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Clinical Cancer Research Imaging, Diagnosis, Prognosis Hypermethylation of the GABREmiR-452miR-224 Promoter in Prostate Cancer Predicts Biochemical Recurrence after Radical Prostatectomy Helle Kristensen1, Christa Haldrup1, Siri Strand1, Kamilla Mundbjerg1, Martin M. Mortensen1,2, Kasper Thorsen1, Marie Stampe Ostenfeld1, Peter J. Wild4, Christian Arsov5, Wolfgang Goering5, € nberg8, Søren Høyer3, Michael Borre2, Tapio Visakorpi6, Lars Egevad7, Johan Lindberg8, Henrik Gro Torben F. rntoft1, and Karina D. Sørensen1 Abstract Purpose: Available tools for prostate cancer diagnosis and prognosis are suboptimal and novel biomarkers are urgently needed. Here, we investigated the regulation and biomarker potential of the GABREmiR-452miR-224 genomic locus. Experimental Design: GABRE/miR-452/miR-224 transcriptional expression was quantified in 80 nonmalignant and 281 prostate cancer tissue samples. GABREmiR-452miR-224 promoter methylation was determined by methylation-specific qPCR (MethyLight) in 35 nonmalignant, 293 prostate cancer [radical prostatectomy (RP) cohort 1] and 198 prostate cancer tissue samples (RP cohort 2). Diagnostic/prognostic biomarker potential of GABREmiR-452miR-224 methylation was evaluated by ROC, Kaplan–Meier, uni- and multivariate Cox regression analyses. Functional roles of miR-224 and miR-452 were investigated in PC3 and DU145 cells by viability, migration, and invasion assays and gene-set enrichment analysis (GSEA) of posttransfection transcriptional profiling data. Results: GABREmiR-452miR-224 was significantly downregulated in prostate cancer compared with nonmalignant prostate tissue and had highly cancer-specific aberrant promoter hypermethylation (AUC ¼ 0.98). Functional studies and GSEA suggested that miR-224 and miR-452 inhibit proliferation, migration, and invasion of PC3 and DU145 cells by direct/indirect regulation of pathways related to the cell cycle and cellular adhesion and motility. Finally, in uni- and multivariate analyses, high GABREmiR-452miR-224 promoter methylation was significantly associated with biochemical recurrence in RP cohort 1, which was successfully validated in RP cohort 2. Conclusion: The GABREmiR-452miR-224 locus is downregulated and hypermethylated in prostate cancer and is a new promising epigenetic candidate biomarker for prostate cancer diagnosis and prognosis. Tumor-suppressive functions of the intronic miR-224 and miR-452 were demonstrated in two prostate cancer cell lines, suggesting that epigenetic silencing of GABREmiR-452miR-224 may be selected for in prostate cancer. Clin Cancer Res; 20(8); 2169–81. 2014 AACR. Authors' Affiliations: Departments of 1Molecular Medicine and 2Urology and 3Institute of Pathology, Aarhus University Hospital, Aarhus, Denmark; 4 Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland; 5Department of Urology, Medical Faculty, Heinrich Heine Uni€sseldorf, Germany; 6Institute of Biomedical Technology and versity, Du BioMediTech, University of Tampere and Tampere University Hospital, Tampere, Finland; Departments of 7Oncology and Pathology and 8Medical Epidemiology and Biostatistics, Karolinska Institute, Stockholm, Sweden Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Karina Dalsgaard Sørensen, Department of Molecular Medicine, Aarhus University Hospital, Brendstrupgaardsvej 100, DK-8200 Aarhus N, Aarhus, Denmark. Phone: (+45) 78455316; Fax: (+45) 86782108; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-13-2642 2014 American Association for Cancer Research. Introduction Prostate cancer is a common malignancy among males in Western countries. Despite its widespread use, prostatespecific antigen (PSA) testing has suboptimal specificity and sensitivity for prostate cancer detection. Another major challenge in prostate cancer management is to distinguish between indolent and aggressive tumors due to the limited accuracy of currently available prognostic indicators (mainly PSA, TNM stage, and Gleason score). Thus, novel biomarkers for prostate cancer are needed to improve the accuracy of diagnosis and prognosis (1). Molecular biomarkers with important biologic functions in prostate cancer could also have potential as therapeutic targets. In this study, we investigated the regulation, function, and biomarker potential for prostate cancer of a genomic www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2014 American Association for Cancer Research. 2169 Kristensen et al. Translational Relevance Management of clinically localized prostate cancer is highly challenging due to absence of accurate prognostic tools. Here, using two independent prostate cancer patient cohorts, including a total of 491 patients from five countries, we found that patients with high GABREmiR-452miR-224 promoter methylation were at significantly higher risk of biochemical recurrence after radical prostatectomy than patients with low methylation. Although further validation is needed, our results indicate that a simple PCR-based test for GABREmiR452miR-224 promoter methylation can add significant prognostic value to currently used routine predictors for prostate cancer patient outcome. We note that our study used radical prostatectomy specimens; however, if transferred to diagnostic prostate biopsies, or urine or blood samples, such a test could be used to aid in treatment decisions, for example, between active surveillance and surgery. Furthermore, we found that hypermethylation was highly cancer-specific, suggesting that GABREmiR452miR-224 may also have potential as a diagnostic biomarker for prostate cancer. locus at Xq28, encompassing the GABRE gene and two intronic microRNAs (miR-224 and miR-452). GABRE encodes the epsilon subunit of gamma-aminobutyric acid (GABA) A receptor (2) and has a promoter-associated CpG island (CGI). Hypermethylation of promoter CGIs is closely linked with gene silencing in cancer, and aberrant promoter hypermethylation of specific genes, such as GSTP1 and PITX2, has shown promising diagnostic and prognostic biomarker potential for prostate cancer (3–7). Here, we focused on the GABREmiR-452miR-224 locus, because integrative analysis of mRNA and miRNA expression and DNA methylation profiling data available in-house indicated significant downregulation accompanied by aberrant promoter hypermethylation in prostate cancer. Moreover, results from several studies (see below) suggested potentially important functional role(s) for this locus in prostate cancer. Deregulation of GABRE expression in prostate cancer has not been reported before this work. The GABAA receptor is a heteropentameric chloride channel that transmits the fastest inhibitory signal of the central nervous system (CNS) with its ligand GABA (8). The GABAA receptor family consists of 19 homologous genes, encoding subunits a1-a6, b1b3, g1-g3, d, r1-r3, q, p, and e (GABRE). The dominant receptor subtype is believed to be 2a:2b:1g. GABRE resembles the g-subunit and may replace this to form a functional receptor (2, 9). Outside the CNS, elevated GABA levels have been associated with prostate cancer metastasis (10) and GABA agonist isoguvacine has been shown to stimulate proliferation of four prostate cancer cell lines (11). In contrast, GABA signaling has been found to inhibit breast cancer cell migration (12), suggesting a highly contextdependent function. 