Hypermethylation of the GABRE$Mir-452$Mir-224 Promoter in Prostate Cancer Predicts Biochemical Recurrence After Radical Prostate

Clinical Cancer Imaging, Diagnosis, Prognosis Research

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, Tapio Visakorpi6, Lars Egevad7, Johan Lindberg8, Henrik Gronberg€ 8, Søren Høyer3, Michael Borre2, 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 Introduction 3 and Institute of Pathology, Aarhus University Hospital, Aarhus, Denmark; Prostate cancer is a common malignancy among males in 4Institute of Surgical Pathology, University Hospital Zurich, Zurich, Swit- zerland; 5Department of Urology, Medical Faculty, Heinrich Heine Uni- Western countries. Despite its widespread use, prostate- versity, Dusseldorf,€ Germany; 6Institute of Biomedical Technology and specific antigen (PSA) testing has suboptimal specificity BioMediTech, University of Tampere and Tampere University Hospital, Tampere, Finland; Departments of 7Oncology and Pathology and 8Med- and sensitivity for prostate cancer detection. Another major ical Epidemiology and Biostatistics, Karolinska Institute, Stockholm, challenge in prostate cancer management is to distinguish Sweden between indolent and aggressive tumors due to the limited

Note: Supplementary data for this article are available at Clinical Cancer accuracy of currently available prognostic indicators (main- Research Online (http://clincancerres.aacrjournals.org/). ly PSA, TNM stage, and Gleason score). Thus, novel biomarkers for prostate cancer are needed to improve the Corresponding Author: Karina Dalsgaard Sørensen, Department of Molecular Medicine, Aarhus University Hospital, Brendstrupgaardsvej accuracy of diagnosis and prognosis (1). Molecular biomar- 100, DK-8200 Aarhus N, Aarhus, Denmark. Phone: (+45) 78455316; kers with important biologic functions in prostate cancer Fax: (+45) 86782108; E-mail: [email protected] could also have potential as therapeutic targets. doi: 10.1158/1078-0432.CCR-13-2642 In this study, we investigated the regulation, function, 2014 American Association for Cancer Research. and biomarker potential for prostate cancer of a genomic

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MicroRNAs (miRNA) are endogenous small noncoding Translational Relevance RNAs (22 nt) that regulate gene expression at the post- Management of clinically localized prostate cancer is transcriptional level, typically by binding to sequence-spe- highly challenging due to absence of accurate prognostic cific sites in the 30UTR of target mRNAs to repress translation tools. Here, using two independent prostate cancer and/or initiate mRNA decay (13). A single miRNA may patient cohorts, including a total of 491 patients from function either as a tumor suppressor or an oncogene, five countries, we found that patients with high GAB- depending on the cellular context and its specific mRNA REmiR-452miR-224 promoter methylation were at target(s) (13). miRNAs have shown promising potential as significantly higher risk of biochemical recurrence after candidate cancer biomarkers, therapeutic targets, and anti- radical prostatectomy than patients with low methyla- cancer drugs (13). Downregulation of miR-224 in prostate tion. Although further validation is needed, our results cancer tissue samples has been reported in two recent indicate that a simple PCR-based test for GABREmiR- studies (14, 15), but another study reported upregulation 452miR-224 promoter methylation can add significant of miR-224 in perineural invasion (PNI) positive versus prognostic value to currently used routine predictors for negative prostate cancer (16). miR-452 expression in pros- prostate cancer patient outcome. We note that our study tate cancer is also poorly characterized, although a study has used radical prostatectomy specimens; however, if trans- suggested that miR-452 is overexpressed in prostate cancer ferred to diagnostic prostate biopsies, or urine or blood progenitor cells (17). Downregulation of miR-224 has been samples, such a test could be used to aid in treatment reported in ovarian, breast, and lung cancer (18–20), while decisions, for example, between active surveillance and upregulation has been reported in medullablastoma, thy- surgery. Furthermore, we found that hypermethylation roid, liver, renal, and colorectal cancer (21–25), suggesting was highly cancer-specific, suggesting that GABREmiR- cell type-specific expression and function. Although miR- 452miR-224 may also have potential as a diagnostic 224 has been linked to proliferation, migration, and inva- biomarker for prostate cancer. sion 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 pos- locus at Xq28, encompassing the GABRE gene and two sible role in progression of these malignancies (26); how- intronic microRNAs (miR-224 and miR-452). GABRE ever, its potential function in prostate cancer has not yet encodes the epsilon subunit of gamma-aminobutyric acid been studied. (GABA) A receptor (2) and has a promoter-associated CpG Here, we found that the entire GABREmiR-452miR- island (CGI). Hypermethylation of promoter CGIs is closely 224 locus was downregulated in prostate cancer compared linked with gene silencing in cancer, and aberrant promoter with nonmalignant prostate tissue samples, and that silenc- hypermethylation of specific genes, such as GSTP1 and ing was associated with frequent aberrant promoter hyper- PITX2, has shown promising diagnostic and prognostic methylation. We also found miR-224 and miR-452 to biomarker potential for prostate cancer (3–7). Here, we inhibit viability, migration, and invasion of two prostate focused on the GABREmiR-452miR-224 locus, because cancer cell lines, suggesting that they hold tumor suppressor integrative analysis of mRNA and miRNA expression and functions in prostatic cells. Finally, high GABREmiR- DNA methylation profiling data available in-house indicat- 452miR-224 methylation was a significant independent ed significant downregulation accompanied by aberrant adverse prognostic factor for biochemical recurrence after promoter hypermethylation in prostate cancer. Moreover, radical prostatectomy in a large prostate cancer patient results from several studies (see below) suggested potentially cohort (n ¼ 293, training cohort), which was successfully important functional role(s) for this locus in prostate cancer. confirmed in an independent validation cohort (n ¼ 198). Deregulation of GABRE expression in prostate cancer has Materials and Methods not been reported before this work. The GABAA receptor is a heteropentameric chloride channel that transmits the fast- Clinical samples est inhibitory signal of the central nervous system (CNS) Fresh-frozen tissue-tek (Bayer Corporation) embedded with its ligand GABA (8). The GABAA receptor family con- radical prostatectomy and transurethral resection of the sists of 19 homologous genes, encoding subunits a1-a6, b1- prostate (TURP) tissue specimens were collected at Depart- b3, g1-g3, d, r1-r3, q, p, and e (GABRE). The dominant ment of Urology, Aarhus University Hospital, Denmark receptor subtype is believed to be 2a:2b:1g. GABRE resem- (Supplementary Table S1) and used for bisulfite sequencing bles the g-subunit and may replace this to form a functional and Infinium analyses. receptor (2, 9). Outside the CNS, elevated GABA levels have For MethyLight analyses, two radical prostatectomy been associated with prostate cancer metastasis (10) and cohorts were used (Table 1). RP cohort 1 (training) con- GABA agonist isoguvacine has been shown to stimulate sisted of consecutive curatively intended radical prostatec- proliferation of four prostate cancer cell lines (11). In tomies of histologically verified clinically localized prostate contrast, GABA signaling has been found to inhibit breast cancer from patients treated at Departments of Urology, cancer cell migration (12), suggesting a highly context- Aarhus University Hospital (Aarhus, Denmark; collected dependent function. 1997–2005), and University Hospital Zurich (Zurich,

