Cancer Res-2012-Majid-6435-46

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Molecular and Cellular Pathobiology miR-23b Represses Proto-oncogene Src Kinase and Functions as Methylation-Silenced Tumor Suppressor with Diagnostic and Prognostic Signicance in Prostate Cancer Shahana Majid 1 , Altaf A. Dar 2 , Sharanjot Saini 1 , Sumit Arora 1 , Varahram Shahryari 1 , Mohd Saif Zaman 1 , Inik Chang 1 , Soichiro Yamamura 1 , Yuichiro Tanaka 1 , Guoren Deng 1 , and Rajvir Dahiya 1 Abstract The miRNAs have great potential as biomarkers and therapeutic agents owing to their ability to control multiple genes and potential to inuence cellular behavior. Here, we identied that miR-23b is a methylation- silenced tumor suppressor in prostate cancer. We showed that miR-23b expression is controlled by promoter methylation and has great promise as a diagnostic and prognostic biomarker in prostate cancer. High levels of miR-23b expression are positively correlated with higher overall and recurrence-free survival in patients with prostate cancer. Furthermore, we elucidated the tumor suppressor role of miR-23b using in vitro and in vivo models. We showed that proto-oncogene Src kinase and Akt are direct targets of miR-23b. Increased expression of miR-23b inhibited proliferation, colony formation, migration/invasion, and triggered G 0 G 1 cell-cycle arrest and apoptosis in prostate cancer. Overexpression of miR-23b inhibited epithelial-to-mesenchymal transition (EMT) causing a decline in mesenchymal markers Vimentin and Snail and increasing the epithelial marker, E-cadherin. Depletion of Src by RNA interference conferred similar functional effects as that of miR-23b reconstitution. miR- 23b expression caused a dramatic decrease in tumor growth in nude mice and attenuated Src expression in excised tumors compared with a control miR. These ndings suggest that miR-23b is a methylation-silenced tumor suppressor that may be a useful biomarker in prostate cancer. Loss of miR-23b may confer proliferative advantage and promote prostate cancer migration and invasion, and reexpression of miR-23b may contribute to the epigenetic therapy for prostate cancer. Cancer Res; 72(24); 643546. Ó2012 AACR. Introduction Prostate cancer is the most frequently diagnosed malig- nant tumor and second leading cause of cancer deaths in American men. It is estimated that 240, 890 newly diagnosed prostate cancer cases and 33,720 attributed deaths will occur in 2011 (1). Current prostate cancer treatments consisting of malignant prostate ablation by radical prostatectomy, radio- therapy, hormonal therapy, and/or neoadjuvant chemother- apy are generally curative for the majority of patients diagnosed with localized and androgen-dependent prostate cancer. However, progression to androgen-independent and metastatic disease states is often accompanied by a recur- rence of prostate cancer and treatment remains a clinical challenge (2, 3). The miRNAs are nonprotein-coding sequences thought to regulate more than 90% of human genes (4). Growing evidence has strongly implicated the involvement of miRNAs in carci- nogenesis (5, 6). From a clinical point of view, miRNAs have great potential as diagnostic and therapeutic agents. Micro- array analysis has shown a general downregulation of miRNAs in tumors when compared with normal tissues (7). Owing to the tissue specicity of miRNAs, they have become a useful tool for dening the origin of tumors in poorly differentiated cancers (8). Prognosis and survival of patients depends on the cancer stage at diagnosis. For this reason, an important issue in clinical cancer research is to identify early biomarkers of the early tumorigenic process. miRNA signatures have been reported to be useful tools for early diagnosis of cancer (9, 10). DNA hypermethylation of CpG sites within CpG islands is known to lead to the inactivation of many tumor-suppressive miRNAs (11). One of the most common causes of the loss for tumor suppressor miRNAs is silencing of their primary tran- scripts by CpG island hypermethylation (1216). The DNA methylation prole of tumors is useful to dene tumor type, clinical prognosis, and treatment response (17, 18). Epigenetic silencing of miRNAs is also involved in the acquisition of an invasive phenotype and the development of metastasis (19). Inactivation of oncogenic miRNAs (20, 21) or restoration of Authors' Afliations: 1 Department of Urology, VA Medical Center and UCSF; and 2 California Pacic Medical Center Research Institute, San Francisco, California Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Note: A.A. Dar and S. Saini both contributed equally to this work. Corresponding Author: Rajvir Dahiya, Professor and Director, Urology Research Center (112F), V A Medical Center and UCSF, 4150 Clement Street, San Francisco, CA 94121. Phone: 415-750-6964; Fax: 415-750- 6639; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-12-2181 Ó2012 American Association for Cancer Research. Cancer Research www.aacrjournals.org 6435 on March 16, 2015. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst October 16, 2012; DOI: 10.1158/0008-5472.CAN-12-2181