2170 Clin Cancer Res; 20(8) April 15, 2014 MicroRNAs (miRNA) are endogenous small noncoding RNAs (22 nt) that regulate gene expression at the posttranscriptional level, typically by binding to sequence-specific sites in the 30 UTR of target mRNAs to repress translation and/or initiate mRNA decay (13). A single miRNA may function either as a tumor suppressor or an oncogene, depending on the cellular context and its specific mRNA target(s) (13). miRNAs have shown promising potential as candidate cancer biomarkers, therapeutic targets, and anticancer drugs (13). Downregulation of miR-224 in prostate cancer tissue samples has been reported in two recent studies (14, 15), but another study reported upregulation of miR-224 in perineural invasion (PNI) positive versus negative prostate cancer (16). miR-452 expression in prostate cancer is also poorly characterized, although a study has suggested that miR-452 is overexpressed in prostate cancer progenitor cells (17). Downregulation of miR-224 has been reported in ovarian, breast, and lung cancer (18–20), while upregulation has been reported in medullablastoma, thyroid, liver, renal, and colorectal cancer (21–25), suggesting cell type-specific expression and function. Although miR224 has been linked to proliferation, migration, and invasion in several cancers (21, 22), its function remains to be investigated in prostate cancer. miR-452 has been found to be upregulated in medulloblastoma (21) and in urothelial carcinomas with lymph node metastasis, suggesting a possible role in progression of these malignancies (26); however, its potential function in prostate cancer has not yet been studied. Here, we found that the entire GABREmiR-452miR224 locus was downregulated in prostate cancer compared with nonmalignant prostate tissue samples, and that silencing was associated with frequent aberrant promoter hypermethylation. We also found miR-224 and miR-452 to inhibit viability, migration, and invasion of two prostate cancer cell lines, suggesting that they hold tumor suppressor functions in prostatic cells. Finally, high GABREmiR452miR-224 methylation was a significant independent adverse prognostic factor for biochemical recurrence after radical prostatectomy in a large prostate cancer patient cohort (n ¼ 293, training cohort), which was successfully confirmed in an independent validation cohort (n ¼ 198). Materials and Methods Clinical samples Fresh-frozen tissue-tek (Bayer Corporation) embedded radical prostatectomy and transurethral resection of the prostate (TURP) tissue specimens were collected at Department of Urology, Aarhus University Hospital, Denmark (Supplementary Table S1) and used for bisulfite sequencing and Infinium analyses. For MethyLight analyses, two radical prostatectomy cohorts were used (Table 1). RP cohort 1 (training) consisted of consecutive curatively intended radical prostatectomies of histologically verified clinically localized prostate cancer from patients treated at Departments of Urology, Aarhus University Hospital (Aarhus, Denmark; collected 1997–2005), and University Hospital Zurich (Zurich, Clinical Cancer Research Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2014 American Association for Cancer Research. Prognostic Value of GABREmiR-452miR-224 Methylation in Prostate Cancer Table 1. Clinicopathologic characteristics of patient sample sets used for MethyLight analyses Prostate cancer samples RP cohort 1, RP cohort 2, training (n ¼ 293) validation (n ¼ 198) MPC (n ¼ 26) Median age, range PSA at diagnosis, n (%) 10 ng/mL >10 ng/mL Unknown Pathologic T stage, n (%) T1 T2a-c T3a-b T4 Unknown Gleason score, n (%) 3–6 7 8–10 Unknown Nodal status, n (%) pN0 pN1 Unknown Surgical margin status, n (%) Negative Positive Unknown Recurrence status, n (%) No recurrence Recurrence Mean follow-up time, months (range) 170 (58.0) 123 (42.0) 49 (2–151) Nonmalignant samples Median age (range) 63 (46–73) 63 (47–75) CRPC (n ¼ 13) LMN (n ¼ 15) 71 (52–89) 64 (49–77) 63 (54–71) 114 (38.9) 178 (60.8) 1 (0.3) 121 (61.1) 69 (34.8) 8 (4.0) 2 (7.7) 24 (92.3) — 1 (3.6) 12 (32.1) — 2 (13.3) 11 (73.3) 2 (13.3) 0 (0.0) 184 (62.8) 106 (36.2) 3 (1.0) 0 (0.0) 1 (0.5) 112 (56.6) 83 (41.9) 0 (0.0) 2 (1.0) NA NA NA NA NA NA NA NA NA NA NA NA 113 (38.6) 143 (48.8) 36 (12.3) 1 (0.3) 72 (36.4) 90 (45.5) 32 (16.2) 4 (2.0) 2 (7.7) 5 (19.2) 19 (73.1) — 0 (0.0) 3 (23.1) 10 (76.9) — NA NA NA 253 (86.3) 5 (1.7) 35 (11.9) 40 (20.2) 1 (0.5) 157 (79.3) NA NA NA NA NA NA — 15 (100) — 196 (66.9) 93 (31.7) 4 (1.4) 71 (35.9) 57 (28.8) 70 (35.4) NA NA NA NA NA NA NA NA NA 103 (52.0) 95 (48.0) 60.4 (1–181) NA NA NA NA NA NA NA NA NA AN (n ¼ 18) BPH (n ¼ 17) PIN (n ¼ 11) — — 63 (56–72) 67 (56–83) 63 (54–68) — — Abbreviation: NA, data not applicable/available. Switzerland; collected 1993–2001). Out of a total of 633 radical prostatectomies, we could retrieve formalin-fixed and paraffin-embedded (FFPE) tissue blocks from 457 patients (see Supplementary Fig. S1A for flow chart of inclusion/exclusion criteria according to REMARK guidelines). All specimens were evaluated by a trained pathologist, representative regions with >90% tumor were marked on hematoxylin and eosin (H&E)-stained sections, and punch biopsies were taken from the corresponding FFPE blocks for DNA and RNA extraction. Of the 457 patients, 164 were excluded due to either lack of follow-up (n ¼ 56), pre-/postoperative endocrine treatment (n ¼ 40), or poor DNA quality (n ¼ 68). Final analyses included 293 radical prostatectomy patients (Table 1). RP cohort 2 (validation) consisted of 310 curatively intended radical prostatectomies of histologically verified clinically localized prostate cancer from Departments of Urology, Heinrich Heine University (D€ usseldorf, Germany; collected 1993–2002), Tampere University Hospital www.aacrjournals.org (Finland; collected 1992–2003), and Karolinska University Hospital (Stockholm, Sweden; collected 2003–2007). A trained pathologist dissected the prostate immediately after radical prostatectomy and tumor samples were snap-frozen. On the basis of H&E-stained sections, DNA was extracted from samples with >70% tumor content. Inclusion/exclusion criteria were employed as for cohort 1 (Supplementary Fig. S1A). Final analyses comprised 198 radical prostatectomy samples (Table 1). FFPE adjacent nonmalignant (AN), benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), hormone-na€ve metastatic prostate cancer (MPC, primary tumor), and castration-resistant metastatic prostate cancer (CRPC, primary tumor) were collected, evaluated, and biopsied as described for cohort 1 (Table 1; Supplementary Fig. S1B). The study was approved by relevant ethical committees in each country. Written informed consent was obtained from all patients. Clin Cancer Res; 20(8) April 15, 2014 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2014 American Association for Cancer Research. 2171 Kristensen et al. DNA extraction and methylation analysis Bisulfite sequencing (biSeq) was performed as described previously (27, 28). A 441 nt region (containing 52 CpG sites) of the GABREmiR-452miR-224 promoter was amplified using PCR primers: 50 -AGATGTTTAGGAGGATTGGAAGAAT-30 and 50 -ACCCCACATCTTATCCCTAAAACAA30 . At least 7 clones were sequenced per sample. MethyLight. For Danish and Swiss FFPE samples, DNA was extracted using gDNA Eliminator columns from the miRneasy FFPE Kit (Qiagen) and the blood and cell culture DNA kit (Qiagen), respectively. For Swedish and Finnish samples, DNA extraction was conducted using the AllPrep Mini DNA/RNA kit (Qiagen), and for German samples the Blood and cell culture DNA kit (Qiagen). In all cases, DNA was bisulfite converted using the EZ-96 DNA Methylation-Gold Kit (Zymo Research). The following primers/ probes were used for quantitative methylation-specific PCR (MethyLight) analysis of the GABREmiR-452miR224 promoter: 50 -GATGTTTAGGAGGATTGGAAGA-30 and 50 -CTCCGCGCAAATAATCG-30 plus FAM-50 -ATATTTTCGCGGAGATCGGC-30 -BHQ1. For normalization and test of input DNA quality and quantity, a CpG-free region of MYOD1 was analyzed in parallel using primers/probe: 50 CCAACTCCAAATCCCCTCTCTAT-30 and 50 -TGGTTTTTTTAGGGAGTAAGTTTGTT-30 plus FAM-50 -TCCCTTCCTATTCCTAAATCCAACCTAAATACCTCC-30 -BHQ1. All reactions were run in triplicates in 384-well plates using the Applied Biosystems 7900HT Sequence Detection System and TaqMan Universal PCR Master Mix without AmpErase UNG (Applied Biosystems). Samples with low input DNA (CT (MYOD1) >36 in at least 2/3 reactions) were excluded from further analysis. The relative GABREmiR-452miR-224 promoter methylation level in each sample was determined as the GABREmiR-452miR-224/MYOD1 ratio. The accuracy of the GABREmiR-452miR-224/MYOD1 assay was validated by direct comparison with biSeq data from several prostate (cancer) tissue samples and cell lines, and by analysis of dilution