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Table 1. Clinicopathologic characteristics of patient sample sets used for MethyLight analyses

RP cohort 1, RP cohort 2, Prostate cancer samples training (n ¼ 293) validation (n ¼ 198) MPC (n ¼ 26) CRPC (n ¼ 13) LMN (n ¼ 15) Median age, range 63 (46–73) 63 (47–75) 71 (52–89) 64 (49–77) 63 (54–71) PSA at diagnosis, n (%) 10 ng/mL 114 (38.9) 121 (61.1) 2 (7.7) 1 (3.6) 2 (13.3) >10 ng/mL 178 (60.8) 69 (34.8) 24 (92.3) 12 (32.1) 11 (73.3) Unknown 1 (0.3) 8 (4.0) —— 2 (13.3) Pathologic T stage, n (%) T1 0 (0.0) 1 (0.5) NA NA NA T2a-c 184 (62.8) 112 (56.6) NA NA NA T3a-b 106 (36.2) 83 (41.9) NA NA NA T4 3 (1.0) 0 (0.0) NA NA NA Unknown 0 (0.0) 2 (1.0) Gleason score, n (%) 3–6 113 (38.6) 72 (36.4) 2 (7.7) 0 (0.0) NA 7 143 (48.8) 90 (45.5) 5 (19.2) 3 (23.1) NA 8–10 36 (12.3) 32 (16.2) 19 (73.1) 10 (76.9) NA Unknown 1 (0.3) 4 (2.0) —— Nodal status, n (%) pN0 253 (86.3) 40 (20.2) NA NA — pN1 5 (1.7) 1 (0.5) NA NA 15 (100) Unknown 35 (11.9) 157 (79.3) NA NA — Surgical margin status, n (%) Negative 196 (66.9) 71 (35.9) NA NA NA Positive 93 (31.7) 57 (28.8) NA NA NA Unknown 4 (1.4) 70 (35.4) NA NA NA Recurrence status, n (%) No recurrence 170 (58.0) 103 (52.0) NA NA NA Recurrence 123 (42.0) 95 (48.0) NA NA NA Mean follow-up time, months (range) 49 (2–151) 60.4 (1–181) NA NA NA

Nonmalignant samples AN (n ¼ 18) BPH (n ¼ 17) PIN (n ¼ 11) —— Median age (range) 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 (Finland; collected 1992–2003), and Karolinska University radical prostatectomies, we could retrieve formalin-fixed Hospital (Stockholm, Sweden; collected 2003–2007). A and paraffin-embedded (FFPE) tissue blocks from 457 trained pathologist dissected the prostate immediately after patients (see Supplementary Fig. S1A for flow chart of radical prostatectomy and tumor samples were snap-frozen. inclusion/exclusion criteria according to REMARK guide- On the basis of H&E-stained sections, DNA was extracted lines). All specimens were evaluated by a trained patholo- from samples with >70% tumor content. Inclusion/exclu- gist, representative regions with >90% tumor were marked sion criteria were employed as for cohort 1 (Supplementary on hematoxylin and eosin (H&E)-stained sections, and Fig. S1A). Final analyses comprised 198 radical prostatec- punch biopsies were taken from the corresponding FFPE tomy samples (Table 1). blocks for DNA and RNA extraction. Of the 457 patients, FFPE adjacent nonmalignant (AN), benign prostatic 164 were excluded due to either lack of follow-up (n ¼ 56), hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), pre-/postoperative endocrine treatment (n ¼ 40), or poor hormone-na€ve metastatic prostate cancer (MPC, primary DNA quality (n ¼ 68). Final analyses included 293 radical tumor), and castration-resistant metastatic prostate cancer prostatectomy patients (Table 1). (CRPC, primary tumor) were collected, evaluated, and biop- RP cohort 2 (validation) consisted of 310 curatively sied as described for cohort 1 (Table 1; Supplementary intended radical prostatectomies of histologically verified Fig. S1B). clinically localized prostate cancer from Departments The study was approved by relevant ethical committees in of Urology, Heinrich Heine University (Dusseldorf,€ Ger- each country. Written informed consent was obtained from many; collected 1993–2002), Tampere University Hospital all patients.

