AR gene and located in the 5'-untranslated region (5'-UTR) of the AR promoter (15, 16), is also present in the human AR gene and that it malfunctions in AI cells (17). A promoter/reporter construct with a deleted ARS element produced an 8-fold increase in AR promoter activity after transfection into AD cells. We also found that nuclear extracts of AD cells contain a repressor protein complex that binds the ARS in a gel shift assay, although this complex was significantly reduced in AI cells (17).

We have now identified the ARS-binding transcriptional repressor complex as containing Pur{alpha} and hnRNP-K (18–20). Based on the insight gained from our mechanistic studies of the Pur{alpha}-containing repressor in AD and AI cell lines, and on observed changes in its expression, localization, and binding to the ARS that take place during human PC progression, we conclude that Pur{alpha} is an important regulator of AR transcription and of AI growth. We also show that Pur{alpha} expression and function can be restored by agents that relieve epigenetic silencing, suggesting that regulation of the transcriptional levels of AR may provide a novel therapeutic strategy to control PC progression and to enhance the efficacy of existing systemic therapies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and tissues. LNCaP cells were maintained in RPMI 1640 with 10% heat-inactivated fetal bovine serum. The AI-LNCaP derivative was isolated by growing LNCaP cells in 10% charcoal-stripped fetal bovine serum and 5 µg/mL of insulin for several months. An initial reduction in the total cell population was followed by a gradual outgrowth of AI cells (8). Formalin-fixed, paraffin-embedded and frozen PC tissues were obtained from the New York University Prostate Cancer Tissue Resource, the Stadtische Kliniken, Offenbach, Germany, and the Mount Sinai School of Medicine. Twelve hormone-naïve (HN), Gleason score 6 frozen PCs were microdissected for quantitative PCR (Q-PCR), four were used for chromatin immunoprecipitation (ChIP), seven HR-PC metastases were processed for Q-PCR: two bone marrows, one bone metastasis protruding into the brain, one cervical lymph node, one epidural metastasis, one malignant ascitis, and one malignant pleural effusion. The latter three were used for ChIP. All patients signed Institutional Review Board–approved consent.

Tissue selection, tissue microarray, immunohistochemistry, and image analysis. Four core samples from 18 HN radical prostatectomy and 18 HR transurethral resection specimens of the prostate after progression following several months of androgen deprivation, matched for Gleason score, were cut in 4-µm-thick sections, histology verified every 10th section and was assembled for tissue microarray (TMA; Beecher Instruments; ref. 21). For immunohistochemistry, TMA slides were soaked in H-3300 solution (Vector Laboratories), microwaved and treated with streptavidin peroxidase. The Pur{alpha} monoclonal antibody 10B12 (IgG1 isotype; 1:1,000; ref. 22) and AR polyclonal antibody, which have been previously described (23), were horseradish peroxidase–conjugated with secondary antibody and diaminobenzidine. Negative controls for Pur{alpha} included an isotype-matched irrelevant monoclonal antibody (mouse IgG1, X0943; Dako); and for AR, a rabbit immunoglobulin fraction (solid-phase absorbed; X09036; Dako) diluted 1:1,000 in PBS. All sections were incubated with horseradish peroxidase–conjugated secondary antibody and diaminobenzidine. Quantitative immunohistochemical analysis was performed using Kodak Molecular Imaging (ver. 4.0) as previously described (24, 25). Positively stained pixel regions-of-interest and the percentage of staining were calculated using Microsoft Excel. Two independent observers determined the percentage of positive Pur{alpha} nuclear staining based on two 40x-magnified TMA images.

Protein isolation, electrophoretic mobility shift assay, and immunoblotting. Nuclear AD and AI cell proteins were processed for electrophoretic mobility shift assay (EMSA) as previously described (17). For EMSA with purified GST-Pur{alpha}, the protein was reacted with [{gamma}-32P]-labeled double-stranded (ds) wt-ARS oligonucleotide 5'-ACC CCG CCT CCC CCC ACC CT-3' +323 and +342 nucleotides; ref. 17, or single-stranded (ss) C-rich or G-rich ARS, or its three mutants: mARS1, ACC CAA CCT CCT TCC ACC CT; mARS2, ACC CCT TCT CAA CCC ACC CT; and mARS3, ACC TTG CCT AAC CCC ACC CT (Bio-synthesis, Inc.), in binding buffer and subjected to 8% PAGE. For the supershift assay, the nuclear proteins were preincubated with anti-Pur{alpha} monoclonal antibody and 10-fold excess mARS3. Anti-Pur{alpha} monoclonal antibody, Pur{alpha} fusion protein pGPur4, and mutant Pur{alpha} proteins (amino acids 167-322/m-GST-Pur{alpha}-1 and amino acids 216-322/m-GST-Pur{alpha}-2) were previously described (22, 26). Southwestern blot was performed as previously described (17).