series of fully methylated/unmethylated control DNA (data not shown). All primers and probes were purchased from Eurofins MWG Operon. Infinium arrays. One microgram of DNA was bisulfite converted, whole genome amplified, and applied to Illumina’s Infinium HumanMethylation27 Beadchip v1.2 (29) or HumanMethylation450 BeadChip (Strand and colleagues, unpublished) according to protocols from the manufacturer. Cell culture Prostatic cell lines PNT1A (European Collection of Cell Cultures), BPH1 (German Collection of Microorganisms and Cell Cultures), DuCaP and VCaP (kindly provided by Dr. Kenneth Pienta, University of Michigan, Ann Arbor, MI), 22rv1, LNCaP, PC3, and DU145 (American Type Culture Collection) were propagated in RPMI-1640 with L-glutamine (Invitrogen) supplemented with 10% heatinactivated fetal calf serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 C in humidified air atmosphere at 5% CO2. PrEC cells were cultured in Prostate 2172 Clin Cancer Res; 20(8) April 15, 2014 Epithelial Cell Basal Medium with added Prostate Epithelial Growth Kit (all from American Type Culture Collection). The authenticity of all cell lines was verified by short tandem repeat analysis (http://IdentiCell.eu). PC3 and DU145 cells were reverse transfected with 20 nmol/L of each miRNA (miRNA mimics purchased from Applied Biosystems/ Ambion) using Lipofectamine 2000 reagent (Invitrogen). Medium was changed 6 hours posttransfection. Transfection efficiencies were examined by fluorescence microscopy after transfection of Cy3-conjugated scrambled miRNA (Ambion) and/or verified by qRT-PCR (see below). Cell viability, migration, and invasion assays were performed as described in Supplementary Methods. Gene Set Enrichment Analysis RNA extraction, qRTPCR, and ChIP analyses For details, see Supplementary Methods Statistical analyses STATA version 11 (StataCorp) was used for all statistical analyses. P values <0.05 were considered statistically significant. Phenotypic effects in transfected cell lines and gene expression differences were assessed by two-sided Student t tests, and methylation differences by the Mann–Whitney U test. Correlations between expression and methylation were analyzed by Spearman rank test. Associations between GABREmiR-452miR-224 methylation and clinicopathologic variables were assessed using Spearman rank test or the Mann–Whitney U test. The diagnostic potential of GABREmiR-452miR-224/MYOD1 methylation was evaluated by receiver operator characteristics (ROC) curve analysis. For survival analysis, biochemical/PSA recurrence (Danish, Swiss, Swedish, and German samples cutoff 0.2 ng/mL, Finnish samples cutoff 0.5 ng/mL, based on local clinical practice) was used as endpoint. Patients not having experienced biochemical recurrence were censored at their last normal PSA measurement. The prognostic value of GABREmiR-452miR-224/MYOD1 methylation was evaluated by Kaplan–Meier analysis and two-sided log-rank test, and by univariate and multivariate Cox regression analysis as a continuous as well as a dichotomous variable. For analysis of GABREmiR-452miR-224/MYOD1 methylation as a dichotomous variable, patients in cohort 1 were divided into high and low methylation groups using a cutoff value determined by ROC analysis of recurrence/nonrecurrence status optimized for high specificity. For validation, patients in cohort 2 were divided into two groups using the cutoff (fraction) defined in cohort 1. The proportional hazards assumption was verified by the log-negative-log survival distribution function for all variables. Prognostic accuracy was estimated using Harrell Concordance Index. Results Silencing of the GABRE/miR-452/miR-224 locus in prostate cancer By microarray and qRT-PCR analyses, we observed significant downregulation of GABRE, miR-224, and miR-452 Clinical Cancer Research Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2014 American Association for Cancer Research. D P = 0.001 * 10 9 7 6 6 8 7 6 5 miR-224 (log2) 5 4 3 2 3 1 miR-224/RNU44 (log2) P = 6.28×10-5 * P = 3.79×10-11 * Nonmalignant (n = 27) Nonmalignant (n = 28) PC (n = 80) P = 4.94×10-5 * 5 6 7 PC (n = 80) 3 4 miR-452 (log2) 10 5 2.6 2.4 2.2 PC (n = 36) P = 9.11×10-7 * 0 miR-452/RNU44 (log2) 15 P = 7.45×10-10 * Nonmalignant (n = 14) PC (n = 150) Nonmalignant (n = 29) PC (n = 15) 0 3 2.8 2.6 PC (n = 36) 2.8 3 8 GABRE (log2) 7.5 7 GABRE (log2) 6.5 6 4 GABRE (log2) 2 C P = 5.89×10-8* Nonmalignant (n = 14) miR-452 (log2) Nonmalignant (n = 10) PC (n = 36) 2.4 miR-224 (log2) 3.2 Nonmalignant (n = 14) P = 1.87×10-8 * 4 B * 8 8 P = 6.56×10-4 8.5 A 11 Prognostic Value of GABREmiR-452miR-224 Methylation in Prostate Cancer Nonmalignant (n = 27) PC (n = 80) Nonmalignant (n = 28) PC (n = 80) Figure 1. Significant downregulation of GABRE, miR-224, and miR-452 in prostate cancer (PC) versus nonmalignant prostate tissue samples. A, mRNA and miRNA profiling (Mortensen and colleagues, unpublished) of 14 nonmalignant and 36 prostate cancer samples (microdissected) revealed significant downregulation of all three genes in prostate cancer. B, microarray data (50) from a distinct set of 10 nonmalignant and 15 prostate cancer samples (macrodissected) showed clearly reduced GABRE expression in prostate cancer. C, qRT-PCR analysis of an independent set of 27 nonmalignant and 80 prostate cancer samples (FFPE punch biopsies) showed significantly lower miR-224 and miR-452 levels in prostate cancer. D, external validation using datasets GSE21032 and GSE21034 from Taylor and colleagues (30) available at the GEO website. Significant P values (two-sided Student t test) are marked by an asterisk ( ). GABRE expression was determined by Affymetrix Human Genome U133 plus 2.0 (A) or Affymetrix Human Exon 1.0 ST array analysis (B and D); miR-224 and miR-452 expression was determined by qRT-PCR using TaqMan miRNA Low Density Arrays (A), single TaqMan miRNA RT-qPCR assays (C), or Agilent microRNA V2 arrays (D). in prostate cancer compared with nonmalignant prostate tissue samples in multiple patient sample sets (Fig. 1A–C). For external validation, expression data from Taylor and colleagues (30) was used, confirming the significant downregulation of all three genes in prostate cancer (Fig. 1D). The findings were further supported by nine transcription profiling datasets from the oncomine database, all showing downregulation of GABRE in prostate cancer (Supplementary Table S2). Moreover, significant (P < 0.001) direct correlations between the expression of GABRE, miR-224, and miR-452 were evident in clinical sample sets (Spearman correlation coefficient, r ¼ 0.42–0.94) and in prostatic cell lines (r ¼ 0.91–0.96; Supplementary Fig. S2A–S2D), consistent with transcriptional coregulation. www.aacrjournals.org The GABRE/miR-452/miR-224 promoter CpG island is hypermethylated in prostate cancer Next, we investigated whether downregulation of the GABREmiR-452miR-224 locus in prostate cancer was associated with hypermethylation of its promoter-associated CGI (Fig. 2A). Two methylation microarray datasets (29), including a total of 30 nonmalignant and 30 prostate cancer tissue samples, showed significant (P < 0.001) promoter hypermethylation in prostate cancer samples (Fig. 2B). This finding was corroborated by genomic biSeq of an independent set of 11 nonmalignant and 21 prostate cancer tissue samples (Fig. 2C and Supplementary Fig. S3). For large-scale validation, we designed a MethyLight assay for a region of the GABREmiR-452miR-224 promoter, Clin Cancer Res; 20(8) April 15, 2014 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2014 American Association for Cancer Research. 