cancer res-2014-kneitz-2591-603

Tumor and Stem Cell Biology

Survival in Patients with High-Risk Prostate Cancer IsPredicted by miR-221, Which Regulates Proliferation,Apoptosis, and InvasionofProstateCancerCells by InhibitingIRF2 and SOCS3

Burkhard Kneitz1, Markus Krebs1, Charis Kalogirou1, Maria Schubert1,5, Steven Joniau8, Hein van Poppel8,Evelyne Lerut9, Susanne Kneitz3, Claus Jurgen Scholz2, Philipp Strobel6, Manfred Gessler4,Hubertus Riedmiller1, and Martin Spahn1,7

AbstractA 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 isinsufcient evidence of its potential utility. Here we report that miR-221 is an independent predictor for cancer-related death, extending and validating earlier ndings. By mechanistic investigations we showed that miR-221regulates cell growth, invasiveness, and apoptosis in prostate cancer at least partially via STAT1/STAT3-mediatedactivation of the JAK/STAT signaling pathway. miR-221 directly inhibits the expression of SOCS3 and IRF2, twooncogenes that negatively regulate this signaling pathway.miR-221 expression sensitized prostate cancer cells forIFN-gmediated growth inhibition. Our ndings suggest that miR-221 offers a novel prognostic biomarker andtherapeutic target in high-risk prostate cancer. Cancer Res; 74(9); 2591603. 2014 AACR.

IntroductionIn Europe, the number of newly diagnosed prostate cancer

cases per year increased from 145,000 in 1996 to 345,000 in2006. Despite this dramatic increase, the number of deathsattributed to the disease over the same time period remainedalmost unchanged (75,000 in 1996 vs. 68,000 in 2006; refs. 1and 2). The current inability to accurately distinguish risk oflife-threatening, aggressive prostate cancer from indolentcases contributes to the dilemma. The identication of factorsthat are specically associated with lethal prostate cancer isurgently needed to reduce overtreatment, as well as to developmore effective targeted therapies.

Several potential prognostic markers have been identiedand there is a plethora of promising biomarkers including

Kallikrein-2, p53, Ki67, PTEN-loss, CCP-scores, and ETS genefusions (3). But, none of these markers has made it into clinicaluse yet. This is mainly because of tumor heterogeneity and thepatient cohorts analyzed (4). One possibility to optimize abiomarker screening strategy is using high-risk prostate cancercohorts. A total of 20% to 35% of all newly diagnosed prostatecancers are classied as high-risk prostate cancer (PSA >20ng/mL; biopsy Gleason score 8, clinical stage T3/4; ref. 5).Up to 30% of these men will develop metastasis and nallydie of their disease (68). Based on these relatively highevent rates, if compared with low-/intermediate-risk studygroups, high-risk prostate cancer represents a good cohortto validate preexisting biomarkers predicting clinical failureand cancer-related death (CRD).

MicroRNAs, small noncoding RNA molecules, play pivotalroles in carcinogenesis and can function as tumor suppressor oroncogene miRs (9). Extensive evidence has indicated that miR-221 dysregulation plays an important role in prostate cancerdevelopment and progression. Several studies showed thatmiR-221 is one of the most strongly and frequently downregulatedmiRNAs in primary prostate cancer (1012). Furthermore, wedemonstrated that miR-221 is progressively downregulated inaggressive prostate cancer, lymph node-metastasis, and haspotential as a biomarker predicting clinical failure in high-riskprostate cancer (13). In contradiction to the observed miR-221downregulation in prostate cancer, miR-221 overexpression hasbeen reported for various other tumor types such as cancer oflung, bladder, thyroid, breast, liver, or pancreas (1416). Over-expressionofmiR-221 in cell lines derived from the latter tumorspromotes proliferation, cell-cycle progression, and inhibits apo-ptosis, indicating an oncogenicmiR-221 function. Consequently,the tumor suppressor p27/kip1, p57kip2, c-kit, Bim, ERa, PTEN,

Authors' Afliations: 1Department of Urology and Paediatric Urology,University Hospital Wuerzburg; 2IZKF Laboratory for Microarray Applica-tions, University Hospital Wuerzburg; Departments of 3PhysiologicalChemistry I; 4Developmental Biochemistry, Biocenter; 5ComprehensiveCancerCenterMainfranken,University ofWuerzburg,Wuerzburg; 6Depart-ment of Pathology, University Hospital Goettingen, Goettingen, Germany;7Department of Urology, University Hospital Bern, Inselspital, Bern, Swit-zerland; and Departments of 8Urology and 9Pathology, University HospitalLeuven, Leuven, Belgium

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Burkhard Kneitz, Department of Urology andPaediatric Urology, University Hospital Wuerzburg, University of Wuerzburg,Oberdurrbacher Str. 8, D-97080 Wuerzburg, Germany. Phone: 49-921-201-32700; Fax: 49-931-201-32719; E-mail: [email protected];and Martin Spahn, [email protected]

doi: 10.1158/0008-5472.CAN-13-1606

2014 American Association for Cancer Research.

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on March 16, 2015. 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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

TIMP3, and PUMA have been reported to be miR-221 targets(1720).