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DNA extraction and methylation analysis Epithelial Cell Basal Medium with added Prostate Epithelial Bisulfite sequencing (biSeq) was performed as described Growth Kit (all from American Type Culture Collection). previously (27, 28). A 441 nt region (containing 52 CpG The authenticity of all cell lines was verified by short tandem sites) of the GABREmiR-452miR-224 promoter was repeat analysis (http://IdentiCell.eu). PC3 and DU145 cells amplified using PCR primers: 50-AGATGTTTAGGAGGATTG- were reverse transfected with 20 nmol/L of each miRNA GAAGAAT-30 and 50-ACCCCACATCTTATCCCTAAAACAA- (miRNA mimics purchased from Applied Biosystems/ 30. At least 7 clones were sequenced per sample. Ambion) using Lipofectamine 2000 reagent (Invitrogen). MethyLight. For Danish and Swiss FFPE samples, DNA Medium was changed 6 hours posttransfection. Transfec- was extracted using gDNA Eliminator columns from the tion efficiencies were examined by fluorescence microscopy miRneasy FFPE Kit (Qiagen) and the blood and cell culture after transfection of Cy3-conjugated scrambled miRNA DNA kit (Qiagen), respectively. For Swedish and Finnish (Ambion) and/or verified by qRT-PCR (see below). Cell samples, DNA extraction was conducted using the AllPrep viability, migration, and invasion assays were performed as Mini DNA/RNA kit (Qiagen), and for German samples described in Supplementary Methods. the Blood and cell culture DNA kit (Qiagen). In all cases, DNA was bisulfite converted using the EZ-96 DNA Meth- Gene Set Enrichment Analysis RNA extraction, qRT- ylation-Gold Kit (Zymo Research). The following primers/ PCR, and ChIP analyses probes were used for quantitative methylation-specific For details, see Supplementary Methods PCR (MethyLight) analysis of the GABREmiR-452miR- 224 promoter: 50-GATGTTTAGGAGGATTGGAAGA-30 and Statistical analyses 50-CTCCGCGCAAATAATCG-30 plus FAM-50-ATATTTTCG- STATA version 11 (StataCorp) was used for all statistical CGGAGATCGGC-30-BHQ1. For normalization and test of analyses. P values <0.05 were considered statistically signif- input DNA quality and quantity, a CpG-free region of icant. Phenotypic effects in transfected cell lines and gene MYOD1 was analyzed in parallel using primers/probe: 50- expression differences were assessed by two-sided Student CCAACTCCAAATCCCCTCTCTAT-30 and 50-TGGTTTTTTT- t tests, and methylation differences by the Mann–Whitney AGGGAGTAAGTTTGTT-30 plus FAM-50-TCCCTTCCTATT- U test. Correlations between expression and methylation CCTAAATCCAACCTAAATACCTCC-30-BHQ1. All reactions were analyzed by Spearman rank test. Associations between were run in triplicates in 384-well plates using the Applied GABREmiR-452miR-224 methylation and clinicopath- Biosystems 7900HT Sequence Detection System and Taq- ologic variables were assessed using Spearman rank test or Man Universal PCR Master Mix without AmpErase UNG the Mann–Whitney U test. The diagnostic potential of (Applied Biosystems). Samples with low input DNA (CT GABRE miR-452 miR-224/MYOD1 methylation was (MYOD1) >36 in at least 2/3 reactions) were excluded from evaluated by receiver operator characteristics (ROC) curve further analysis. The relative GABREmiR-452miR-224 pro- analysis. moter methylation level in each sample was determined as For survival analysis, biochemical/PSA recurrence (Dan- the GABREmiR-452miR-224/MYOD1 ratio. The accuracy ish, Swiss, Swedish, and German samples cutoff 0.2 of the GABREmiR-452miR-224/MYOD1 assay was vali- ng/mL, Finnish samples cutoff 0.5 ng/mL, based on local dated by direct comparison with biSeq data from several clinical practice) was used as endpoint. Patients not having prostate (cancer) tissue samples and cell lines, and by analysis experienced biochemical recurrence were censored at their of dilution series of fully methylated/unmethylated control last normal PSA measurement. The prognostic value of DNA (data not shown). GABREmiR-452miR-224/MYOD1 methylation was eval- All primers and probes were purchased from Eurofins uated by Kaplan–Meier analysis and two-sided log-rank test, MWG Operon. and by univariate and multivariate Cox regression analysis Infinium arrays. One microgram of DNA was bisulfite as a continuous as well as a dichotomous variable. For converted, whole genome amplified, and applied to Illumi- analysis of GABREmiR-452miR-224/MYOD1 methyla- na’s Infinium HumanMethylation27 Beadchip v1.2 (29) or tion as a dichotomous variable, patients in cohort 1 were HumanMethylation450 BeadChip (Strand and colleagues, divided into high and low methylation groups using a cutoff unpublished) according to protocols from the manufacturer. value determined by ROC analysis of recurrence/nonrecur- rence status optimized for high specificity. For validation, Cell culture patients in cohort 2 were divided into two groups using the Prostatic cell lines PNT1A (European Collection of Cell cutoff (fraction) defined in cohort 1. The proportional Cultures), BPH1 (German Collection of Microorganisms hazards assumption was verified by the log-negative-log and Cell Cultures), DuCaP and VCaP (kindly provided by survival distribution function for all variables. Prognostic Dr. Kenneth Pienta, University of Michigan, Ann Arbor, accuracy was estimated using Harrell Concordance Index. MI), 22rv1, LNCaP, PC3, and DU145 (American Type Culture Collection) were propagated in RPMI-1640 with Results L-glutamine (Invitrogen) supplemented with 10% heat- Silencing of the GABRE/miR-452/miR-224 locus in inactivated fetal calf serum, 100 U/mL penicillin, and prostate cancer 100 mg/mL streptomycin at 37C in humidified air atmo- By microarray and qRT-PCR analyses, we observed sig- sphere at 5% CO2. PrEC cells were cultured in Prostate nificant downregulation of GABRE, miR-224, and miR-452