2173 TSS –270 Exon 1 miR-452 miR-224 Intron 6 +1 (ATG) +470 1.0 Nonmalignant (n = 21) 0.4 PC (n = 9) CpG island cg12204574 100 AN (n = 7) BPH LPC MPC (n = 4) (n = 10) (n = 11) Nonmalignant (n = 35) PC (n = 245) 0.50 Sensitivity 1 AN PIN LPC CRPC MPC BPH LNM (n = 18) (n = 17) (n = 11) (n = 191) (n = 13) (n = 26) (n = 15) 0.25 NS AUC = 0.98 Sensitivity = 95.5% 0.00 2 0.75 1.00 3 MethyLight dataset 0 0 1 2 3 Methylation GABRE~miR-224~miR-452/MYOD1 4 4 P = 3.56 × 10–19 D Methylation GABRE~miR-224~miR-452/MYOD1 cg12204574 Nonmalignant (n = 9) cg01480550 0.0 0 cg08783090 0.2 P = 6.98 × 10–7 80 0.6 P = 0.26 60 β-value β-value 0.2 P = 1.00 PC (n = 21) 0.8 0.4 BiSeq dataset 40 P = 0.006 cg07053880 0.6 C 450K dataset 27K dataset cg25528646 B CpG island (83 CpGs) 0 20 –404 % GABRE~miR-452~miR-224 methylation –351 +144 +16101 GABRE MethyLight (7 CpGs) +16021 Bisulfite sequencing (52 CpGs) –351 +14967 A +15051 Kristensen et al. Specificity = 94.3% 0.00 0.25 0.50 0.75 1.00 1 – Specificity Figure 2. A, schematic structure of GABREmiR-224miR-452 genomic locus (not to scale). miR-224 and miR-452 are located in intron 6. Location of promoter-associated CpG island, containing 83 CpG sites, is shown by a gray box. Regions analyzed by bisulfite sequencing and MethyLight are marked with arrows, indicating PCR primer positions. TSS marks the putative transcription start site. B–D, GABREmiR-452miR-224 promoter methylation levels in four independent clinical sample sets determined by (B) Illumina 27 K and 450 K microarray analysis, (C) bisulfite sequencing (percent methylated CpG sites/ total number of CpG sites; see Supplementary Fig. S3 for more details), or (D) MethyLight. The number of patients in each group is given in brackets. B, an asterisk ( ) indicates significant P values (P < 0.001; Mann–Whitney U Test) for Illumina probes covering GABREmiR-452miR-224. C and D, horizontal gray lines mark median methylation levels in each group and significant P values (P < 0.05 vs. ANþBPH; Mann–Whitney U test) are marked by an asterisk ( ). D, ROC curve analysis (right) showing accurate discrimination between nonmalignant and prostate cancer tissue samples based on GABREmiR-452miR-224 methylation. AUC, area under the curve. AN, adjacent nonmalignant prostate; BPH, benign prostatic hyperplasia; LPC, clinically localized prostate cancer (radical prostatectomy specimen); MPC, hormone-naïve metastatic prostate cancer (primary tumor); CRPC, castrationresistant prostate cancer (primary tumor); LMN, lymph node metastasis. NS, not significant. including 7 CpG sites also analyzed by biSeq (Fig. 2A). A CpG-free region of MYOD1 was used for normalization (represents total input of bisulfite-converted DNA). GABREmiR-452miR-224/MYOD1 methylation was determined in 291 FFPE tissue samples (191 LPC/RP samples from cohort 1, plus several nonmalignant, premalignant, 2174 Clin Cancer Res; 20(8) April 15, 2014 and advanced prostate cancer samples; Table 1). Overall, GABREmiR-452miR-224 was significantly (P ¼ 3.56 1019; Mann–Whitney U test) hypermethylated in prostate cancer compared with nonmalignant tissue samples (Fig. 2D, left). Also, GABREmiR-452miR-224 was hypermethylated in 3 of 11 PIN samples, suggesting it could be an Clinical Cancer Research Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2014 American Association for Cancer Research. Prognostic Value of GABREmiR-452miR-224 Methylation in Prostate Cancer early event in prostate carcinogenesis. Methylation levels were similar between subgroups of nonmalignant (AN/ BPH) and malignant (LPC/MPC/CRPC/LNM) samples, respectively (Fig. 2D, middle). Together, these results strongly indicate that aberrant promoter hypermethylation is associated with coordinated downregulation of the entire GABREmiR-452miR-224 locus in prostate cancer. Consistent with epigenetic silencing, a significant (P < 0.001) inverse correlation between promoter methylation and miR224 (Spearman rho, r ¼ 0.41) or miR-452 expression (r ¼ 0.43), respectively, was seen in the clinical samples (Supplementary Fig. S2C). Likewise, expression of GABRE, miR-224, and miR-452 was strongly inversely correlated with promoter methylation (r ¼ 0.83 to 0.88) in 9 prostatic cell lines (Supplementary Fig. S2E). GABREmiR-452miR224 was hypermethylated in 4 of 6 prostate cancer cell lines and in 0 of 3 nonmalignant prostate cell lines (Supplementary Fig. S4A–S4C). Treatment with the demethylating drug 5-aza-dC was insufficient for reactivation of transcription in hypermethylated DU145 and PC3 cells, which may be explained by presence of high levels of the repressive H3K27me3 mark at the inactive GABREmiR-452miR224 promoter (Supplementary Fig. S4D and S4E). To assess the diagnostic potential of GABREmiR452miR-224 methylation, we performed ROC curve analysis using the large MethyLight dataset (Fig. 2D, left). The preliminary sensitivity and specificity of GABREmiR452miR-224 methylation for prostate cancer detection was 95.5% and 94.3%, respectively (Fig. 2D, right). Together, our findings strongly indicate that aberrant promoter hypermethylation of the GABREmiR-452miR-224 promoter-associated CGI is a frequent event in clinical prostate cancer samples as well as in prostate cancer cell lines. We note that the GABREmiR-452miR-224 locus is located at Xq28 within a cluster of MAGE (cancer-testis antigens) genes known to be hypomethylated in prostate cancer and recently described as a long range epigenetic activation region (31). Focal hypermethylation of the GABREmiR452miR-224 promoter within this otherwise activated and hypomethylated region (Supplementary Fig. S5) suggests that epigenetic silencing of GABREmiR-452miR-224 is highly specific and may be selected for during prostate cancer development and/or progression. miR-224 and miR-452 inhibit viability, migration, and invasion of prostate cancer cells Next, we investigated the potential functional roles of miR224 and miR-452 in prostate cancer (GABRE was not analyzed as we were unable to validate a good antibody; data not shown). miR-224 or miR-452 mimics were transfected into two prostate cancer cell lines (PC3 and DU145) with low endogenous expression (Supplementary Fig. S4B). As positive controls, we used miR-145 in cell viability assays (known to reduce PC3 and DU145 cell viability; ref. 32) and miR-101 in cell migration/invasion assays (known to inhibit migration and invasion of PC3 and DU145 cells; ref. 33). Scrambled miRNA was used as a negative control. Transfection efficiencies were confirmed by qRT-PCR (data not shown). www.aacrjournals.org Figure 3. Ectopic expression of miR-224 or miR-452 inhibited viability, migration, and invasion in two prostate cancer cell lines. A, cell viability at 48 and 72 hours posttransfection of miR-224 or miR-452 mimics (20 nmol/L) in PC3 and DU145 cells, as determined by an MTT assay. All results are presented relative to scrambled (scr) miRNA transfected cells. B and C, Trevigen Cultrex Migration and Invasion assays performed 48 hours posttransfection. Background migration levels for nontransfected cells without chemoattractant (no serum) are also given (B). miR-145 and miR-101 were used as positive controls for inhibition of cell viability (A) and migration/invasion (B and C), respectively. The presented results (A-C) are representative of a minimum of three experiments performed in triplicate; all significant phenotypic effects, that is, P < 0.05 (two-sided Student t test) and >25% reduction, are marked by an asterisk ( ). Reintroduction of either miR-224 or miR-452 had a moderate (25%) and statistically significant (P < 0.05) inhibitory effect on cell viability in both PC3 and DU145 cells, as evident at 72 hours posttransfection but not at 48 hours (Fig. 3A). Furthermore, ectopic expression of miR224 or miR-452 significantly (P < 0.05) reduced migration and invasion of PC3 and DU145 cells at 48 hours posttransfection (Fig. 3B and C), and hence was not simply ascribed to miRNA-induced reduction in cell viability, as this occurred at 72 hours. Cotransfection of miR-224 and Clin Cancer Res; 20(8) April 15, 2014 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2014 American Association for Cancer Research. 