Androgen Receptor Overexpression in Prostate Cancer Linked to Pura Loss from a Novel Repressor Complex

Research Article

Androgen Receptor Overexpression in Prostate Cancer Linked to PurA Loss from a Novel Repressor Complex

Longgui G. Wang,1 Edward M. Johnson,2 Yayoi Kinoshita,3 James S. Babb,1 Michael T. Buckley,1 Leonard F. Liebes,1 Jonathan Melamed,1 Xiao-Mei Liu,1 Ralf Kurek,4 Liliana Ossowski,3 and Anna C. Ferrari1

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

Abstract through several pathways, it is apparent that the AR is essential to PC progression in the presence or absence of the androgen Increased androgen receptor (AR) expression and activity are ligand (7). pivotal for androgen-independent (AI) prostate cancer (PC) Increased AR expression is also a feature of the emergent AI progression and resistance to androgen-deprivation therapy. phenotype in PC models. To mimic the AI phenotype that emerges We show that a novel transcriptional repressor complex that during androgen deprivation therapy, we chronically deprived an binds a specific sequence (repressor element) in the AR gene androgen-dependent (AD) cell line (LNCaP) of androgen (8). We 5¶-untranslated region contains PurA and hnRNP-K. PurA found that, similar to some advanced PC xenografts (9, 10), the expression, its nuclear localization, and its AR promoter emergent AI cell line had 2-fold to 4-fold more AR than the parental association, as determined by chromatin immunoprecipita- AD cells (8) resulting from transcriptional up-regulation of the AR. tion analysis, were found to be significantly diminished in AI- Increased AR was also described in another pair of LNCaP-AD cells LNCaP cells and in hormone-refractory human PCs. Transfec- and their AI derivatives (9). More recently, a microarray analysis of tion of AI cells with a plasmid that restored PurA expression seven HR/hormone-sensitive isogenic pairs of human PC xeno- reduced AR at the transcription and protein levels. PurA grafts showed that among 12,500 gene probe sets tested, only AR knockdown in androgen-dependent cells yielded higher AR mRNA was differentially expressed in all seven cases (10). In the and reduced p21, a gene previously shown to be under latter study, the HR-PC had more AR protein, and even a modest negative control of AR. These changes were linked to change in AR level was able to shift the relative abundance of increased proliferation in androgen-depleted conditions. coactivators and corepressors assembled on the promoters of Treatment of AI cells with histone deacetylase and DNA androgen target genes. Moreover, an increase in the AR level of a methylation inhibitors restored PurA protein and binding magnitude similar to that observed in our AI-LNCaP cells caused to the AR repressor element. This correlated with decreased AR AR antagonists to function as agonists (10). mRNA and protein levels and inhibition of cell growth. PurA AR Overall, this body of evidence indicates that AR expression level is therefore a key repressor of transcription and its loss is an important determinant of biological outcomes and provides a from the transcriptional repressor complex is a determinant compelling reason to study the mechanisms responsible for AR of AR overexpression and AI progression of PC. The success overexpression (10–12). Indeed, although multiple alterations at A in restoring Pur and the repressor complex function by the gene and protein level have been described in AI PC (13), there pharmacologic intervention opens a promising new therapeu- is only limited information regarding alterations in the regulation tic approach for advanced PC. [Cancer Res 2008;68(8):2678–88] of AR transcription that may account for increased AR mRNA levels (14). We found that a suppressor element (ARS), originally Introduction identified in a mouse AR gene and located in the 5¶-untranslated Recurrent prostate cancer (PC) remains a therapeutic challenge region (5¶-UTR) of the AR promoter (15, 16), is also present in in part because the mechanisms of progression to androgen the human AR gene and that it malfunctions in AI cells (17). A independence (AI) and the reasons for the development of promoter/reporter construct with a deleted ARS element produced resistance to androgen deprivation therapy in hormone-refractory an 8-fold increase in AR promoter activity after transfection into (HR) PC patients are still unknown. A large proportion of patients AD cells. We also found that nuclear extracts of AD cells contain a with high-grade localized cancer (1), metastatic disease (2), and repressor protein complex that binds the ARS in a gel shift assay, HR-PC (3) show increased expression of the androgen receptor although this complex was significantly reduced in AI cells (17). (AR), suggesting that this plays a key role in disease progression. We have now identified the ARS-binding transcriptional rep- Approximately 30% of cases have AR gene amplification (4) and ressor complex as containing Pura and hnRNP-K (18–20). Based 10% have mutations (5), but the vast majority of cases have on the insight gained from our mechanistic studies of the Pura- increased AR gene transcription and/or altered receptor protein containing repressor in AD and AI cell lines, and on observed stability (6). Although PC cells can derive proliferative signals changes in its expression, localization, and binding to the ARS that take place during human PC progression, we conclude that Pura is an important regulator of AR transcription and of AI growth. We also show that Pura expression and function can be restored by Requests for reprints: Anna C. Ferrari, New York University Cancer Institute, New agents that relieve epigenetic silencing, suggesting that regulation York University School of Medicine, 8th Floor, 160 East 34th Street, New York, NY of the transcriptional levels of AR may provide a novel therapeutic 10016. Phone: 212-731-5389; Fax: 212-731-5455; E-mail: [email protected]. I2008 American Association for Cancer Research. strategy to control PC progression and to enhance the efficacy of doi:10.1158/0008-5472.CAN-07-6017 existing systemic therapies.