On the basis of our previous report we evaluated miR-221 asa prognostic marker in high-risk prostate cancer in a largerpatient cohort and an external validation. Furthermore, we arethe rst to demonstrate a tumor suppressor function of miR-221 in prostate cancer analyzing the mechanism by which thismicroRNA promotes tumor cell growth, invasiveness, andapoptosis in prostate cancer.

Patients and MethodsPatients and samples

Consecutive men with high-risk prostate cancer [prostate-specic antigen (PSA) >20 ng/mL and/or clinical stage T3/4and/or biopsy Gleason score 810], who had undergone radicalprostatectomy between 1987 and 2005 at the Community Hos-pital of Karlsruhe, Germany (cohort 1) and the UniversityHospital Leuven, Belgium (cohort 2), were identied in theEuropeanClinical andTranslationalHigh-Risk ProstateCancerResearchGroupdatabase(EMPaCT)andwereincludedintothisstudy. Clinical stage was assigned according to the 2002 TNMsystem, prostate biopsy cores were obtained under transrectal-ultrasound guidance, and pretreatment PSA was measuredbefore digital rectal examination (DRE) or prostate ultrasound.

All patients were staged preoperatively with DRE, abdomi-nopelvic computed tomography scan, and bone scan. Clinicalnode positive disease was not considered as exclusion criteria.None of the patients had received neo-adjuvant hormonal,radiation, or chemotherapy. Prostate specimens were stagedand graded according to the 2002 TNM classication and theGleason grading system by two senior pathologists (P. Strobel,E. Lerut). Follow-upwas performed every 3months for the rst2 years after surgery, every 6 months in the following 3 years,and annually thereafter. Clinical failure was dened either ashistologically proven local recurrence or distant metastasisconrmed by computed tomography or bone-scan. Cause ofdeath was veried by physician correspondence and/or deathcerticates and CRD was dened as death because of prostatecancer. Overall survival (OS) was dened as time from radicalprostatectomy to death of any cause, cancer-specic survival(CSS) as the time from radical prostatectomy to death attrib-uted to prostate cancer or complications of the disease.

Prostate cancer samples were parafn-embedded tissue spe-cimens from radical prostatectomy (regions with >90% cancer-ous tissue were used for the RNA extraction and quantitativereal-time PCR).

Clinical and pathologic characteristics, clinical failurefreesurvival, and OS for both cohorts were comparable. After amedian follow-up of 76 months (1154) for cohort 1 and 108months (1200) for cohort 2, a total of 16 men (11.9%) and 15men (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 comparablefor both patient groups (89% and 74% for cohort 1 and 87% and78% for cohort 2). This study was approved by the local ethicalcommittees (No. 59/04 and B322201214832). All includedpatients provided written, informed consent.

RNA extraction of prostate cancer samples andquantitative real-time PCR

Total RNA for real-time PCR was extracted from the paraf-n-embedded prostate cancer tissues with a Total RNAExtrac-tion Kit (Applied Biosystems) as described previously (13).The RNA quality and concentration was determined with aBioAnalyzer (Agilent). cDNA was synthesized from total RNAwith stem-loop reverse transcription primers for miR-221according to the TaqMan MicroRNA Assay protocol. MaturemicroRNA expression was quantied in tissue samples withTaqMan microRNA assay kits and an Applied Biosystems7900HT system according to the protocol provided inthe manufacturer's instructions (Applied Biosystems). Theexpression of miR-151-3p was used for normalization.Relative miR expression was calculated with the comparativeDCt-method (DCt sample Ct sample Ct miR-151-3p; DCt BPH Ct BPH Ct miR-151-3p). mRNA analysis of SOCS3 and IRF2expression was performed according to standard qRT-PCRprocedures. The expression of both glyceraldehyde-3-phos-phate dehydrogenase and b-actin was used for normalization.All primer sequences are available under request. Mean Ct wasalways determined from triplicate PCRs.