2175 Kristensen et al. miR-452 did not cause further inhibition of cell viability, migration, or invasion (Fig. 3A–C), suggesting at least some functional redundancy between these miRNAs. Subsequently, we performed GSEA of microarray transcriptional profiling data obtained at 48 hours posttransfection. For genes (mRNA) downregulated upon miR-224 or miR-452 transfection, the top ten most significantly enriched "Canonical Pathways" in both DU145 and PC3 cells (Supplementary Fig. S6A and S6B) were primarily associated with positive regulation of the cell cycle, DNA replication, and mitosis, consistent with the observed negative effect of these miRNAs on prostate cancer cell viability (Fig. 3A). GSEA was also performed for genes significantly upregulated upon miR-224 or miR-452 transfection, likely representing indirect effects. Several "Canonical Pathways" related to cellular adhesion and motility were enriched for both miR-224 and miR-452 (Supplementary. Fig. S6C and S6D) in agreement with their effects on migration and invasion (Fig. 3B and C). Among the genes most heavily downregulated in both cell lines after miR-224 and miR-452 overexpression, respectively (Supplementary Tables S3 and S4), we shortlisted potential target genes using the following criteria: (i) mRNAs containing at least one 7mer target site predicted by both TargetScan (www.targetscan.org) and PicTar (http://pictar. mdc-berlin.de), and (ii) mRNAs significantly upregulated in prostate cancer versus nonmalignant prostate tissue samples in multiple patient sample sets (Supplementary Table S5). Among eight shortlisted potential miR-224 target genes, expression of C1orf116 (chromosome 1 open reading frame 116, also known as specifically androgen-regulated gene, SARG), GOLM1 (golgi membrane protein 1), and FAM64A (family with sequence similarity 64, member A) was significantly inversely correlated with miR-224 expression in the datasets from Taylor and colleagues (30) and Mortensen and colleagues (Spearman r: 0.49 to 0.25; P < 0.05; Supplementary Table S5), suggesting that these genes could be clinically relevant. Moreover, among eight shortlisted potential miR-452 target genes, expression of DR1 (downregulator of transcription 1) and IGF2BP2 [insulin-like growth factor 2 mRNA binding protein 2 (p62)] was significantly inversely correlated with miR-452 in the dataset from Mortensen and colleagues (r: 0.47 to 0.30; P < 0.05), but no significant correlations were seen in the Taylor and colleagues dataset (Supplementary Table S5). Prognostic value of GABRE/miR-452/miR-224 methylation and association with clinicopathological parameters Next, we investigated possible correlations between clinicopathologic variables and GABREmiR-452miR-224 methylation in two independent radical prostatectomy cohorts (Table 1; Supplementary Fig. S1A). There was no significant correlation between GABREmiR-452miR-224 methylation and age at diagnosis (Supplementary. Fig. S7A), but high methylation was significantly (P < 0.05) associated with the established adverse prognostic factors high PSA, high T-stage, high Gleason score, and positive 2176 Clin Cancer Res; 20(8) April 15, 2014 surgical margin status in at least one cohort (Supplementary Fig. S7B–S7E). To assess the prognostic value of GABREmiR-452miR224 methylation, we performed PSA-based recurrence-free survival (RFS) analysis. By univariate Cox regression analysis of GABREmiR-452miR-224 methylation (as a continuous variable) in RP cohort 1 (training cohort, n ¼ 293; see Table 1), short RFS time was significantly associated with high GABREmiR-452miR-224 methylation, as well as with the established prognostic factors high PSA, high Gleason score, advanced T-stage, and positive surgical margin status (P < 0.001; Table 2). All variables remained significant in a multivariate model (Table 2), indicating that GABREmiR-452miR-224 methylation is a significant independent prognostic factor for prostate cancer patients treated by radical prostatectomy. The prognostic value of GABREmiR-452miR-224 methylation was successfully validated by univariate as well as multivariate analysis in cohort 2 (validation cohort, n ¼ 198; see Table 1; Table 2). In cohort 2, surgical margin status was left out in the final model, because of missing data for 70 patients. Predictive accuracies were estimated by Harrell C-index, which increased from 0.73 to 0.74 in cohort 1, and from 0.75 to 0.77 in cohort 2 by addition of GABREmiR452miR-224 methylation, suggesting moderately improved model performance. The contribution of GABREmiR-452miR-224 methylation to the multivariate model was comparable with that of a single clinicopathologic variable: Harrell C-index of the multivariate model (cohort 1/cohort 2) when leaving out either PSA (0.72/ 0.76), T-stage (0.72/0.74), Gleason score (0.73/0.73), or surgical margin status (0.73/not applicable). Similar results were obtained when GABREmiR452miR-224 methylation was analyzed as a dichotomous variable in uni- and multivariate Cox regression analyses (Supplementary Table S6). For these analyses, patients in cohort 1 were divided into low/high methylation subgroups using a cutoff value determined by ROC curve analysis (optimized for high specificity). Likewise, patients in cohort 2 were divided into low/high methylation subgroups using the cutoff (fraction) defined in cohort 1. Finally, by Kaplan– Meier analysis, high GABREmiR-452miR-224 methylation was significantly (P < 0.001, log-rank test) associated with short RFS in cohort 1 (Fig. 4A), which was successfully validated (P < 0.001) in cohort 2 (Fig. 4B). In summary, we found GABREmiR-452miR-224 methylation to be a significant independent prognostic predictor of biochemical recurrence in two radical prostatectomy patient cohorts. Finally, to assess the possible prognostic value of GABRE, miR-452, and miR-224 expression, we used the large radical prostatectomy patient cohort from Taylor and colleagues (30). A multigene model including the expression of all three genes was a significant independent predictor of biochemical recurrence after radical prostatectomy (HR, 2.37; P ¼ 0.013; Supplementary Table S7). Expression of either GABRE, miR-224, or miR-452 individually was not significant in multivariate analysis, but GABRE and miR224 were significant in univariate analysis (Supplementary Clinical Cancer Research Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2014 American Association for Cancer Research. Prognostic Value of GABREmiR-452miR-224 Methylation in Prostate Cancer Table 2. Uni- and multivariate Cox regression analyses of biochemical recurrence-free survival in two RP cohorts Univariate Variable Cohort 1 (training), n ¼ 293 Age at diagnosis Preoperative PSA Tumor stage Surgical margin status Gleason score GABREmiR-452miR-224 methylation Cohort 2 (validation), n ¼ 198 Age at diagnosis Preoperative PSA Tumor stage Surgical margin statusb Gleason score GABREmiR-452miR-224 methylation Multivariate Characteristics HR (95% CI) P C-index HR (95% CI) Continuous 10 vs. >10 ng/mL pT2a-c vs. pT3a-c Negative vs. positive Continuous Continuous 0.98 (0.95–1.01) 2.67 (1.75–408) 3.37 (2.35- 4.84) 3.03 (2.10–4.36) 1.48 (1.22–1.80) 1.75 (1.37–2.23) 0.211 <0.001 <0.001 <0.001 <0.001 <0.001 0.55 0.61 0.65 0.63 0.59 0.61 — — 2.37 (1.53–3.69) <0.001 0.74 0.73 2.06 (1.37–3.10) 0.001 1.99 (1.33–2.98) 0.001 1.27 (1.02–1.58) 0.033 1.38 (1.06–1.81) 0.019 Continuous 10 vs. >10 ng/mL pT2a-c vs. pT3a-c Negative vs. positive Continuous Continuous 1.02 (0.99–1.06) 2.20 (1.45–3.34) 3.60 (2.36–5.49) 2.50 (1.44–4.37) 1.84 (1.50–2.25) 2.99 (1.71–5.21) 0.206 <0.001 <0.001 0.001 <0.001 <0.001 0.53 0.59 0.66 0.62 0.69 0.65 — 2.22 (1.45–3.41) 2.58 (1.63–4.08) — 1.71 (1.45–3.41) 2.45 (1.26–4.75) a P C-indexa — <0.001 0.77 0.75 <0.001 — <0.001 0.008 a Predictive accuracy, estimated by the Harrell concordance index (c-index). Left column, C-index based on all variables; right column, C-index based on clinicopathological variables only (i.e. excluding GABREmiR-452miR-224 methylation). b Margin status was excluded from the final multivariate analysis, because of missing data for 70 of 198 patients in cohort 2. Significant P values (P < 0.05) are marked in bold. Table S7). Finally, among the shortlisted miR-224 and miR452 potential target genes (Supplementary Table S5), high expression of OAZ2 (Ornithine Decarboxylase Antizyme 2; A predicted miR-452 target) had significant adverse prognostic value in multivariate analysis in the Taylor and colleagues dataset (Supplementary Table S8). A multigene model B P < 0.001 0 25 50 75 100 Recurrence-free survival time (months) 0.75 0.50 Low meth. High meth. 0.25 0.25 High meth. Cumulative survival 1.00 Cohort 2 0.00 0.75 0.50 Low meth. 0.00 Cumulative survival 1.00 Cohort 1 P < 0.001 125 0 8 1 130 68 25 50 75 100 Recurrence-free survival time (months) 125 Number at risk Low meth. 193 High meth. 100 141 67 93 40 33 18 21 5 107 40 88 29 64 16 27 1 12 0 Figure 4. Kaplan–Meier survival analysis of RFS based on GABREmiR-452miR-224/MYOD1 methylation levels (low vs. high) in radical prostatectomy samples. A, patients in cohort 1 (training) were divided into high versus low methylation groups after ROC analysis. B, patients in validation cohort 2 were divided into low/high methylation groups according to the cutoff (fraction) defined in cohort 1. Significant ( ) P values for two-sided log-rank test are given. High GABREmiR-452miR224 methylation was significantly associated with early biochemical recurrence after radical prostatectomy in two independent cohorts. www.aacrjournals.org Clin Cancer Res; 20(8) April 15, 2014 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2014 American Association for Cancer Research. 