Cancer Res-2014-Kneitz-2591-603

Published OnlineFirst March 7, 2014; DOI: 10.1158/0008-5472.
CAN-13-1606
Cancer
Research
Tumor and Stem Cell Biology
Survival in Patients with High-Risk Prostate Cancer Is

Predicted by miR-221, Which Regulates Proliferation,
Apoptosis, and Invasion of Prostate Cancer Cells by Inhibiting
IRF2 and SOCS3
Burkhard Kneitz1, Markus Krebs1, Charis Kalogirou1, Maria Schubert1,5, Steven Joniau8, Hein van Poppel8,
bel6, Manfred Gessler4,
rgen Scholz2, Philipp Stro
Evelyne Lerut9, Susanne Kneitz3, Claus Ju
1
1,7
Hubertus Riedmiller , and Martin Spahn
Abstract
A lack of reliably informative biomarkers to distinguish indolent and lethal prostate cancer is one reason this
disease is overtreated. miR-221 has been suggested as a biomarker in high-risk prostate cancer, but there is
insufcient evidence of its potential utility. Here we report that miR-221 is an independent predictor for cancerrelated death, extending and validating earlier ndings. By mechanistic investigations we showed that miR-221
regulates cell growth, invasiveness, and apoptosis in prostate cancer at least partially via STAT1/STAT3-mediated
activation of the JAK/STAT signaling pathway. miR-221 directly inhibits the expression of SOCS3 and IRF2, two
oncogenes that negatively regulate this signaling pathway. miR-221 expression sensitized prostate cancer cells for
IFN-gmediated growth inhibition. Our ndings suggest that miR-221 offers a novel prognostic biomarker and
therapeutic target in high-risk prostate cancer. Cancer Res; 74(9); 2591603. 2014 AACR.
Introduction
In Europe, the number of newly diagnosed prostate cancer
cases per year increased from 145,000 in 1996 to 345,000 in
2006. Despite this dramatic increase, the number of deaths
attributed to the disease over the same time period remained
almost unchanged (75,000 in 1996 vs. 68,000 in 2006; refs. 1
and 2). The current inability to accurately distinguish risk of
life-threatening, aggressive prostate cancer from indolent
cases contributes to the dilemma. The identication of factors
that are specically associated with lethal prostate cancer is
urgently needed to reduce overtreatment, as well as to develop
more effective targeted therapies.
Several potential prognostic markers have been identied
and there is a plethora of promising biomarkers including
Authors' Afliations: 1Department of Urology and Paediatric Urology,

University Hospital Wuerzburg; 2IZKF Laboratory for Microarray Applications, University Hospital Wuerzburg; Departments of 3Physiological
Chemistry I; 4Developmental Biochemistry, Biocenter; 5Comprehensive
Cancer Center Mainfranken, University of Wuerzburg, Wuerzburg; 6Department of Pathology, University Hospital Goettingen, Goettingen, Germany;
7
Department of Urology, University Hospital Bern, Inselspital, Bern, Switzerland; and Departments of 8Urology and 9Pathology, University Hospital
Leuven, Leuven, Belgium
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Authors: Burkhard Kneitz, Department of Urology and
Paediatric Urology, University Hospital Wuerzburg, University of Wuerzburg,
rrbacher Str. 8, D-97080 Wuerzburg, Germany. Phone: 49-921-201Oberdu
32700; Fax: 49-931-201-32719; E-mail: Kneitz_B@klinik.uni-wuerzburg.de;
and Martin Spahn, martin.spahn@insel.ch
doi: 10.1158/0008-5472.CAN-13-1606
2014 American Association for Cancer Research.
Kallikrein-2, p53, Ki67, PTEN-loss, CCP-scores, and ETS gene

fusions (3). But, none of these markers has made it into clinical
use yet. This is mainly because of tumor heterogeneity and the
patient cohorts analyzed (4). One possibility to optimize a
biomarker screening strategy is using high-risk prostate cancer
cohorts. A total of 20% to 35% of all newly diagnosed prostate
cancers are classied as high-risk prostate cancer (PSA >20
ng/mL; biopsy Gleason score 8, clinical stage T3/4; ref. 5).
Up to 30% of these men will develop metastasis and nally
die of their disease (68). Based on these relatively high
event rates, if compared with low-/intermediate-risk study
groups, high-risk prostate cancer represents a good cohort
to validate preexisting biomarkers predicting clinical failure
and cancer-related death (CRD).
MicroRNAs, small noncoding RNA molecules, play pivotal
roles in carcinogenesis and can function as tumor suppressor or
oncogene miRs (9). Extensive evidence has indicated that miR221 dysregulation plays an important role in prostate cancer
development and progression. Several studies showed that miR221 is one of the most strongly and frequently downregulated
miRNAs in primary prostate cancer (1012). Furthermore, we
demonstrated that miR-221 is progressively downregulated in
aggressive prostate cancer, lymph node-metastasis, and has
potential as a biomarker predicting clinical failure in high-risk
prostate cancer (13). In contradiction to the observed miR-221
downregulation in prostate cancer, miR-221 overexpression has
been reported for various other tumor types such as cancer of
lung, bladder, thyroid, breast, liver, or pancreas (1416). Overexpression of miR-221 in cell lines derived from the latter tumors
promotes proliferation, cell-cycle progression, and inhibits apoptosis, indicating an oncogenic miR-221 function. Consequently,
the tumor suppressor p27/kip1, p57kip2, c-kit, Bim, ERa, PTEN,
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on March 16, 2015. 2014 American Association for Cancer Research.
2591
Published OnlineFirst March 7, 2014; DOI: 10.1158/0008-5472.CAN-13-1606
Kneitz et al.
TIMP3, and PUMA have been reported to be miR-221 targets

(1720).
On the basis of our previous report we evaluated miR-221 as
a prognostic marker in high-risk prostate cancer in a larger
patient cohort and an external validation. Furthermore, we are
the rst to demonstrate a tumor suppressor function of miR221 in prostate cancer analyzing the mechanism by which this
microRNA promotes tumor cell growth, invasiveness, and
apoptosis in prostate cancer.
Patients and Methods