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Transcript of Cancer Res-2012-Majid-6435-46

  • Molecular and Cellular Pathobiology

    miR-23b Represses Proto-oncogene Src Kinase andFunctions as Methylation-Silenced Tumor Suppressor withDiagnostic and Prognostic Signicance in Prostate Cancer

    Shahana Majid1, Altaf A. Dar2, Sharanjot Saini1, Sumit Arora1, Varahram Shahryari1, Mohd Saif Zaman1,Inik Chang1, Soichiro Yamamura1, Yuichiro Tanaka1, Guoren Deng1, and Rajvir Dahiya1

    AbstractThe miRNAs have great potential as biomarkers and therapeutic agents owing to their ability to control

    multiple genes and potential to inuence cellular behavior. Here, we identied that miR-23b is a methylation-silenced tumor suppressor in prostate cancer. We showed that miR-23b expression is controlled by promotermethylation and has great promise as a diagnostic and prognostic biomarker in prostate cancer. High levels ofmiR-23b expression are positively correlated with higher overall and recurrence-free survival in patients withprostate cancer. Furthermore, we elucidated the tumor suppressor role of miR-23b using in vitro and in vivomodels.We showed that proto-oncogene Src kinase andAkt are direct targets ofmiR-23b. Increased expression ofmiR-23b inhibited proliferation, colony formation, migration/invasion, and triggered G0G1 cell-cycle arrest andapoptosis in prostate cancer. Overexpression of miR-23b inhibited epithelial-to-mesenchymal transition (EMT)causing a decline in mesenchymal markers Vimentin and Snail and increasing the epithelial marker, E-cadherin.Depletion of Src by RNA interference conferred similar functional effects as that of miR-23b reconstitution. miR-23b expression caused a dramatic decrease in tumor growth in nude mice and attenuated Src expression inexcised tumors compared with a control miR. These ndings suggest that miR-23b is a methylation-silencedtumor suppressor that may be a useful biomarker in prostate cancer. Loss of miR-23b may confer proliferativeadvantage and promote prostate cancer migration and invasion, and reexpression of miR-23b may contribute tothe epigenetic therapy for prostate cancer. Cancer Res; 72(24); 643546. 2012 AACR.

    IntroductionProstate cancer is the most frequently diagnosed malig-

    nant tumor and second leading cause of cancer deaths inAmerican men. It is estimated that 240, 890 newly diagnosedprostate cancer cases and 33,720 attributed deaths will occurin 2011 (1). Current prostate cancer treatments consisting ofmalignant prostate ablation by radical prostatectomy, radio-therapy, hormonal therapy, and/or neoadjuvant chemother-apy are generally curative for the majority of patientsdiagnosed with localized and androgen-dependent prostatecancer. However, progression to androgen-independent andmetastatic disease states is often accompanied by a recur-

    rence of prostate cancer and treatment remains a clinicalchallenge (2, 3).

    The miRNAs are nonprotein-coding sequences thought toregulate more than 90% of human genes (4). Growing evidencehas strongly implicated the involvement of miRNAs in carci-nogenesis (5, 6). From a clinical point of view, miRNAs havegreat potential as diagnostic and therapeutic agents. Micro-array analysis has shown a general downregulation of miRNAsin tumors when compared with normal tissues (7). Owing tothe tissue specicity ofmiRNAs, they have become a useful toolfor dening the origin of tumors in poorly differentiatedcancers (8). Prognosis and survival of patients depends on thecancer stage at diagnosis. For this reason, an important issue inclinical cancer research is to identify early biomarkers of theearly tumorigenic process. miRNA signatures have beenreported to be useful tools for early diagnosis of cancer (9, 10).

