Elevated E2F1 Inhibits Transcription of the Androgen Receptor in Metastatic Hormone-Resistant Prostate Cancer

Research Article

Joanne N. Davis,1 Kirk J. Wojno,1 Stephanie Daignault,1 Matthias D. Hofer,2,3 Rainer Kuefer,3 Mark A. Rubin,3,4 and Mark L. Day1

1Department of Urology, University of Michigan, Ann Arbor, Michigan; 2Department of Urology, University of Ulm, Ulm, Germany; 3Department of Pathology, Brigham and Women’s Hospital; and 4Harvard University, School of Medicine, Boston, Massachusetts

Abstract disease recurs in an estimated 15% to 30% of patients (2). The androgen receptor (AR) is the mediator of the physiologic effects of Activation of E2F transcription factors, through disruption of androgen. It regulates the growth of normal and malignant the retinoblastoma (Rb) tumor-suppressor gene, is a key event prostate epithelial cells. Upon ligand binding, AR translocates to in the development of many human cancers. Previously, we the nucleus, binds to DNA recognition sequences, and activates showed that homozygous deletion of Rb in a prostate tissue transcription of target genes, including genes involved in cell recombination model exhibits increased E2F activity, acti- proliferation, apoptosis, and differentiation (reviewed in ref. 3). vation of E2F-target genes, and increased susceptibility to Androgen ablation therapy is highly successful for the treatment of hormonal carcinogenesis. In this study, we examined the hormone-sensitive prostate cancer; however, hormone resistance expression of E2F1 in 667 prostate tissue cores and compared significantly limits its benefits. Hormonal ablation therapy will it with the expression of the androgen receptor (AR), a marker control metastatic disease for 18 to 24 months (4), but once of prostate epithelial differentiation, using tissue microarray metastatic prostate cancer ceases to respond to hormonal therapy, analysis. We show that E2F1 expression is low in benign and median survival decreases significantly (5). Although the focus of localized prostate cancer, modestly elevated in metastatic rigorous study, the molecular mechanisms underlying the progres- lymph nodes from hormone-naı¨ve patients, and significantly sion of prostate cancer to hormone-independent disease remain elevated in metastatic tissues from hormone-resistant pros- unclear. tate cancer patients (P = 0.0006). In contrast, strong AR The E2F family of transcription factors consists of at least eight expression was detected in benign prostate (83%), localized characterized family members (E2Fs 1–8), which can form prostate cancer (100%), and lymph node metastasis (80%), but heterodimers with differentiating protein family members (DP1, decreased to 40% in metastatic hormone-resistant prostate DP2), giving rise to functional E2F activity (6, 7). E2F controls the cancer (P = 0.004). Semiquantitative reverse transcription- progression through the cell cycle by regulating the transcription of PCR analysis showed elevated E2F1 mRNA levels and genes that are essential for DNA synthesis and cell cycle increased levels of the E2F-target genes dihyrofolate reductase progression (8). Several E2Fs, including E2F1, behave as oncogenes, and proliferating cell nuclear antigen in metastatic hormone– and are able to induce quiescent cells to enter the cell cycle, independent prostate cancer cases compared with benign override various growth-arrest signals, and transform primary cells tissues. To identify a role of E2F1 in hormone-independent (9–11). Elevated E2F1 can induce hyperproliferation, hyperplasia, prostate cancer, we examined whether E2F1 can regulate AR and p53-dependent apoptosis in E2F1 transgenic mice (12). E2Fs expression. We show that exogenous expression of E2F1 also act like tumor suppressors by activating apoptotic pathways significantly inhibited AR mRNA and AR protein levels in (13), suggesting that E2Fs affect multiple downstream targets prostate epithelial cells. E2F1 also inhibited an AR promoter- depending on cell type and environmental context. luciferase construct that was dependent on the transactivation Aberrant expression of E2F1 has been documented in human domain of E2F1. Furthermore, using chromatin immunopre- cancers, including amplification of the E2F1 gene in erythroleuke- cipitation assays, we show that E2F1 and the pocket protein mia cell lines (14), and overexpression of E2F1 protein in breast family members p107 and p130 bind to the AR promoter cancer cell lines (15) and head and neck carcinoma cell lines (16). in vivo. Taken together, these results show that elevated E2F1, Elevated E2F1 expression has been reported in invasive ductal through its ability to repress AR transcription, may contribute breast carcinomas (15) and non–small cell lung carcinomas (17), to the progression of hormone-independent prostate cancer. where high levels of E2F1 were associated with advanced disease (Cancer Res 2006; 66(24): 11897-906) and poor prognosis. E2F3 was found to be elevated in localized prostate cancer and is associated with decreased overall survival Introduction (18). Recently, Rhodes et al. (19) reported that genes with E2F Prostate cancer is the number one diagnosed cancer in males in binding sites are significantly overexpressed in a wide variety of the United States other than skin cancer and is the second leading human cancers, further supporting the view that activation of the cause of cancer-related deaths (1). Locally defined disease is often E2F pathway is a prevalent event in certain human cancers. successfully treated with surgery and/or radiotherapy; however, We have previously shown that disruption of the retinoblastoma (Rb)-E2F complex by homozygous deletion of Rb in a prostate tissue recombination model exhibited increased E2F activity and Requests for reprints: Mark L. Day, Department of Urology, University of activation of E2F-target genes in vitro and predisposed prostatic Michigan, 6131 CCGC, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0944. epithelium to hormonal carcinogenesis in vivo (20–22). We Phone: 734-763-9968; Fax: 734-647-9271; E-mail: [email protected]. I2006 American Association for Cancer Research. hypothesized that enhanced E2F1 expression may be a common doi:10.1158/0008-5472.CAN-06-2497 event in human prostate cancer and may contribute to prostate www.aacrjournals.org 11897 Cancer Res 2006; 66: (24). December 15, 2006

