Download Hypermethylation of the GABRE$miR-452$miR

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
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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.
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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,
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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
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(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.
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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
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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
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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.
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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,
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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,
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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
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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
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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
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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
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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.
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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/
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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
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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.
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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.
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