    DNA hypermethylation of CpG sites within CpG islands isknown to lead to the inactivation of many tumor-suppressivemiRNAs (11). One of the most common causes of the loss fortumor suppressor miRNAs is silencing of their primary tran-scripts by CpG island hypermethylation (1216). The DNAmethylation prole of tumors is useful to dene tumor type,clinical prognosis, and treatment response (17, 18). Epigeneticsilencing of miRNAs is also involved in the acquisition of aninvasive phenotype and the development of metastasis (19).Inactivation of oncogenic miRNAs (20, 21) or restoration of

    Authors' Afliations: 1Department of Urology, VA Medical Center andUCSF; and 2California Pacic Medical Center Research Institute, SanFrancisco, California

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

    Note: A.A. Dar and S. Saini both contributed equally to this work.

    Corresponding Author: Rajvir Dahiya, Professor and Director, UrologyResearch Center (112F), V A Medical Center and UCSF, 4150 ClementStreet, San Francisco, CA 94121. Phone: 415-750-6964; Fax: 415-750-6639; E-mail: [email protected]

    doi: 10.1158/0008-5472.CAN-12-2181

    2012 American Association for Cancer Research.

    CancerResearch

    www.aacrjournals.org 6435

    on March 16, 2015. 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

    Published OnlineFirst October 16, 2012; DOI: 10.1158/0008-5472.CAN-12-2181

  • tumor suppressor miRNAs (1214) has great potential forcancer treatment.

    Here, we report that (i) miR-23b is frequently silencedthrough tumor-specic DNA methylation in prostate cancer,(ii) miR-23b acts as a tumor suppressor miRNA and hasdiagnostic/prognostic implication in prostate cancer, (iii)miR-23b directly targets proto-oncogene Src kinase and Akt,(iv) miR-23b has anti-proliferative/-migratory/-invasive effectsby downregulating molecules involved in these pathways, (v)miR-23b inhibits epithelial-to-mesenchymal transition (EMT)markers, and (vi) miR-23b decreased in vivo tumor growth andSrc kinase expression in nude mice xenografts.

    Materials and MethodsCell culture, plasmids, and probes/primers

    Human prostate cancer cell lines PC3, DU145, LNCaP and anonmalignant prostate cell line RWPE1 were obtained fromthe American Type Culture Collection (ATCC) and grownaccording to ATCC protocol. These human-derived cell lineswere authenticated by DNA short-tandem repeat analysis byATCC. The experiments with cell lines were carried out within6 months of their procurement/resuscitation. Plasmids pEZX-MT01 miRNA 30-untranslated region (UTR) target expressionclones for Src (HmiT017696-MT01), AKT (HmiT004995-MT01)and miRNA Target clone control vector for pEZX-MT01(CmiT000001-MT01) were purchased from GeneCopoeia. Taq-Man probes for hsa-miR-23b (miR-23b) and negative controlpre-miR were purchased from Applied Biosystems. siRNAduplexes [(Src (Human)-3 unique 27mer siRNA duplexes(SR304574)] were purchased from Origene Technologies, Inc.

    Quantitative real-time PCRTissue samples from radical prostatectomy were obtained

    from the Veterans Affairs Medical Center (San Francisco, CA).Total RNA was extracted and assayed for mature miRNAs andmRNAs using the TaqManMicroRNAAssays andGene Expres-sion Assays, respectively, in accordance with the manufac-turer's instructions (Applied Biosystems). All real-time reac-tions were run in a 7500 Fast Real Time PCR system (AppliedBiosystems). Relative expression was calculated using thecomparative Ct.

    Methylation analysis of miR-23b by quantitativemethylation-specic PCR

    To investigate the mechanism involved in reduced levels ofmiR-23b in prostate cancer, we conducted methylation anal-ysis in the 1.0-kb upstream sequence of miR-23b. Two CpGislands: CG-1 (120 to 3 bp) and CG-2 (820 to 650 bp)were located in 1.0 kb upstream sequence and further analyzedformethylation status in cell lines and tissue samples. DNAwasavailable only for 38 (19 pairs) of laser-capturedmicrodissected(LCM) tissue samples, and these samples were from the samecohort of 118 samples for which RNA expression was available.DNA was bisulfate-converted using EZ DNAMethylation-GoldKit (ZymoResearch) according to themanufacturer's protocol.The converted DNA was amplied by PCR with 400 pmol/L ofeither primer set F1/R1, or F2/R2, and HotStar Taq Plus DNA