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Cancer Research cancer development and progression. In this study, we used tissue Cell lines. LNCaP and 293 cells were purchased from the American Type microarray (TMA) analysis to examine protein expression of E2F1 Culture Collection (Manassas, VA). All cells were maintained in RPMI 1640 and AR during prostate cancer progression. A TMA containing 667 supplemented with 5% fetal bovine serum (FBS), 0.1% penicillin/ tissue cores designed to examine prostate cancer progression to streptomycin, and 0.1% L-glutamine. Semiquantitative reverse transcription-PCR analysis. Total RNA hormone-independent disease was used to assess the relationship from cell lines was isolated using Qiagen RNA Easy kit using the between E2F1 protein expression and prostate cancer progression. manufacturer’s protocol (Qiagen, Valencia, CA). Frozen human prostate We observed that E2F1 was significantly elevated in hormone- tissue samples used for semiquantitative reverse transcription-PCR (RT- resistant prostate cancer. Interestingly, we also observed that PCR) analysis were evaluated by a pathologist to confirm pathologic overall AR levels were significantly down-regulated in hormone- disease. Two to three 10-Am sections were used to isolate total RNA using resistant prostate cancer. To define a role for elevated E2F1 in Qiagen RNA Easy kit using the manufacturer’s protocol. RNA was hormone-resistant prostate cancer, we investigated whether E2F1 quantitated spectrophotometrically and 2 Ag of RNA were used to make could regulate the expression of the AR and found that E2F1 acts as cDNA using Thermoscript RT-PCR Reaction System (Invitrogen, Carlsbad, a transcriptional repressor of the AR gene. This is the first report CA) per manufacturer’s protocol. PCR reaction was done using the ¶ ¶ investigating E2F1 expression during prostate cancer progression following primers: human E2F1, 5 -CATCCAGCTCATTGCCAAGAAG-3 and 5¶-GATCCCACCTACGGTCTCCTCA-3¶; hypoxanthine phosphoribosyltrans- using TMA and the first study to show that E2F1 may be a critical ferase, 5¶-CAGTACAGCCCCAAAATGGT-3¶ and 5¶-TTACTAGGCAGATGGC- component of AR transcriptional regulation. CACA-3¶; proliferating cell nuclear antigen (PCNA), 5¶-GCCACTCCGCCAC- CATGTTCG-3¶ and 5¶-GCCTAAGATCCTTCTTCATCCTC-3¶; dihydrofolate reductase (DHFR), 5¶-GGTTGGTTCGCTAAACTGCATC-3¶ and 5¶-CCTTT- Materials and Methods CTCCTCCTGGACATCAG-3¶; and actin, 5¶-CCTGGACTTCGAGCAAGA- TMA construction. The tissue donation program and rapid autopsy GATG-3¶ and 5¶-GTAACGCAACTAAGTCATAGTCCG-3¶. PCR was done program was approved by the Institutional Review Board of the University using a Thermocycler with an annealing temperature of 59jC. Ten- of Michigan. Benign and localized prostate cancer tissues were constructed microliter aliquots were removed at cycles 25, 28, 31, and 34. Samples were from transurethral resection of the prostate and prostatectomy samples run in triplicate and reaction products were electrophoresed in a 0.8% taken from 28 patients with prostate cancer seen at the University of agarose gel as previously described (20). Densitometric analysis was done Michigan from 1994. Regional lymph node metastases from hormone-naı¨ve using Image J Software downloaded from NIH (Bethesda, MD).6 patients were derived from radical prostatectomy series at the University of RNA isolation and Northern blot analysis. Total RNA was prepared Ulm (Ulm, Germany) from 1997 to 2000. In this time interval, 49 patients using Qiagen RNA Easy kit per manufacturer’s protocol (Qiagen). Twenty had lymph node metastases, and, in nine cases, the representative paraffin micrograms of RNA were resolved by gel electrophoresis under denaturing block of the lymph node provided enough metastatic tissue suitable for conditions and RNA was transferred to a Duralon-UV membrane TMA construction. Metastatic tissues from hormone-resistant prostate (Stratagene, La Jolla, CA) overnight by capillary action in 20Â SSC buffer cancer patients were acquired from the rapid autopsy procurement (3 mol/L NaCl and 0.3 mol/L Na citrate). RNA was cross-linked to the program at the University of Michigan. Formalin-fixed, paraffin-embedded membrane by Stratagene UV cross-linking. A 1.6 kb human AR cDNA tissue blocks with tumor samples from each case were identified. One fragment was isolated from CMV3-hAR3.1 (kindly provided by D. Robins, pathologist (M.A.R.) reviewed the H&E-stained slides and circled areas of University of Michigan, Ann Arbor, MI) using a HindIII and NheI prostate cancer, which were then used as template for the TMA. To take restriction enzyme sites. The human and mouse AR cDNA fragments tumor heterogeneity into account, four TMA cores (0.6-mm diameter) were were gel purified using Qiagen Gel Purification kit, according to the sampled from each donor block using a validated sampling method. The manufacturer’s protocol, and subsequently labeled with [a-32P]dATP using TMA was constructed using a manual tissue arrayer (Beecher Instruments, the random oligonucleotide primer labeling kit (Stratagene) and purified Silver Spring, MD) as previously described (23). on Stratagene Nucleotide Push Columns following the manufacturer’s Evaluation of E2F1 and AR protein expression by immunohisto- protocol. The [a-32P]dATP-labeled probes were hybridized to a Duralon- chemistry. Standard biotin-avidin complex immunohistochemistry was UV membrane (Stratagene) at 65jC overnight in hybridization buffer done using an E2F1 antibody (PharMingen, Franklin Lakes, NJ) and an AR [0.25 mol/L Na2HPO4 (pH 7.2) and 7% SDS] while rotating. The membrane antibody (Santa Cruz Biotechnology, Santa Cruz, CA). All cores on the TMA was subsequently washed twice for 45 minutes each in 20 mmol/L 5 were assigned at diagnosis and evaluated using TMA Profile, an internet- Na2HPO4 (pH 7.2) and 5% SDS followed by two additional washes for based image evaluation tool that uses zoomable TMA images generated by 45 minutes each in 20 mmol/L Na2HPO4 (pH 7.2) and 1% SDS. The the BLISS Imaging System (Bacus Lab, Lombard, IL). Tissue cores were membranes were exposed to X-ray film (Kodak, Rochester, NY) overnight examined by one pathologist (K.J.W.) blinded to each patient’s identity and and the RNA was visualized by autoradiography. clinical features. Based on previous work, E2F1 staining intensities were Western blot analysis. Cells were trypsinized, centrifuged, and scored as negative (0), weak (1), moderate (2), and strong (3). Specimens washed once with PBS. Cell pellets were lysed in radioimmunoprecipi- were considered positive only when at least 2% of the contained epithelial tation assay buffer [50 mmol/L Tris (pH 8.0), 120 mmol/L NaCl, 0.5% cells unequivocally expressed E2F1 or AR staining. For each tissue core, the NP40, 1.0 mmol/L EGTA, 200 Ag/mL phenylmethylsulfonyl fluoride percentage of nuclei within the epithelial component clearly staining (PMSF), 50 Ag/mL aprotinin, 5 Ag/mL leupeptin, and 200 Amol/L sodium positive was recorded. orthovanidate] and protein concentrations were determined using Statistical analysis. A comparison of expression levels between different Bradford Protein Assay Reagent (Bio-Rad, Hercules, CA), following the cohorts was done using the Mann-Whitney test of the Wilcoxon signed- manufacturer’s protocol. For Western blot analysis, 50 Ag of protein extract ranks test for dependent samples. For metastatic hormone-resistant were subjected to gel electrophoresis on a Tris-glycine polyacrylamide gel samples, the protein expression was investigated for associations with (Invitrogen). The gel was transferred to Optitran nitrocellulose membrane clinical and pathologic variables using the t test and the Mantel-Haenszel (Schleicher & Schuell Biosciences, Inc., Keene, NH) by electrophoresis for m2 test. Protein expression scores are presented in a graphical format using 1 hour at 43 V. The membrane was blocked in 10% nonfat dry milk in TBST error bars with 95% confidence intervals. P values <0.05 were considered (10 mmol/L Tris, 250 mmol/L NaCl, 1% Tween 20) for 1 hour at room statistically significant. temperature and immunoblotted with primary antibodies for AR (Santa