Transfection, luciferase assay, and Western blot. Cells were cotransfected with 1 µg of "empty" vector DNA (pVector), AR luciferase reporter pLARS-1, or its ARS-deleted mutant pLARS-del (17), with or without expression plasmids for phnRNP-K (27, 28), or Pur{alpha} vector pHApur1 (26) or Pur{alpha}-small interfering RNA (siRNA; 1–2 µg/well) and 0.5 µg of pSV-β-galactosidase (Promega) control, using Effectene (Qiagen). Luciferase and β-galactosidase activity (Promega) of cell lysates were measured 48 h later. Total and subcellular proteins from parallel cultures were processed for Western blotting 48 h after cotransfection using Pur{alpha} 10B12 (22), AR, β-actin (Santa Cruz), hnRNP-K monoclonal (ImmunQuest, Ltd.), and p21 (Dako) antibodies as described (29).

Pull-down assays using biotinylated DS-ARS-streptavidin beads. Nuclear extracts of AD cells were incubated with streptavidin/magnetic beads (BioLab) bound to three repeats of DS-ARS [sense, 5'-CCC GCC TCC CCC CAC CCG CCT CCC CCC ACC CGC CTC CCC CCA-3'; antisense, (BioTEG)5'-TGG GGG GAG GCG GGT GGG GGG AGG CGG GTG GGG GGA GGC GGG-3'; and its mutant, sense, 5'-CCA ACC TCC TTC CAC CAA CCT CCT TCC ACC AAC CTC CTT CCA-3'; and antisense (BioTEG)5'-TGG AAG GAG GTT GGT GGA AGG AGG TTG GTG GAA GGA GGT TGG-3']. The beads were then eluted and the protein fractions processed for Western blot.

ChIP assays. The protein/DNA complexes from intact AD and AI cells or from minced frozen PC tissues were cross-linked and then lysed as previously described (30). Equal amounts of protein or tissue lysates (50–130 mg total weight) were incubated with the primary antibody, Pur{alpha} 5B11 and 1A12 (31), followed by Protein G PLUS-Agarose beads (Santa Cruz), washed twice with high-salt buffer (0.5 mol/L NaCl), followed by LiCl/detergent solution and TE buffer. The DNA-protein immunoprecipitates were eluted, proteinase K–treated, and the DNA extracted. PCR (Expand High-Fidelity PCR System; Roche) was performed using primers to sequence +248 to +487 nucleotides of the AR 5'-UTR region and the Sp1 sites: 5'-AGC TGC TAA AGA CTC GGA GG-3' and 5'-GGA GTT ACC TCT CTG CAA AC-3'. The images of PCR products stained with SYBR Gold (Molecular Probes) in agarose gels were captured with Fotodyne, imported into Photoshop, and the black bands scanned and quantified using NIH Image. For clinical samples, Pur{alpha} binding was corrected for input DNA.

Pur{alpha} knockdown. AD cells seeded at 5,000 cells/well were either fixed 24 h later (day 0) with 10% trichloroacetic acid or transfected with Pur{alpha}-siRNA (85 ng/well), AR siRNA (85 ng/well), Pur{alpha}-siRNA plus AR siRNA (85 ng each/well), or control (scrambled) siRNA (labeled NC, 85 ng/well; Ambion) using Effectene in serum-free medium. Cell growth was measured at baseline, 48, and 72 h posttransfection by sulforhodamine B assay. AI cells in AI-medium served as positive control. AD cells seeded at 0.5 to 1 x 106 cells/well were transfected with 1 µg of siRNA per well and processed for protein extraction and Western blotting with AR and Pur{alpha}.

Analysis of AR and Pur{alpha} expression using Oncomine. We searched Oncomine5 for PURA cDNA and PC. The original raw data and results including the median, 75/25 percentiles, t test, and P values for each study were collected and linked to the original publications. Only one (32) of two studies described HN PC and metastatic HR-PC as defined in our study and probed for Pur{alpha}.

Q-PCR. Frozen PC specimen were microdissected and processed for RNA extraction, cDNA, and Q-PCR as previously described (33). Primers and VIC-labeled probe for glyceraldehyde-3'-phosphate dehydrogenase, AR, and PURA (ABI) were used to measure copy numbers by reference to log-linear standard curves (2 to 2 x 107 copy range) of serial dilutions of linearized plasmid DNAs containing the respective gene inserts. The positive controls were LNCaP cells, the negative control lacked a cDNA template. The mean copy number of triplicate Q-PCR assays were used to compare samples.