2177 Kristensen et al. including GABRE, miR-224, miR452, and OAZ2 expression also remained significant in a multivariate model (Supplementary Table S8), further supporting a clinically relevant role of the GABREmiR-224miR-452 locus and its expression circuit in prostate cancer. Discussion This study showed that the GABREmiR-452miR-224 locus is downregulated in prostate cancer and that aberrant GABREmiR-452miR-224 promoter hypermethylation is a very common event in prostate cancer, suggesting it is a key molecular mechanism for coordinated silencing of this locus in prostate cancer cells. Moreover, GABREmiR-452miR224 hypermethylation was found to be a significant independent predictor of biochemical recurrence in two radical prostatectomy patient cohorts from five European countries. To the best of our knowledge, this is the first comprehensive study of GABREmiR-452miR-224 expression in prostate cancer as well as the first report to identify GABREmiR452miR-224 as a promising diagnostic and prognostic methylation marker candidate for prostate cancer. Consistent with our results, downregulation of miR-224 in prostate cancer tissue samples has been reported in two recent studies (14, 15). Another study has reported miR-224 upregulation in PNI-positive versus negative prostate cancer (16), but used only a small sample set without nonmalignant prostate samples for comparison. PNI status was not determined in our sample set. Moreover, although one earlier study has reported upregulation of miR-452 in prostate cancer progenitor cells (17), differential expression in prostate cancer patient samples has not been previously described. There are no prior reports of GABRE deregulation in prostate cancer, but upregulation has been reported in non–small cell lung cancer (34). In this study, GABREmiR-452miR-224 promoter methylation was found to be highly cancer-specific (AUC ¼ 0.98) and thereby comparable to previously reported top candidate methylation markers for prostate cancer detection, including GSTP1 (5). Furthermore, our findings indicated that GABREmiR-452miR-224 methylation has significant independent prognostic value for prediction of biochemical recurrence after radical prostatectomy, beyond routine clinicopathologic predictors. The prognostic potential of GABREmiR-452miR-224 methylation was identified in a training cohort from Denmark and Switzerland and successfully validated in an independent patient set from Germany, Finland, and Sweden, supporting the validity of our finding. Before the present study, PITX2 (3, 4) and C1orf114 (29) were the only single genes with independent prognostic value reported in two radical prostatectomy cohorts. Further studies including large prospective cohorts are needed to assess the clinical utility of GABREmiR-452miR-224 methylation as a prognostic candidate marker alone and in combination with other candidate molecular markers. The significant prognostic value of GABREmiR-452miR224 methylation was also reflected at the expression level, when we analyzed the radical prostatectomy cohort from Taylor and colleagues (30). Thus, a three-gene GABRE/ 2178 Clin Cancer Res; 20(8) April 15, 2014 miR-224/miR-452 expression signature was a significant independent predictor of PSA recurrence after radical prostatectomy in this cohort. Low expression was associated with higher recurrence risk, consistent with an important role of coordinated epigenetic silencing of the GABREmiR452miR-224 locus in prostate cancer. Our work showed that miR-224 and miR-452 inhibit proliferation, migration, and invasion of two prostate cancer cell lines. The GABREmiR-452miR-224 locus has not been functionally linked to prostate cancer before, but earlier studies have shown involvement of miR-224 in both repression and stimulation of cell viability, migration, and/ or invasion in other cancer types (20, 22, 35). None of the validated miR-224 targets from these studies (e.g., KLK10 in ovarian cancer and RKIP in breast cancer; refs. 20, 35) were significantly affected, when we overexpressed miR-224 in PC3 or DU145 cells (data not shown), consistent with a cell type and context-dependent function of this miRNA. To our knowledge, there are no previous reports of a functional role nor validated target for miR-452. Using an integrative bioinformatic approach, we shortlisted 16 predicted miR224 or miR-452 target genes of potential clinical importance in prostate cancer (Supplementary Table S5). Among these, we identified the three predicted miR-224 target genes C1orf116, GOLM1, and FAM64A that were not only downregulated in PC3 and DU145 cells upon miR-224 overexpression, but also significantly upregulated in prostate cancer tissue versus nonmalignant tissue samples in multiple patient sets. Similarly, we identified predicted miR-452 target genes, including IGF2BP2, DR1, and OAZ2. These genes have all been previously linked to cancer and in some cases prostate cancer specifically. Thus, GOLM1 has been suggested as a putative tissue and urine biomarker of prostate cancer (36–39) and shown to induce migration and invasion of prostate cancer cells (40). C1orf116 has been reported as highly expressed in prostate cancer tissue (41), whereas FAM64A is highly expressed in leukemia and lymphoma, and known to promote cell division (42, 43). Moreover, we found that high expression of the predicted miR-452 target gene OAZ2 was associated with high recurrence risk after radical prostatectomy in the patient cohort from Taylor and colleagues. Expression of OAZ2 has previously been associated with aggressive behavior in neuroblastoma (44). IGF2BP2 expression has been associated with liver cancer, liposarcoma, and endometrial adenocarcinomas, and with promotion of cell migration and metastasis (45, 46). High expression of DR1 is associated with basal cell carcinoma in renal transplant patients (47). Another interesting potential miR-452 target gene, ARF4 (ADP-ribosylation factor 4), has been shown to promote breast cancer cell migration (48) and to inhibit apoptosis in glioblastoma (49). Additional studies are needed to investigate the molecular pathways and target genes regulated by miR-224 and miR-452 in prostatic cells in more details. Moreover, the potential functional role of the host gene GABRE remains to be investigated. Despite several attempts to overexpress GABRE in prostate cancer cell lines, we observed no Clinical Cancer Research Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2014 American Association for Cancer Research. Prognostic Value of GABREmiR-452miR-224 Methylation in Prostate Cancer phenotypic effects (data not shown); however, we were unable to validate GABRE expression at the protein level due to lack of a specific antibody. Elevated GABA levels have previously been associated with prostate cancer metastasis (10) and GABA agonist isoguvacine has been shown to stimulate proliferation of some prostate cancer