5 http://Profiler.tch.harvard.edu. 6 http://rsb.info.nih.gov/ij/.

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Cruz Biotechnology), E2F1 (PharMingen), Rb (PharMingen), prostate- following primers for AR promoter: 5¶-CCCGAGTTTGCAGAGAGGTAA-3¶, specific antigen (PSA; DAKO, Carpinteria, CA), cyclin E (Santa Cruz 5¶-TCCTTGAGCTTGGCTGAATC-3¶, with an annealing temperature of 59jC Biotechnology), PCNA (Santa Cruz Biotechnology), or h-actin (Santa Cruz for 36 cycles. PCR samples were electrophoresed on a 1.0% agarose gel in Biotechnology). The membrane was incubated with a secondary antibody 0.5Â TE buffer and visualized under UV light. conjugated to horseradish peroxidase, and the bands were detected using enhanced chemiluminescence (Pierce, Rockford, IL) detection system Results following the manufacturer’s protocol. Luciferase assay. LNCaP cells were plated at 2 Â 105 per six-well dish Clinical features of patient population. Two TMAs con- and incubated at 37jC overnight. Cells were cotransfected with 1 Ag of the taining 667 tissue cores were evaluated for E2F1 and AR following promoter-luciferase reporter constructs: DHFR-Luc, E2F-Luc, expression by immunohistochemistry. One array contained 188 CRE-Luc (kindly provided by G. Denis, Boston University, Boston, MA; tissue cores representing benign prostate, localized prostate ref. 24), and 2.0 kb human AR promoter-Luc (kindly provided by F.H. Sarkar, cancer, metastatic lymph nodes from hormone-naı¨ve prostate Wayne State University, Detroit, MI). The promoter-reporter constructs cancer patients, and metastatic tissues from men who died from were cotransfected with either empty pcDNA3 vector, wild-type E2F1, or the hormone-resistant prostate cancer. The second array contained E2F1 mutants E2F1-284 or Eco132 (gifts from W.D. Cress, Moffitt Cancer 479 tissue cores representing benign prostate and metastatic Center, Tampa, FL; refs. 25, 26). A dominant-negative E2F1 was kindly hormone-resistant prostate cancer from rapid autopsies of 30 provided by W. Kaelin (Harvard University, Boston, MA; ref. 27), and 0.1 Ag of pSV-h-galactosidase (Promega) was added to each sample as an internal men who died of hormone-resistant prostate cancer. Benign and control. DNA was transfected using Tfx50 transfection reagent (Promega) localized prostate cancer tissues were constructed from tran- at a ratio of 3:1 (DNA/Tfx50) following the manufacturer’s protocol. After surethral resection of the prostate and prostatectomy samples 72 hours of transfection, whole-cell lysates were collected in lysis buffer. from 28 patients, with a median age of 65 years and median PSA Luciferase expression was determined by adding 50 AL luciferase substrate value of 5.8 ng/mL. Pathologic stage was T1 in 26 patients and A (Promega) to 50 L of lysate, and luciferase was monitored using a T2 in two patients. Regional lymph node metastases were con- Monolight 2010 luminometer. h-Galactosidase expression was monitored structed from radical prostatectomy samples from nine patients, using a h-galactosidase detection system (Tropix, Bedford, MA) following with a median age of 64 years and median PSA value of 43.1 the manufacturer’s protocol using Monolight 2010 luminometer. Samples ng/mL. All patients had a pathologic stage T3, and eight patients were assayed in triplicate and luciferase activity was normalized to had seminal vesicle involvement. Metastatic hormone-resistant h-galactosidase activity. Data were analyzed by two-tailed Student’s t test. prostate cancer tissues were acquired from rapid autopsies done P < 0.05 was accepted as the level of significance. Chromatin immunoprecipitation assay. Human 293 cells were on 30 men who died of advanced hormone-resistant prostate maintained in RPMI 1640 supplemented with 10% FBS, 0.1% penicillin/ cancer. The median age at time of death was 71 years. Twenty- 7 streptomycin, and 0.1% L-glutamine. For each immunoprecipitation, 1 Â 10 eight men were initially diagnosed with clinically localized cells were used. Cells were cross-linked with 1% formaldehyde for 10 prostate cancer, but developed widely disseminated disease after minutes at room temperature on a shaking platform. Cross-linking was 5 to 10 years. Two men were initially diagnosed with metastatic stopped by the addition of 0.125 mol/L glycine. Cells were subsequently prostate cancer. All patients were treated with androgen washed twice with cold PBS, harvested using a cell scraper, and pelleted by deprivation therapy. centrifugation. Cells were resuspended in lysis buffer [5 mmol/L PIPES Immunohistochemistry and quantitative analysis of E2F1 A A (pH 8.0), 85 mmol/L KCl, 05% NP40, 200 g/mL PMSF, 50 g/mL aprotinin, staining in prostate tissues. The staining intensity of E2F1 was 5 Ag/mL leupeptin] and