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Materials and Methods 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, Cells and tissues. LNCaP cells were maintained in RPMI 1640 with 10% (BioTEG)5¶-TGG GGG GAG GCG GGT GGG GGG AGG CGG GTG GGG GGA heat-inactivated fetal bovine serum. The AI-LNCaP derivative was isolated GGC GGG-3¶; and its mutant, sense, 5¶-CCA ACC TCC TTC CAC CAA CCT by growing LNCaP cells in 10% charcoal-stripped fetal bovine serum and CCT TCC ACC AAC CTC CTT CCA-3¶; and antisense (BioTEG)5¶-TGG AAG A 5 g/mL of insulin for several months. An initial reduction in the total cell GAG GTT GGT GGA AGG AGG TTG GTG GAA GGA GGT TGG-3¶]. The population was followed by a gradual outgrowth of AI cells (8). Formalin- beads were then eluted and the protein fractions processed for Western fixed, paraffin-embedded and frozen PC tissues were obtained from the blot. New York University Prostate Cancer Tissue Resource, the Stadtische ChIP assays. The protein/DNA complexes from intact AD and AI cells Kliniken, Offenbach, Germany, and the Mount Sinai School of Medicine. or from minced frozen PC tissues were cross-linked and then lysed as Twelve hormone-naı¨ve (HN), Gleason score 6 frozen PCs were micro- previously described (30). Equal amounts of protein or tissue lysates dissected for quantitative PCR (Q-PCR), four were used for chromatin (50–130 mg total weight) were incubated with the primary antibody, Pura immunoprecipitation (ChIP), seven HR-PC metastases were processed for 5B11 and 1A12 (31), followed by Protein G PLUS-Agarose beads (Santa Q-PCR: two bone marrows, one bone metastasis protruding into the brain, Cruz), washed twice with high-salt buffer (0.5 mol/L NaCl), followed by one cervical lymph node, one epidural metastasis, one malignant ascitis, LiCl/detergent solution and TE buffer. The DNA-protein immunoprecipi- and one malignant pleural effusion. The latter three were used for ChIP. All tates were eluted, proteinase K–treated, and the DNA extracted. PCR patients signed Institutional Review Board–approved consent. (Expand High-Fidelity PCR System; Roche) was performed using primers to Tissue selection, tissue microarray, immunohistochemistry, and sequence +248 to +487 nucleotides of the AR 5¶-UTR region and the Sp1 image analysis. Four core samples from 18 HN radical prostatectomy and sites: 5¶-AGC TGC TAA AGA CTC GGA GG-3¶ and 5¶-GGA GTT ACC TCT 18 HR transurethral resection specimens of the prostate after progression CTG CAA AC-3¶. The images of PCR products stained with SYBR Gold following several months of androgen deprivation, matched for Gleason (Molecular Probes) in agarose gels were captured with Fotodyne, imported A score, were cut in 4- m-thick sections, histology verified every 10th section into Photoshop, and the black bands scanned and quantified using NIH and was assembled for tissue microarray (TMA; Beecher Instruments; Image. For clinical samples, Pura binding was corrected for input DNA. ref. 21). For immunohistochemistry, TMA slides were soaked in H-3300 PurA knockdown. AD cells seeded at 5,000 cells/well were either fixed solution (Vector Laboratories), microwaved and treated with streptavidin 24 h later (day 0) with 10% trichloroacetic acid or transfected with Pura- peroxidase. The Pura monoclonal antibody 10B12 (IgG1 isotype; 1:1,000; siRNA (85 ng/well), AR siRNA (85 ng/well), Pura-siRNA plus AR siRNA ref. 22) and AR polyclonal antibody, which have been previously described (85 ng each/well), or control (scrambled) siRNA (labeled NC, 85 ng/well; (23), were horseradish peroxidase–conjugated with secondary antibody and Ambion) using Effectene in serum-free medium. Cell growth was measured diaminobenzidine. Negative controls for Pura included an isotype-matched at baseline, 48, and 72 h posttransfection by sulforhodamine B assay. irrelevant monoclonal antibody (mouse IgG1, X0943; Dako); and for AR, a AI cells in AI-medium served as positive control. AD cells seeded at 0.5 to rabbit immunoglobulin fraction (solid-phase absorbed; X09036; Dako) 1 Â 106 cells/well were transfected with 1 Ag of siRNA per well and diluted 1:1,000 in PBS. All sections