Statistical analysis. Average values of multiple assessments of AR and Pur{alpha} regarding the percentage of positive staining intensities and expression levels by Q-PCR and ChIP among HN HR-PC's were compared by the Mann-Whitney test. The P values were exact significance levels and two-sided, except where indicated, and statistically significant at <0.05 by SAS version 9.0 (SAS Institute) analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of ARS nuclear binding complex. We previously reported that high AR expression in LNCaP-AI cells might be due to loss of binding of a repressor complex to an ARS element in the 5'-UTR of the AR gene (17). Gel blotting analysis of nuclear extracts from LNCaP-AI and AD cells with a labeled dsARS probe (Southwestern) revealed several bands (~105 to ~30 kDa; results not shown). To further identify these bands, nuclear extracts of AD cells were affinity-purified on an ARS-oligonucleotide column (dswt-ARS, 20 bp), the protein was eluted with a linear gradient of KCl and separated on duplicate SDS-PAGE gels; one gel was analyzed by Southwestern analysis whereas the other was used to elute proteins from the areas corresponding to the bands (a total of five) detected by Southwestern analysis. One of the bands was identified as the nuclear protein hnRNP-K (CAA51267.1; ref. 34), by Bio-Mass Spectrometry (results not shown). This protein has multiple functions (35), including the ability to bind ssDN (28, 36) and to repress transcription (34). Because hnRNP-K can shuttle between the nuclear and cytoplasmic compartments (37), we tested nuclear and cytoplasmic extracts of AD and AI cells for proteins that bind to the ssARS. In Southwestern analysis with a C-rich oligonucleotide probe, a single, equal-intensity band of ~55 to 65 kDa (Fig. 1A, left ) was produced by nuclear extracts of AD and AI cells. When the membrane was stripped of the radioactive probe and tested by immunoblotting with specific anti–hnRNP-K antibody, a doublet, with molecular weight corresponding to hnRNP-K was detected (Fig. 1B, left). Several additional faint bands were also noted (Fig. 1A, left). Using G-strand ARS as a probe, several distinct bands, some of high intensity, were noted (Fig. 1A, right) including a weak 40 kDa band, which was more intense in AD extracts. Immunoblotting with anti–Pur{alpha} antibodies revealed a band of similar molecular weight (Fig. 1B, right). Pur{alpha} was reported to cooperate with hnRNP-K in transcriptional repression (27). Direct immunoblotting of whole cell extracts showed that AR is elevated and confirmed that Pur{alpha} is reduced in AI cells, whereas hnRNP-K was equally expressed in AI and AD cells (Fig. 1C, left). Moreover, Pur{alpha} was shown to be reduced in the cytoplasm of AI cells and was almost undetectable in their nuclei (Fig. 1C, right). Finally, analysis of AD and AI cells using an Affymetrix gene expression array (U133A GeneChip) showed in three independent arrays that AI cells express ~2.5-fold more AR mRNA, similar levels of hnRNP-K mRNA, and ~4-fold less Pur{alpha} mRNA compared with AD cells (Fig. 1D).


Figure 1
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  		Figure 1. hnRNP-K and Pur{alpha} in the ARS-binding complexes. A and B, nuclear and cytoplasmic proteins (50 µg/lane) from AD and AI cells were subjected to Southwestern blotting (A) using [32P]-labeled ARS-C-strand or ARS-G-strand as probes. The same membranes were stripped and subjected to Western blotting (B) with monoclonal antibodies against hnRNP-K or Pur{alpha}. C, AR, hnRNP-K, and Pur{alpha} levels and subcellular localization in AD and AI cells. Total extracts (50 µg of proteins/lane) or nuclear and cytoplasmic proteins (50 µg/lane) from AD and AI cells, obtained as described in Materials and Methods, were analyzed by Western blotting with antibodies against AR, hnRNP-K, or Pur{alpha} or actin for loading control. D, mRNA levels of AR (black column), Pur{alpha} (light gray column), and hnRNP-K (dark gray column) obtained from an Affymetrix array using human U133A GeneChip. Columns, mean from three independent experiments; bars, SD; *, P < 0.01; **, P < 0.001.

 
Pur{alpha} and hnRNP-K form an ARS-binding complex in vitro. We previously showed that nuclear extracts of AD cells incubated with a dsARS probe produced a retarded complex in gel shift (EMSA) assays that were greatly diminished in AI cells (17). We show here that the intensity of this complex is strongly reduced by anti–hnRNP-K blocking antibody, whereas nonimmune IgG have no effect (Fig. 2A, lanes 3 and 4 ). Anti-Pur{alpha} antibody "supershifted" most of this complex (Fig. 2A, lane 7), as well as a complex produced by purified GST-Pur{alpha} fusion protein (Fig. 2B, lane 3), and this complex could be competed off with an excess of unlabeled dsARS (ref. 17; results not shown). Two Pur{alpha} truncation mutants (mGST-Pur{alpha}-1 and mGST-Pur{alpha}-2, amino acids 167–322 and 216–322, respectively), shown previously to bind only very weakly to a Pur{alpha} element in the c-MYC promoter (30), did not produce retarded complexes with the ARS (Fig. 2B, lanes 6 and 7). The complex pattern was different when three mutated ARS oligonucleotides (mARS1–3) were used as probes: AD nuclear extracts did not bind mARS1 and mARS3 at all, and with mARS2, produced patterns of bands different from those observed with wtARS (ref. 17; results not shown). Purified GST-Pur{alpha} protein was bound to neither mARS1 nor to mARS3 (Fig. 2C) and was indistinguishable from the wtARS when bound to mARS2. Moreover, excess of unlabeled dsARS reduced the ARS-Pur{alpha} complex (Fig. 2C) indicating specificity of interaction. These results suggest that Pur{alpha} and hnRNP-K are present in the complex and that they directly bind to the ARS.


Figure 2
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  		Figure 2. Binding specificity of nuclear proteins to ARS in EMSA. A, nuclear extracts (5 µg) or purified GST-Pur{alpha} fusion protein (0.2 µg; B and C) were incubated for 30 min at room temperature with [32P]-labeled, double-stranded, dsARS (A, lanes 1–9) or with ARS mutants (B), or unlabeled wt-ARS (C) or single stranded, ssC-rich sense, CSS (A, lanes 10–14) or G-rich antisense, GSS (A, lanes 15–20)-ARS oligonucleotide in the presence or absence of the indicated antibody. The reaction mixtures were subjected to native 8% PAGE as described in Materials and Methods. B and D, positions of anti-Pur{alpha} antibody–shifted bands (*). Changes in complex intensities in B (lanes 4 and 5) were deemed to be nonspecific. D, pull-down assays using biotinylated dsARS-streptavidin beads, followed by Western blots, using monoclonal antibodies against hnRNP-K, Pur{alpha}, and Sp1 (undetected), respectively.