incubated on ice for 10 minutes. Nuclei were evaluated in 61 benign, 56 localized prostate cancer, 36 regional pelleted by centrifugation, resuspended in nuclei lysis buffer [50 mmol/L Tris-HCl (pH 8.1), 10 mmol/L EDTA, 1% SDS, 200 Ag/mL PMSF, 50 Ag/mL lymph node metastasis, and 35 hormone-resistant prostate aprotinin, 5 Ag/mL leupeptin] and then incubated on ice for 10 minutes. cancer cores. Nuclear E2F1 staining was observed in 27.1% of Chromatin was sonicated to an average length of 600 bp with four rounds of benign, 14.3% of localized prostate, 38.9% of hormone-naı¨ve 15-second pulse using a Misonix 300 Sonicator while keeping samples on lymph nodes, and 54.8% of metastatic hormone-resistant prostate ice. Samples were subsequently centrifuged at 14,000 rpm for 10 minutes at cancer (m2 P = 0.0006). The number of positive E2F1 nuclei was 4jC. Chromatin was precleared with protein A–agarose beads containing low in benign and localized prostate cancer (Fig. 1A) with mean sonicated salmon sperm DNA and bovine serum albumin for 15 minutes at expression values of 1.77% [SD, 3.18; 95% confidence interval j j 4 C. Samples were centrifuged for 5 minutes at 4 C and diluted in IP (95% CI), 0.98–2.58] for benign and 1.07% (SD, 3.06; 95% CI, dilution buffer [0.01% SDS, 1.1% Triton X-100, 1.2 mmol/L EDTA, 16.7 0.217–1.93) for localized prostate cancer (Fig. 1B). The number of mmol/L Tris (pH 8.1), 167 mmol/L NaCl, plus protease inhibitors]. Samples positive E2F1 nuclei increased in lymph nodes from hormone- were incubated overnight at 4jCwith5Ag of the respective antibody: E2F1 (Santa Cruz Biotechnology), E2F4 (Santa Cruz Biotechnology), Rb naı¨ve patients with a mean expression value of 3.61% (SD, 5.6; (PharMingen), p107 (Santa Cruz Biotechnology), p130 (Santa Cruz 95% CI, 1.79–5.43; Fig. 1A and B). Metastatic hormone-resistant Biotechnology), and IgG (Santa Cruz Biotechnology). Immunocomplexes prostate cancer had the greatest number of E2F1-positive nuclei, were collected by adding protein A–agarose beads, and pellets were washed with a mean expression value of 13.04% (SD, 8.5; 95% CI, 8.45– twice with a low salt buffer [0.1% SDS, 1% Triton X-100, 2 mmol/L EDTA, 17.62). There was wide variation in the percentage of nuclei 20 mmol/L Tris (pH 8.1), 150 mmol/L NaCl], twice in a high salt buffer exhibiting staining in hormone-resistant metastatic tissues. The [0.1% SDS, 1% Triton X-100, 2 mmol/L EDTA, 20 mmol/L Tris (pH 8.1), data were, therefore, stratified into eight categories: negative, 500 mmol/L NaCl], twice with LiCl buffer [0.25 mol/L LiCl, 1% NP40, 1% V5%, 6% to 10%, 11% to 20%, 21% to 40%, 41% to 60%, 61% to deoxycholate, 1 mmol/L EDTA, 10 mmol/L Tris (pH 8.0)], and twice with TE. 80%, and 81% to 100%. The distributions of staining for benign, Antibody-protein-DNA complexes were eluted with IP elution buffer (1% localized prostate cancer, hormone-naı¨ve, and hormone-resistant SDS and 0.1 mol/L NaHCO3). Formaldehyde cross-links were reversed by adding 0.3 mol/L NaCl and RNase A and by incubating samples at 65jC for metastatic prostate cancer are shown in Fig. 1C. These analyses z 4 to 5 hours. Protein-DNA complexes were precipitated with ethanol show that high levels of nuclear E2F1 staining ( 20%) are overnight at À20jC and centrifuged at 14,000 rpm for 20 minutes at 4jC. restricted to hormone-resistant metastatic prostate cancer. Two Proteinase K was added for 1 to 2 hours at 45jC and DNA was purified patients with liver metastasis had over 60% of cells staining using Qiagen DNA purification columns. PCR reaction was done using the positive for nuclear E2F1. www.aacrjournals.org 11899 Cancer Res 2006; 66: (24). December 15, 2006

Figure 1. E2F1 expression during prostate cancer progression. A, immunohistochemical staining of E2F1 in benign prostate (Benign), localized prostate cancer (Tumor), metastatic lymph nodes from hormone-naı¨ve patients (HN Mets), and metastatic hormone-resistant prostate cancer (HR Mets). Magnification, Â5(a, c, e, g) and Â20 (b, d, f, h). B, E2F1 expression in benign prostate, localized prostate cancer, metastatic lymph nodes from hormone-naı¨ve patients, and metastatic hormone-resistant prostate cancer. Points, mean nuclear E2F1 protein expression; bars, 95% CI. N, number of cases indicated for each tissue type. C, distribution of E2F1 staining in benign prostate, localized prostate cancer, metastatic lymph nodes from hormone-naı¨ve patients, and metastatic hormone-resistant prostate cancer. Columns, percentage of samples in each category. D, stain intensity of nuclear E2F1 for benign prostate, localized prostate cancer, metastatic lymph nodes from hormone-naı¨ve patients, and metastatic hormone-resistant prostate cancer. Points, nuclear E2F1 intensity; bars, 95% CI.