were incubated with horseradish processed for protein extraction and Western blotting with AR and Pura. peroxidase–conjugated secondary antibody and diaminobenzidine. Quan- Analysis of AR and PurA expression using Oncomine. We searched titative immunohistochemical analysis was performed using Kodak Oncomine5 for PURA cDNA and PC. The original raw data and results Molecular Imaging (ver. 4.0) as previously described (24, 25). Positively including the median, 75/25 percentiles, t test, and P values for each study stained pixel regions-of-interest and the percentage of staining were were collected and linked to the original publications. Only one (32) of two calculated using Microsoft Excel. Two independent observers determined studies described HN PC and metastatic HR-PC as defined in our study and the percentage of positive Pura nuclear staining based on two 40Â- probed for Pura. magnified TMA images. Q-PCR. Frozen PC specimen were microdissected and processed for Protein isolation, electrophoretic mobility shift assay, and immu- RNA extraction, cDNA, and Q-PCR as previously described (33). Primers and noblotting. Nuclear AD and AI cell proteins were processed for VIC-labeled probe for glyceraldehyde-3¶-phosphate dehydrogenase, AR, and electrophoretic mobility shift assay (EMSA) as previously described (17). PURA (ABI) were used to measure copy numbers by reference to log-linear For EMSA with purified GST-Pura, the protein was reacted with [g-32P]- standard curves (2 to 2 Â 107 copy range) of serial dilutions of linearized labeled double-stranded (ds) wt-ARS oligonucleotide 5¶-ACC CCG CCT CCC plasmid DNAs containing the respective gene inserts. The positive controls CCC ACC CT-3¶ +323 and +342 nucleotides; ref. 17, or single-stranded (ss) were LNCaP cells, the negative control lacked a cDNA template. The mean C-rich or G-rich ARS, or its three mutants: mARS1, ACC CAA CCT CCT TCC copy number of triplicate Q-PCR assays were used to compare samples. ACC CT; mARS2, ACC CCT TCT CAA CCC ACC CT; and mARS3, ACC TTG Statistical analysis. Average values of multiple assessments of AR and CCT AAC CCC ACC CT (Bio-synthesis, Inc.), in binding buffer and subjected Pura regarding the percentage of positive staining intensities and to 8% PAGE. For the supershift assay, the nuclear proteins were pre- expression levels by Q-PCR and ChIP among HN HR-PC’s were compared incubated with anti-Pura monoclonal antibody and 10-fold excess mARS3. by the Mann-Whitney test. The P values were exact significance levels and Anti-Pura monoclonal antibody, Pura fusion protein pGPur4, and mutant two-sided, except where indicated, and statistically significant at <0.05 by Pura proteins (amino acids 167-322/m-GST-Pura-1 and amino acids 216- SAS version 9.0 (SAS Institute) analysis. 322/m-GST-Pura-2) were previously described (22, 26). Southwestern blot was performed as previously described (17). Results Transfection, luciferase assay, and Western blot. Cells were cotransfected with 1 Ag of ‘‘empty’’ vector DNA (pVector), AR luciferase reporter Identification of ARS nuclear binding complex. We previ- pLARS-1, or its ARS-deleted mutant pLARS-del (17), with or without ously reported that high AR expression in LNCaP-AI cells might be expression plasmids for phnRNP-K (27, 28), or Pura vector pHApur1 (26) or due to loss of binding of a repressor complex to an ARS element in Pura-small interfering RNA (siRNA; 1–2 Ag/well) and 0.5 Ag of pSV-h- the 5¶-UTR of the AR gene (17). Gel blotting analysis of nuclear galactosidase (Promega) control, using Effectene (Qiagen). Luciferase and extracts from LNCaP-AI and AD cells with a labeled dsARS probe h-galactosidase activity (Promega) of cell lysates were measured 48 h later. (Southwestern) revealed several bands (f105 to f30 kDa; results Total and subcellular proteins from parallel cultures were processed for not shown). To further identify these bands, nuclear extracts of AD Western blotting 48 h after cotransfection using Pura 10B12 (22), AR, h-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 5 http://www.oncomine.org/ www.aacrjournals.org 2679 Cancer Res 2008; 68: (8). April 15, 2008