Patients and samples
Consecutive men with high-risk prostate cancer [prostatespecic antigen (PSA) >20 ng/mL and/or clinical stage T3/4
and/or biopsy Gleason score 810], who had undergone radical
prostatectomy between 1987 and 2005 at the Community Hospital of Karlsruhe, Germany (cohort 1) and the University
Hospital Leuven, Belgium (cohort 2), were identied in the
European Clinical and Translational High-Risk Prostate Cancer
Research Groupdatabase (EMPaCT) andwereincluded intothis
study. Clinical stage was assigned according to the 2002 TNM
system, prostate biopsy cores were obtained under transrectalultrasound guidance, and pretreatment PSA was measured
before digital rectal examination (DRE) or prostate ultrasound.
All patients were staged preoperatively with DRE, abdominopelvic computed tomography scan, and bone scan. Clinical
node positive disease was not considered as exclusion criteria.
None of the patients had received neo-adjuvant hormonal,
radiation, or chemotherapy. Prostate specimens were staged
and graded according to the 2002 TNM classication and the
Gleason grading system by two senior pathologists (P. Str
obel,
E. Lerut). Follow-up was performed every 3 months for the rst
2 years after surgery, every 6 months in the following 3 years,
and annually thereafter. Clinical failure was dened either as
histologically proven local recurrence or distant metastasis
conrmed by computed tomography or bone-scan. Cause of
death was veried by physician correspondence and/or death
certicates and CRD was dened as death because of prostate
cancer. Overall survival (OS) was dened as time from radical
prostatectomy to death of any cause, cancer-specic survival
(CSS) as the time from radical prostatectomy to death attributed to prostate cancer or complications of the disease.
Prostate cancer samples were parafn-embedded tissue specimens from radical prostatectomy (regions with >90% cancerous tissue were used for the RNA extraction and quantitative
real-time PCR).
Clinical and pathologic characteristics, clinical failurefree
survival, and OS for both cohorts were comparable. After a
median follow-up of 76 months (1154) for cohort 1 and 108
months (1200) for cohort 2, a total of 16 men (11.9%) and 15
men (16.9%) developed clinical failure and 11 (8.2%) and 12
(13.5%) of the men died prostate cancer related, respectively.
Also the estimated 10 and 15 years CSS rates were comparable
for both patient groups (89% and 74% for cohort 1 and 87% and
78% for cohort 2). This study was approved by the local ethical
committees (No. 59/04 and B322201214832). All included
patients provided written, informed consent.
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Cancer Res; 74(9) May 1, 2014
RNA extraction of prostate cancer samples and

quantitative real-time PCR
Total RNA for real-time PCR was extracted from the parafn-embedded prostate cancer tissues with a Total RNA Extraction Kit (Applied Biosystems) as described previously (13).
The RNA quality and concentration was determined with a
BioAnalyzer (Agilent). cDNA was synthesized from total RNA
with stem-loop reverse transcription primers for miR-221
according to the TaqMan MicroRNA Assay protocol. Mature
microRNA expression was quantied in tissue samples with
TaqMan microRNA assay kits and an Applied Biosystems
7900HT system according to the protocol provided in
the manufacturer's instructions (Applied Biosystems). The
expression of miR-151-3p was used for normalization.
Relative miR expression was calculated with the comparative
DCt-method (DCt sample Ct sample Ct miR-151-3p; DCt BPH
Ct BPH Ct miR-151-3p). mRNA analysis of SOCS3 and IRF2
expression was performed according to standard qRT-PCR
procedures. The expression of both glyceraldehyde-3-phosphate dehydrogenase and b-actin was used for normalization.
All primer sequences are available under request. Mean Ct was
always determined from triplicate PCRs.
Cell cultures, generation of stable miR-221
overexpressing PC-3 clones, commercial growth assay,
and miRNA transfections
DU-145, PC-3, and LNCaP cells were purchased from the
American Type Culture Collection (ATCC) and were grown
in medium as indicated by ATCC instructions. Cells were
transfected with human precursor miR-221 or negative
control oligonucleotides using Lipofectamine following the
manufacturer's instructions (Applied Biosystems). The optimal miRNA oligonucleotide concentrations were titrated for
optimal transfection results. In all experiments, the nal
miRNA concentration was 10 nmol/L. To stably overexpress
miR-221, we transfected PC-3 cells with a transposon vector
and the pCMV(CAT)T7-SB100 expression plasmid for encoding the sleeping beauty transposase (21). Selection of the
transgene was performed with puromycin (0.5 mg/mL). The
transposon vector was cloned by inserting the TurboRFPmiR-221 Fragment (Ecl136II/AfeI) of Tripz-miR-221 into
AfeI-cut pSB-ET (M. Gessler, unpublished), which allows
tetracycline regulated expression of the TurboRFP-miR221 cassette. Puromycin-resistant PC-3/miR-221 clones were
picked and analyzed for doxycycline (0.5 mg/mL) induced
expression of the TurboRFP-miR-221 cassette detecting RFP
by uorescence microscopy. miR-221 overexpression was
tested in doxycycline treated cells by qRT-PCR. Cell growth
was analyzed by MTS assay (Promega) as indicated by the
manufactures instructions as triplicates of 96-well cultures.
Two days posttransfection, total RNA was extracted for RTPCR and microarrays from cells cultured on 6-well plates
using TRIzol reagent (Invitrogen) or PhosphoSave (Novagen)
and used for expression analysis.
siRNA-mediated knockdown of messenger RNA
Cells were grown in 96-well plates for MTS assays or in 6-well
plates for total RNA and protein isolation. siRNA transfections
Cancer Research
mir-221 Is a Biomarker in Prostate Cancer and Inhibits IRF2 and SOCS3
were performed with Lipofectamine 2000 (Invitrogen) according to the manufactures instructions. Cells were transfected
with 5 nmol/L siRNA or control siRNA. Sequences for SOCS3
siRNA and IRF2 siRNA for targeting human SOCS3 or respectively human IRF2 were synthesized as published previously
(22, 23). Control siRNA was purchased from Qiagen. DU-145
and PC-3 cells were cultured at a density of 4 105 cells/
well and LNCaP cell were cultured at 8 105 cells/well in 6well plates. At day 2, posttransfection cells on 6-well plates
were harvested and total RNA or protein was isolated as
described.
Microarray analysis
Before labeling RNA quality was checked using a BioAnalyzer (Agilent). RNA integrity numbers (RIN) of the RNAs were 9.4
and 9.8. Total RNA was labeled according to Affymetrix standard protocols (IVT-Express Kit), without modication starting from 100 ng and hybridized to a GeneChip HG U 133 A 2.0
array. (Affymetrix). For the analysis of the resulting data,
different R packages from the Bioconductor project (www.
bioconductor.org) were used. Signal intensities were normalized by variance stabilization normalization (vsn package,
Bioconductor) and differential regulation of genes was
assessed by a modied t test (Limma package, Bioconductor)
as described previously (13). A gene was regarded as being
differentially expressed, if its log-fold change >1 and P-value
<0.05. For functional clustering, The Database for Annotation,
Visualization and Integrated Discovery (http://david.abcc.
ncifcrf.gov/home.jsp) has been used. Additional functional
clusters and text mining for gene interactions were generated
through the use of IPA (Ingenuity Systems). The data discussed
in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession
number GSE45627.
Apoptosis assay
Caspase-3/7 activity was analyzed using the Caspase-GLO
3/7 Kit (Promega) as recommended by the manufacturer's
instructions. Cells were transfected with miRNAs or siRNAs
in a 96-well plate as described. After 24 hours, cells were
incubated with medium supplemented with caspase-3/7
reagent for 4 hours at room temperature. Cells were lysated
and transferred to a white-walled 96 plate for measurement
of luminescence. Resulting data were expressed as OD values
and normalized to untransfected control cells. Experiments
were performed as triplicates.
In vitro invasion assay
A modied Boyden chamber assay was performed as
described previously (24). PC-3 cells were cultivated in medium
and transfected with premiR-221 or premiR-ctrl as
described. After 48 hours incubation and overnight starving
in Dulbecco's Modied Eagle Medium with 0.5% fetal calf
serum, cells were seeded in the upper chamber of BSA-coated
8 mmol/L pore size Transwell Boyden chambers (Corning star).
Normal growth medium supplemented with 10% fetal calf
serum was added to the bottom chamber as a chemo attractant
and cells were allowed to migrate through the membrane for 6
hours. After removal of all cells remaining at the upper surface