    Polymerase (Qiagen). PCR was carried out by denaturation at95C for 5minutes, followed by 15 cycles of 94C for 30 seconds,56C for 30 seconds, and 72C for 30 seconds. Two microlitersof the PCR product was added to 40 mL solution containing 20mL TaqMan Fast Universal PCR Master Mix (2; AppliedBiosystems), 500 pmol/L primers F1/R1 or F2/R2. The mixedsolution was aliquoted evenly into 2 tubes and was added 1 mL,5 mmol/L probe for methylation reaction (PM) probe formethylation (M) reaction and 1 mL, 5 mmol/L probe forunmethylation reaction (PU) probe for unmethylation (U)reaction, respectively. Methylation in CGI-1 and CGI-2 wasmeasured by quantitative real-time PCR (qRT-PCR) with anApplied Biosystems 7500 Fast Sequence Detection. For eachsample, the percentage of methylation was calculated by thedifference of Ct inM reaction (Ct_M) andCt in U reaction (Ct_U).

    In situ hybridizationIn situ hybridization (ISH) was conducted as described

    previously (22). Briey, cell lines and tissues were stained usingDIG-labeled locked nucleic acid (LNA)-based probes specicfor mir-23b and U6 following the manufacturer's protocol(Exiqon, Inc.) and detected using anti-DIG-Fluorescein, FabFragments (Roche Applied Science for cell lines and BMPurpleAP Substrate (Roche Applied Science) for tissues. ISH resultsfor tissue array were graded according to quick score (per-centage of cells stained intensity of stain) and normalized toU6 levels.

    Flow cytometry, cell viability, migration, clonability, andinvasion assays

    Fluorescence-activated cell-sorting (FACS) analysis for cellcycle and apoptosis was done 72 hours posttransfection usingnuclear stain, 40,6-diamidino-2-phenylindole (DAPI), for cell-cycle analysis or Annexin V-FITC/7-AAD Kit (Beckman Coul-ter, Inc.) for apoptosis analysis according to themanufacturer'sprotocol. Cell viability was determined at 24, 48, and 72 hoursby using the CellTiter 96 AQueous One Solution Cell Prolifer-ation Assay Kit (Promega) according to the manufacturer'sprotocol. For colony formation assay, cells were seeded at lowdensity (1,000 or 200 cells per plate) and allowed to grow untilvisible colonies appeared. Then, cells were stainedwithGiemsaand colonies were counted. Cytoselect 24-well cell migrationand invasion assay kit (Cell Biolabs, Inc.) was used for migra-tion and invasion assays according tomanufacturer's protocol.

    Immunoblotting and immunouorescenceImmunoblotting was conducted as described previously

    (23). The antibodies used were specic for Src (2123; CellSignaling), pSrc (2101; Cell Signaling), MEK1/2 (4694; CellSignaling), pMEK1/2 (Ser217/221; 9154; Cell Signaling), p44/42 MAPK (Erk1/2; 4695; Cell Signaling), p-p44/42 MAPK(Thr2021/Tyr204; 4370; Cell Signaling), STAT3 (9132), p-STAT3(Tyr705; 9145; Cell Signaling), Akt (4685; Cell Signaling), p-Akt(Ser473; 4060; Cell Signaling), Bad (9292; Cell Signaling), p-Bad(Ser136; 4366; Cell Signaling), FAK (3285; Cell Signaling), c-Jun(9165; Cell Signaling), and GAPDH (sc-32233; Santa Cruz Bio-technology, Inc.). Blots were visualized using Western blottingluminal reagent (sc-2048; Santa Cruz Biotechnology, Inc.).

    Majid et al.

    Cancer Res; 72(24) December 15, 2012 Cancer Research6436

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  • For immunouorescence, cells were transfected with pre-cursors ofmiR-23b or cont-miR for 72 hours, washed, and xedwith acetone-methanol (1:1) mixture and hybridized with thespecic primary antibodies against EMT markers. Cells werewashed and hybridizedwith uorescein-conjugated secondaryantibody (1:1,000) and mounted with ProLong Gold antifadereagent with DAPI (Invitrogen Life Technologies).