 
Both Pur{alpha} (38–40) and hnRNP-K (34, 41) can strand-separate dsDNA and subsequently bind to the individual DNA strands. Nuclear extracts of AD and AI cells retarded the single C- and G-rich ssARS probes (Fig. 2A, lanes 10–14 and 15–20, respectively), and the anti-Pur{alpha} antibody supershifted the G-rich band (Fig. 2A, lanes 18 and 19), whereas the anti–hnRNP-K antibody reduced the intensity of the C-rich band (Fig. 2A, lane 13). Thus, it seems that when faced with dsARS, each of the proteins could strand-separate it and bind to the C-rich (hnRNP-K) or G-rich (Pur{alpha}) strand, producing a retarded protein-DNA complex.

A pull-down experiment in which AD cell extracts were incubated with biotinylated wt-dsARS oligonucleotide bound to streptavidin beads showed that both hnRNP-K (Fig. 2D, top) and Pur{alpha} (middle) were eluted at 0.25 and 0.75 mol/L KCl, respectively. Beads with mutated dsARS bound much less hnRNP-K and no Pur{alpha} (Fig. 2D). Sp1, also present in the nuclear extracts (see below), did not bind to the ARS probe (bottom). This suggests that both hnRNP-K and Pur{alpha} interact specifically with dsARS.

hnRNP-K and Pur{alpha} bind to the ARS in vivo. We used ChIP assays to test whether Pur{alpha} and hnRNP-K interact with the ARS in vivo. As the PCR primers used for ChIP were positioned to amplify the ARS and the two nearby SP1 binding sites of the AR gene, anti-Sp1 antibody was used as a positive control. A sequence located between +248 to +487 nucleotides, which contains the putative ARS (+323 to +342 nucleotides), was amplified from the DNA immunoprecipitated by two different anti-Pur{alpha} antibodies, anti–hnRNP-K antibody or anti-Sp1 antibody in AD and AI cells (Fig. 3, top ). No DNA amplification was found with irrelevant IgG, beads alone or lysates alone. To quantify the amount of Pur{alpha} associated with the ARS, we performed quantitative ChIP (Q-ChIP), using strictly controlled reaction inputs for AD and AI cells. With the two anti-Pur{alpha} antibodies (22), 1.9-fold to 2.5-fold more Pur{alpha} was associated with the ARS-containing 248- to 487-nucleotide sequences in the AD cells (Fig. 3, bottom).


Figure 3
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  		Figure 3. In vivo interaction of hnRNP-K and Pur{alpha} with an ARS containing AR gene sequence (ChIP assay) in AD and AI LNCaP cells. Top, ChIP assays; bottom, Q-ChIP assay for Pur{alpha}. Proteins cross-linked to chromatin were processed as described in Materials and Methods using two different antibodies for Pur{alpha} (5B11 and 1A12), and antibody to hnRNP-K and SP1. Lysates, beads alone, and irrelevant IgG served as negative controls. The PCR product corresponds to the 5'-UTR sequence from +248 to +487 nucleotides containing the putative ARS and Sp1 sites. In Q-ChIP, the same amounts of proteins and DNA under the same conditions were used for both cell lines. The PCR was repeated thrice, the density of each DNA band was measured using densitometry, normalized for DNA input, and expressed as mean ± SD for AD (white columns) and AI (black columns) LNCaP cells. A DNA sequence within the proliferating cell nuclear antigen promoter served as a negative control for the anti-Pur{alpha} antibodies (results not shown).

 
Down-regulation of AR transcription by Pur{alpha}. To test whether Pur{alpha} and hnRNP-K affect transcription of the AR gene, AD and AI cells were cotransfected with an ARS-containing AR promoter/luciferase reporter (pLARS-1) or with a reporter containing a deleted ARS (pLARS-del; ref. 17) and with expression plasmids for Pur{alpha} or hnRNP-K or both. Forced expression of Pur{alpha} in pLARS-transfected AI cells produced a >50% decrease in luciferase activity, and a significantly (P < 0.05) lesser effect in pLARS-del–transfected cells (Fig. 4A ). Importantly, this transcriptional effect was accompanied by a reduced expression of endogenous AR protein (Fig. 4A, right). Forced expression of hnRNP-K, which produced only a very slight increase in the protein level, did not further enhance the effect of Pur{alpha} (Fig. 4A), suggesting that the level of hnRNP-K was not rate-limiting.