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2008 American Association for Cancer Research. Cancer Research cells were affinity-purified on an ARS-oligonucleotide column Both Pura (38–40) and hnRNP-K (34, 41) can strand-separate (dswt-ARS, 20 bp), the protein was eluted with a linear gradient of dsDNA and subsequently bind to the individual DNA strands. KCl and separated on duplicate SDS-PAGE gels; one gel was Nuclear extracts of AD and AI cells retarded the single C- and analyzed by Southwestern analysis whereas the other was used to G-rich ssARS probes (Fig. 2A, lanes 10–14 and 15–20, respectively), elute proteins from the areas corresponding to the bands (a total of and the anti-Pura antibody supershifted the G-rich band (Fig. 2A, five) detected by Southwestern analysis. One of the bands was lanes 18 and 19), whereas the anti–hnRNP-K antibody reduced the identified as the nuclear protein hnRNP-K (CAA51267.1; ref. 34), by intensity of the C-rich band (Fig. 2A, lane 13). Thus, it seems that Bio-Mass Spectrometry (results not shown). This protein has when faced with dsARS, each of the proteins could strand-separate multiple functions (35), including the ability to bind ssDN (28, 36) it and bind to the C-rich (hnRNP-K) or G-rich (Pura) strand, and to repress transcription (34). Because hnRNP-K can shuttle producing a retarded protein-DNA complex. between the nuclear and cytoplasmic compartments (37), we tested A pull-down experiment in which AD cell extracts were nuclear and cytoplasmic extracts of AD and AI cells for proteins incubated with biotinylated wt-dsARS oligonucleotide bound to that bind to the ssARS. In Southwestern analysis with a C-rich streptavidin beads showed that both hnRNP-K (Fig. 2D, top) and oligonucleotide probe, a single, equal-intensity band of f55 to Pura (middle) were eluted at 0.25 and 0.75 mol/L KCl, respectively. 65 kDa (Fig. 1A, left) was produced by nuclear extracts of AD and AI Beads with mutated dsARS bound much less hnRNP-K and no cells. When the membrane was stripped of the radioactive probe Pura (Fig. 2D). Sp1, also present in the nuclear extracts (see below), and tested by immunoblotting with specific anti–hnRNP-K did not bind to the ARS probe (bottom). This suggests that both antibody, a doublet, with molecular weight corresponding to hnRNP-K and Pura interact specifically with dsARS. hnRNP-K was detected (Fig. 1B, left). Several additional faint bands hnRNP-K and PurA bind to the ARS in vivo. We used ChIP were also noted (Fig. 1A, left). Using G-strand ARS as a probe, assays to test whether Pura and hnRNP-K interact with the ARS several distinct bands, some of high intensity, were noted (Fig. 1A, in vivo. As the PCR primers used for ChIP were positioned to right) including a weak 40 kDa band, which was more intense in amplify the ARS and the two nearby SP1 binding sites of the AR AD extracts. Immunoblotting with anti–Pura antibodies revealed a gene, anti-Sp1 antibody was used as a positive control. A sequence band of similar molecular weight (Fig. 1B, right). Pura was reported located between +248 to +487 nucleotides, which contains the to cooperate with hnRNP-K in transcriptional repression (27). putative ARS (+323 to +342 nucleotides), was amplified from the Direct immunoblotting of whole cell extracts showed that AR is DNA immunoprecipitated by two different anti-Pura antibodies, elevated and confirmed that Pura is reduced in AI cells, whereas anti–hnRNP-K antibody or anti-Sp1 antibody in AD and AI cells hnRNP-K was equally expressed in AI and AD cells (Fig. 1C, left). (Fig. 3, top). No DNA amplification was found with irrelevant IgG, Moreover, Pura was shown to be reduced in the cytoplasm of AI beads alone or lysates alone. To quantify the amount of Pura cells and was almost undetectable in their nuclei (Fig. 1C, right). associated with the ARS, we performed quantitative ChIP (Q-ChIP), Finally, analysis of AD and AI cells using an Affymetrix gene using strictly controlled reaction inputs for AD and AI cells. With expression array (U133A GeneChip) showed in three independent the two anti-Pura antibodies (22), 1.9-fold to 2.5-fold more Pura arrays that AI cells express f2.5-fold more AR mRNA, similar was associated with the ARS-containing 248- to 487-nucleotide levels of hnRNP-K mRNA, and f4-fold less Pura mRNA compared sequences in the AD cells (Fig. 3, bottom). with AD cells (Fig. 1D). Down-regulation of AR transcription by PurA. To test PurA and hnRNP-K form an ARS-binding complex in vitro. whether Pura and hnRNP-K affect transcription of the AR gene, We previously showed that nuclear extracts of AD cells incubated AD and AI cells were cotransfected with an ARS-containing with a dsARS probe produced a retarded complex in gel shift AR promoter/luciferase reporter (pLARS-1) or with a reporter (EMSA) assays that were greatly diminished in AI cells (17). We containing a deleted ARS (pLARS-del; ref. 17) and with expression show here that the intensity of this complex is strongly reduced by plasmids for Pura or hnRNP-K or both. Forced expression of anti–hnRNP-K blocking antibody, whereas nonimmune IgG have Pura in pLARS-transfected AI cells produced a >50% decrease in no effect (Fig. 2A, lanes 3 and 4). Anti-Pura antibody ‘‘supershifted’’ luciferase activity, and a significantly (P < 0.05) lesser effect in most of this complex (Fig. 2A, lane 7), as well as a complex pLARS-del–transfected cells (Fig. 4A). Importantly, this transcrip- produced by purified GST-Pura fusion protein (Fig. 2B, lane 3), and tional effect was accompanied by a reduced expression of endo- this complex could be competed off with an excess of unlabeled genous AR protein (Fig. 4A, right). Forced expression of hnRNP-K, dsARS (ref. 17; results not shown). Two Pura truncation mutants which produced only a very slight increase in the protein level, did (mGST-Pura-1 and mGST-Pura-2, amino acids 167–322 and not further enhance the effect of Pura (Fig. 4A), suggesting that the 216–322, respectively), shown previously to bind only very weakly level of hnRNP-K was not rate-limiting. to a Pura element in the c-MYC promoter (30), did not produce As an alternative experimental approach, we tested Pura and retarded complexes with the ARS (Fig. 2B, lanes 6 and 7). The hnRNP-K knockdown by siRNAs in AD and AI cells for their complex pattern was different when three mutated ARS oligonu- potential to derepress AR transcription, using the AR promoter/ cleotides (mARS1–3) were used as probes: AD nuclear extracts did luciferase assay or AR protein levels. Transfection of AD cells, not bind mARS1 and mARS3 at all, and with mARS2, produced which have higher endogenous levels of Pura, with 1 or 2 Agof patterns of bands different from those observed with wtARS Pura/siRNA increased AR/luciferase activity by 6-fold and 8-fold, (ref. 17; results not shown). Purified GST-Pura protein was bound respectively (Fig. 4C), and transfection with 1 Ag of Pura/siRNA to neither mARS1 nor to mARS3 (Fig. 2C) and was indistinguish- induced a strong increase in endogenous AR protein (Fig. 4D). able from the wtARS when bound to mARS2. Moreover, excess of Transfection with two individual hnRNP-K siRNAs produced unlabeled dsARS reduced the ARS-Pura complex (Fig. 2C) only an f1.5-fold increase in AR/luciferase activity (Fig. 4B), and indicating specificity of interaction. These results suggest that a slight enhancement of the siPura effect in AI cells (Fig. 4D). As Pura and hnRNP-K are present in the complex and that they expected, the endogenous Pura protein was very low in AI cells, directly bind to the ARS. and was further reduced to a barely detectable level by Pura-siRNA