2172 Clin Cancer Res; 20(8) April 15, 2014 Clinical Cancer Research

P -8 A P = 6.56×10-4 B D = 1.87×10 * * P = 0.001 * 8.5 ) ) ) 2 2 2 8 9 10 11 468 GABRE (log GABRE (log GABRE (log 6.5 7 7.5 8 6 67 2 Nonmalignant PC Nonmalignant PC Nonmalignant PC (n = 14) (n = 36) (n = 10) (n = 15) (n = 29) (n = 150)

C P = 6.28×10-5 * P = 5.89×10-8* P = 3.79×10-11 * ) 2 3 3.2 ) ) 2 2 2.6 2.8 miR-224 (log miR-224 (log 12345 345678 2.4 miR-224/RNU44 (log 0 Nonmalignant PC Nonmalignant PC Nonmalignant PC n n n (n = 14) ( = 36) (n = 27) (n = 80) ( = 28) ( = 80)

P = 7.45×10-10 * P -5 3 = 4.94×10 * P = 9.11×10-7* ) 2 2.8 ) ) 2 2 10 15 2.4 2.6 miR-452 (log miR-452 (log miR-452/RNU44 (log 05 34567 2.2 Nonmalignant PC Nonmalignant PC Nonmalignant PC (n = 14) (n = 36) (n = 27) (n = 80) (n = 28) (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 The GABRE/miR-452/miR-224 promoter CpG island is tissue samples in multiple patient sample sets (Fig. 1A–C). hypermethylated in prostate cancer For external validation, expression data from Taylor and Next, we investigated whether downregulation of the colleagues (30) was used, confirming the significant down- GABREmiR-452miR-224 locus in prostate cancer was regulation of all three genes in prostate cancer (Fig. 1D). The associated with hypermethylation of its promoter-associat- findings were further supported by nine transcription pro- ed CGI (Fig. 2A). Two methylation microarray datasets filing datasets from the oncomine database, all showing (29), including a total of 30 nonmalignant and 30 prostate downregulation of GABRE in prostate cancer (Supplemen- cancer tissue samples, showed significant (P < 0.001) pro- tary Table S2). Moreover, significant (P < 0.001) direct moter hypermethylation in prostate cancer samples (Fig. correlations between the expression of GABRE, miR-224, 2B). This finding was corroborated by genomic biSeq of an and miR-452 were evident in clinical sample sets (Spearman independent set of 11 nonmalignant and 21 prostate cancer correlation coefficient, r ¼ 0.42–0.94) and in prostatic cell tissue samples (Fig. 2C and Supplementary Fig. S3). lines (r ¼ 0.91–0.96; Supplementary Fig. S2A–S2D), con- For large-scale validation, we designed a MethyLight assay sistent with transcriptional coregulation. for a region of the GABREmiR-452miR-224 promoter,

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A Bisulfite sequencing –351 +144 (52 CpGs) MethyLight TSS –270 –351 (7 CpGs) +14967 +15051 +16021 +16101 GABRE Exon 1 +1 (ATG) miR-452 miR-224 Intron 6

–404 CpG island +470 (83 CpGs)

BC27K dataset 450K dataset BiSeq dataset

0.6 P = 0.006 1.0 Nonmalignant (n = 21) P = 1.00 P = 0.26 PC (n = 21) 0.8

0.4 100806040 –7 0.6 P = 6.98 × 10

0.4 β -value β -value 0.2 0.2

0.0

0 methylation Non- PC malignant n 20 n ( = 9) 0

( = 9) cg08783090 cg25528646 cg07053880 cg01480550 cg12204574 AN BPH LPC MPC % GABRE~miR-452~miR-224 cg12204574 CpG island (n = 7) (n = 4) (n = 10) (n = 11)

D P = 3.56 × 10–19

MethyLight dataset

NS Methylation Methylation

Sensitivity AUC = 0.98 Sensitivity = 95.5% GABRE~miR-224~miR-452/MYOD1

GABRE~miR-224~miR-452/MYOD1 Specificity = 94.3% 01 234 01 234

Nonmalignant PC 0.00 0.25 0.50 0.75 1.00 AN BPH PIN LPC CRPC MPC LNM 0.00 0.25 0.50 0.75 1.00 (n = 35) (n = 245) (n = 18) (n = 17) (n = 11)(n = 191) (n = 13) (n = 26) (n = 15) 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, castration- resistant prostate cancer (primary tumor); LMN, lymph node metastasis. NS, not significant.

including 7 CpG sites also analyzed by biSeq (Fig. 2A). A and advanced prostate cancer samples; Table 1). Overall, CpG-free region of MYOD1 was used for normalization GABREmiR-452miR-224 was signiﬁcantly (P ¼ 3.56 (represents total input of bisulﬁte-converted DNA). GAB- 10 19; Mann–Whitney U test) hypermethylated in prostate REmiR-452miR-224/MYOD1 methylation was deter- cancer compared with nonmalignant tissue samples mined in 291 FFPE tissue samples (191 LPC/RP samples (Fig. 2D, left). Also, GABREmiR-452miR-224 was hyper- from cohort 1, plus several nonmalignant, premalignant, methylated in 3 of 11 PIN samples, suggesting it could be an