Figure 4
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  		Figure 4. Effect of hnRNP-K and Pur{alpha} on AR transcription/expression in AI and/or AD cells. A, effect of forced hnRNP-K and Pur{alpha} expression on AR expression: AI cells were cotransfected with AR luciferase reporter, pLARS-1 (black columns), or its ARS-deleted mutant pLARS-del (white columns) plus pVector, or expression plasmids phnRNP-K or pHApur1, or both, and pSV β-galactosidase as internal control, and the AR luciferase reporter activities were measured 48 h after transfection. The AR protein levels were also determined by Western blot. Note that the Pur{alpha} transcript from pHApur1 is of higher molecular weight than the wild-type Pur{alpha}. B, effects of knockdown of hnRNP-K and Pur{alpha} on AR transcription: AI cells were cotransfected with pLARS-1 and 1 µg of two hnRNP-K-siRNAs (11155 and 11059), or Pur{alpha} siRNA, or with a scrambled version (negative control, NC) of siRNA. Columns, AR luciferase activity measured 48 h after transfection, as a ratio of β-galactosidase activity (see Materials and Methods). C, comparison of AR transcription in AD (black columns) and AI (white columns) cells in response to Pur{alpha}-siRNA. AD and AI cells were transfected and processed as in B. Columns, mean from three independent experiments; bars, SD; *, P < 0.05; **, P < 0.01 as compared with control (pVector) or with ARS-deleted mutant pLARS-del (A) or to scrambled (NC) siRNA (B and C). D, AD and AI cells transfected with Pur{alpha}-siRNA and tested by Western blotting for AR, Pur{alpha}, and actin as loading control 48 h after transfection.

 
As an alternative experimental approach, we tested Pur{alpha} and hnRNP-K knockdown by siRNAs in AD and AI cells for their potential to derepress AR transcription, using the AR promoter/luciferase assay or AR protein levels. Transfection of AD cells, which have higher endogenous levels of Pur{alpha}, with 1 or 2 µg of Pur{alpha}/siRNA increased AR/luciferase activity by 6-fold and 8-fold, respectively (Fig. 4C), and transfection with 1 µg of Pur{alpha}/siRNA induced a strong increase in endogenous AR protein (Fig. 4D). Transfection with two individual hnRNP-K siRNAs produced only an ~1.5-fold increase in AR/luciferase activity (Fig. 4B), and a slight enhancement of the siPur{alpha} effect in AI cells (Fig. 4D). As expected, the endogenous Pur{alpha} protein was very low in AI cells, and was further reduced to a barely detectable level by Pur{alpha}-siRNA (Fig. 4D), causing a 2.0-fold increase in luciferase activity (Fig. 4B) and a slight increase in AR protein (Fig. 4D). These results reveal the fine transcriptional tuning of AR by Pur{alpha} expression.

Reduction of Pur{alpha} level converts AD cells to androgen independence for growth. We reasoned that AI proliferation and higher AR expression (AI phenotype), similar to that achieved by chronic maintenance of LNCaP-AD cells in androgen-poor medium (8, 12), might be mimicked by acute reduction of Pur{alpha} levels. Thus, we transfected AD cells with Pur{alpha}/siRNA and cultured the cells in androgen-depleted medium for 2 and 3 days. Compared with cells transfected with a scrambled siRNA, the reduced Pur{alpha} led to increased AR protein and resulted in a 1.6-fold and 2.5-fold increase in cell growth on days 2 and 3, respectively (Fig. 5A ). Importantly, proliferation was entirely AR-dependent because reduction of AR by knockdown in Pur{alpha} siRNA-treated cells blocked androgen-induced growth (Fig. 5B). We and others have previously shown (12, 42) that high AR levels inhibit p21/WAF1 expression. A similar effect was then found in cells in which AR expression was increased by Pur{alpha}-siRNA treatment (Fig. 5A and B), indicating that the restored AR is functional. These results indicate an inverse relationship between Pur{alpha} and AR levels and cell growth.


Figure 5
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  		Figure 5. Down-regulation of Pur{alpha} by siRNA and increase in active AR induce AI growth of AD cells. A, AD and AI cells were seeded into 96-well dishes at 5,000 cells per well in AD-containing medium (full serum) and AI-containing medium (charcoal-stripped serum), respectively. After 24 h, cell growth was determined (day 0) and the AD cells were transfected with 85 ng/well of Pur{alpha}-siRNA (dark gray columns) or the same amount of scrambled-siRNA, negative control (NC; light gray columns), and 5 h after the transfection, medium with charcoal-stripped serum was added to all cells and their growth was measured on days 2 and 3. The AI cells that served as positive controls (black columns) were maintained in the same medium. Parallel cultures of AD cells plated in 12-well dishes were transfected with Pur{alpha}-siRNA and examined, at the indicated times, for AR, Pur{alpha}, p21, and actin (loading control) by Western blotting. B, the growth of AD cells, seeded overnight as in A in serum-containing medium, was determined after 24 h (day 0) and the cells were transfected with 85 ng/well of Pur{alpha}-siRNA (light gray columns), AR siRNA alone (dark gray columns) or Pur{alpha}-siRNA plus AR siRNA (white columns), or the same amount of scrambled siRNA (NC; black columns). Five hours after the transfection, medium with charcoal-stripped serum was added to all cells and their growth was measured on days 2 and 3. Parallel cultures of cells were lysed after 3 d of treatment and the content of AR, Pur{alpha}, p21/WAF1, and β-actin was analyzed by Western blotting. C, nuclear extracts of AI cells treated with SAHA for 1 d (days 5 to 6 of culture), or with 5-AzaC for 6 d, or with 5-AzaC for 5 d, followed by a day of SAHA, were prepared and 5 µg of respective proteins were analyzed by EMSA using ssG-rich ARS as probe. Anti-Pur{alpha} antibody (2 µg/sample) was used for supershifting. Five micrograms of protein from AD cell nuclear extract served as a positive control. D, AI cells were seeded at 2,500 cells/well in charcoal-stripped serum and, after 24 h of incubation, were treated for 7 d with the indicated concentrations of SAHA ({circ}), 5-AzaC (bullet) or the two drugs together ({blacktriangledown}). Cell growth was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and expressed as a percentage of untreated control. Inset, Western blot shows AR and Pur{alpha} levels in AI cells, treated 16 h after plating, with 5.0 µmol/L of 5-Aza for 6 d, or 7.5 µmol/L of SAHA for 1 day, or 5.0 µmol/L of 5-Aza for 5 days followed by 7.5 µmol/L of SAHA for 1 d. Bottom, parallel results of AR Q-PCR analysis.