using a cotton carrier the lower surfaces of the membranes
were stained for 30 seconds in a solution of 1% (w/v) crystal
violet. Membranes were washed then with distilled water. Cellassociated crystal violet was extracted in 10% acetic acid and
measured at 595 nm absorbance. The experiments were performed in triplicates.
Western blotting
After harvesting, cells were washed twice by PBS and lysed
in PhosphoSafe (Novagen) as recommended by the manufacturer's instructions. Total protein concentrations were
quantied (Bradford). Protein isolates were loaded on 12%
SDS-PAGE gel with a concentration of 50 mg per lane
resolved and transferred onto nitrocellulose membranes
(Bio-Rad). The membranes were blocked using starting
Block buffer (Invitrogen) and incubated at 4 C with primary
antibody following the manufacturers' instructions. For
protein expression by Western blot analysis, we used following antibodies: SOCS3 (1:1,000, Abcam), IRF2, STAT1,
pSTAT1, pSTAT3, and ERK-2 (all from Ambion) as loading
control. We used horseradish peroxidasecoupled secondary
antibodies and the ECL Plus system (GE Healthcare) to
visualize the protein expression and quantied band intensities using Image J program.
Luciferase assays
We used the Dual Luciferase Reporter Assay System (Promega) as indicated by the manufactures instructions and
analyzed the luciferase activity 48 hours after transfection.
Prostate cancer cell lines were transiently transfected with
premiR-221 as described. The 30 untranslated regions of IRF2
and SOCS3 containing miR-221 binding sites were cloned into
the pMIR-REPORT luciferase reporter vector (pMRL, Ambion).
The constructs of the resulting pMRL-IRF2 or pMRL-SOCS3
vectors were cotransfected together with premiR-221 or premiR ctrl. miR-221 binding sites were identied by a bioinformatics search (Pictar, Targetscan, miRanda) and conrmed by
alignment to the complementary miR-221 sequence. The 30 UTR
of human SOCS3 and IRF2 were amplied by PCR using
following primers: SOCS3 Fw 50 -ACCAAGCTTGCCCACAGCCAGGGAAGTG-30 and SOCS3 Rw 50 -ACAACTAGTCTGTCCAGCCCAATACCTG-30 ; IRF2 Fw 50 -TCACTAGTGTTATTACATCCTTGTGGCAC-30 and IRF2 Rw 50 -GAACTAGTGAAGTCATGCAAAACGCTCA-30 . Mutagenesis of miR-221 binding
sites in the 30 UTRs of IRF2 or SOCS3 was performed using
the Site-Directed Mutagenesis Kit (QuickChange, Agilent Technologies). Primers for mutagenesis are as followed: SOCS3mut
Fw 50 -GTGACAATTTACAGGAATCGATCAGCGATGGAATTACCTGGAACAG-30 and SOCS3mut Rw 50 -CTGTTCCAGGTAATTCCATCGCTGATCGATTCCTGTAAATTGTCAC-30 ; IRF2mut
Fw 50 -GGTGAAAAAAGCTTTTCGGCAACACTGTAGCAATCAGA-30 and IRF2mut Rw 50 -TCTGATTGCTACATGAGTTGCCGAAAAGCTTTTTTCACC-30 .
For all reporter assays, cells were transfected with 50 ng
pMRL containing the mutated or wild-type SOCS3 30 UTR or
IRF2 30 UTR and 50 nmol/L of premiR-221 or premiRcontrol.
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Kneitz et al.
Statistical analysis of microRNA expression in the study

cohort
Relative miR-221 expression values display sample-specic
characteristics. Based on the normalized miR-221 expression
values, we determined receiver operating characteristics
(ROC) for various endpoints, precisely CRD and clinical failure.
Endpoint-specic high/low miR-221 expression thresholds
were determined based on ROC analysis such that cutoff values
represent the optimal tradeoff between specicity and sensitivity. Survival was illustrated by KaplanMeier curves; survival
differences between groups were examined with log-rank tests.
The inuence of miR-221 expression values as well as that of
various clinical and epidemiological parameters was analyzed
with univariate and multivariate Cox proportional hazard
regression. The best tting COX model was selected by measuring the relative goodness of t with the Akaike information
criterion (AIC). Differences in the mean values of miR expression in 2 risk groups were analyzed by the 2-sided Mann
Whitney test.
Results
miR-221 as prognostic marker in high-risk prostate
cancer
On the basis of our previous report indicating that miR-221 is
a prognostic marker in prostate cancer, we analyzed two
independent high-risk prostate cancer cohorts (cohort 1,
n 134; cohort 2, n 89) to validate this nding. Patient
selection and characteristics of both cohorts is provided in
Supplementary Fig. S1 and Table S1, respectively. In both
cohorts, we determined the miR-221 expression by RT-PCR
and found downregulation in the large majority of the analyzed
prostate cancer samples as compared with expression in BPH
samples (data not shown). Reductions in mean miR-221
expression levels were identied between risk groups split by
CRD but not for clinically used prognostic parameters, indicating an association between progressive miR-221 downregulation and tumor aggressiveness in both cohorts (Fig. 1A and
Supplementary Fig. S2). The prognostic value of miR-221 for
predicting CRD in cohort 1 (learning cohort) was analyzed
using ROC analysis. The ROC analysis for CRD dened an
optimal cutoff level (DCt miR-221 < 0.32) to dichotomize the
patients into risk groups (Fig. 1B). The calculated area under
the curve (AUC) for CRD was 0.903 (Fig. 1B). Using this miR-221
cut off level, we observed correct classication of 23.0% among
the high-risk cases (11 of 37) and respectively 100% among lowrisk cases (87 of 87). In KaplanMeier analysis, low miR-221
expression (DCt miR-221 < 0.32) was signicantly correlated
with CRD (Fig. 1C; P < 0.0001). Cox proportional hazards
regression analysis for time to CRD showed that miR-221
expression, Gleason score, and lymph node invasion predicted
CRD in univariate analysis. By stepwise regression analysis we
generated a multivariate model for predicting CRD (determined by AIC), which contained miR-221, Gleason score, and
lymph node invasion, indicating that miR-221 functions as an
independent predictor for CRD (Fig. 1D) in this model. The
estimate of an HR for miR-221 was innity because there were
100% correct classication in one group.
2594
To validate the predictive potential of the determined miR221 cutoff level, we used cohort 2 (test cohort). Using the same
cutoff level for miR-221 (DCt 0.32) as for cohort 1, we
dichotomized the test cohort and performed KaplanMeier
estimates. Also in cohort 2, low miR-221 expression correlated
signicantly with CRD (P < 0.001; Fig. 1C). Among the high-risk
group 10 of 20 (50.0%) and among the low-risk group 68 of 69
(97%) cases were correctly classied by miR-221. Samples of
the test cohort with miR-221 expression under the previously
determined cutoff level were found to be associated with CRD
by univariate Cox regression analysis [HR (95% CI) 0.026
(0.0030.201); P < 0.0001].
As expected, miR-221 is also correlated with clinical failure,
indicating that miR-221 can independently predict this outcome parameter either (Supplementary Fig. S3).
Expression of miR-221 in prostate cancer cells causes
growth inhibition, apoptosis, and reduced invasive
capabilities
To analyze a tumor suppressor function of miR-221, we
transiently transfected LNCaP, DU-145, and PC-3 cells with
precursor-miR-221. We observed an efcient and strong miR221 expression on day 2 posttransfection by qRT-PCR in all
three cell lines (Supplementary Fig. S4A). DU-145 and PC-3
cells responded to miR-221 reexpression by a signicant
decrease in cell proliferation (48% decrease in DU-145 and
69% in PC-3; P < 0.01), whereas the androgen-dependent
LNCaP cells showed a moderate increase in proliferation (Fig.
2A). In concordance with the observed inhibition in proliferation in DU-145 and PC-3 cells, we also found reduced viability
and changes in cell morphology after premiR-221 transfection
(Fig. 2B). To prove that the decrease in cell viability is linked to
induction of apoptosis, we analyzed the activity of caspase-3/7.
The caspase-3/7 activity was signicantly increased after miR221 transfection in DU-145 and PC-3 cells, whereas LNCaP cells
did not show increased apoptosis (Fig. 2C). We next assessed
whether the expression of miR-221 had an impact on the
invasive activities. Boyden chamber invasion assays showed
reduced invasion levels in miR-221transfected PC-3 cells (Fig.
2D). These results indicate that miR-221 acts as tumor suppressor in PC-3 and DU-145 prostate cancer cells by regulating
cell growth, apoptosis, and invasiveness.
Global gene expression analysis of miR-221 reexpressing
PC-3 cells
To search for molecular changes responsible for the
observed biological effects, we performed a microarray study
on mRNA isolated from premiR-221-transfected PC-3 cells.
This analysis revealed a set of signicantly up- or downregulated genes in miR-221 reexpressing PC-3 cells (Fig. 3A). We
found that from 54,675 genes on the array, 282 genes were
upregulated and 64 downregulated (>2-fold; P < 0.05; Supplementary Table S2). Many of the upregulated genes were known
to be also upregulated by interferons. Validation of the array
data using qRT-PCR assays conrmed this upregulation after
miR-221 transfection for STAT-1, IRF1, IRF9, OSA1, IFI27, and
IFI44 in PC-3 (Fig. 3B and Supplementary Fig. S4B). Moreover,
we found downregulation of several potential oncogenes
Cancer Research
1
0
1
No
100
2
80
1
0
1
60
40
Threshold: 0.325
Area under the curve:
90.28% (83.95%96.61%)
20
Yes
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Yes
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Cancer-specific survival (%)
80
60
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100
80
60
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100
miR-221
*
Sensitivity (%)
Group II
Cancer-related death
miR-221 expression Ct
miR-221 expression Ct
Group I
Cancer-related death
Log-rank test: P < 0.0001