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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 miR- 224 (Spearman rho, r ¼0.41) or miR-452 expression (r ¼ 0.43), respectively, was seen in the clinical samples (Supple- mentary Fig. S2C). Likewise, expression of GABRE, miR-224, and miR-452 was strongly inversely correlated with promoter methylation (r ¼0.83 to 0.88) in 9 prostatic cell lines (Supplementary Fig. S2E). GABREmiR-452miR- 224 was hypermethylated in 4 of 6 prostate cancer cell lines and in 0 of 3 nonmalignant prostate cell lines (Supplemen- tary 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-452miR- 224 promoter (Supplementary Fig. S4D and S4E). To assess the diagnostic potential of GABREmiR- 452miR-224 methylation, we performed ROC curve analysis using the large MethyLight dataset (Fig. 2D, left). The preliminary sensitivity and specificity of GABREmiR- 452miR-224 methylation for prostate cancer detection was 95.5% and 94.3%, respectively (Fig. 2D, right). Togeth- er, 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 Figure 3. Ectopic expression of miR-224 or miR-452 inhibited viability, region (31). Focal hypermethylation of the GABREmiR- 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 (20 452 miR-224 promoter within this otherwise activated and nmol/L) in PC3 and DU145 cells, as determined by an MTT assay. All hypomethylated region (Supplementary Fig. S5) suggests results are presented relative to scrambled (scr) miRNA transfected cells. that epigenetic silencing of GABREmiR-452miR-224 is B and C, Trevigen Cultrex Migration and Invasion assays performed 48 highly specific and may be selected for during prostate hours posttransfection. Background migration levels for nontransfected cancer development and/or progression. 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 miR-224 and miR-452 inhibit viability, migration, and (A-C) are representative of a minimum of three experiments performed in invasion of prostate cancer cells triplicate; all significant phenotypic effects, that is, P < 0.05 (two-sided t Next, we investigated the potential functional roles of miR- Student test) and >25% reduction, are marked by an asterisk ( ). 224 and miR-452 in prostate cancer (GABRE was not analyzed as we were unable to validate a good antibody; data not Reintroduction of either miR-224 or miR-452 had a shown). miR-224 or miR-452 mimics were transfected into moderate (25%) and statistically significant (P < 0.05) two prostate cancer cell lines (PC3 and DU145) with low inhibitory effect on cell viability in both PC3 and DU145 endogenous expression (Supplementary Fig. S4B). As posi- cells, as evident at 72 hours posttransfection but not at 48 tive controls, we used miR-145 in cell viability assays (known hours (Fig. 3A). Furthermore, ectopic expression of miR- to reduce PC3 and DU145 cell viability; ref. 32) and miR-101 224 or miR-452 significantly (P < 0.05) reduced migration in cell migration/invasion assays (known to inhibit migra- and invasion of PC3 and DU145 cells at 48 hours post- tion and invasion of PC3 and DU145 cells; ref. 33). Scram- transfection (Fig. 3B and C), and hence was not simply bled miRNA was used as a negative control. Transfection ascribed to miRNA-induced reduction in cell viability, as efficiencies were confirmed by qRT-PCR (data not shown). this occurred at 72 hours. Cotransfection of miR-224 and

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miR-452 did not cause further inhibition of cell viability, surgical margin status in at least one cohort (Supplementary migration, or invasion (Fig. 3A–C), suggesting at least some Fig. S7B–S7E). functional redundancy between these miRNAs. Subsequent- To assess the prognostic value of GABREmiR-452miR- ly, we performed GSEA of microarray transcriptional profil- 224 methylation, we performed PSA-based recurrence-free ing data obtained at 48 hours posttransfection. For genes survival (RFS) analysis. By univariate Cox regression anal- (mRNA) downregulated upon miR-224 or miR-452 trans- ysis of GABREmiR-452miR-224 methylation (as a con- fection, the top ten most significantly enriched "Canon- tinuous variable) in RP cohort 1 (training cohort, n ¼ 293; ical Pathways" in both DU145 and PC3 cells (Supple- see Table 1), short RFS time was significantly associated with mentary Fig. S6A and S6B) were primarily associated with high GABREmiR-452miR-224 methylation, as well as positive regulation of the cell cycle, DNA replication, and with the established prognostic factors high PSA, high mitosis, consistent with the observed negative effect of Gleason score, advanced T-stage, and positive surgical mar- these miRNAs on prostate cancer cell viability (Fig. 3A). gin status (P < 0.001; Table 2). All variables remained GSEA was also performed for genes significantly upregu- significant in a multivariate model (Table 2), indicating lated upon miR-224 or miR-452 transfection, likely repre- that GABREmiR-452miR-224 methylation is a signifi- senting indirect effects. Several "Canonical Pathways" cant independent prognostic factor for prostate cancer related to cellular adhesion and motility were enriched patients treated by radical prostatectomy. The prognostic for both miR-224 and miR-452 (Supplementary. Fig. S6C value of GABREmiR-452miR-224 methylation was suc- and S6D) in agreement with their effects on migration cessfully validated by univariate as well as multivariate and invasion (Fig. 3B and C). analysis in cohort 2 (validation cohort, n ¼ 198; Among the genes most heavily downregulated in both cell see Table 1; Table 2). In cohort 2, surgical margin status lines after miR-224 and miR-452 overexpression, respective- was left out in the final model, because of missing data for ly (Supplementary Tables S3 and S4), we shortlisted poten- 70 patients. Predictive accuracies were estimated by Harrell tial target genes using the following criteria: (i) mRNAs C-index, which increased from 0.73 to 0.74 in cohort 1, and containing at least one 7mer target site predicted by both from 0.75 to 0.77 in cohort 2 by addition of GABREmiR- TargetScan (www.targetscan.org) and PicTar (http://pictar. 452miR-224 methylation, suggesting moderately mdc-berlin.de), and (ii) mRNAs significantly upregulated in improved model performance. The contribution of GAB- prostate cancer versus nonmalignant prostate tissue samples REmiR-452miR-224 methylation to the multivariate in multiple patient sample sets (Supplementary Table S5). model was comparable with that of a single clinicopatho- Among eight shortlisted potential miR-224 target genes, logic variable: Harrell C-index of the multivariate model expression of C1orf116 (chromosome 1 open reading frame (cohort 1/cohort 2) when leaving out either PSA (0.72/ 116, also known as specifically androgen-regulated gene, 0.76), T-stage (0.72/0.74), Gleason score (0.73/0.73), or SARG), GOLM1 (golgi membrane protein 1), and FAM64A surgical margin status (0.73/not applicable). (family with sequence similarity 64, member A) was signif- Similar results were obtained when GABREmiR- icantly inversely correlated with miR-224 expression in the 452miR-224 methylation was analyzed as a dichotomous datasets from Taylor and colleagues (30) and Mortensen and variable in uni- and multivariate Cox regression analyses colleagues (Spearman r: 0.49 to 0.25; P < 0.05; Supple- (Supplementary Table S6). For these analyses, patients in mentary Table S5), suggesting that these genes could be cohort 1 were divided into low/high methylation subgroups clinically relevant. Moreover, among eight shortlisted poten- using a cutoff value determined by ROC curve analysis tial miR-452 target genes, expression of DR1 (downregulator (optimized for high specificity). Likewise, patients in cohort of transcription 1) and IGF2BP2 [insulin-like growth factor 2 were divided into low/high methylation subgroups using 2 mRNA binding protein 2 (p62)] was significantly inversely the cutoff (fraction) defined in cohort 1. Finally, by Kaplan– correlated with miR-452 in the dataset from Mortensen and Meier analysis, high GABREmiR-452miR-224 methyla- colleagues (r: 0.47 to 0.30; P < 0.05), but no significant tion was significantly (P < 0.001, log-rank test) associated correlations were seen in the Taylor and colleagues dataset with short RFS in cohort 1 (Fig. 4A), which was successfully (Supplementary Table S5). validated (P < 0.001) in cohort 2 (Fig. 4B). In summary, we found GABREmiR-452miR-224 methylation to be a sig- Prognostic value of GABRE/miR-452/miR-224 nificant independent prognostic predictor of biochemical methylation and association with clinicopathological recurrence in two radical prostatectomy patient cohorts. parameters Finally, to assess the possible prognostic value of GABRE, Next, we investigated possible correlations between clin- miR-452, and miR-224 expression, we used the large radical icopathologic variables and GABREmiR-452miR-224 prostatectomy patient cohort from Taylor and colleagues methylation in two independent radical prostatectomy (30). A multigene model including the expression of all cohorts (Table 1; Supplementary Fig. S1A). There was no three genes was a significant independent predictor of significant correlation between GABREmiR-452miR-224 biochemical recurrence after radical prostatectomy (HR, methylation and age at diagnosis (Supplementary. Fig. 2.37; P ¼ 0.013; Supplementary Table S7). Expression of S7A), but high methylation was significantly (P < 0.05) either GABRE, miR-224, or miR-452 individually was not associated with the established adverse prognostic factors significant in multivariate analysis, but GABRE and miR- high PSA, high T-stage, high Gleason score, and positive 224 were significant in univariate analysis (Supplementary