 
To assess the potential clinical value of this dependence, we tested whether the repressor complex was amenable to up-regulation by pharmacologic agents known to have epigenetic effects on gene expression. AI cells were treated with suberoylanilide hydroxamic acid (SAHA), 5-azacytidine (5-AzaC), or the two drugs together, and examined using a gel shift assay for the presence of a functional (ARS binding) repressor complex. Individual treatments increased the intensity of the bands (Fig. 5C, lanes 5 and 6 compared with lane 4), and the combination of the two drugs produced a more intense band (lane 7), which was supershifted by anti-Pur{alpha} antibody (Fig. 5C, lane 8), in a pattern similar to that of AD cells (Fig. 5C, lanes 2 and 3). Treatment of AI cells with the same dose of SAHA (7 µmol/L) increased Pur{alpha} and strongly reduced the AR mRNA and protein levels (Fig. 5D, inset). Treatment with 5-AzaC (5 µmol/L) also restored Pur{alpha} expression and in vitro binding to ARS (gel shift) but had no effect on AR levels. Most importantly, SAHA and 5-AzaC individually, and in combination, had a dose-dependent growth-inhibitory effect on AI cells (Fig. 5D). The isobologram showed that the combination was synergistic (results not shown).

Higher AR levels and lower or delocalized Pur{alpha} characterize HR human PCs. To examine whether human PCs recapitulate the reciprocal relationship between Pur{alpha} and AR levels observed in the AI and AD cell lines, we compared the AR and Pur{alpha} content and/or their subcellular localization in TMA. Gleason score–matched 18 localized HN and 18 HR-PCs, were immunostained for AR and Pur{alpha}. Figure 6A shows the results of representative sections of HN and HR tumors. In all tumors, AR was found to be predominantly nuclear (Fig. 6A, top), whereas Pur{alpha} was both nuclear and cytoplasmic in HN tumors (Fig. 6A, bottom left) but was mostly cytoplasmic in the majority of HR tumors (Fig. 6A, bottom middle). The mean staining intensity of nuclear AR was significantly lower (P = 0.011) in HN tumors (mean ± SD, 15.4 ± 16.4) than in HR tumors (24.4 ± 17.8). The overall Pur{alpha} level in the immunohistochemical analysis was not significantly different between HN and HR tumors (P = 0.52), possibly due to the difficulty in direct comparison of immunostaining intensities of nuclear and cytosolic antigens. However, Pur{alpha} was localized almost exclusively to the nucleus in the majority (12 of 15) of the HN tumors, but in only 5 of 15 HR tumors, and the percentage of Pur{alpha}-positive nuclei per tumor was significantly lower (P = 0.005) for HR tumors (mean ± SD, 34.1 ± 37.9; median, 12.0) than for HN tumors (mean ± SD, 80.5 ± 37.2; median, 100).


Figure 6
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  		Figure 6. AR and Pur{alpha} levels in samples of HN and HR human PC. A, representative sections of TMA showing HN PC and HR-PC tumors stained, respectively, for AR (top left and middle), Pur{alpha} antibody 10B12 (bottom left and middle), and negative controls (right). Insets, higher magnification (x40) of the indicated areas. Note greater intensity of AR nuclear staining and predominantly cytoplasmic Pur{alpha} staining in the HR tumors. Pur{alpha} in HN tumor is mostly nuclear. The quantification of nuclear localization of Pur{alpha} in TMAs is represented by the graph in which each column represents an individual tumor. The medians of nuclear Pur{alpha} were 12.0% for HR (red columns), and 100.0% for HN (blue columns), respectively. B, the levels of Pur{alpha} and AR mRNAs in clinical specimens by Q-PCR. C, Pur{alpha}-RNA content of HN (n = 59; blue column) and HR (n = 20; red column) tumors derived from Oncomine (http://www.oncomine.org/). The line in each column represents the median of the group. D, Q-ChIP assay of human HN- and HR-PCs. The PCR product of input DNA, amplified with primers within the 5'-UTR of AR, and products of three individual PCR reactions (ChIP-Pur{alpha} with Pur{alpha} antibodies 5B11 and 1A12) on 1-HN PC and 1-HR-PC. Q-ChIP analysis of four HN PCs (blue column) and three HR-PCs (red column). Columns, mean of triplicate PCR for each tumor were quantified as described in Materials and Methods, and are corrected for input DNA values; bars, SD; P = 0.029 as determined by one-sided Mann-Whitney test.