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The staining intensity of E2F1 was quantified on a scale of 0 with intense E2F1 stain, suggesting a role of E2F1 in prostate to 3; 0 representing absence of stain, 1 representing weak stain, cancer progression. 2 representing moderate stain, and 3 representing intense stain. AR expression in prostate cancer progression. A central issue Mean expression values for nuclear E2F1 stain intensity to understanding hormone-resistant prostate cancer is to identify according to diagnosis is shown in Fig. 1D. E2F1 immunoreac- the role of AR during prostate cancer progression; we, therefore, tivity was low in benign prostate and localized prostate cancer assessed AR expression during prostate progression in the same with a mean stain intensity score of 0.215 (SD, 0.816; 95% CI, TMA described above. Nuclear AR expression was detected in 83% 0.052–0.378) and 0.214 (SD, 0.63; 95% CI, 0.05–0.38) for benign of benign, 100% of localized prostate cancer, 80% of metastatic and localized prostate cancer, respectively. Metastatic lymph lymph nodes, and 40% of metastatic hormone-resistant prostate nodes from hormone-naı¨ve patients had the strongest nuclear cancer (Fig. 2A). The number of positive AR nuclei was similar E2F1 stain intensity, with 28% having intense nuclear E2F1 between benign, tumor, and hormone-naı¨ve lymph nodes with stain and a mean stain intensity score of 1.03 (SD, 1.07; 95% CI, mean expression values of 45.5%, 52.21%, and 45.83%, respectively 1.89–2.6; Fig. 1D). Metastatic tissues from hormone-resistant (Fig. 2B). Expression of AR was down-regulated in metastatic patients had a wide distribution of E2F1 staining intensity, tissues from hormone-resistant patients with a mean expression with 29% with low stain intensity, 16% with moderate stain value of 21.2% (Fig. 2B). AR stain intensity was greatest in benign intensity, and 10% with intense staining. These results show and localized prostate cancer, decreased slightly in hormone-naı¨ve that benign and localized prostate cancer have low E2F1 stain lymph nodes, and was lowest in metastatic hormone-resistant intensity and metastatic tissues from hormone-naı¨ve and prostate cancer (Fig. 2C). Sixty percent of hormone-resistant hormone-resistant patients have the greatest percentage of cells tissues had <10% of tumor cells staining positive for AR.

Figure 2. AR expression during prostate cancer progression. A, AR expression detected by immunohistochemistry. Example of positive AR staining in benign prostate (a, e), localized prostate cancer (b, f), and metastatic lymph nodes from hormone-naı¨ve patients (c, g). Example of negative AR staining in a liver metastasis from a hormone-resistant patient (d, h). Magnification, Â5(a–d) and Â20 (e–h). B, AR expression for benign prostate, localized prostate cancer, metastatic lymph nodes from hormone-naı¨ve patients, and metastatic hormone-resistant prostate cancer. Points, mean nuclear AR protein expression; bars, 95% CI. C, stain intensity of nuclear AR expression for benign prostate, localized prostate cancer, metastatic lymph nodes from hormone-naı¨ve patients, and metastatic hormone-resistant prostate cancer. Points, mean AR protein expression; bars, 95% CI. www.aacrjournals.org 11901 Cancer Res 2006; 66: (24). December 15, 2006

E2F1 and AR expression in metastatic hormone-resistant well as the prostate gland. Our results are similar to the study by prostate cancer. We wanted to expand our assessment of E2F1 Shah et al. (28), which reported a wide variation of AR and PSA expression in metastatic prostate tissues from hormone-resistant expression in hormone-resistant prostate cancer from rapid patients; therefore, we stained a second prostate TMA containing autopsy patients. 479 metastatic tissue cores from 30 patients who had died from Elevated E2F1 mRNA and E2F-target genes in metastatic hormone-resistant prostate cancer. Metastatic tissues included hormone-resistant prostate cancer. Because E2F1 protein bone, liver, lung, dura, bladder, lymph node, adrenal gland, expression was significantly elevated in metastatic hormone- pancreas, and soft tissues. Nuclear E2F1 stain was detected in resistant prostate cancer compared with benign prostate tissue, 80% (382 of 479) of hormone-resistant cases and the mean we wondered whether E2F1 mRNA transcripts were also increased expression value of E2F1 was 28% (range 0–90%). The distribution to the same degree as protein expression. We analyzed E2F1 of nuclear E2F1 staining in hormone-resistant cases based on transcripts from nine metastatic hormone-resistant prostate tissue type is shown in Fig. 3A. E2F1 staining was evaluated in the cancer patients and four benign cases by semiquantitative RT- prostate gland from 13 hormone-resistant patients and showed PCR analysis. As shown in Fig. 4A, E2F1 transcripts were elevated increased nuclear E2F1 expression with a mean value of 32.5% in hormone-resistant prostate cancer when compared with benign (SD, 23.23, 95% CI, 26.36–39.24) compared with 4.4% (SD, 10.14, 95% prostate tissues. Densitometric analysis of mRNA transcripts CI, 1.2–10.1) for benign prostate. Figure 3B (a and b) shows low revealed up to 47-fold activation of E2F1 mRNA expression (after E2F1 staining in benign prostate tissue versus strong E2F1 staining normalizing to actin) in metastatic hormone-resistant prostate in the prostate of a patient with hormone-resistant disease. In cancer compared with benign tissues (Fig. 4B). When we compared some cases, perinuclear E2F1 staining was observed, as shown in the band intensity of E2F1 mRNA transcripts with the expression a metastatic bladder sample (Fig. 3B, c) and a bone metastasis value of nuclear E2F1 stain reported on the TMA for each (Fig. 3B, d). We observed wide heterogeneity of E2F1 expression individual case, we found a statistically significant correlation between different organ sites and patients; however, there was no between E2F1 mRNA and E2F1 protein expression (Fig. 4C; Pearson pattern to the differences in E2F1 expression between the different correlation coefficient r = 0.87393, P < 0.01). These results showed organ sites that could be distinguished. that both E2F1 mRNA and protein expression are significantly AR expression was evaluated in the same tissue samples (Fig. 3B, elevated in metastatic hormone-resistant prostate cancer com- f–j). Nuclear AR expression varied across tumor samples with a pared with benign tissues. median expression value of 63.3% (SD, 38.5; 95% CI, 58.78–67.81) To investigate whether increased E2F1 expression resulted in and median stain intensity value of 2.15 (SD, 1.22; 95% CI, 2.04– increased E2F1 transcriptional activity, the same set of benign and 2.26). One patient had a complete loss of AR in all metastatic sites hormone-resistant prostate cancer cases was evaluated for mRNA examined, including lymph node, liver, pancreas, and soft tissue, as expression of two established E2F-target genes, DHFR and PCNA.