2176 Clin Cancer Res; 20(8) April 15, 2014 Clinical Cancer Research

Table 2. Uni- and multivariate Cox regression analyses of biochemical recurrence-free survival in two RP cohorts Univariate Multivariate

Variable Characteristics HR (95% CI) P C-indexa HR (95% CI) P C-indexa Cohort 1 (training), n ¼ 293 Age at diagnosis Continuous 0.98 (0.95–1.01) 0.211 0.55 —— Preoperative PSA 10 vs. >10 ng/mL 2.67 (1.75–408) <0.001 0.61 2.37 (1.53–3.69) <0.001 0.74 0.73 Tumor stage pT2a-c vs. pT3a-c 3.37 (2.35- 4.84) <0.001 0.65 2.06 (1.37–3.10) 0.001 Surgical margin status Negative vs. positive 3.03 (2.10–4.36) <0.001 0.63 1.99 (1.33–2.98) 0.001 Gleason score Continuous 1.48 (1.22–1.80) <0.001 0.59 1.27 (1.02–1.58) 0.033 GABREmiR-452miR-224 Continuous 1.75 (1.37–2.23) <0.001 0.61 1.38 (1.06–1.81) 0.019 methylation

Cohort 2 (validation), n ¼ 198 Age at diagnosis Continuous 1.02 (0.99–1.06) 0.206 0.53 —— Preoperative PSA 10 vs. >10 ng/mL 2.20 (1.45–3.34) <0.001 0.59 2.22 (1.45–3.41) <0.001 0.77 0.75 Tumor stage pT2a-c vs. pT3a-c 3.60 (2.36–5.49) <0.001 0.66 2.58 (1.63–4.08) <0.001 Surgical margin statusb Negative vs. positive 2.50 (1.44–4.37) 0.001 0.62 —— Gleason score Continuous 1.84 (1.50–2.25) <0.001 0.69 1.71 (1.45–3.41) <0.001 GABREmiR-452miR-224 Continuous 2.99 (1.71–5.21) <0.001 0.65 2.45 (1.26–4.75) 0.008 methylation

aPredictive 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). bMargin 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 miR- predicted miR-452 target) had signiﬁcant adverse prognos- 452 potential target genes (Supplementary Table S5), high tic value in multivariate analysis in the Taylor and colleagues expression of OAZ2 (Ornithine Decarboxylase Antizyme 2; dataset (Supplementary Table S8). A multigene model

ABCohort 1 Cohort 2

Low meth. Low meth.