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(Fig. 4D), causing a 2.0-fold increase in luciferase activity (Fig. 4B) nilide hydroxamic acid (SAHA), 5-azacytidine (5-AzaC), or the two and a slight increase in AR protein (Fig. 4D). These results reveal drugs together, and examined using a gel shift assay for the presence the fine transcriptional tuning of AR by Pura expression. of a functional (ARS binding) repressor complex. Individual Reduction of PurA level converts AD cells to androgen treatments increased the intensity of the bands (Fig. 5C, lanes 5 independence for growth. We reasoned that AI proliferation and and 6 compared with lane 4), and the combination of the two drugs higher AR expression (AI phenotype), similar to that achieved by produced a more intense band (lane 7), which was supershifted by chronic maintenance of LNCaP-AD cells in androgen-poor medium anti-Pura antibody (Fig. 5C, lane 8), in a pattern similar to that of (8, 12), might be mimicked by acute reduction of Pura levels. Thus, AD cells (Fig. 5C, lanes 2 and 3). Treatment of AI cells with the same we transfected AD cells with Pura/siRNA and cultured the cells in dose of SAHA (7 Amol/L) increased Pura and strongly reduced the androgen-depleted medium for 2 and 3 days. Compared with cells AR mRNA and protein levels (Fig. 5D, inset). Treatment with 5-AzaC transfected with a scrambled siRNA, the reduced Pura led to (5 Amol/L) also restored Pura expression and in vitro binding to increased AR protein and resulted in a 1.6-fold and 2.5-fold increase ARS (gel shift) but had no effect on AR levels. Most importantly, in cell growth on days 2 and 3, respectively (Fig. 5A). Importantly, SAHA and 5-AzaC individually, and in combination, had a dose- proliferation was entirely AR-dependent because reduction of AR dependent growth-inhibitory effect on AI cells (Fig. 5D). The by knockdown in Pura siRNA-treated cells blocked androgen- isobologram showed that the combination was synergistic (results induced growth (Fig. 5B). We and others have previously shown not shown). (12, 42) that high AR levels inhibit p21/WAF1 expression. A similar Higher AR levels and lower or delocalized PurA character- effect was then found in cells in which AR expression was increased ize HR human PCs. To examine whether human PCs recapitulate by Pura-siRNA treatment (Fig. 5A and B), indicating that the the reciprocal relationship between Pura and AR levels observed restored AR is functional. These results indicate an inverse in the AI and AD cell lines, we compared the AR and Pura content relationship between Pura and AR levels and cell growth. and/or their subcellular localization in TMA. Gleason score– To assess the potential clinical value of this dependence, we matched 18 localized HN and 18 HR-PCs, were immunostained for tested whether the repressor complex was amenable to up- AR and Pura. Figure 6A shows the results of representative sections regulation by pharmacologic agents known to have epigenetic of HN and HR tumors. In all tumors, AR was found to be pre- effects on gene expression. AI cells were treated with suberoyla- dominantly nuclear (Fig. 6A, top), whereas Pura was both nuclear