cell lines (11), but the exact composition(s) of the GABA receptor(s) mediating these effects are not known. Furthermore, GABA signaling has been found to inhibit breast cancer cell migration (12), suggesting a context-dependent function. We note that all methylation analyses were performed using radical prostatectomy tissue specimens. A prognostic test based on GABREmiR-452miR-224 methylation would currently have limited clinical utility at this stage, as there are no established adjuvant therapies after radical prostatectomy. In future studies, it will therefore be important to investigate if GABREmiR-452miR-224 methylation levels measured in prostate biopsies, or in a urine or blood sample, taken at the time of diagnosis can predict prostate cancer aggressiveness and thus guide treatment decisions. Conceivably, a high level of GABREmiR452miR-224 methylation in a biopsy may favor intervention (e.g., surgery) over active surveillance. Likewise, an increase in GABREmiR-452miR-224 methylation in biopsies taken during active surveillance could indicate disease progression, and hence the need for intervention. Similarly, to assess the clinical utility of GABREmiR452miR-224 methylation for prostate cancer diagnosis, future studies should investigate whether detection of tumor-derived hypermethylated GABREmiR-452miR224 DNA in biofluids, such as urine or blood, can be used to identify patients with prostate cancer. Given the very high frequency and cancer specificity of GABREmiR-452miR224 promoter hypermethylation that we observed in radical prostatectomy tissue samples, it is possible that a methylation test for GABREmiR-452miR-224 in biofluids could be used as a supplement to for example, a PSA test (high sensitivity, low specificity) to increase the accuracy of diagnosis. Furthermore, premalignant and/or cancer field effects that are not evident histopathologically may be detected by DNA methylation analysis of prostate biopsies, as demonstrated by others for e.g. GSTP1 (6, 7). Accordingly, GABREmiR-452miR-224 methylation analysis could also potentially be used to guide repeat biopsy in men with initial cancer-negative biopsies. The use of PSA recurrence as clinical endpoint is also a limitation of our study. Notably, due to lower sensitivity of the specific PSA kit applied, a higher cutoff value was used to define biochemical recurrence in the Finnish patients (0.5 ng/mL) compared with other patients (0.2 ng/mL). Thus, it cannot be excluded that detection of PSA recurrence was slightly delayed for some of the Finnish patients. However, time to recurrence is also influenced by the frequency and exact timing of PSA testing after radical prostatectomy, which despite regulation by clinical guidelines also may vary between patients in real life. Future studies should investigate other clinical endpoints such as cancer-specific and overall survival. Given the long disease course of prostate cancer this would require large cohorts with at least 10 to 20 years of follow-up. In conclusion, our results showed that the GABREmiR-452miR-224 locus is epigenetically silenced in prostate cancer compared with nonmalignant prostate tissue samples. Furthermore, our findings indicated that miR-224 and miR-452 hold important tumor suppressor functions in prostate cancer. Finally, we identified the GABREmiR-452miR-224 promoter as a promising new candidate methylation marker for prostate cancer detection and for prediction of biochemical recurrence after radical prostatectomy. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Authors' Contributions Conception and design: H. Kristensen, S. Høyer, M. Borre, T.F. rntoft, K.D. Sørensen Development of methodology: H. Kristensen, C. Haldrup, S. Høyer, K.D. Sørensen Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Kristensen, S. Strand, K. Mundbjerg, M.M. Mortensen, P.J. Wild, C. Arsov, T. Visakorpi, L. Egevad, J. Lindberg, H. Gr€ onberg, S. Høyer, M. Borre, K.D. Sørensen Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Kristensen, K. Mundbjerg, M.M. Mortensen, K. Thorsen, M.S. Ostenfeld, S. Høyer, T.F. rntoft, K.D. Sørensen Writing, review, and/or revision of the manuscript: H. Kristensen, C. Haldrup, K. Mundbjerg, M.S. Ostenfeld, P.J. Wild, C. Arsov, W. Goering, T. Visakorpi, H. Gr€ onberg, S. Høyer, M. Borre, T.F. rntoft, K.D. Sørensen Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Haldrup, M.M. Mortensen, P.J. Wild, W. Goering, J. Lindberg Study supervision: C. Haldrup, M.M. Mortensen, K.D. Sørensen Acknowledgments The authors thank Anne Slotsdal, Pamela Celis, Maria Mark, Gitte Høj, Mette Rasmussen, Conni Sørensen and Susanne Skou for excellent technical assistance and Dr. Wolfgang A. Schulz for comments on the article. The Danish Cancer Biobank (DCB) is acknowledged for biologic material and for information regarding handling and storage. Grant Support This work was supported by The Danish Cancer Society, The Lundbeck Foundation, The John and Birthe Meyer Foundation, The Danish Council for Strategic Research, The Danish Advanced Technology Foundation, and The Deutsche Forschungsgemeinschaft (Schu604/16-3). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received September 25, 2013; revised January 6, 2014; accepted February 5, 2014; published online April 15, 2014. References 1. Prensner JR, Rubin MA, Wei JT, Chinnaiyan AM. Beyond PSA: the next generation of prostate cancer biomarkers. Sci Transl Med 2012; 4:127rv3. www.aacrjournals.org 2. Wilke K, Gaul R, Klauck SM, Poustka A. A gene in human chromosome band Xq28 (GABRE) defines a putative new subunit class of the GABAA neurotransmitter receptor. Genomics 1997;45:1–10. Clin Cancer Res; 20(8) April 15, 2014 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2014 American Association for Cancer Research. 2179 Kristensen et al. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 2180 Weiss G, Cottrell S, Distler J, Schatz P, Kristiansen G, Ittmann M, et al. DNA methylation of the PITX2 gene promoter region is a strong independent prognostic marker of biochemical recurrence in patients with prostate cancer after radical prostatectomy. J Urol 2009;181:1678–85. Banez LL, Sun L, van Leenders GJ, Wheeler TM, Bangma CH, Freedland SJ, et al. Multicenter clinical validation of PITX2 methylation as a prostate specific antigen recurrence predictor in patients with postradical prostatectomy prostate cancer. J Urol 2010;184:149–56. Goering W, Kloth M, Schulz WA. DNA methylation changes in prostate cancer. Methods Mol Biol 2012;863:47–66. Trock BJ, Brotzman MJ, Mangold LA, Bigley JW, Epstein JI, McLeod D, et al. Evaluation of GSTP1 and APC methylation as indicators for repeat biopsy in a high-risk cohort of men with negative initial prostate biopsies. BJU Int 2012;110:56–62. Troyer DA, Lucia MS, de Bruine AP, Mendez-Meza R, Baldewijns MM, Dunscomb N, et al. Prostate cancer detected by methylated gene markers in histopathologically cancer-negative tissues from men with subsequent positive biopsies. Cancer Epidemiol Biomarkers Prev 2009;18:2717–22. D'Hulst C, Atack JR, Kooy RF. The complexity of the GABAA receptor shapes unique pharmacological profiles. Drug Discov Today 2009;14: 866–75. Neelands TR, Fisher JL, Bianchi M, Macdonald RL. Spontaneous and gamma-aminobutyric acid (GABA)-activated GABA(A) receptor channels formed by epsilon subunit-containing isoforms. Mol Pharmacol 1999;55:168–78. Azuma H, Inamoto T, Sakamoto T, Kiyama S, Ubai T, Shinohara Y, et al. Gamma-aminobutyric acid as a promoting factor of cancer metastasis; induction of matrix metalloproteinase production is potentially its underlying mechanism. Cancer Res 2003;63:8090–6. Abdul M, McCray SD, Hoosein NM. Expression of gamma-aminobutyric acid receptor (subtype A) in prostate cancer. Acta Oncol 2008;47:1546–50. Drell TLt, Joseph J, Lang K, Niggemann B, Zaenker KS, Entschladen F. Effects of neurotransmitters on the chemokinesis and chemotaxis of MDA-MB-468 human breast carcinoma cells. Breast Cancer Res Treat 2003;80:63–70. Jansson MD, Lund AH. MicroRNA and cancer. Mol Oncol 2012;6: 590–610. Mavridis K, Stravodimos K, Scorilas A. Downregulation