High meth. High meth. Cumulative survival Cumulative survival Cumulative

P < 0.001 P < 0.001 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0 25 50 75 100 125 0 25 50 75 100 125 Recurrence-free survival time (months) Recurrence-free survival time (months)

Number at risk

Low meth. 193 141 93 33 21 8 130 107 88 64 27 12 High meth. 100 67 40 18 5 1 68 40 29 16 1 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-452miR- 224 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 2177

including GABRE, miR-224, miR452, and OAZ2 expression miR-224/miR-452 expression signature was a significant inde- also remained significant in a multivariate model (Supple- pendent predictor of PSA recurrence after radical prostatec- mentary Table S8), further supporting a clinically relevant tomy in this cohort. Low expression was associated with role of the GABREmiR-224miR-452 locus and its expres- higher recurrence risk, consistent with an important role sion circuit in prostate cancer. of coordinated epigenetic silencing of the GABREmiR- 452miR-224 locus in prostate cancer. Discussion Our work showed that miR-224 and miR-452 inhibit This study showed that the GABREmiR-452miR-224 proliferation, migration, and invasion of two prostate can- locus is downregulated in prostate cancer and that aberrant cer cell lines. The GABREmiR-452miR-224 locus has not GABREmiR-452miR-224 promoter hypermethylation is a been functionally linked to prostate cancer before, but very common event in prostate cancer, suggesting it is a key earlier studies have shown involvement of miR-224 in both molecular mechanism for coordinated silencing of this locus repression and stimulation of cell viability, migration, and/ in prostate cancer cells. Moreover, GABREmiR-452miR- or invasion in other cancer types (20, 22, 35). None of the 224 hypermethylation was found to be a significant inde- validated miR-224 targets from these studies (e.g., KLK10 in pendent predictor of biochemical recurrence in two radical ovarian cancer and RKIP in breast cancer; refs. 20, 35) were prostatectomy patient cohorts from five European countries. significantly affected, when we overexpressed miR-224 in To the best of our knowledge, this is the first comprehensive PC3 or DU145 cells (data not shown), consistent with a cell study of GABREmiR-452miR-224 expression in prostate type and context-dependent function of this miRNA. To our cancer as well as the first report to identify GABREmiR- knowledge, there are no previous reports of a functional role 452miR-224 as a promising diagnostic and prognostic nor validated target for miR-452. Using an integrative methylation marker candidate for prostate cancer. bioinformatic approach, we shortlisted 16 predicted miR- Consistent with our results, downregulation of miR-224 224 or miR-452 target genes of potential clinical importance in prostate cancer tissue samples has been reported in two in prostate cancer (Supplementary Table S5). Among these, recent studies (14, 15). Another study has reported miR-224 we identified the three predicted miR-224 target genes upregulation in PNI-positive versus negative prostate cancer C1orf116, GOLM1, and FAM64A that were not only down- (16), but used only a small sample set without nonmalig- regulated in PC3 and DU145 cells upon miR-224 over- nant prostate samples for comparison. PNI status was not expression, but also significantly upregulated in prostate determined in our sample set. Moreover, although one cancer tissue versus nonmalignant tissue samples in mul- earlier study has reported upregulation of miR-452 in tiple patient sets. Similarly, we identified predicted miR-452 prostate cancer progenitor cells (17), differential expression target genes, including IGF2BP2, DR1, and OAZ2. in prostate cancer patient samples has not been previously These genes have all been previously linked to cancer and described. There are no prior reports of GABRE deregulation in some cases prostate cancer specifically. Thus, GOLM1 has in prostate cancer, but upregulation has been reported in been suggested as a putative tissue and urine biomarker of non–small cell lung cancer (34). prostate cancer (36–39) and shown to induce migration In this study, GABREmiR-452miR-224 promoter meth- and invasion of prostate cancer cells (40). C1orf116 has ylation was found to be highly cancer-specific (AUC ¼ 0.98) been reported as highly expressed in prostate cancer tissue and thereby comparable to previously reported top candidate (41), whereas FAM64A is highly expressed in leukemia and methylation markers for prostate cancer detection, including lymphoma, and known to promote cell division (42, 43). GSTP1 (5). Furthermore, our findings indicated that GAB- Moreover, we found that high expression of the predicted REmiR-452miR-224 methylation has significant indepen- miR-452 target gene OAZ2 was associated with high recur- dent prognostic value for prediction of biochemical recurrence risk after radical prostatectomy in the patient cohort rence after radical prostatectomy, beyond routine clinico- from Taylor and colleagues. Expression of OAZ2 has pre- pathologic predictors. The prognostic potential of GAB- viously been associated with aggressive behavior in neuro- REmiR-452miR-224 methylation was identified in a blastoma (44). IGF2BP2 expression has been associated training cohort from Denmark and Switzerland and success- with liver cancer, liposarcoma, and endometrial adenocar- fully validated in an independent patient set from Germany, cinomas, and with promotion of cell migration and metas- Finland, and Sweden, supporting the validity of our finding. tasis (45, 46). High expression of DR1 is associated with Before the present study, PITX2 (3, 4) and C1orf114 (29) were basal cell carcinoma in renal transplant patients (47). the only single genes with independent prognostic value Another interesting potential miR-452 target gene, ARF4 reported in tworadical prostatectomy cohorts.Further studies (ADP-ribosylation factor 4), has been shown to promote including large prospective cohorts are needed to assess the breast cancer cell migration (48) and to inhibit apoptosis in clinical utility of GABREmiR-452miR-224 methylation as glioblastoma (49). a prognostic candidate marker alone and in combination Additional studies are needed to investigate the molec- with other candidate molecular markers. ular pathways and target genes regulated by miR-224 and The significant prognostic value of GABREmiR-452miR- miR-452 in prostatic cells in more details. Moreover, the 224 methylation was also reflected at the expression level, potential functional role of the host gene GABRE remains to when we analyzed the radical prostatectomy cohort from be investigated. Despite several attempts to overexpress Taylor and colleagues (30). Thus, a three-gene GABRE/ GABRE in prostate cancer cell lines, we observed no