Figure 1. hnRNP-K and Pura in the ARS-binding complexes. A and B, nuclear and cytoplasmic proteins (50 Ag/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 Pura. C, AR, hnRNP-K, and Pura levels and subcellular localization in AD and AI cells. Total extracts (50 Ag of proteins/ lane) or nuclear and cytoplasmic proteins (50 Ag/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 Pura or actin for loading control. D, mRNA levels of AR (black column), Pura (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. www.aacrjournals.org 2681 Cancer Res 2008; 68: (8). April 15, 2008

Figure 2. Binding specificity of nuclear proteins to ARS in EMSA. A, nuclear extracts (5 Ag) or purified GST-Pura fusion protein (0.2 Ag; 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-Pura 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, Pura, and Sp1 (undetected), respectively.

and cytoplasmic in HN tumors (Fig. 6A, bottom left) but was mostly tumor was significantly lower (P = 0.005) for HR tumors (mean F cytoplasmic in the majority of HR tumors (Fig. 6A, bottom middle). SD, 34.1 F 37.9; median, 12.0) than for HN tumors (mean F SD, The mean staining intensity of nuclear AR was significantly lower 80.5 F 37.2; median, 100). (P = 0.011) in HN tumors (mean F SD, 15.4 F 16.4) than in HR We also compared AR and Pura mRNA levels by Q-PCR in frozen tumors (24.4 F 17.8). The overall Pura level in the immunohisto- tissues from 12 localized PCs (HN tumors) and 7 HR metastatic chemical analysis was not significantly different between HN and tissues (Fig. 6B). HN tumors had significantly (P = 0.0317) lower HR tumors (P = 0.52), possibly due to the difficulty in direct AR mRNA levels (mean F SD, 4,172.1 F 5,992.0 and 52,341.0 F comparison of immunostaining intensities of nuclear and cytosolic 102.570.0, respectively) and significantly (P = 0.0317) higher Pura- antigens. However, Pura was localized almost exclusively to the mRNA levels (5,282.3 F 6,498.4 and 1,764.5 F 1,055.6, respectively) nucleus in the majority (12 of 15) of the HN tumors, but in only than HR metastases. Moreover, a study published in Oncomine À 5 of 15 HR tumors, and the percentage of Pura-positive nuclei per reported a significant (t = 10.924, P =8.7Â 10 13) decrease of Pura

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2008 American Association for Cancer Research. Androgen Receptor Gene Repressor mRNA level in metastatic HR-PC compared with HN primary provide evidence for a causal relationship between decreased Pura PC (Fig. 6C; ref. 32). The box plot shows that the median for Pura binding and increased AR expression in the AI progression of was À1.28788 in the HR group and 0.50424 in the HN group. No human PC. data were available for AR and hnRNP-K expression for this study, but published evidence supports increased AR expression in advanced tumors (32, 43). Discussion Finally, we performed Q-ChIP analyses of Pura on tissue samples We have now identified a mechanism to explain the previously from four primary HN PCs and three HR metastases. In Fig. 6D confirmed association of increased AR and PC progression to (top), we show three independent PCR amplifications for a hormone resistance. Results of gel shift assays of nuclear extracts representative HN and HR tumor. Similar results were obtained and in vivo ChIP analyses in an AD and AI LNCaP cell model for each of the tumors tested (data not shown), and the gel bands indicate that a previously identified cis-acting suppressor element were scanned, quantified using NIH Image, and corrected to in the 5¶-UTR of the human AR gene (17) binds a novel tran- account for the DNA input (Fig. 6D, bar graph). These results show scriptional repressor complex that contains Pura and hnRNP-K that the amount of Pura bound to the ARS-containing DNA (Fig. 1). Compared with the parental LNCaP-AD cells, the level of sequence isolated from the HR tumors was nearly 4-fold lower expression (Fig. 1C and D) and the binding of Pura to ARS was (one-sided P = 0.029) than Pura bound to HN-derived DNA. markedly decreased in the LNCaP-AI cells, which are androgen- Together with the gene expression data, these results are consistent independent for growth, and which overexpress AR (Fig. 1A–C; with the role of Pura in AR regulation in human cancer and ref. 8). HnRNP-K levels were similar in both cell lines and the

Figure 3. In vivo interaction of hnRNP-K and Pura with an ARS containing AR gene sequence (ChIP assay) in AD and AI LNCaP cells. Top, ChIP assays; bottom, Q-ChIP assay for Pura. Proteins cross-linked to chromatin were processed as described in Materials and Methods using two different antibodies for Pura (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 F 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-Pura antibodies (results not shown).