miR-221>-0.32, n = 87; alive: 87 death: 0
miR-221<-0.32, n = 47; alive: 36 death: 11
80
60
40
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0
20
60
100
Time since radical prostatectomy (mo)
Variable
Log-rank test: P < 0.0001

miR-221>-0.32, n = 69; alive: 68 death: 1
miR-221<-0.32, n = 20; alive: 10 death: 10
50
100
150
200
Time since radical prostatectomy (mo)
Univariate
HR
Multivariate AIC
95% CI
HR
0.13
miR-221
(dichotomized)
Gleason
P < 0.0001
1.97
1.113.48
P = 0.015
PSA
1.08
0.991.02
P = 0.32
Lymph node invasion
0.44
0.021.13
P = 0.018
Age
0.93
0.911.08
P = 0.88
pT
0.98
0.293.22
P = 0.97
95% CI
P < 0.001
1.59
0.882.85
0.12
0.0160.99
0.049
Figure 1. miR-221 downregulation predicts cancer-related death in high-risk prostate cancer. The analysis was performed in two independent patient
cohorts originating from Germany (Group I) and Belgium (Group II). A, relative miR-221 expression levels (DCt) of prostate cancer samples of both cohorts were
analyzed by qRT-PCR and subsequently divided into risk groups based on CRD. Signicant reductions in the median expression levels were identied
between the two groups in both cohorts and indicated by , P < 0.001 using 2-sided MannWhitney test. The cutoff level dened in the ROC analysis is
indicated by the horizontal black line. B, ROC analysis for predicting CRD by miR-221 expression (DCt). The prognostic value of miR-221 for prostate
CRD was evident from AUC of 0.9028 (learning cohort). An optimal ROC-derived threshold value to dichotomize the patients by normalized miR-221
expression was 0.325. C, KaplanMeier analysis of patients with high-risk prostate cancer. Patients were grouped by the miR-221 expression cutoff dened
in the ROC analysis; survival curves are shown for both groups. Low miR-221 expression is associated with earlier CRD (log-rank P < 0.0001 in both groups).
D, Cox proportional hazards regression analysis for time to CRD revealed that dichotomized miR-221 expression level predicted cancer-related death. , the
actual HR is innity, because one of the groups had no events (100% correct classication).
including PMEPA1 or PRUNE by qRT-PCR (Fig. 3C and Supplementary Fig. S4C), which might function as potential target
genes for miR-221.
Pathway analysis revealed that miR-221 reexpression
seemed to be preferentially associated with the TOLL-like
receptor-, RIG-like receptor-, or the JAK/STAT pathways and
that specically interactions of the JAK/STAT pathway listed in
the KEGG pathway showed changes (Supplementary Fig. S5).
Because it was shown by several studies that transfection
with synthetic small RNA molecules might randomly induce
inammatory cytokines like interferons (25, 26), we decided to
generate PC-3 cells stable overexpressing miR-221. Using a