Cell cultures, generation of stable miR-221overexpressing PC-3 clones, commercial growth assay,and miRNA transfections

DU-145, PC-3, and LNCaP cells were purchased from theAmerican Type Culture Collection (ATCC) and were grownin medium as indicated by ATCC instructions. Cells weretransfected with human precursor miR-221 or negativecontrol oligonucleotides using Lipofectamine following themanufacturer's instructions (Applied Biosystems). The opti-mal miRNA oligonucleotide concentrations were titrated foroptimal transfection results. In all experiments, the nalmiRNA concentration was 10 nmol/L. To stably overexpressmiR-221, we transfected PC-3 cells with a transposon vectorand the pCMV(CAT)T7-SB100 expression plasmid for encod-ing the sleeping beauty transposase (21). Selection of thetransgene was performed with puromycin (0.5 mg/mL). Thetransposon vector was cloned by inserting the TurboRFP-miR-221 Fragment (Ecl136II/AfeI) of Tripz-miR-221 intoAfeI-cut pSB-ET (M. Gessler, unpublished), which allowstetracycline regulated expression of the TurboRFP-miR-221 cassette. Puromycin-resistant PC-3/miR-221 clones werepicked and analyzed for doxycycline (0.5 mg/mL) inducedexpression of the TurboRFP-miR-221 cassette detecting RFPby uorescence microscopy. miR-221 overexpression wastested in doxycycline treated cells by qRT-PCR. Cell growthwas analyzed by MTS assay (Promega) as indicated by themanufactures instructions as triplicates of 96-well cultures.Two days posttransfection, total RNA was extracted for RT-PCR and microarrays from cells cultured on 6-well platesusing TRIzol reagent (Invitrogen) or PhosphoSave (Novagen)and used for expression analysis.

siRNA-mediated knockdown of messenger RNACells were grown in 96-well plates forMTS assays or in 6-well

plates for total RNA and protein isolation. siRNA transfections

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were performed with Lipofectamine 2000 (Invitrogen) accord-ing to the manufactures instructions. Cells were transfectedwith 5 nmol/L siRNA or control siRNA. Sequences for SOCS3siRNA and IRF2 siRNA for targeting human SOCS3 or respec-tively human IRF2 were synthesized as published previously(22, 23). Control siRNA was purchased from Qiagen. DU-145and PC-3 cells were cultured at a density of 4 105 cells/well and LNCaP cell were cultured at 8 105 cells/well in 6-well plates. At day 2, posttransfection cells on 6-well plateswere harvested and total RNA or protein was isolated asdescribed.

Microarray analysisBefore labeling RNA quality was checked using a BioAnaly-

zer (Agilent). RNA integrity numbers (RIN) of the RNAswere 9.4and 9.8. Total RNA was labeled according to Affymetrix stan-dard protocols (IVT-Express Kit), without modication start-ing from 100 ng and hybridized to a GeneChip HG U 133 A 2.0array. (Affymetrix). For the analysis of the resulting data,different R packages from the Bioconductor project (www.bioconductor.org) were used. Signal intensities were normal-ized by variance stabilization normalization (vsn package,Bioconductor) and differential regulation of genes wasassessed by a modied t test (Limma package, Bioconductor)as described previously (13). A gene was regarded as beingdifferentially expressed, if its log-fold change >1 and P-value

Statistical analysis of microRNA expression in the studycohort

Relative miR-221 expression values display sample-speciccharacteristics. Based on the normalized miR-221 expressionvalues, we determined receiver operating characteristics(ROC) for various endpoints, precisely CRD and clinical failure.Endpoint-specic high/low miR-221 expression thresholdswere determined based onROCanalysis such that cutoff valuesrepresent the optimal tradeoff between specicity and sensi-tivity. Survival was illustrated byKaplanMeier curves; survivaldifferences between groupswere examinedwith log-rank tests.The inuence of miR-221 expression values as well as that ofvarious clinical and epidemiological parameters was analyzedwith univariate and multivariate Cox proportional hazardregression. The best tting COX model was selected by mea-suring the relative goodness of t with the Akaike informationcriterion (AIC). Differences in the mean values of miR expres-sion in 2 risk groups were analyzed by the 2-sided MannWhitney test.

ResultsmiR-221 as prognostic marker in high-risk prostatecancer

On the basis of our previous report indicating thatmiR-221 isa prognostic marker in prostate cancer, we analyzed twoindependent high-risk prostate cancer cohorts (cohort 1,n 134; cohort 2, n 89) to validate this nding. Patientselection and characteristics of both cohorts is provided inSupplementary Fig. S1 and Table S1, respectively. In bothcohorts, we determined the miR-221 expression by RT-PCRand found downregulation in the largemajority of the analyzedprostate cancer samples as compared with expression in BPHsamples (data not shown). Reductions in mean miR-221expression levels were identied between risk groups split byCRD but not for clinically used prognostic parameters, indi-cating an association between progressive miR-221 downre-gulation and tumor aggressiveness in both cohorts (Fig. 1A andSupplementary Fig. S2). The prognostic value of miR-221 forpredicting CRD in cohort 1 (learning cohort) was analyzedusing ROC analysis. The ROC analysis for CRD dened anoptimal cutoff level (DCt miR-221 < 0.32) to dichotomize thepatients into risk groups (Fig. 1B). The calculated area underthe curve (AUC) for CRDwas 0.903 (Fig. 1B). Using thismiR-221cut off level, we observed correct classication of 23.0% amongthe high-risk cases (11 of 37) and respectively 100% among low-risk cases (87 of 87). In KaplanMeier analysis, low miR-221expression (DCt miR-221 < 0.32) was signicantly correlatedwith CRD (Fig. 1C; P < 0.0001). Cox proportional hazardsregression analysis for time to CRD showed that miR-221expression, Gleason score, and lymph node invasion predictedCRD in univariate analysis. By stepwise regression analysis wegenerated a multivariate model for predicting CRD (deter-mined by AIC), which contained miR-221, Gleason score, andlymph node invasion, indicating that miR-221 functions as anindependent predictor for CRD (Fig. 1D) in this model. Theestimate of an HR for miR-221 was innity because there were100% correct classication in one group.