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Figure 4. Effect of hnRNP-K and Pura on AR transcription/expression in AI and/or AD cells. A, effect of forced hnRNP-K and Pura 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 h-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 Pura transcript from pHApur1 is of higher molecular weight than the wild-type Pura. B, effects of knockdown of hnRNP-K and Pura on AR transcription: AI cells were cotransfected with pLARS-1 and 1 Ag of two hnRNP-K-siRNAs (11155 and 11059), or Pura siRNA, or with a scrambled version (negative control, NC) of siRNA. Columns, AR luciferase activity measured 48 h after transfection, as a ratio of h-galactosidase activity (see Materials and Methods). C, comparison of AR transcription in AD (black columns) and AI (white columns) cells in response to Pura-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 Pura-siRNA and tested by Western blotting for AR, Pura, and actin as loading control 48 h after transfection.

binding of hnRNP-K to the ARS was similar in both cell types suppressed growth through other mechanisms (24). Thus, it is (Fig. 1A and B), suggesting that deregulation of Pura activity is the likely that decreased binding of Pura to the ARS, which allows pathogenic event. overexpression of active AR, provides the AI-derivative of LNCaP- Indeed, we have determined that regulation of Pura, and not AD cells with a mechanism for thriving in the androgen-deprived hnRNP-K, is crucial for the repression of AR levels in this model. conditions, a requirement for AI progression in patients on SiRNA knockdown of Pura in LNCaP-AD cells produced higher AR hormonal treatment. levels and activation (Fig. 4C and D), resulting in the inhibition of It is remarkable that the interrelations and alterations observed its downstream target p21 (Fig. 5A; refs. 12, 42) and AI growth in this model seem to be directly relevant to HN and HR-PC (Fig. 5A). Increased expression of an active AR was shown to be the specimens from patients. Compared with HN specimens, HR-PCs direct cause of AI growth of the LNCaP-AD Pura knockdown cells with higher AR levels had reduced Pura-mRNA, cytosolic rather because concomitant knockdown of AR abrogated AI growth in than nuclear localization of Pura protein, and occupancy by Pura these cells (Fig. 5B). Conversely, forced expression of Pura in the of the suppressor element in the 5¶-UTR of the human AR gene AI-derivative of LNCaP cells, which had markedly decreased (P = 0.029; Fig. 6A–D). Although the number of HR-PC specimens expression and binding of Pura to ARS, was sufficient to reduce available for analysis by both Q-PCR and ChIP was small, the AR expression and to decrease transcriptional activation of an AR consistency of the results in this sequential, nonselected set of promoter/reporter construct (Fig. 4A). Our studies also suggest samples, and their correlation with similar results from an inde- that the interaction between Pura and AR is quite specific. Forced pendent gene expression array (Oncomine) on a similar group of expression of Pura in AI cells reduced ARS-containing AR pro- patients (Fig. 6C), supports the notion that this mechanism is a moter/reporter activity only when the ARS was intact (Fig. 4A), frequent determinant of AI progression in patients with PC. indicating that the cis-element is specific in mediating this effect The mechanisms that regulate Pura expression and its and in lowering the expression of endogenous AR. Moreover, Pura localization in PC cells have not yet been definitively elucidated. mutants, in which two or three of the five central repeat modules The fact that a histone deacetylase inhibitor or an inhibitor of DNA involved in binding to a single-stranded Pura response element methylation restored the expression and function of the Pura- (26) were deleted, lost their ability to bind to the dsARS (Fig. 2C). containing repressor complex indicates that the genes coding for The specificity and the dependence of this effect on AR regulation proteins constituting the repressor complex are epigenetically are noteworthy in view of the observation that overexpressed Pura silenced. Also, there are reports that Pura can down-regulate its

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Figure 5. Down-regulation of Pura 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 Pura-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 Pura-siRNA and examined, at the indicated times, for AR, Pura, 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 Pura-siRNA (light gray columns), AR siRNA alone (dark gray columns) or Pura-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, Pura, p21/WAF1, and h-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 Ag of respective proteins were analyzed by EMSA using ssG-rich ARS as probe. Anti-Pura antibody (2 Ag/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 (o), 5-AzaC (.) or the two drugs together (!). 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 Pura levels in AI cells, treated 16 h after plating, with 5.0 Amol/L of 5-Aza for 6 d, or 7.5 Amol/L of SAHA for 1 day, or 5.0 Amol/L of 5-Aza for 5 days followed by 7.5 Amol/L of SAHA for 1 d. Bottom, parallel results of AR Q-PCR analysis. own expression, and that it can be transactivated by E2F-1 (44). these observations, we speculate that the loss of nuclear localiza- Our data suggest that both a reduced level of Pura and its tion is crucial for up-regulation of AR transcription and expression exclusion from the nucleus can account for the loss of Pura from during hormone-refractory progression. Therefore, identification of the AR 5¶-UTR in HR-PCs. This conclusion is further strengthened the mechanism that regulates the subcellular localization of Pura by the results of biochemical analyses of Pura levels, which showed in human PC might emerge as an important future outcome of an overall reduction with absence in the nucleus in LNCaP-AI cells this work. (Fig. 1C), and of the ChIP assays which showed a major decrease in Overall, our finding of the combined presence of Pura and Pura binding in HR tumors compared with HN tumors (Fig. 6D). It hnRNPK in the repressor complex of the AR gene is similar to that has been previously shown that the nuclear form of Pura migrate recently reported for the CD43 gene promoter (27). In this instance, slower in PAGE and that a domain within Pura, required for however, both proteins bind upstream of the transcription nuclear transport or retention, might be a substrate for phosphor- initiation site to the same DNA strand with hnRNP-K binding to ylation (45). There is also evidence that Pura shuttles between the a cruciform DNA structure with a single-stranded loop. In contrast, nucleus and the cytoplasm during the cell cycle (39, 46). Based on our findings show the binding of two proteins to opposing strands www.aacrjournals.org 2685 Cancer Res 2008; 68: (8). April 15, 2008