transposon vector containing a TurboRFP-miR-221 fragment
and the pCMV(CAT)T7-SB100 expression plasmid for encoding the sleeping beauty transposase we generated PC-3/miR221 cell clones. Three of 3 PC-3/miR-221 clones treated with
doxycycline showed >4 times overexpression of miR-221 (Supplementary Fig. S6A). Analyzing 2 PC-3/miR-221 clones we
conrmed miR-221mediated growth inhibition and activation of interferon-regulated genes (Supplementary Fig. 6B and
C), conrming that miR-221 might specically regulate the
interferon-signaling pathway in PC-3 cells.
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Kneitz et al.
PC-3
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1,600
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Figure 2. Expression of miR-221 in prostate cancer cells cause antitumorigenic effects. A, MTS assay for the growth of indicated prostate cancer cell
lines that were transfected with premiR-221 or premiR-precursor negative control (ctrl) and analyzed at day 2, 4, and 6 posttransfection. Mocktransfected cells showed no signicant differences to control cells and were excluded from the graph for better overview. Experiments were performed
as triplicates. Data, mean SD from ve independent experiments. B, PC-3 cells were transfected with premiR-221, pre-miR precursor negative,
control (ctrl), or mock control. At day 6, posttransfection pictures were captured (magnication, 40). C, indicated prostate cancer cell lines were
transfected with premiR-221 or pre-miR precursor negative control (ctrl). Caspase-3/7 activity was analyzed and was increased in PC-3 and
DU145 cells transfected with premiR-221 when compared with control cells, but not in LNCaP cells. Results are presented in relation to the values
measured in cells transfected with pre-miR precursor negative control that was arbitrarily set as 1. Data, mean values SD of ve independent
experiments; , P < 0.01, Wilcoxon rank sum test. D, miR-221 upregulation reduces cell migration of prostate cancer cells. PC-3 cells were transfected
with premiR-221 or control siRNA. Migration of PC-3 cells was measured more than 6 hours in a Transwell cell culture chamber. Four chambers from
three different experiments were analyzed (t test; P < 0.001). Each bar represents the mean SD. miR-221 overexpression in premiR-221-transfected
PC-3 cells as shown in Supplementary Fig. S7.
miR-221 expression induces STAT1 and STAT3

phosphorylation and sensitizes prostate cancer cells for
the antiproliferative effects of IFN-g
To elucidate, if miR-221 expression is sufcient for STAT1
and STAT3 phosphorylation, we analyzed the expression of
STAT1, pSTAT1, and pSTAT3 in premiR-221-transfected cells.
miR-221 reexpression activates STAT1 in PC-3 and DU-145 and
induced STAT3 phosphorylation in DU-145 cell, but not in the
STAT3-negative PC-3 cells (Fig. 4A).
It was previously shown that IFNs mediate their antiproliferative function by phosphorylation and activation of the JAK/
STAT pathway in prostate cancer cells. In fact, we could
2596
observe that IFN-g treatment of miR-221transfected cells

resulted in a signicantly reduced proliferation (DU-145 cells
78%, PC-3 cells 81% reduction, P < 0.01) compared with single
IFN-g treatment or untreated miR-221transfected cells (Fig.
4B). In context with this IFN-g sensitization, we observed
activation of STAT1 and/or STAT3 in miR-221 reexpressing
cells treated with IFN-g (Fig. 4A). Thus, we observed additive
effects in IFN-mediated growth inhibition by miR-221 expression in prostate cancer cells. In contrast, IFN-gresistant
LNCaP cells, that are known to be SOCS3 negative, did not
show activation of STAT1 or inhibition of proliferation after
IFN-g treatment independent of miR-221 expression (Fig. 4A).
Cancer Research
1A
22
iR-
Pre
Pre
1B
22
iR-
Pre
ctr
iR-
lA
ctr
iR-
Pre
lB
x-time underexpression compared with ctrl
PremiR-221
IFI44
IFI27
IRF-9
OAS1
0
IRF-1
Ctrl
4
Stat1
x-time overexpression compared with ctrl
Ctrl
PremiR-221
2
Pr
un
MT
HN
1
PA
E
PM
Figure 3. Comparison of mRNA expression patterns in PC-3 cells transfected with premiR-221. A, heat plot of genes showing a log-fold change >2 and
a P value < 0.001 in a comparison of premiR-221 and pre-miR precursor negative control transfected PC-3 samples. PremiR-221 A and premiR-221 B,
respectively, ctrl A and ctrl B, represent two independently performed experiments. B and C, for technical validation of array data, we analyzed
relative expression of selected genes, which were shown to be upregulated (B) or downregulated (C) on the array. Expression of indicated genes was
signicantly dysregulated in miR-221-transfected PC-3 cells. Normalized qRT-PCR results from miR-221-transfected cells were calculated as x-time
expression changes in comparison to PC-3 cells transfected with pre-miR precursor negative control. Data represent mean values SD of ve independent
experiments. The relative expression level of each gene in control transfected PC-3 cells was arbitrarily set as 1. Signicant differences (P < 0.01) between
expression in control and miR-221-transfected cells are indicated by the asterisk ( ). P values were calculated by Student t test.
miR-221 targets IRF2 and SOCS3 and inhibits expression

of IRF2 and SOCS3
Next we searched for miR-221 target genes, whose miR-221
mediated downregulation might be responsible for the observed
biological effects. By in silico analysis we identied potential
target sites in the 30 UTR mRNA regions of IRF2 and SOCS3.
Both genes are known negative regulators of the JAK/STAT
signaling cascade. Therefore, we analyzed the expression levels
in miR-221transfected cells and found moderate decrease in
IRF2 or SOCS3 mRNA levels (Supplementary Fig. S7) and a
strong reduction in protein levels (Figs. 4A and 5B).
To demonstrate a direct interaction between miR-221 and
IRF2 or SOCS3, we generated pGF-IRF2 and pGF-SOCS3 luciferase constructs, containing the miR-221 binding sites. In
addition, vectors with mutations at putative binding sites were

cloned and used as controls. These vectors were cotransfected
together with premiR-221 or scrambled miRNAs as negative
controls followed by measurement of luciferase activity 48
hours after transfection. As shown in Fig. 5A, the luciferase
activity in PC-3 cells cotransfected with constructs containing
the 30 UTR of IRF2 or SOCS3 and premiR-221 was decreased
by 62% (IRF2) and 41% (SOCS3).
Downregulation of IRF2 or SOCS3 recapitulate the
biological effects of miR-221 reexpression in prostate
carcinoma cells
To test whether IRF2 and SOCS3 are involved in the miR221mediated regulation of the JAK/STAT pathway, we
2597
Kneitz et al.
PC-3
DU-145
LNCaP
Premir-221
IFN-
STAT1
pSTAT1
pSTAT3
SOCS3
1,000
Ctrl
PC-3
PremiR-221
800
Ctrl+IFN-
PremiR-221+IFN-
600
400
200
0
2
4
6
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1,600
Ctrl
PremiR-221
Ctrl+IFN-
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1,400
1,200
1,000
800
600
400
200
0
2
4
6
ERK
LNCaP
Ctrl
PremiR-221
Ctrl+IFN-
PremiR-221+IFN-
1,200
1,000
800
600
400
200
0
2
4
6
Figure 4. miR-221 expression induces STAT1 and STAT3 phosphorylation and sensitizes prostate cancer cells for antiproliferative effects of IFN-g. A, PC-3,
DU-145, and LNCaP cells were transfected with premiR-221 and pre-miR precursor negative control as indicated. On day 1 posttransfection, IFN-g (10 ng/
mL) was added to the cell culture as indicated. At day 2 posttransfection, cells were harvested and Western blots for STAT1 pSTAT1, STAT3, pSTAT3 SOCS3,
and Erk (loading control) were performed. Results show induction of pSTAT1 in PC-3 and DU-145 cells and induction of pSTAT3 in DU-145 cells after
transfection with premiR-221 or by IFN-g treatment. B, MTS assay for the growth of indicated prostate cancer cell lines that were transfected with premiR221 or pre-miR precursor negative control (ctrl) in the presence or absence of IFN-g (10 ng/mL). IFN-g was added to the cell culture at day 1 posttransfection.
Cell cultures replicates were analyzed at day 2, 4, and 6 posttransfection. Mock-transfected cells showed no signicant differences compared with control
cells and were not added to the graph for better overview. Experiments were performed as triplicates. Data, mean SD from ve independent experiments.
inhibited the expression of both genes in PC-3 cells. siRNA