To validate the predictive potential of the determined miR-221 cutoff level, we used cohort 2 (test cohort). Using the samecutoff level for miR-221 (DCt 0.32) as for cohort 1, wedichotomized the test cohort and performed KaplanMeierestimates. Also in cohort 2, low miR-221 expression correlatedsignicantly with CRD (P < 0.001; Fig. 1C). Among the high-riskgroup 10 of 20 (50.0%) and among the low-risk group 68 of 69(97%) cases were correctly classied by miR-221. Samples ofthe test cohort with miR-221 expression under the previouslydetermined cutoff level were found to be associated with CRDby 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 out-come parameter either (Supplementary Fig. S3).

Expression of miR-221 in prostate cancer cells causesgrowth inhibition, apoptosis, and reduced invasivecapabilities

To analyze a tumor suppressor function of miR-221, wetransiently transfected LNCaP, DU-145, and PC-3 cells withprecursor-miR-221. We observed an efcient and strong miR-221 expression on day 2 posttransfection by qRT-PCR in allthree cell lines (Supplementary Fig. S4A). DU-145 and PC-3cells responded to miR-221 reexpression by a signicantdecrease in cell proliferation (48% decrease in DU-145 and69% in PC-3; P < 0.01), whereas the androgen-dependentLNCaP cells showed a moderate increase in proliferation (Fig.2A). In concordance with the observed inhibition in prolifer-ation in DU-145 and PC-3 cells, we also found reduced viabilityand changes in cellmorphology after premiR-221 transfection(Fig. 2B). To prove that the decrease in cell viability is linked toinduction of apoptosis, we analyzed the activity of caspase-3/7.The caspase-3/7 activity was signicantly increased after miR-221 transfection inDU-145 and PC-3 cells, whereas LNCaP cellsdid not show increased apoptosis (Fig. 2C). We next assessedwhether the expression of miR-221 had an impact on theinvasive activities. Boyden chamber invasion assays showedreduced invasion levels in miR-221transfected PC-3 cells (Fig.2D). These results indicate that miR-221 acts as tumor sup-pressor in PC-3 and DU-145 prostate cancer cells by regulatingcell growth, apoptosis, and invasiveness.

Global gene expression analysis of miR-221 reexpressingPC-3 cells

To search for molecular changes responsible for theobserved biological effects, we performed a microarray studyon mRNA isolated from premiR-221-transfected PC-3 cells.This analysis revealed a set of signicantly up- or downregu-lated genes in miR-221 reexpressing PC-3 cells (Fig. 3A). Wefound that from 54,675 genes on the array, 282 genes wereupregulated and 64 downregulated (>2-fold; P < 0.05; Supple-mentary Table S2). Many of the upregulated genes were knownto be also upregulated by interferons. Validation of the arraydata using qRT-PCR assays conrmed this upregulation aftermiR-221 transfection for STAT-1, IRF1, IRF9, OSA1, IFI27, andIFI44 in PC-3 (Fig. 3B and Supplementary Fig. S4B). Moreover,we found downregulation of several potential oncogenes

Kneitz et al.




including PMEPA1 or PRUNE by qRT-PCR (Fig. 3C and Sup-plementary Fig. S4C), which might function as potential targetgenes for miR-221.

Pathway analysis revealed that miR-221 reexpressionseemed to be preferentially associated with the TOLL-likereceptor-, RIG-like receptor-, or the JAK/STAT pathways andthat specically interactions of the JAK/STATpathway listed inthe KEGG pathway showed changes (Supplementary Fig. S5).

Because it was shown by several studies that transfectionwith synthetic small RNA molecules might randomly induceinammatory cytokines like interferons (25, 26), we decided to

generate PC-3 cells stable overexpressing miR-221. Using atransposon vector containing a TurboRFP-miR-221 fragmentand the pCMV(CAT)T7-SB100 expression plasmid for encod-ing the sleeping beauty transposase we generated PC-3/miR-221 cell clones. Three of 3 PC-3/miR-221 clones treated withdoxycycline showed >4 times overexpression of miR-221 (Sup-plementary Fig. S6A). Analyzing 2 PC-3/miR-221 clones weconrmed miR-221mediated growth inhibition and activa-tion of interferon-regulated genes (Supplementary Fig. 6B andC), conrming that miR-221 might specically regulate theinterferon-signaling pathway in PC-3 cells.