    Luciferase assaysThe complimentary sites in 30UTR of Src and Akt for miR-

    23b and mutated sequences are given in Supplementary TableS7. The Src, Akt, and control vectors were purchased fromGeneCopoeia and named Src-30UTR and Akt 30UTR. Mutated30UTR sequences of Src and Akt were cloned and named MutSrc30UTR and Mut Akt30UTR. For reporter assays, cells weretransiently transfected with wild-type or mutated reporterplasmid and miR-23b or control-miR. Firey luciferase activ-itiesweremeasured using theDual Luciferase Assay (Promega)18 hours after transfection, and the results were normalizedwith Renilla luciferase. Each reporter plasmid was transfectedat least 3 times, and each sample was assayed in triplicate.

    In vivo intratumoral delivery of miR-23b and genisteinThe antitumor effect of miR-23b was determined by local

    administration of miR-23b precursor in established tumors.Each mouse was injected subcutaneously with 5.0 106 PC3prostate cancer cells. Once palpable tumors developed (aver-age volume 55 mm3), 6.25 mg of synthetic miRNA complexedwith 1.6 mL siPORT Amine transfection reagent (Ambion) in 50mL PBS was delivered 8 times intratumorally at 3-day intervals.Tumor growthwas followed for 21 days from the rst injection.All animal care was in accordance with the institutionalguidelines.

    Statistical analysisStatistical analyses were conducted with StatView for Win-

    dows (SAS Institute Inc.), GraphPad-Prism5 and MedCalc. Allquantied data represent average of at least triplicate samplesor as indicated. Error bars represent SD of mean. All testsconducted were 2-tailed, and P < 0.05 was considered statis-tically signicant. Receiver-operating curves (ROC) were cal-culated to determine potential ofmiR-23b or itsmethylation todiscriminate between malignant and nonmalignant samples.The c2 tests were conducted to determine correlation betweenmiR-23b expression and clinicopathologic characteristics. Fordisease progression analysis, KaplanMeier approach (log-rank test) and multiple regression analysis were conducted.

    ResultsmiR-23b is signicantly downregulated in prostatecancer

    To identify silenced miRNAs in prostate cancer, we con-ducted microarray screening using cancer cell lines and anonmalignant cell line. Mir-23b was found to be a signicantlydownregulated miRNA in cancer compared with the nonma-lignant cell line. We validated the microarray data by miRNAqRT-PCR analysis. The results conrmed that miR-23b wasdownregulated in cancer cell lines (Fig. 1A). In situ hybridiza-

    tion also conrmed the presence of miR-23b expression (greensignal) in RWPE1 cells compared with cancer cell lines (Fig.1B). DU145 cells express higher levels of miR-23b but thegreen signal is absent because we have to keep the exposuretime same across the cell lines. To examine the biologicsignicance of miR-23b, its expression was analyzed byqRT-PCR in 236 (118 pairs) LCM matched tissue samples(Fig. 1C; Supplementary Fig. S1) and an unmatched group of27 benign prostatic hyperplasia (BPH) and 20 tumor samples(Fig. 1D) along with another cohort of 48 samples whereexpression was analyzed by ISH (Fig. 1E). Among all samples,miR-23b expression was signicantly downregulated in can-cer samples compared with normal or BPH (Fig. 1CE). Theseresults indicate a putative tumor suppressor role of miR-23bin prostate cancer.