knockdown of both genes caused a strong and efcient
decrease of protein levels (8090%; Fig. 5B). We also observed
signicantly reduced proliferation, induction of apoptosis
and activation of STAT1 in response to siRNA-mediated
IRF2 or SOCS3 downregulation (Fig. 5C and D). We concluded that the biological effects caused by miR-221 overexpression are mediated at least partially by downregulation of
SOCS3 and IRF2.
In vivo regulation of IRF2 and SOCS3 by miR-221 in
prostate cancer
To assess the role of miR-221mediated inhibition of IRF2
and SOCS3 mRNA expression in primary prostate cancer, we
selected a group of fresh frozen tumor samples on the basis of
their miR-221 expression. In this series of prostate cancer
samples we correlated the expression of IRF2 and SOCS3 in
response to miR-221 downregulation. As Fig. 6 shows, we found
an inverse correlation between miR-221 downregulation and
upregulation of IRF2 or SOCS3 by Spearman rank correlation
2598
analysis, whereas the mRNA expression of SOCS3 and IRF2 was

not correlated. We concluded that miR-221 is also in vivo
critically involved in the expression of both potential target
genes.
Discussion
Based on the lack of prognostic models to accurately predict
survival, the need to better identify patients with lethal disease
is one of the main challenges in prostate cancer research. We
previously demonstrated that miR-221 downregulation hallmarks lymph node metastasis and possesses potential as a
prognostic marker in high-risk prostate cancer (13). Here we
demonstrated that miR-221 predicted clinical failure and
survival of patients with high-risk prostate cancer and determined a specic miR-221 expression level as independent
predictive marker for CRD and clinical failure. Using an
independent test cohort we successfully validated the predictive power of miR-221 in predicting CRD and clinical failure.
The role of miR-221 as a prognostic biomarker is further
Cancer Research
0.2
0
trl
C
0.2
3 SO
U CS
TR 3
m ut
S
3 OC
U S
TR 3
w
t
0.4
IR
F
TR 2m
ut
0.4
0.6
0.6
0.8
1
0.8
3 IR
U F2
TR w
t
Relative luciferase activity
trl
trl
si
iR
Pr
iR
1,000
900
800
700
600
500
400
300
200
100
0
Ctrl
siRNA IRF2
IFN-
IFN-+siRNA IRF2
3
2.5
2
1.5
1
0.5
siIRF2
siSOCS3
0
PremiR-221
Ctrl
siRNA SOCS3
IFN-
IFN-+siRNA SOCS3
3.5
Ctrl
1,000
900
800
700
600
500
400
300
200
100
0
siRNA ctrl
C
m
e
Pr
si
x-fold change in caspase-3/7 activity
ERK
-2
21
IR
F2
ERK
4.5
pSTAT1
pSTAT1
-2
2
SO 1
C
S3
IRF2
trl
SOCS3
PremiR ctrl
Relative luciferase activity
Figure 5. miR-221 expression inhibits expression of IRF2 and SOCS3 and siRNA-mediated downregulation of IRF2 and SOCS3 mimics effects of
miR-221 reexpression in prostate cancer cells. A, SOCS3 and IRF2 are targets of miR-221. SOCS3 and IRF2 luciferase constructs, containing a wild-type or
mutated SOCS3 or IRF2 30 UTR, were cotransfected with premiR-221 in PC-3 cells. SOCS3 30 UTR or IRF2 30 UTR containing a mutation in the miR-221
binding site showed no signicant difference in reporter activity compared with control transfected cells. Relative expression of rey luciferase was
standardized to control transfections. Luciferase activities were analyzed 48 hours after transfection. Reporter activities of cells cotransfected with miRprecursor negative control (black bars) were arbitrarily set as 1. The results were obtained from three independent experiments and are presented as mean
SD. B, miR-221 reexpression or siRNA treatment decreased expression levels of SOCS3 or IRF2 and activated STAT1. PC-3 cells were transfected with
negative control, premiR-221, and SOCS3 siRNA or IRF2 siRNA for 48 hours. Western blots were performed to analyze the expression of pSTAT1 and SOCS3
or pSTAT1 and IRF2. For both plots, we used antiERK-2 as loading control. Western blots were repeated at least three times, showing comparable results.
C, effect of siRNA-mediated knockdown of SOCS3 or IRF2 on the growth of PC-3 cells. MTS assay analysis for the growth of PC-3 cells. Cells were transfected
with SOCS3 or IRF2 siRNA and control siRNA. On day 1 posttransfection, 10 ng/mL IFN-g was added to the cultures when indicated. Cell culture
replicates were analyzed at day 2, 4, and 6 posttransfection Experiments were performed in triplicates. Presented data are mean values SD from three
independent experiments. D, PC-3 cells (ctrl) were compared with 1 PC-3 cells transfected with premiR-ctrl, siRNA ctrl, premiR-221, SOCS3 siRNA, or IRF2
siRNA. Caspase-3/7 activity was analyzed 24 hours after transfection. Results are presented in relation to the values measured in nontransfected PC-3 cells,
which was arbitrarily set as 1. Data represent mean values SD of three independent experiments; , P < 0.01, Wilcoxon rank sum test.
supported by a recent report showing a correlation of miR-221

downregulation on BCR and clinical failure in TMPRSS:ERG
fusion positive prostate cancer (27). The results presented here
provide the groundwork to prospectively test miR-221 as a
tissue-based biomarker in patients with high-risk prostate
cancer and to develop new treatment strategies within new
clinical trial concepts (28).
Such new therapies are strongly related to the biological
function of each individual microRNA. Several studies including our biomarker analysis clearly suggested a tumor suppressive function of miR-221 in prostate cancer. Therefore, we
analyzed the effects of reexpressing miR-221 in androgen