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miR-221 expression induces STAT1 and STAT3phosphorylation and sensitizes prostate cancer cells forthe antiproliferative effects of IFN-g

To elucidate, if miR-221 expression is sufcient for STAT1and STAT3 phosphorylation, we analyzed the expression ofSTAT1, pSTAT1, andpSTAT3 in premiR-221-transfected cells.miR-221 reexpression activates STAT1 in PC-3 andDU-145 andinduced STAT3 phosphorylation in DU-145 cell, but not in theSTAT3-negative PC-3 cells (Fig. 4A).

It was previously shown that IFNs mediate their antiproli-ferative function by phosphorylation and activation of the JAK/STAT pathway in prostate cancer cells. In fact, we could

observe that IFN-g treatment of miR-221transfected cellsresulted in a signicantly reduced proliferation (DU-145 cells78%, PC-3 cells 81% reduction, P < 0.01) compared with singleIFN-g treatment or untreated miR-221transfected cells (Fig.4B). In context with this IFN-g sensitization, we observedactivation of STAT1 and/or STAT3 in miR-221 reexpressingcells treated with IFN-g (Fig. 4A). Thus, we observed additiveeffects in IFN-mediated growth inhibition by miR-221 expres-sion in prostate cancer cells. In contrast, IFN-gresistantLNCaP cells, that are known to be SOCS3 negative, did notshow activation of STAT1 or inhibition of proliferation afterIFN-g treatment independent of miR-221 expression (Fig. 4A).

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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 celllines that were transfected with premiR-221 or premiR-precursor negative control (ctrl) and analyzed at day 2, 4, and 6 posttransfection. Mock-transfected cells showed no signicant differences to control cells and were excluded from the graph for better overview. Experiments were performedas 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 weretransfected with premiR-221 or pre-miR precursor negative control (ctrl). Caspase-3/7 activity was analyzed and was increased in PC-3 andDU145 cells transfected with premiR-221 when compared with control cells, but not in LNCaP cells. Results are presented in relation to the valuesmeasured in cells transfected with pre-miR precursor negative control that was arbitrarily set as 1. Data, mean values SD of ve independentexperiments; , P < 0.01, Wilcoxon rank sum test. D, miR-221 upregulation reduces cell migration of prostate cancer cells. PC-3 cells were transfectedwith premiR-221 or control siRNA. Migration of PC-3 cells was measured more than 6 hours in a Transwell cell culture chamber. Four chambers fromthree different experiments were analyzed (t test; P < 0.001). Each bar represents the mean SD. miR-221 overexpression in premiR-221-transfectedPC-3 cells as shown in Supplementary Fig. S7.

Kneitz et al.




miR-221 targets IRF2 and SOCS3 and inhibits expressionof IRF2 and SOCS3

Next we searched for miR-221 target genes, whose miR-221mediated downregulationmight be responsible for the observedbiological effects. By in silico analysis we identied potentialtarget sites in the 30 UTR mRNA regions of IRF2 and SOCS3.Both genes are known negative regulators of the JAK/STATsignaling cascade. Therefore, we analyzed the expression levelsin miR-221transfected cells and found moderate decrease inIRF2 or SOCS3 mRNA levels (Supplementary Fig. S7) and astrong reduction in protein levels (Figs. 4A and 5B).

To demonstrate a direct interaction between miR-221 andIRF2 or SOCS3, we generated pGF-IRF2 and pGF-SOCS3 lucif-erase constructs, containing the miR-221 binding sites. In

addition, vectors withmutations at putative binding sites werecloned and used as controls. These vectors were cotransfectedtogether with premiR-221 or scrambled miRNAs as negativecontrols followed by measurement of luciferase activity 48hours after transfection. As shown in Fig. 5A, the luciferaseactivity in PC-3 cells cotransfected with constructs containingthe 30 UTR of IRF2 or SOCS3 and premiR-221 was decreasedby 62% (IRF2) and 41% (SOCS3).

Downregulation of IRF2 or SOCS3 recapitulate thebiological effects of miR-221 reexpression in prostatecarcinoma cells

To test whether IRF2 and SOCS3 are involved in the miR-221mediated regulation of the JAK/STAT pathway, we

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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 anda 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 analyzedrelative expression of selected genes, which were shown to be upregulated (B) or downregulated (C) on the array. Expression of indicated genes wassignicantly dysregulated in miR-221-transfected PC-3 cells. Normalized qRT-PCR results from miR-221-transfected cells were calculated as x-timeexpression changes in comparison to PC-3 cells transfected with pre-miR precursor negative control. Data represent mean values SD of ve independentexperiments. The relative expression level of each gene in control transfected PC-3 cells was arbitrarily set as 1. Signicant differences (P < 0.01) betweenexpression in control and miR-221-transfected cells are indicated by the asterisk (). P values were calculated by Student t test.