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2008 American Association for Cancer Research. Cancer Research of the AR promoter. Using the Gene Runner algorithm, several is possible that the repressor complex interferes with the proper secondary structures were found surrounding putative binding function of Sp1 or other transactivators (47). sites of Pura and hnRNP-K in the CD43 promoter, including the Sequencing of the Pura transcript from AI cells did not reveal structure previously reported (27), but a similar analysis of the any mutations, suggesting that epigenetic changes might define the 60mer oligo surrounding the ARS detected no secondary altered function of Pura and, under the selective pressure of structures. Because three Sp1-binding sites surround the ARS, it androgen deprivation, lead to AR overexpression and AI growth.

Figure 6. AR and Pura 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), Pura antibody 10B12 (bottom left and middle), and negative controls (right). Insets, higher magnification (Â40) of the indicated areas. Note greater intensity of AR nuclear staining and predominantly cytoplasmic Pura staining in the HR tumors. Pura in HN tumor is mostly nuclear. The quantification of nuclear localization of Pura in TMAs is represented by the graph in which each column represents an individual tumor. The medians of nuclear Pura were 12.0% for HR (red columns), and 100.0% for HN (blue columns), respectively. B, the levels of Pura and AR mRNAs in clinical specimens by Q-PCR. C, Pura-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-Pura with Pura 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.

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As mentioned above, we found that normal function of the In summary, this is the first report that links the loss of a Pura-containing repressor can be restored by treatment of AI cells defined transcriptional repressor complex to increased AR levels with the HDAC inhibitor SAHA alone or combined with 5-AzaC in an AI PC cell line and human HR-PC tissues. Specifically, we (Fig. 5C). This was not achieved by treatment with 5-AzaC alone have established that decreased Pura level and/or its displace- suggesting that its effect on Pura expression and binding to the ment from the nucleus to the cytoplasm are critical to the loss of ARS element is insufficient to restore full activity of the repressor transcriptional repressor complex function and AI growth. complex. Therefore, its growth inhibition of AI cells must be Furthermore, we have shown that therapeutic restoration of mediated via a different mechanism which, when combined with Pura repressor function with agents that relieve epigenetic SAHA, becomes synergistic. Epigenetic regulators are known to silencing can reduce AR transcription and inhibit AI growth, affect the activity of multiple genes in diverse cells (48), but our providing a novel strategy to control HR-PC progression. data suggests that in PCs, inhibition of AR transcription (17) through Pura may be the dominant effect that determines the AD phenotype. Therefore, although further studies are needed to Acknowledgments understand the effect of these agents on the regulation of Pura Received 10/26/2007; revised 12/10/2007; accepted 1/10/2008. expression and trafficking, we have identified a potential thera- Grant support: USPHS Research grants CA-98135-04 (A.C. Ferrari), the peutic strategy to reverse the effect of Pura loss on AR over- Chemotherapy Foundation (A.C. Ferrari and L.G. Wang), CA55219 (E. Johnson), USPHS Research grant CA-40578, and the Samuel Waxman Cancer Research expression and AI growth of HR-PC (Fig. 5D). Furthermore, Foundation (L. Ossowski). because the activity of the AR protein can be altered by several 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 posttranslational modifications through a variety of signaling with 18 U.S.C. Section 1734 solely to indicate this fact. pathways (13), reducing the levels of AR (17) may enhance the We thank Drs. Charles Hauer and X. Ding from the Wadsworth Center, New York efficacy of agents targeted to ligand-independent pathways that 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 activate AR, inhibitors of androgen-binding, or cytotoxic agents samples; and Drs. Robert Gallagher and Arthur Zelent for critical reading of the (12, 49). manuscript.

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Longgui G. Wang, Edward M. Johnson, Yayoi Kinoshita, et al.

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