2178 Clin Cancer Res; 20(8) April 15, 2014 Clinical Cancer Research

phenotypic effects (data not shown); however, we were time to recurrence is also influenced by the frequency and unable to validate GABRE expression at the protein level exact timing of PSA testing after radical prostatectomy, due to lack of a specific antibody. Elevated GABA levels have which despite regulation by clinical guidelines also may previously been associated with prostate cancer metastasis vary between patients in real life. Future studies should (10) and GABA agonist isoguvacine has been shown to investigate other clinical endpoints such as cancer-specific stimulate proliferation of some prostate cancer cell lines and overall survival. Given the long disease course of (11), but the exact composition(s) of the GABA receptor(s) prostate cancer this would require large cohorts with at mediating these effects are not known. Furthermore, GABA least 10 to 20 years of follow-up. signaling has been found to inhibit breast cancer cell In conclusion, our results showed that the GAB- migration (12), suggesting a context-dependent function. REmiR-452miR-224 locus is epigenetically silenced in We note that all methylation analyses were performed prostate cancer compared with nonmalignant prostate using radical prostatectomy tissue specimens. A prognostic tissue samples. Furthermore, our findings indicated that test based on GABREmiR-452miR-224 methylation miR-224 and miR-452 hold important tumor suppressor would currently have limited clinical utility at this stage, functions in prostate cancer. Finally, we identified the as there are no established adjuvant therapies after radical GABREmiR-452miR-224 promoter as a promising new prostatectomy. In future studies, it will therefore be impor- candidate methylation marker for prostate cancer detec- tant to investigate if GABREmiR-452miR-224 methylation and for prediction of biochemical recurrence after tion levels measured in prostate biopsies, or in a urine or radical prostatectomy. blood sample, taken at the time of diagnosis can predict prostate cancer aggressiveness and thus guide treatment Disclosure of Potential Conflicts of Interest decisions. Conceivably, a high level of GABREmiR- No potential conflicts of interest were disclosed. 452miR-224 methylation in a biopsy may favor intervention (e.g., surgery) over active surveillance. Likewise, an Authors' Contributions Conception and design: H. Kristensen, S. Høyer, M. Borre, T.F. rntoft, increase in GABRE miR-452 miR-224 methylation in K.D. Sørensen biopsies taken during active surveillance could indicate Development of methodology: H. Kristensen, C. Haldrup, S. Høyer, K.D. disease progression, and hence the need for intervention. Sørensen Acquisition of data (provided animals, acquired and managed patients, Similarly, to assess the clinical utility of GABRE miR- provided facilities, etc.): H. Kristensen, S. Strand, K. Mundbjerg, M.M. 452miR-224 methylation for prostate cancer diagnosis, Mortensen, P.J. Wild, C. Arsov, T. Visakorpi, L. Egevad, J. Lindberg, future studies should investigate whether detection of H. Gronberg,€ S. Høyer, M. Borre, K.D. Sørensen Analysis and interpretation of data (e.g., statistical analysis, biosta- tumor-derived hypermethylated GABRE miR-452 miR- tistics, computational analysis): H. Kristensen, K. Mundbjerg, M.M. 224 DNA in biofluids, such as urine or blood, can be used Mortensen, K. Thorsen, M.S. Ostenfeld, S. Høyer, T.F. rntoft, K.D. Sørensen Writing, review, and/or revision of the manuscript: H. Kristensen, to identify patients with prostate cancer. Given the very high C. Haldrup, K. Mundbjerg, M.S. Ostenfeld, P.J. Wild, C. Arsov, W. Goering, frequency and cancer specificity of GABREmiR-452miR- T. Visakorpi, H. Gronberg,€ S. Høyer, M. Borre, T.F. rntoft, K.D. Sørensen 224 promoter hypermethylation that we observed in radical Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): C. Haldrup, M.M. Mortensen, P.J. prostatectomy tissue samples, it is possible that a methyl- Wild, W. Goering, J. Lindberg ation test for GABREmiR-452miR-224 in biofluids could Study supervision: C. Haldrup, M.M. Mortensen, K.D. Sørensen be used as a supplement to for example, a PSA test (high sensitivity, low specificity) to increase the accuracy of diag- Acknowledgments nosis. Furthermore, premalignant and/or cancer field effects The authors thank Anne Slotsdal, Pamela Celis, Maria Mark, Gitte Høj, Mette Rasmussen, Conni Sørensen and Susanne Skou for excellent technical that are not evident histopathologically may be detected by assistance and Dr. Wolfgang A. Schulz for comments on the article. The DNA methylation analysis of prostate biopsies, as demon- Danish Cancer Biobank (DCB) is acknowledged for biologic material and for strated by others for e.g. GSTP1 (6, 7). Accordingly, GAB- information regarding handling and storage. REmiR-452miR-224 methylation analysis could also potentially be used to guide repeat biopsy in men with Grant Support This work was supported by The Danish Cancer Society, The Lundbeck initial cancer-negative biopsies. Foundation, The John and Birthe Meyer Foundation, The Danish Council for The use of PSA recurrence as clinical endpoint is also a Strategic Research, The Danish Advanced Technology Foundation, and The limitation of our study. Notably, due to lower sensitivity of Deutsche Forschungsgemeinschaft (Schu604/16-3). The costs of publication of this article were defrayed in part by the the specific PSA kit applied, a higher cutoff value was used to payment of page charges. This article must therefore be hereby marked define biochemical recurrence in the Finnish patients (0.5 advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate ng/mL) compared with other patients (0.2 ng/mL). Thus, it this fact. cannot be excluded that detection of PSA recurrence was Received September 25, 2013; revised January 6, 2014; accepted February slightly delayed for some of the Finnish patients. However, 5, 2014; published online April 15, 2014.

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Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2014 American Association for Cancer Research. Hypermethylation of the GABRE∼miR-452∼miR-224 Promoter in Prostate Cancer Predicts Biochemical Recurrence after Radical Prostatectomy

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