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

www.aacrjournals.org Cancer Res; 74(9) May 1, 2014 2597



inhibited the expression of both genes in PC-3 cells. siRNAknockdown of both genes caused a strong and efcientdecrease of protein levels (8090%; Fig. 5B). We also observedsignicantly reduced proliferation, induction of apoptosisand activation of STAT1 in response to siRNA-mediatedIRF2 or SOCS3 downregulation (Fig. 5C and D). We conclud-ed that the biological effects caused by miR-221 overexpres-sion are mediated at least partially by downregulation ofSOCS3 and IRF2.

In vivo regulation of IRF2 and SOCS3 by miR-221 inprostate cancer

To assess the role of miR-221mediated inhibition of IRF2and SOCS3 mRNA expression in primary prostate cancer, weselected a group of fresh frozen tumor samples on the basis oftheir miR-221 expression. In this series of prostate cancersamples we correlated the expression of IRF2 and SOCS3 inresponse tomiR-221 downregulation. As Fig. 6 shows, we foundan inverse correlation between miR-221 downregulation andupregulation of IRF2 or SOCS3 by Spearman rank correlation

analysis, whereas themRNA expression of SOCS3 and IRF2wasnot correlated. We concluded that miR-221 is also in vivocritically involved in the expression of both potential targetgenes.

DiscussionBased on the lack of prognostic models to accurately predict

survival, the need to better identify patients with lethal diseaseis one of the main challenges in prostate cancer research. Wepreviously demonstrated that miR-221 downregulation hall-marks lymph node metastasis and possesses potential as aprognostic marker in high-risk prostate cancer (13). Here wedemonstrated that miR-221 predicted clinical failure andsurvival of patients with high-risk prostate cancer and deter-mined a specic miR-221 expression level as independentpredictive marker for CRD and clinical failure. Using anindependent test cohort we successfully validated the predic-tive power of miR-221 in predicting CRD and clinical failure.The role of miR-221 as a prognostic biomarker is further

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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 andWestern 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 aftertransfection with premiR-221 or by IFN-g treatment. B, MTS assay for the growth of indicated prostate cancer cell lines that were transfected with premiR-221 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 controlcells and were not added to the graph for better overview. Experiments were performed as triplicates. Data, mean SD from ve independent experiments.

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supported by a recent report showing a correlation of miR-221downregulation on BCR and clinical failure in TMPRSS:ERGfusion positive prostate cancer (27). The results presented hereprovide the groundwork to prospectively test miR-221 as atissue-based biomarker in patients with high-risk prostatecancer and to develop new treatment strategies within newclinical trial concepts (28).

Such new therapies are strongly related to the biologicalfunction of each individual microRNA. Several studies includ-ing our biomarker analysis clearly suggested a tumor suppres-sive function of miR-221 in prostate cancer. Therefore, we

analyzed the effects of reexpressing miR-221 in androgenindependent prostate cancer cell lines and demonstrated thatmiR-221 reexpression reduced proliferation, invasiveness, andinduced apoptotic cell death, indicating a tumor suppressorrole of miR-221 in androgen independent prostate cancer cells.

To elucidate molecular pathways regulated by miR-221, weanalyzed global mRNA expression proles in miR-221 expres-sing prostate cancer cells. Interestingly, besides the down-regulation of oncogenic target genes such as PMEPA1 andPRUNE (29, 30), we found an increased expression of genes,known to be associated with cell exposure to interferons.

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Figure 5. miR-221 expression inhibits expression of IRF2 and SOCS3 and siRNA-mediated downregulation of IRF2 and SOCS3 mimics effects ofmiR-221 reexpression in prostate cancer cells. A, SOCS3 and IRF2 are targets of miR-221. SOCS3 and IRF2 luciferase constructs, containing a wild-type ormutated 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-221binding site showed no signicant difference in reporter activity compared with control transfected cells. Relative expression of rey luciferase wasstandardized to control transfections. Luciferase activities were analyzed 48 hours after transfection. Reporter activities of cells cotransfected with miR-precursor negative control (black bars) were arbitrarily set as 1. The results were obtained from three independent experiments and are presented asmeanSD. B, miR-221 reexpression or siRNA treatment decreased expression levels of SOCS3 or IRF2 and activated STAT1. PC-3 cells were transfected withnegative control, premiR-221, andSOCS3siRNAor IRF2siRNA for 48hours.Western blotswereperformed to analyze theexpressionof pSTAT1andSOCS3or 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 knockdownofSOCS3or IRF2on thegrowth ofPC-3 cells.MTSassay analysis for thegrowth ofPC-3 cells. Cellswere transfectedwith SOCS3 or IRF2 siRNA and control siRNA. On day 1 posttransfection, 10 ng/mL IFN-g was added to the cultures when indicated. Cell culturereplicates were analyzed at day 2, 4, and 6 posttransfection Experiments were performed in triplicates. Presented data are mean values SD from threeindependent experiments. D, PC-3 cells (ctrl) were comparedwith 1PC-3 cells transfectedwith premiR-ctrl, siRNA ctrl, premiR-221, SOCS3 siRNA, or IRF2siRNA. 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.