and prognostic performance of microRNA 224 expression in prostate cancer. Clin Chem 2013;59:261–9. Martens-Uzunova ES, Jalava SE, Dits NF, van Leenders GJ, Moller S, Trapman J, et al. Diagnostic and prognostic signatures from the small non-coding RNA transcriptome in prostate cancer. Oncogene 2012;31:978–91. Prueitt RL, Yi M, Hudson RS, Wallace TA, Howe TM, Yfantis HG, et al. Expression of microRNAs and protein-coding genes associated with perineural invasion in prostate cancer. Prostate 2008;68:1152–64. Liu C, Kelnar K, Vlassov AV, Brown D, Wang J, Tang DG. Distinct microRNA expression profiles in prostate cancer stem/progenitor cells and tumor-suppressive functions of let-7. Cancer Res 2012;72:3393–404. Giricz O, Reynolds PA, Ramnauth A, Liu C, Wang T, Stead L, et al. HsamiR-375 is differentially expressed during breast lobular neoplasia and promotes loss of mammary acinar polarity. J Pathol 2012;226:108–19. Yanaihara N, Caplen N, Bowman E, Seike M, Kumamoto K, Yi M, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell 2006;9:189–98. White NM, Chow TF, Mejia-Guerrero S, Diamandis M, Rofael Y, Faragalla H, et al. Three dysregulated miRNAs control kallikrein 10 expression and cell proliferation in ovarian cancer. Br J Cancer 2010;102:1244–53. Gokhale A, Kunder R, Goel A, Sarin R, Moiyadi A, Shenoy A, et al. Distinctive microRNA signature of medulloblastomas associated with the WNT signaling pathway. J Cancer Res Ther 2010;6:521–9. Li Q, Wang G, Shan JL, Yang ZX, Wang HZ, Feng J, et al. MicroRNA224 is upregulated in HepG2 cells and involved in cellular migration and invasion. J Gastroenterol Hepatol 2010;25:164–71. Boguslawska J, Wojcicka A, Piekielko-Witkowska A, Master A, Nauman A. MiR-224 targets the 30 UTR of type 1 50 -iodothyronine deiodi- Clin Cancer Res; 20(8) April 15, 2014 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. nase possibly contributing to tissue hypothyroidism in renal cancer. PloS ONE 2011;6:e24541. Arndt GM, Dossey L, Cullen LM, Lai A, Druker R, Eisbacher M, et al. Characterization of global microRNA expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer. BMC Cancer 2009;9:374. Liu H, Brannon AR, Reddy AR, Alexe G, Seiler MW, Arreola A, et al. Identifying mRNA targets of microRNA dysregulated in cancer: with application to clear cell Renal Cell Carcinoma. BMC Syst Biol 2010;4:51. Veerla S, Lindgren D, Kvist A, Frigyesi A, Staaf J, Persson H, et al. MiRNA expression in urothelial carcinomas: important roles of miR10a, miR-222, miR-125b, miR-7 and miR-452 for tumor stage and metastasis, and frequent homozygous losses of miR-31. Int J Cancer 2009;124:2236–42. Vestergaard EM, Nexo E, Torring N, Borre M, Orntoft TF, Sorensen KD. Promoter hypomethylation and upregulation of trefoil factors in prostate cancer. Int J Cancer 2010;127:1857–65. Sorensen KD, Abildgaard MO, Haldrup C, Ulhoi BP, Kristensen H, Strand S, et al. Prognostic significance of aberrantly silenced ANPEP expression in prostate cancer. Br J Cancer 2013;108:420–8. Haldrup C, Mundbjerg K, Vestergaard EM, Lamy P, Wild P, Schulz WA, et al. DNA methylation signatures for prediction of biochemical recurrence after radical prostatectomy of clinically localized prostate cancer. J Clin Oncol 2013;31:3250–8. Taylor BS, Schultz N, Hieronymus H, Gopalan A, Xiao Y, Carver BS, et al. Integrative genomic profiling of human prostate cancer. Cancer Cell 2010;18:11–22. Bert SA, Robinson MD, Strbenac D, Statham AL, Song JZ, Hulf T, et al. Regional activation of the cancer genome by long-range epigenetic remodeling. Cancer Cell 2013;23:9–22. Fuse M, Nohata N, Kojima S, Sakamoto S, Chiyomaru T, Kawakami K, et al. Restoration of miR-145 expression suppresses cell proliferation, migration and invasion in prostate cancer by targeting FSCN1. Int J Oncol 2011;38:1093–101. Cao P, Deng Z, Wan M, Huang W, Cramer SD, Xu J, et al. MicroRNA-101 negatively regulates Ezh2 and its expression is modulated by androgen receptor and HIF-1alpha/HIF-1beta. Mol Cancer 2010;9:108. Zhang X, Zhang R, Zheng Y, Shen J, Xiao D, Li J, et al. Expression of gamma-aminobutyric acid receptors on neoplastic growth and prediction of prognosis in non-small cell lung cancer. J Transl Med 2013;11:102. Huang L, Ting D, Lin X, Zhao XH, Chen XT, Wang CJ, et al. MicroRNA224 targets RKIP to control cell invasion and expression of metastasis genes in human breast cancer cells. Biochem Biophys Res Commun 2012;24;425:127–33. Li W, Wang X, Li B, Lu J, Chen G. Diagnostic significance of overexpression of Golgi membrane protein 1 in prostate cancer. Urology 2012;80:952 e1–7. Varambally S, Laxman B, Mehra R, Cao Q, Dhanasekaran SM, Tomlins SA, et al. Golgi protein GOLM1 is a tissue and urine biomarker of prostate cancer. Neoplasia 2008;10:1285–94. Laxman B, Morris DS, Yu J, Siddiqui J, Cao J, Mehra R, et al. A firstgeneration multiplex biomarker analysis of urine for the early detection of prostate cancer. Cancer Res 2008;68:645–9. Kristiansen G, Fritzsche FR, Wassermann K, Jager C, Tolls A, Lein M, et al. GOLPH2 protein expression as a novel tissue biomarker for prostate cancer: implications for tissue-based diagnostics. Br J Cancer 2008;99:939–48. Kojima S, Enokida H, Yoshino H, Itesako T, Chiyomaru T, Kinoshita T, et al. The tumor-suppressive microRNA-143/145 cluster inhibits cell migration and invasion by targeting GOLM1 in prostate cancer. J Hum Genet 2014;59:78–87. Steketee K, Ziel-van der Made AC, van der Korput HA, Houtsmuller AB, Trapman J. A bioinformatics-based functional analysis shows that the specifically androgen-regulated gene SARG contains an active direct repeat androgen response element in the first intron. J Mol Endocrinol 2004;33:477–91. Zhao JJ, Yang J, Lin J, Yao N, Zhu Y, Zheng J, et al. Identification of miRNAs associated with tumorigenesis of retinoblastoma by miRNA microarray analysis. Childs Nerv Syst 2009;25:13–20. Clinical Cancer Research Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2014 American Association for Cancer Research. Prognostic Value of GABREmiR-452miR-224 Methylation in Prostate Cancer 43. Archangelo LF, Greif PA, Holzel M, Harasim T, Kremmer E, Przemeck GK, et al. The CALM and CALM/AF10 interactor CATS is a marker for proliferation. Mol Oncol 2008;2:356–67. 44. Geerts D, Koster J, Albert D, Koomoa DL, Feith DJ, Pegg AE, et al. The polyamine metabolism genes ornithine decarboxylase and antizyme 2 predict aggressive behavior in neuroblastomas with and without MYCN amplification. Int J Cancer 2010;126:2012–24. 45. Bell JL, Wachter K, Muhleck B, Pazaitis N, Kohn M, Lederer M, et al. Insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs): posttranscriptional drivers of cancer progression? Cell Mol Life Sci 2013;70:2657–75. 46. Png KJ, Halberg N, Yoshida M, Tavazoie SF. A microRNA regulon that mediates endothelial recruitment and metastasis by cancer cells. Nature 2012;481:190–4. www.aacrjournals.org 47. de Carvalho AV, Bonamigo RR, da Silva CM, Pinto AC. Positivity for HLA DR1 is associated with basal cell carcinoma in renal transplant patients in southern Brazil. Int J Dermatol 2012;51:1448–53. 48. Jang SY, Jang SW, Ko J. Regulation of ADP-ribosylation factor 4 expression by small leucine zipper protein and involvement in breast cancer cell migration. Cancer Lett 2012;314:185–97. 49. Woo IS, Eun SY, Jang HS, Kang ES, Kim GH, Kim HJ, et al. Identification of ADP-ribosylation factor 4 as a suppressor of N(4-hydroxyphenyl)retinamide-induced cell death. Cancer Lett 2009; 276:53–60. 50. Thorsen K, Sorensen KD, Brems-Eskildsen AS, Modin C, Gaustadnes M, Hein AM, et al. Alternative splicing in colon, bladder, and prostate cancer identified by exon array analysis. Mol Cell Proteomics 2008;7:1214–24. Clin Cancer Res; 20(8) April 15, 2014 Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2014 American Association for Cancer Research. 2181 Hypermethylation of the GABRE∼miR-452∼miR-224 Promoter in Prostate Cancer Predicts Biochemical Recurrence after Radical Prostatectomy Helle Kristensen, Christa Haldrup, Siri Strand, et al. Clin Cancer Res 2014;20:2169-2181. 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