Figure 3. E2F1 and AR expression in benign prostate and tissues from hormone-resistant prostate cancer patients. A, distribution of mean nuclear E2F1 expression in benign prostate and all metastatic tissues from 30 hormone-resistant cancer patients. Numbers in parentheses, number of tissue cores for each tissue type. B, E2F1 staining represented in benign prostate (a) and hormone-resistant prostate cancer tissues in prostate (b), bladder (c), bone (d), and dura (e). AR staining is shown in benign prostate (f) and hormone-resistant prostate cancer tissues in prostate (g), bladder (h), bone (i), and dura (j). E2F1 and AR staining was evaluated in the same tissue cores from the same patient.

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we hypothesized that elevated levels of E2F1 may down-regulate AR expression. To test this hypothesis, we examined whether overexpression of E2F1 could inhibit endogenous AR mRNA levels in prostate epithelial cells. LNCaP cells were transiently transfected with increasing amounts of a plasmid-containing human E2F1 or empty pcDNA3 control vector. Total RNA was harvested and subjected to Northern blot analysis for the detection of AR and E2F1 mRNA. As shown in Fig. 5A, AR mRNA levels decrease with increasing amounts of E2F1 in LNCaP cells. The 28S and 18S rRNAs are shown as a loading control. Figure 5B shows decreased AR protein expression in E2F1-overexpressing cells. As a positive control to show increased E2F activity, we show increased protein expression of cyclin E and PCNA, two well-characterized E2F-target genes (Fig. 5B). We also show decreased protein expression of the AR-target gene, PSA, which is most likely due to decreased AR protein expression. These results suggest that E2F1 down-regulates AR expression in prostate epithelial cells. E2F1 expression results in repression of both mouse and human AR promoters. To assess whether E2F1 can regulate AR promoter activity, we used a 2 kb human AR promoter construct containing À900 to +1,129 bp relative to the transcription start site cloned upstream of a luciferase reporter gene (29). The human AR promoter/luciferase construct (hAR-Luc) was cotransfected into human LNCaP cells with either empty pcDNA3 vector (control), wild-type E2F1, or a dominant-negative E2F1 construct, along with a cytomegalovirus (CMV) promoter-driven h-galactosidase reporter plasmid as an internal control (Fig. 6A). E2F1 repressed AR promoter activity by 62% in LNCaP cells when compared with cells transfected with empty pCDNA3 plasmid. The dominant- negative E2F1 did not significantly inhibit AR promoter activity. As a positive control, we show that E2F1 activates an E2F-inducible promoter, which contains four adjacent E2F consensus binding sites (E2F-Luc). To show promoter specificity, we show that E2F1 does not have a similar effect on a CRE-Luc promoter, which contains four adjacent cyclic AMP (cAMP) regulatory elements in front of a luciferase reporter construct. These results show that Figure 4. Molecular analysis of E2F1 mRNA and E2F-target genes in benign E2F1 inhibits AR promoter activity. and metastatic hormone-resistant prostate cancer. A, results of semiquantitative We next examined the effects of different E2F1 mutants on AR RT-PCR analysis for evaluation of mRNA levels of E2F1, DHFR, PCNA, and actin in four benign prostate tissues (lanes 1–4) and nine metastatic promoter activity in LNCaP cells. A mutation in the DNA binding hormone-resistant prostate cancer cases (lanes 5–13). Actin, loading control. domain (Eco132) slightly relieved E2F-mediated inhibition, whereas B, band intensities for E2F1 (white columns), DHFR (gray columns), and deletion of the transactivation domain of E2F1 (E2F1 ) PCNA (black columns) transcripts. Each sample was normalized to actin and 1-284 represented as arbitrary units Â102. C, semiquantitative RT-PCR analysis of completely abrogated the inhibitory effect of E2F1 (Fig. 6B). As E2F1 mRNA in benign and metastatic hormone-resistant prostate cancer expected, the E2F1 mutants did not activate the E2F-Luc promoter (white columns). Levels are normalized to corresponding actin values for each case. Black columns, mean expression value of nuclear E2F1 protein for activity, which requires both the E2F1 transactivation and DNA each tissue as determined by TMA. Note the different scales. binding domains for activation. These results indicate that the transactivation domain of E2F1 is essential for E2F1-mediated repression of the AR promoter. DHFR and PCNA transcripts were high in metastatic hormone- We next examined the human AR promoter for potential E2F1 resistant prostate cancer compared with benign tissues with up to consensus binding sites using MatInspector computational anal- 10-fold activation of DHFR and 30-fold activation of PCNA in ysis. We identified two potential E2F1 binding sites located at metastatic prostate cancer (Fig. 4A and B). There was a statistically positions 617 to 630 and 961 to 977 relative to the transcription significant (P < 0.01) positive correlation between E2F1 and DHFR start site, located on the complimentary DNA strand. To investigate expression (Pearson correlation coefficient, r = 0.852219) and E2F1 whether E2F1 regulates the AR promoter in vivo, we used the and PCNA expression (Pearson correlation coefficient, r = 0.76305). chromatin immunoprecipitation (ChIP) assay using primers These results suggest that increased E2F1 expression in hormone- designed to include the two potential E2F1 binding sites identified resistant prostate cancer results in increased E2F1 transcriptional at positions 617 to 630 and 961 to 977 on the AR promoter. The activity. ChIP assays were done in the human 293 cell line because these E2F1 down-regulates AR expression. Overall, our TMA results cells routinely work well in ChIP assays and have been used to clearly show increased E2F1 protein expression and decreased AR easily show the binding of E2F1 to endogenous promoters. As expression in hormone-resistant prostate cancer compared with shown in Fig. 6C, a strong PCR signal was seen when samples were benign and localized prostate cancer. Based on these observations, incubated with an antibody to E2F1. No signal was detected with www.aacrjournals.org 11903 Cancer Res 2006; 66: (24). December 15, 2006