Pathway analysis revealed activation of the Toll-like receptor,the RIG-like receptor, and most impressively the JAK/STATpathways. IFN-mediated JAK/STAT activation in cancer devel-opment is not unexpected, because IFN usually functions as acytokine with antitumor activity (31), moreover, there is agrowing body of evidence that activation of the JAK/STATpathway can inhibit proliferation and induce apoptosis incertain microenvironmental conditions. STAT1 is a knowntumor suppressor involved in tumor development and expan-sion by switching on antiproliferative and proapoptotic path-ways (32, 33). We detected STAT1 and STAT3 phosphorylationin miR-221 reexpressing cells, indicating a strong JAK/STATpathway activation. An antitumorigenic activity by miR-221mediated STAT3 activation is conicting, because of its onco-genic function in some tumor entities (3436). However,

STAT3 activation can induce growth arrest and apoptosisunder certain conditions in various cancer types includingprostate cancer (22, 37, 38). The activation of both STAT1 andSTAT3might explain at least partially the antiproliferative andproapoptotic 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 knownnegative regulator genes of the JAK/STAT pathway, asmiR-221targets. The role of SOCS3 as inhibitor of the JAK/STATpathway in prostate cancer is documented by the observationthat STAT1 and STAT3 phosphorylation is inversely correlatedwith SOCS3 expression (39). Moreover, SOCS3 downregulationdetermined reduced proliferation rates and an increased apo-ptotic response by converting the antiapoptotic STAT3 func-tion into proapoptotic (40). It was also shown that reduced

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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, andIRF2 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 nontumorigenicprostate sample. Subsequently, the samples of the cohort were divided into subgroups based on amore than 2-fold downregulation (log-fold change >1 or 1). B, plots showing the coefcient of correlation for relative expression levels ofmiR-221, SOCS3, and IRF2 from the samples described above.

Kneitz et al.




SOCS3 protein expression enhanced the IFN-g responsiveness,indicating a regulation of IFN-g sensitivity in prostate cancercells and other tumors by SOCS3 (22, 41).

In various cancer types, IRF2 overexpressionwas found to beassociatedwith the development and progression ofmalignantphenotypes. IRF2 acts as an antagonist to the tumor suppres-sor IRF1 and it is known that the IRF1/IRF2 balance is criticallyinvolved in the immunomodulatory, antiproliferative, andproapoptotic IFN-g effects (42, 43). Previous studies haveshown that IRF1 and IRF2 are regulating transcription of thesame IFN-ginducible genes (44), but with entirely opposingeffects for cell growth and tumorigenicity (23). Thus, there isgrowing body of evidence that the IRF2 expression determinesthe cellular response to JAK/STAT pathway. Here we demon-strated that miR-221 reexpression induced IRF1 and down-regulated IRF2 expression. In addition, we found that, similaras recently described for pancreatic cells, IRF2 knockdownleads to growth inhibition and apoptosis in prostate cancercells. Based on these results it is very likely that the down-regulation of SOCS3 and IRF2 is responsible for the antitu-morigenic biological effects in miR-221 reexpressing prostatecancer cells. The relevance of these results for tumor devel-opment and tumor progression is further supported by theinverse correlation between miR-221 and SOCS3 or IRF2expression in primary prostate cancer probes. Figure 7summarizes a model how miR-221 regulates the JAK/STATpathway and how miR-221 downregulation inhibits IFN-gmediated antiproliferative and proapoptotic signals inprostate cancer cells.

Our present study also provides evidence for a possible roleof miR-221 to overcome the problem of low sensitivity against

cytokine therapies in prostate cancer. Interferon therapy wasdiscussed for clinically advanced prostate cancer (45). How-ever, systemic IFN-g therapy in prostate cancer has shown onlylimited efciency (46). We now demonstrate that miR-221expression mediates the responsiveness against the antitu-morigenic effects of IFN-g in vitro. Moreover, we show evidencethat miR-221 downregulation might reduce the IFN-g respon-siveness in primary prostate cancer by upregulation of twoindependent negative regulator proteins (SOCS3 and IRF2) ofthis cytokine pathway. It is well known that the interaction ofvarious negative regulatory proteins involved in cytokine sig-naling is very complex. Here we show that miR-221 is able tocontrol such a signaling pathway by regulating various com-ponents and therefore it might be a good candidate fortherapeutic use.