independent prostate cancer cell lines and demonstrated that
miR-221 reexpression reduced proliferation, invasiveness, and
induced apoptotic cell death, indicating a tumor suppressor
role of miR-221 in androgen independent prostate cancer cells.
To elucidate molecular pathways regulated by miR-221, we
analyzed global mRNA expression proles in miR-221 expressing prostate cancer cells. Interestingly, besides the downregulation of oncogenic target genes such as PMEPA1 and
PRUNE (29, 30), we found an increased expression of genes,
known to be associated with cell exposure to interferons.
2599
cor= 0.784
miR-221 <(1)
miR-221 <(1)
10
20
30
40
50
40
50
40
50
miR-221 >(1)
miR-221
miR-221 >(1)
0 10 20 30 40 50
SOCS3
cor= 0.549
10
20
30
IRF2
IRF2
0 10 20 30 40 50
iR IR
-2 F2
21
>
1
m
iR IR
-2 F2
21
<
1
m
S
iR OC
-2 S
21 3
>
1
m
S
iR OC
-2 S
21 3
<
1
Relative expression SOCS3 or IRF2
miR-221
0 10 20 30 40 50
Kneitz et al.
cor= 0.419
10
20
30
SOCS3
Figure 6. Expression of miR-221 and SOCS3 or IRF2 is inversely regulated in human prostate cancer. A, relative expression levels of miR-221, SOCS3, and
IRF2 were analyzed by qRT-PCR in RNA extracts from fresh frozen human prostate cancer samples and adjacent nontumorigenic prostate tissue (n 30).
Expression of miR-221, SOCS3, and IRF2 was calculated as x-fold overexpression in the cancer sample compared with the corresponding nontumorigenic
prostate sample. Subsequently, the samples of the cohort were divided into subgroups based on a more than 2-fold downregulation (log-fold change >1 or
<1) of miR-221 and plotted against expression (calculated as log-fold changes) of SOCS3 or IRF2, respectively. Results show signicant increased
expression of SOCS3 and IRF2 in miR-221downregulated prostate cancer samples (black blots/miR-221 < 1) when compared with prostate cancer
samples with no miR-221 downregulation (gray plots/miR-221 > 1). B, plots showing the coefcient of correlation for relative expression levels of
miR-221, SOCS3, and IRF2 from the samples described above.
Pathway analysis revealed activation of the Toll-like receptor,

the RIG-like receptor, and most impressively the JAK/STAT
pathways. IFN-mediated JAK/STAT activation in cancer development is not unexpected, because IFN usually functions as a
cytokine with antitumor activity (31), moreover, there is a
growing body of evidence that activation of the JAK/STAT
pathway can inhibit proliferation and induce apoptosis in
certain microenvironmental conditions. STAT1 is a known
tumor suppressor involved in tumor development and expansion by switching on antiproliferative and proapoptotic pathways (32, 33). We detected STAT1 and STAT3 phosphorylation
in miR-221 reexpressing cells, indicating a strong JAK/STAT
pathway activation. An antitumorigenic activity by miR-221
mediated STAT3 activation is conicting, because of its oncogenic function in some tumor entities (3436). However,
2600
STAT3 activation can induce growth arrest and apoptosis

under certain conditions in various cancer types including
prostate cancer (22, 37, 38). The activation of both STAT1 and
STAT3 might explain at least partially the antiproliferative and
proapoptotic activity of miR-221 in prostate cancer cells.
To elucidate how miR-221 expression activates the JAK/
STAT pathway we identied SOCS3 and IRF2, both known
negative regulator genes of the JAK/STAT pathway, as miR-221
targets. The role of SOCS3 as inhibitor of the JAK/STAT
pathway in prostate cancer is documented by the observation
that STAT1 and STAT3 phosphorylation is inversely correlated
with SOCS3 expression (39). Moreover, SOCS3 downregulation
determined reduced proliferation rates and an increased apoptotic response by converting the antiapoptotic STAT3 function into proapoptotic (40). It was also shown that reduced
Cancer Research
Figure 7. Model of miR-221 tumor suppressor function in prostate cancer. We propose a model in which miR-221 downregulates SOCS3 and IRF2, which in
turn, leads to an activation of the JAK-STAT pathway by STAT1 phosphorylation and increased IRF1-induced gene expression, resulting in the activation of an
apoptotic pathway, cell growth inhibition, and decreased invasive activity. Modied based on: 20002011 Ingenuity Systems, Inc. All rights reserved.
SOCS3 protein expression enhanced the IFN-g responsiveness,

indicating a regulation of IFN-g sensitivity in prostate cancer
cells and other tumors by SOCS3 (22, 41).
In various cancer types, IRF2 overexpression was found to be
associated with the development and progression of malignant
phenotypes. IRF2 acts as an antagonist to the tumor suppressor IRF1 and it is known that the IRF1/IRF2 balance is critically
involved in the immunomodulatory, antiproliferative, and
proapoptotic IFN-g effects (42, 43). Previous studies have
shown that IRF1 and IRF2 are regulating transcription of the
same IFN-ginducible genes (44), but with entirely opposing
effects for cell growth and tumorigenicity (23). Thus, there is
growing body of evidence that the IRF2 expression determines
the cellular response to JAK/STAT pathway. Here we demonstrated that miR-221 reexpression induced IRF1 and downregulated IRF2 expression. In addition, we found that, similar
as recently described for pancreatic cells, IRF2 knockdown
leads to growth inhibition and apoptosis in prostate cancer
cells. Based on these results it is very likely that the downregulation of SOCS3 and IRF2 is responsible for the antitumorigenic biological effects in miR-221 reexpressing prostate
cancer cells. The relevance of these results for tumor development and tumor progression is further supported by the
inverse correlation between miR-221 and SOCS3 or IRF2
expression in primary prostate cancer probes. Figure 7
summarizes a model how miR-221 regulates the JAK/STAT
pathway and how miR-221 downregulation inhibits IFNgmediated antiproliferative and proapoptotic signals in
prostate cancer cells.
Our present study also provides evidence for a possible role
of miR-221 to overcome the problem of low sensitivity against
cytokine therapies in prostate cancer. Interferon therapy was

discussed for clinically advanced prostate cancer (45). However, systemic IFN-g therapy in prostate cancer has shown only
limited efciency (46). We now demonstrate that miR-221
expression mediates the responsiveness against the antitumorigenic effects of IFN-g in vitro. Moreover, we show evidence
that miR-221 downregulation might reduce the IFN-g responsiveness in primary prostate cancer by upregulation of two
independent negative regulator proteins (SOCS3 and IRF2) of
this cytokine pathway. It is well known that the interaction of
various negative regulatory proteins involved in cytokine signaling is very complex. Here we show that miR-221 is able to
control such a signaling pathway by regulating various components and therefore it might be a good candidate for
therapeutic use.
However, microRNAtargeted therapy is challenging. Tissue-specic delivery, stability, cellular uptake, and off-target
effects might be overcome by technical solutions in the future,
but safety might remain a major concern in microRNAbased
therapy. Several cancerassociated miRNA showed pivotal
roles in tumor development and progression because a miRNA
can function either as an oncogene if in a given cell type its
critical target is a tumor suppressor or the same miRNA can be
a tumor suppressor if in a different cell type its target is an
oncogene (9). This seems to be true also for miR-221. Overexpression and regulation of tumor suppressor genes (i.e.,
p27kip1, Pten, etc.) were described for miR-221 in several
tumor entities (1417, 19, 20), whereas we and others showed
that miR-221 is one of the most strongly and frequently downregulated miRNA in primary prostate cancer inhibiting the
expression of the potential oncogenes IRF2 and SOCS3 (1013).
2601
Kneitz et al.
However, in prostate cancer the situation seems to be even