Figure 5. E2F1 inhibits AR mRNA and protein expression. A, Northern blot analysis to detect AR and E2F1 gene expression in LNCaP cells transfected with 0, 5, 10, or 15 Ag E2F1. Total RNA was harvested for Northern blot analysis using the indicated probes. Equivalent loading of RNA was confirmed by 28S and 18S rRNAs. B, Western blot analysis of whole-cell lysates harvested from LNCaP cells transfected with 0, 5, 10, and 15 Ag E2F1 for the detection of AR, E2F1, cyclin E, PCNA, and PSA. PARP, poly(ADP)ribose polymerase: protein loading control.

other antibodies, including IgG, which was used as a control for The oncogenic capacity of E2Fs has been attributed to their ability nonspecific precipitation of protein-DNA complexes or E2F4. The to transactivate genes involved in DNA synthesis and cell cycle samples were also subjected to PCR reactions using primers to progression. In this study, we have shown that human metastatic actin to ensure that the immunoprecipitations were not binding hormone-resistant prostate cancer tissues had increased levels of nonspecifically. Because E2F1 can transcriptionally repress pro- E2F-target genes DHFR and PCNA, which are essential components moters by recruiting Rb pocket protein family members, we for DNA replication. DHFR is required for purine biosynthesis decided to perform ChIP assays with antibodies to Rb and the and is thus important for the production of nucleotides required pocket protein family members p107 and p130. Strong PCR bands for DNA synthesis and S-phase progression (29). PCNA is a nuclear were detected when anti-p107 and anti-p130 antibodies were used, cofactor for DNA polymerase y and is widely used as a marker of but not when anti-Rb antibodies were used. These results suggest cell proliferation. PCNA expression in prostate cancer has been that E2F1, p107, and p130, but not Rb, bind to the AR promoter correlated with disease progression and negatively correlated in vivo and may suggest that p107 and p130 cooperate with E2F1 to with survival (31, 32). High levels of E2F1 may provide tumor cells mediate repression of the AR. with a proliferative advantage by transcriptionally activating downstream targets involved in DNA synthesis and thus contribute to tumor progression. With the use of cDNA microarray techno- Discussion logy, hundreds of genes have been identified as E2F targets, Activation of E2F transcription factors by disruption of the Rb including genes involved in cell cycle regulation, mRNA splicing, pathway is thought to be a key event in human cancer; however, chromatin modification, angiogenesis, apoptosis, invasion, and detailed assessment of E2F1 expression has not been previously metastasis (33–35). We have recently shown that cyclooxygenase 2 examined in human prostate cancer. Using TMA technology, we and DNA methyltransferase 1 are downstream targets of E2F1 in have shown that E2F1 is low in benign and localized prostate prostate epithelial cells and may contribute to hormonal car- cancer, modestly elevated in hormone-naı¨ve metastatic lymph cinogenesis (20, 21), suggesting that E2Fs play multiple roles in nodes, and significantly elevated in metastatic tissues from men tumorigenesis. who died of hormone-resistant prostate cancer (P = 0.0006). On a Androgens and AR are critical components for prostate gland larger TMA, E2F1 immunoreactivity was detected in 80% of metas- development, prostatic function, and are involved in prostate tatic tissues from 30 men who died of hormone-resistant prostate tumorigenesis. AR expression has been observed in primary cancer with an overall mean expression value of 28% (range 0–90%) prostate cancer and can be detected throughout progression in compared with benign tissues with a mean expression value of both hormone-sensitive and hormone-resistant cancers (36, 37); 4.4% (range 0–15%). Elevated E2F1 protein expression correlated however, the importance of AR during late stages of prostate with increased E2F1 mRNA and increased expression of E2F1-target cancer is not entirely clear. Initial studies showed that AR mRNA is genes DHFR and PCNA, suggesting that E2F1 expression is elevated present in androgen-sensitive prostate cancer cell lines, but is in advanced prostate cancer. absent or expressed at low levels in androgen-independent cell E2F1 expression has been evaluated in other human cancers and lines (38). Recent studies have shown heterogenous expression of high levels of E2F1 have been associated with advanced-stage AR throughout prostate cancer. Sadi and Barrack (39) showed that disease and poor prognosis in breast cancer and non–small cell cancer cells in patients with advanced prostate cancer who lung cancer patients (15, 17). Elevated E2F1 expression has been responded poorly to therapy exhibited a significant heterogeneity reported in lymph node metastasis of breast cancer patients and in the staining intensity of AR. de Vere White et al. (40) showed a amplification of the E2F1 gene has been reported in metastatic loss of AR expression in 33% of prostate cancer patients following tumors of human colorectal cancer (15, 30). These studies are in combined androgen blockade therapy and Hobisch et al. (41) agreement with our study, which demonstrates low E2F1 expres- showed complete loss of AR in prostate cancer lymph node sion in benign tissues and elevated levels in late-stage disease. metastases. Kinoshita et al. (42) showed a significant loss of AR