However, microRNAtargeted therapy is challenging. Tis-sue-specic delivery, stability, cellular uptake, and off-targeteffects might be overcome by technical solutions in the future,but safety might remain a major concern in microRNAbasedtherapy. Several cancerassociated miRNA showed pivotalroles in tumor development and progression because amiRNAcan function either as an oncogene if in a given cell type itscritical target is a tumor suppressor or the samemiRNA can bea tumor suppressor if in a different cell type its target is anoncogene (9). This seems to be true also for miR-221. Over-expression and regulation of tumor suppressor genes (i.e.,p27kip1, Pten, etc.) were described for miR-221 in severaltumor entities (1417, 19, 20), whereas we and others showedthat miR-221 is one of the most strongly and frequently down-regulated miRNA in primary prostate cancer inhibiting theexpression of the potential oncogenes IRF2 and SOCS3 (1013).

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 inturn, leads to an activation of the JAK-STATpathway bySTAT1phosphorylation and increased IRF1-inducedgene expression, resulting in the activation of anapoptotic pathway, cell growth inhibition, and decreased invasive activity. Modied based on: 20002011 Ingenuity Systems, Inc. All rights reserved.





However, in prostate cancer the situation seems to be evenmore complex, because miR-221 expression levels were shownto be increased in tumor tissue derived frombonemetastasis ofcastration-resistant prostate cancer (CRPC; refs. 47 and 48).Nevertheless, this observation is not per se mutually exclusivewith the ndings in this study. Although Sun and colleaguesobserved miR-221 overexpression only in CRPC, we detectedmiR-221 downregulation in hormone nave tumors. Theseobserved differences implicate a specic function of miR-221 in the development of androgen resistance. Recent studiesby Sun and colleagues supported this suggestion showing thatthe development of androgen independence in LNCaP cellswas promoted via miR-221mediated downregulation ofHECTD2 and RAB1A re-programming the androgen signalingpathway (47). In contrast to these resultswedemonstrated thatmiR-221 overexpression activated the antiproliferative andproapoptotic JAK/STAT pathway only in androgen-indepen-dent, SOCS3-positive DU-145 and PC-3, but not in androgen-dependent, SOCS3-negative LNCaP-cells. One possible expla-nation for the diverging results might be a pivotal function ofmiR-221 in the regulation of androgen-independent growthand interferon signaling in the presence or absence of SOCS3,because it was shown that sensitivity against androgen andinterferon signaling in prostate cancer cells depends on SOCS3expression (49). Therefore, we suggest that the different func-tion of miR-221 in prostate cancer cells at least partiallydepends on a SOCS3-mediated regulation of the androgenreceptor- or the interferon-signaling pathway in prostate can-cer cells. Future in vitro and in vivo analysis describing apossible role of miR-221 in controlling various signaling path-way via posttranscriptional regulation of SOCS3 and otherpotential target genes might clarify this clinically relevantquestion.

In summary, we demonstrated for the rst time that miR-221 has tumor suppressive function in prostate cancer con-trolling apoptotic pathways, cell growth, and invasiveness. The

antitumorigenic effect of miR-221 expression is mediated atleast partially by activation of the JAK/STAT pathway. Wecould show that miR-221 regulates two of the most importantnegative regulator proteins, SOCS3 and IRF2, of the JAK/STATsignaling pathway, indicating a role of miR-221 as a masterregulator of IFN-g sensitivity in prostate cancer cells. More-over, we demonstrated that miR-221 expression is progres-sively decreased during prostate cancer development andprogression in clinical specimens and is an independentprognostic marker to predict cancer-related death in high-riskprostate cancer. On the basis of our results, we think that miR-221 has potential as a prognostic biomarker and as a target forfuture therapies of high-risk prostate cancer.

Disclosure of Potential Conicts of InterestNo potential conicts of interest were disclosed.

Authors' ContributionsConception and design: B. Kneitz, S. Joniau, M. SpahnDevelopment of methodology: B. Kneitz, M. SpahnAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. Kneitz, M. Krebs, C. Kalogirou, M. Schubert, H. vanPoppel, E. Lerut, C.J. Scholz, P. Strobel, M. Gessler, M. SpahnAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B. Kneitz, M. Krebs, C. Kalogirou, S. Joniau, S. Kneitz,M. Gessler, M. SpahnWriting, review, and/or revision of the manuscript: B. Kneitz, S. Joniau,H. van Poppel, S. Kneitz, P. Strobel, M. SpahnAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): H. RiedmillerStudy supervision: B. Kneitz, H. van Poppel, H. Riedmiller, M. Spahn

AcknowledgmentsThe 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 ofpage charges. This article must therefore be hereby marked advertisement inaccordance 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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2014;74:2591-2603. Published OnlineFirst March 7, 2014.Cancer Res

Burkhard Kneitz, Markus Krebs, Charis Kalogirou, et al.

Prostate Cancer Cells by Inhibiting IRF2 and SOCS3ofmiR-221, Which Regulates Proliferation, Apoptosis, and Invasion

Survival in Patients with High-Risk Prostate Cancer Is Predicted by

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