 
We also compared AR and Pur{alpha} mRNA levels by Q-PCR in frozen tissues from 12 localized PCs (HN tumors) and 7 HR metastatic tissues (Fig. 6B). HN tumors had significantly (P = 0.0317) lower AR mRNA levels (mean ± SD, 4,172.1 ± 5,992.0 and 52,341.0 ± 102.570.0, respectively) and significantly (P = 0.0317) higher Pur{alpha}-mRNA levels (5,282.3 ± 6,498.4 and 1,764.5 ± 1,055.6, respectively) than HR metastases. Moreover, a study published in Oncomine reported a significant (t = 10.924, P = 8.7 x 10–13) decrease of Pur{alpha} mRNA level in metastatic HR-PC compared with HN primary PC (Fig. 6C; ref. 32). The box plot shows that the median for Pur{alpha} was –1.28788 in the HR group and 0.50424 in the HN group. No data were available for AR and hnRNP-K expression for this study, but published evidence supports increased AR expression in advanced tumors (32, 43).

Finally, we performed Q-ChIP analyses of Pur{alpha} on tissue samples from four primary HN PCs and three HR metastases. In Fig. 6D (top), we show three independent PCR amplifications for a representative HN and HR tumor. Similar results were obtained for each of the tumors tested (data not shown), and the gel bands were scanned, quantified using NIH Image, and corrected to account for the DNA input (Fig. 6D, bar graph). These results show that the amount of Pur{alpha} bound to the ARS-containing DNA sequence isolated from the HR tumors was nearly 4-fold lower (one-sided P = 0.029) than Pur{alpha} bound to HN-derived DNA. Together with the gene expression data, these results are consistent with the role of Pur{alpha} in AR regulation in human cancer and provide evidence for a causal relationship between decreased Pur{alpha} binding and increased AR expression in the AI progression of human PC.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have now identified a mechanism to explain the previously confirmed association of increased AR and PC progression to hormone resistance. Results of gel shift assays of nuclear extracts and in vivo ChIP analyses in an AD and AI LNCaP cell model indicate that a previously identified cis-acting suppressor element in the 5'-UTR of the human AR gene (17) binds a novel transcriptional repressor complex that contains Pur{alpha} and hnRNP-K (Fig. 1). Compared with the parental LNCaP-AD cells, the level of expression (Fig. 1C and D) and the binding of Pur{alpha} to ARS was markedly decreased in the LNCaP-AI cells, which are androgen-independent for growth, and which overexpress AR (Fig. 1A–C; ref. 8). HnRNP-K levels were similar in both cell lines and the binding of hnRNP-K to the ARS was similar in both cell types (Fig. 1A and B), suggesting that deregulation of Pur{alpha} activity is the pathogenic event.

Indeed, we have determined that regulation of Pur{alpha}, and not hnRNP-K, is crucial for the repression of AR levels in this model. SiRNA knockdown of Pur{alpha} in LNCaP-AD cells produced higher AR levels and activation (Fig. 4C and D), resulting in the inhibition of its downstream target p21 (Fig. 5A; refs. 12, 42) and AI growth (Fig. 5A). Increased expression of an active AR was shown to be the direct cause of AI growth of the LNCaP-AD Pur{alpha} knockdown cells because concomitant knockdown of AR abrogated AI growth in these cells (Fig. 5B). Conversely, forced expression of Pur{alpha} in the AI-derivative of LNCaP cells, which had markedly decreased expression and binding of Pur{alpha} to ARS, was sufficient to reduce AR expression and to decrease transcriptional activation of an AR promoter/reporter construct (Fig. 4A). Our studies also suggest that the interaction between Pur{alpha} and AR is quite specific. Forced expression of Pur{alpha} in AI cells reduced ARS-containing AR promoter/reporter activity only when the ARS was intact (Fig. 4A), indicating that the cis-element is specific in mediating this effect and in lowering the expression of endogenous AR. Moreover, Pur{alpha} mutants, in which two or three of the five central repeat modules involved in binding to a single-stranded Pur{alpha} response element (26) were deleted, lost their ability to bind to the dsARS (Fig. 2C). The specificity and the dependence of this effect on AR regulation are noteworthy in view of the observation that overexpressed Pur{alpha} suppressed growth through other mechanisms (24). Thus, it is likely that decreased binding of Pur{alpha} to the ARS, which allows overexpression of active AR, provides the AI-derivative of LNCaP-AD cells with a mechanism for thriving in the androgen-deprived conditions, a requirement for AI progression in patients on hormonal treatment.

It is remarkable that the interrelations and alterations observed in this model seem to be directly relevant to HN and HR-PC specimens from patients. Compared with HN specimens, HR-PCs with higher AR levels had reduced Pur{alpha}-mRNA, cytosolic rather than nuclear localization of Pur{alpha} protein, and occupancy by Pur{alpha} of the suppressor element in the 5'-UTR of the human AR gene (P = 0.029; Fig. 6A–D). Although the number of HR-PC specimens available for analysis by both Q-PCR and ChIP was small, the consistency of the results in this sequential, nonselected set of samples, and their correlation with similar results from an independent gene expression array (Oncomine) on a similar group of patients (Fig. 6C), supports the notion that this mechanism is a frequent determinant of AI progression in patients with PC.