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. E2F1 Expression in Prostate Cancer expression in advanced prostate cancer and found that methyla- tumors. These results suggest that loss of AR expression during tion of the AR promoter was tightly linked to metastatic hormone- prostate cancer progression may be associated with the develop- resistant tumors. In our study, 60% of hormone-resistant prostate ment of androgen independence in a subset of patients. It is tumors had <10% of tumor cells staining positive for AR and a important to note that these studies do not rule out the importance complete loss of AR expression was observed in several metastatic of AR amplification and mutated forms of AR, which have shown to confer androgen independence of prostate cancer cells (reviewed in refs. 43–45). A variety of AR mutations have been identified in prostate cancer cell lines and human tumors with the most common mutations occurring in the ligand binding domain. Mutations in the ligand binding domain alter the specificity of the AR; enhancing the binding of estrogens, progesterone, and anti- androgens; and thereby decreasing its dependency on androgens while stimulating cell growth (46, 47). In addition to AR mutations, a variety of growth factors, including insulin-like growth factor I, epidermal growth factor, and keratinocyte growth factor, can activate androgen-responsive genes via the AR, suggesting that androgen independence could arise from overexpression of growth factors in the local environment (48). Clearly, mutations of the AR and activation of AR via growth factors play a role in androgen- independent growth. In this study, we are suggesting that decreased expression of AR may also play a role in the selection of androgen-independent tumors. Mechanisms for AR down- regulation in prostate cancer have been proposed, including promoter silencing by DNA methylation (49) and transcriptional repression by transcription factors nuclear factor-nB and nuclear factor-1 (50, 51). This is the first study to show that E2F1 represses AR mRNA and protein expression and inhibits AR promoter activity. We have also shown that physiologic levels of E2F1 can bind to the AR promoter in vivo in human 293 cells. E2F1 primarily functions as a positive regulator; nevertheless, in addition to the present report, a negative regulatory role of E2F1 has also been described for a number of genes, including urokinase-type plasminogen activator (uPA; ref. 52), the antiapoptotic protein Mcl-1 (26), and human telomerase reverse transcriptase (hTERT; ref. 53). Unlike its positive regulatory function, which is mediated by direct interaction of E2F1 to DNA binding sites, the mecha- nism(s) for E2F1-mediated negative regulation are still largely unknown. Crowe et al. (53) identified two putative E2F binding sites in the hTERT promoter that were important for E2F1- mediated repression. The E2F1 mutant (E132), which lacks the DNA binding domain of E2F1, was inefficient at repressing hTERT promoter activity, suggesting that the DNA binding domain was essential for repression. In another study by Croxton et al. (26), direct repression of the Mcl-1 promoter by E2F1 also required the DNA binding domain, but not the transactivation domain. Koziczak et al. (52) showed that both the DNA and transactivation binding domains of E2F1 were necessary for the negative regul- Figure 6. E2F1 inhibits AR promoter activity. A, LNCaP cells were ation of the uPA and plasminogen activator inhibitor 1 genes; cotransfected with 1 Ag E2F-Luc, hAR-Luc, or CRE-Luc luciferase reporter constructs with 0.5 Ag of empty vector (pcDNA3), wild-type E2F1, however, E2F repressed promoter activity independently of the dominant-negative E2F1 (DN E2F1), or E2F4. After 72 hours, cells were pocket protein Rb. Our data show that the carboxyl-terminal harvested and assayed for luciferase expression. Results are averages of three transactivation domain was essential for E2F1 suppression of the AR independent experiments. Assays were done in triplicate. Columns, mean; bars, SD. The results were normalized to h-galactosidase expression from a promoter (Fig. 5). Several proteins are known to bind to this region cotransfected CMV promoter-driven h-galactosidase reporter construct. and regulate transcription, including cAMP-responsive element B, effects of E2F1 mutations on negative regulation of the AR promoter. LNCaP binding protein–binding protein (54), MDM2 (55), and TRRAP-Tip60 cells were cotransfected with either 1 Ag of AR-Luc or E2F-Luc and 500 ng of either wild-type E2F1, E2F11-284, or Eco132 mutant constructs. Columns, complex (56). The transactivation domain also interacts with the mean value of three independent experiments; bars, SD. Results were basal transcription factor IIH and the Rb pocket protein family normalized to h-galactosidase expression from a cotransfected CMV promoter-driven h-galactosidase reporter construct. C, ChIP assays were members (57). Our ChIP analysis show that p107 and p130 are done on human 293 cells using antibodies to E2F1, E2F4, Rb, p107, p130, recruited to the AR promoter along with E2F1 and suggest that p107 and control IgG. PCR reactions were carried out with chromatin samples and and p130 may cooperate with E2F1 to mediate AR transcriptional primers to the AR promoter and actin (negative control). An input sample containing 0.2% of total input chromatin and a mock sample containing no repression. Future studies are being done to determine if p107 and chromatin were included as controls. p130 are required for E2F1-mediated repression of the AR. www.aacrjournals.org 11905 Cancer Res 2006; 66: (24). December 15, 2006

Acknowledgments 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 Received 7/7/2006; revised 9/5/2006; accepted 9/25/2006. with 18 U.S.C. Section 1734 solely to indicate this fact. Grant support: Specialized Program of Research Excellence for Prostate Cancer We thank the patients and families, who, through their generous participation National Cancer Institute grant P50CA69569, the American Cancer Society Clyde-Dixon in the tissue donor program, have facilitated the study of prostate cancer and Dr. Postdoctoral Fellowship PF-03-252-01-TBE (J.N. Davis), and National Institutes of Dia- Kenneth J. Pienta for his establishment and participation of the rapid autopsy betes, Digestive and Kidney Diseases grants R01-DK61488 and R01-DK066610 (M.L. Day). program.

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