more complex, because miR-221 expression levels were shown
to be increased in tumor tissue derived from bone metastasis of
castration-resistant prostate cancer (CRPC; refs. 47 and 48).
Nevertheless, this observation is not per se mutually exclusive
with the ndings in this study. Although Sun and colleagues
observed miR-221 overexpression only in CRPC, we detected
miR-221 downregulation in hormone nave tumors. These
observed differences implicate a specic function of miR221 in the development of androgen resistance. Recent studies
by Sun and colleagues supported this suggestion showing that
the development of androgen independence in LNCaP cells
was promoted via miR-221mediated downregulation of
HECTD2 and RAB1A re-programming the androgen signaling
pathway (47). In contrast to these results we demonstrated that
miR-221 overexpression activated the antiproliferative and
proapoptotic JAK/STAT pathway only in androgen-independent, SOCS3-positive DU-145 and PC-3, but not in androgendependent, SOCS3-negative LNCaP-cells. One possible explanation for the diverging results might be a pivotal function of
miR-221 in the regulation of androgen-independent growth
and interferon signaling in the presence or absence of SOCS3,
because it was shown that sensitivity against androgen and
interferon signaling in prostate cancer cells depends on SOCS3
expression (49). Therefore, we suggest that the different function of miR-221 in prostate cancer cells at least partially
depends on a SOCS3-mediated regulation of the androgen
receptor- or the interferon-signaling pathway in prostate cancer cells. Future in vitro and in vivo analysis describing a
possible role of miR-221 in controlling various signaling pathway via posttranscriptional regulation of SOCS3 and other
potential target genes might clarify this clinically relevant
question.
In summary, we demonstrated for the rst time that miR221 has tumor suppressive function in prostate cancer controlling apoptotic pathways, cell growth, and invasiveness. The
antitumorigenic effect of miR-221 expression is mediated at

least partially by activation of the JAK/STAT pathway. We
could show that miR-221 regulates two of the most important
negative regulator proteins, SOCS3 and IRF2, of the JAK/STAT
signaling pathway, indicating a role of miR-221 as a master
regulator of IFN-g sensitivity in prostate cancer cells. Moreover, we demonstrated that miR-221 expression is progressively decreased during prostate cancer development and
progression in clinical specimens and is an independent
prognostic marker to predict cancer-related death in high-risk
prostate cancer. On the basis of our results, we think that miR221 has potential as a prognostic biomarker and as a target for
future therapies of high-risk prostate cancer.
Disclosure of Potential Conicts of Interest
No potential conicts of interest were disclosed.
Authors' Contributions
Conception and design: B. Kneitz, S. Joniau, M. Spahn
Development of methodology: B. Kneitz, M. Spahn
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): B. Kneitz, M. Krebs, C. Kalogirou, M. Schubert, H. van
Poppel, E. Lerut, C.J. Scholz, P. Strobel, M. Gessler, M. Spahn
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): B. Kneitz, M. Krebs, C. Kalogirou, S. Joniau, S. Kneitz,
M. Gessler, M. Spahn
Writing, review, and/or revision of the manuscript: B. Kneitz, S. Joniau,
H. van Poppel, S. Kneitz, P. Strobel, M. Spahn
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H. Riedmiller
Study supervision: B. Kneitz, H. van Poppel, H. Riedmiller, M. Spahn
Acknowledgments
The authors thank K. Borschert, A. Winkler, V. Schwarz, and B. Dexler for
skillful technical assistance.
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 June 7, 2013; revised December 19, 2013; accepted January 22, 2014;
published OnlineFirst March 7, 2014.
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2603
Survival in Patients with High-Risk Prostate Cancer Is Predicted by

miR-221, Which Regulates Proliferation, Apoptosis, and Invasion of
Prostate Cancer Cells by Inhibiting IRF2 and SOCS3
Burkhard Kneitz, Markus Krebs, Charis Kalogirou, et al.
Cancer Res 2014;74:2591-2603. Published OnlineFirst March 7, 2014.
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Published OnlineFirst March 7, 2014; DOI: 10.1158/0008-5472.

Tumor and Stem Cell Biology

Survival in Patients with High-Risk Prostate Cancer Is

Authors' Afliations: 1Department of Urology and Paediatric Urology,

Kallikrein-2, p53, Ki67, PTEN-loss, CCP-scores, and ETS gene

Published OnlineFirst March 7, 2014; DOI: 10.1158/0008-5472.CAN-13-1606

TIMP3, and PUMA have been reported to be miR-221 targets

Patients and Methods

Cancer Res; 74(9) May 1, 2014

RNA extraction of prostate cancer samples and

Published OnlineFirst March 7, 2014; DOI: 10.1158/0008-5472.CAN-13-1606

mir-221 Is a Biomarker in Prostate Cancer and Inhibits IRF2 and SOCS3

hours. After removal of all cells remaining at the upper surface

Cancer Res; 74(9) May 1, 2014

Published OnlineFirst March 7, 2014; DOI: 10.1158/0008-5472.CAN-13-1606

Statistical analysis of microRNA expression in the study

Cancer Res; 74(9) May 1, 2014

Published OnlineFirst March 7, 2014; DOI: 10.1158/0008-5472.CAN-13-1606

mir-221 Is a Biomarker in Prostate Cancer and Inhibits IRF2 and SOCS3

Cancer-specific survival (%)

Cancer-specific survival (%)

Log-rank test: P < 0.0001

Log-rank test: P < 0.0001

Lymph node invasion

generate PC-3 cells stable overexpressing miR-221. Using a

Cancer Res; 74(9) May 1, 2014

Published OnlineFirst March 7, 2014; DOI: 10.1158/0008-5472.CAN-13-1606

MTS absorbance (490 nm)

x fold increase in caspase-3/7 activity

miR-221 expression induces STAT1 and STAT3

Cancer Res; 74(9) May 1, 2014

observe that IFN-g treatment of miR-221transfected cells

Published OnlineFirst March 7, 2014; DOI: 10.1158/0008-5472.CAN-13-1606

mir-221 Is a Biomarker in Prostate Cancer and Inhibits IRF2 and SOCS3

x-time underexpression compared with ctrl

x-time overexpression compared with ctrl

miR-221 targets IRF2 and SOCS3 and inhibits expression

addition, vectors with mutations at putative binding sites were

Cancer Res; 74(9) May 1, 2014

Published OnlineFirst March 7, 2014; DOI: 10.1158/0008-5472.CAN-13-1606

MTS absorbance (490 nm)

MTS absorbance (490 nm)

MTS absorbance (490 nm)

inhibited the expression of both genes in PC-3 cells. siRNA

Cancer Res; 74(9) May 1, 2014

analysis, whereas the mRNA expression of SOCS3 and IRF2 was

Published OnlineFirst March 7, 2014; DOI: 10.1158/0008-5472.CAN-13-1606

Relative luciferase activity

x-fold change in caspase-3/7 activity

Relative luciferase activity

mir-221 Is a Biomarker in Prostate Cancer and Inhibits IRF2 and SOCS3

supported by a recent report showing a correlation of miR-221

analyzed the effects of reexpressing miR-221 in androgen

Cancer Res; 74(9) May 1, 2014

Published OnlineFirst March 7, 2014; DOI: 10.1158/0008-5472.CAN-13-1606

Relative expression SOCS3 or IRF2

Pathway analysis revealed activation of the Toll-like receptor,

Cancer Res; 74(9) May 1, 2014

STAT3 activation can induce growth arrest and apoptosis

Published OnlineFirst March 7, 2014; DOI: 10.1158/0008-5472.CAN-13-1606