The mechanisms that regulate Pur{alpha} expression and its localization in PC cells have not yet been definitively elucidated. The fact that a histone deacetylase inhibitor or an inhibitor of DNA methylation restored the expression and function of the Pur{alpha}-containing repressor complex indicates that the genes coding for proteins constituting the repressor complex are epigenetically silenced. Also, there are reports that Pur{alpha} can down-regulate its own expression, and that it can be transactivated by E2F-1 (44). Our data suggest that both a reduced level of Pur{alpha} and its exclusion from the nucleus can account for the loss of Pur{alpha} from the AR 5'-UTR in HR-PCs. This conclusion is further strengthened by the results of biochemical analyses of Pur{alpha} levels, which showed an overall reduction with absence in the nucleus in LNCaP-AI cells (Fig. 1C), and of the ChIP assays which showed a major decrease in Pur{alpha} binding in HR tumors compared with HN tumors (Fig. 6D). It has been previously shown that the nuclear form of Pur{alpha} migrate slower in PAGE and that a domain within Pur{alpha}, required for nuclear transport or retention, might be a substrate for phosphorylation (45). There is also evidence that Pur{alpha} shuttles between the nucleus and the cytoplasm during the cell cycle (39, 46). Based on these observations, we speculate that the loss of nuclear localization is crucial for up-regulation of AR transcription and expression during hormone-refractory progression. Therefore, identification of the mechanism that regulates the subcellular localization of Pur{alpha} in human PC might emerge as an important future outcome of this work.

Overall, our finding of the combined presence of Pur{alpha} and hnRNPK in the repressor complex of the AR gene is similar to that recently reported for the CD43 gene promoter (27). In this instance, however, both proteins bind upstream of the transcription initiation site to the same DNA strand with hnRNP-K binding to a cruciform DNA structure with a single-stranded loop. In contrast, our findings show the binding of two proteins to opposing strands of the AR promoter. Using the Gene Runner algorithm, several secondary structures were found surrounding putative binding sites of Pur{alpha} and hnRNP-K in the CD43 promoter, including the structure previously reported (27), but a similar analysis of the 60mer oligo surrounding the ARS detected no secondary structures. Because three Sp1-binding sites surround the ARS, it is possible that the repressor complex interferes with the proper function of Sp1 or other transactivators (47).

Sequencing of the Pur{alpha} transcript from AI cells did not reveal any mutations, suggesting that epigenetic changes might define the altered function of Pur{alpha} and, under the selective pressure of androgen deprivation, lead to AR overexpression and AI growth. As mentioned above, we found that normal function of the Pur{alpha}-containing repressor can be restored by treatment of AI cells with the HDAC inhibitor SAHA alone or combined with 5-AzaC (Fig. 5C). This was not achieved by treatment with 5-AzaC alone suggesting that its effect on Pur{alpha} expression and binding to the ARS element is insufficient to restore full activity of the repressor complex. Therefore, its growth inhibition of AI cells must be mediated via a different mechanism which, when combined with SAHA, becomes synergistic. Epigenetic regulators are known to affect the activity of multiple genes in diverse cells (48), but our data suggests that in PCs, inhibition of AR transcription (17) through Pur{alpha} may be the dominant effect that determines the AD phenotype. Therefore, although further studies are needed to understand the effect of these agents on the regulation of Pur{alpha} expression and trafficking, we have identified a potential therapeutic strategy to reverse the effect of Pur{alpha} loss on AR overexpression and AI growth of HR-PC (Fig. 5D). Furthermore, because the activity of the AR protein can be altered by several posttranslational modifications through a variety of signaling pathways (13), reducing the levels of AR (17) may enhance the efficacy of agents targeted to ligand-independent pathways that activate AR, inhibitors of androgen-binding, or cytotoxic agents (12, 49).

In summary, this is the first report that links the loss of a defined transcriptional repressor complex to increased AR levels in an AI PC cell line and human HR-PC tissues. Specifically, we have established that decreased Pur{alpha} level and/or its displacement from the nucleus to the cytoplasm are critical to the loss of transcriptional repressor complex function and AI growth. Furthermore, we have shown that therapeutic restoration of Pur{alpha} repressor function with agents that relieve epigenetic silencing can reduce AR transcription and inhibit AI growth, providing a novel strategy to control HR-PC progression.


   Acknowledgments
 
Grant support: USPHS Research grants CA-98135-04 (A.C. Ferrari), the Chemotherapy Foundation (A.C. Ferrari and L.G. Wang), CA55219 (E. Johnson), USPHS Research grant CA-40578, and the Samuel Waxman Cancer Research Foundation (L. Ossowski).

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.

We thank Drs. Charles Hauer and X. Ding from the Wadsworth Center, New York State Department of Health (Albany, NY), and Dr. R. Wang at Mount Sinai School of Medicine (New York, NY) for partial mass spectrometry analysis of purified protein samples; and Drs. Robert Gallagher and Arthur Zelent for critical reading of the manuscript.


   Footnotes
 
5 http://www.oncomine.org/ Back

Received 10/26/07. Revised 12/10/07. Accepted 1/10/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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