    Diagnostic and prognostic signicance of miR-23b inprostate cancer

    Clinical demographics of the study cohort are summarizedin Supplementary Table S1. ROC analyses were conducted toevaluate the ability of miR-23b expression to discriminatebetween normal and tumor cases using 151 pairs of tissuesamples. An area under the ROC curve (AUC) of 0.975 [P 0.8-fold) groups and conducted KaplanMeier survival analysisandmultiple regression analysis. In KaplanMeier analysis, themiR-23b high group displayed signicantly higher overallsurvival (OS) probability than themiR-23b low group (log-ranktest: P < 0.0001; HR, 3.3; 95% CI, 419; Fig. 2B). KaplanMeiersurvival analysis for recurrence-free survival (RFS) was con-ducted using 105 cases. Cases with high miR-23b expressionhad better RFS than with low miR-23b expression cases (log-rank test: P < 0.002; HR, 6; 95% CI, 313; Fig. 2C). We conductedmultiple regression analysis for the same set of patients usingentry, forward, backward, and stepwise methods one by one(Supplementary Tables S2S5). Multiple regression analysisrevealed that miR-23b expression is an independent predictorof biochemical recurrence (P < 0.02) as determined in entry,forward, backward and stepwise methods (SupplementaryTable S2). We also determined the correlation of miR-23bexpression with clinicopathologic variables such as Gleasongrade, pathologic stage (pT), and biochemical recurrence (Fig.2D) and details are described in detail in SupplementaryResults. Correlation tests revealed that cases with low miR-23b expression increased from low-grade, low pathologic stageto high-grade and high pathologic stage (Fig. 2D). Patients whohad prostate-specic antigen (PSA) recurrence also had sig-nicantly lowmiR-23b expression. These ndings suggest thatmiR-23b has a potential to be a diagnostic and prognosticmarker for predicting the biochemical recurrence of patientswith prostate cancer, although addition of more samples maystrengthen these results.

    miR-23b in Prostate Cancer

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  • Mechanism of silencing of miR-23b in prostate cancer isthrough CpG hypermethylation: correlation betweenmiR-23b methylation status with expression and use asdiagnostic marker

    Two CpG islands, CG-1 and CG-2, were located in 1.0 kbupstream sequence (Fig. 3A). CG-1 was the methylation "hotspot" that was methylated in cancer cell lines and in tumors.Our results showed a hypermethylated sequence in matchedtumor tissue samples compared with their normal counter-parts (Fig. 3B). Similarly, prostate cancer cell lines were alsohypermethylated compared with the nonmalignant RWPE1cell line (Fig. 3C). These results indicate that miR-23b issilenced by hypermethylation in prostate cancer. To investi-gate whethermiR-23bmethylation status affects its expressionin prostate cancer cell lines, we treated cells with the demethy-lating agent 5-aza-deoxycitidine (5-Aza, 1 mmol/L) for 1 weekand then determined methylation status and miR-23b expres-sion by methylation-specic qRT-PCR and qRT-PCR, respec-tively. The methylation percentage in 5-Azatreated cells wassignicantly decreased compared with untreated controlsalong with a concomitant increase in miR-23b expression (Fig.3C and D). To determine the correlation between percentmethylation of miR-23b and expression levels of its target Srckinase, we analyzed the expression of miR-23b, Src kinase, andthe methylation of miR-23b in a different set of matchedpatient samples (12 pairs). A negative correlationwas observedbetween miR-23b and Src expression (P < 0.01), whereas a

    positive correlation was found between percentage of meth-ylation of miR-23b and Src expression (P < 0.03; Fig. 3H) in thesame patient samples.

    To conrm that promoter methylation is an importantmechanism of miR-23b suppression in patients with prostatecancer, we investigated the correlation between methylationstatus of miR-23b and its expression in the same samples.Therewas a signicant inverse correlation between percentageof methylation and miR-23b expression such that cases withhigher miR-23b expression had a lower percentage of meth-ylation (P < 0.01; Fig. 3B and E; Supplementary Table S6).Sequences of primers and probes are given in SupplementaryTable S7. We also conducted ROC analysis to evaluate thepercentage of miR-23b methylation to distinguish malignantfromnormal cases inmatched tissue samples and cell lines. Fortissues, the AUC was 0.742 (P < 0.001; 95% CI, 0.5750.870; Fig.3G), whereas for cell lines (PC3 and DU145 vs. RWPE1), theAUC was a perfect 1.00 (P < 0.000; 95% CI, 0.6641.000; Fig. 3H),indicating that miR-23b methylation can distinguish betweendisease and normal cases.

    miR-23b overexpression suppresses prostate cancer cellproliferation, migration/invasion and colony formationand induces G0G1 cell-cycle arrest and apoptosis

    We determined the functional signicance of miR-23boverexpressing prostate cancer. A signicant decrease in cellproliferation was observed over time in miR-23btransfected

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  • PC3 and DU145 cells (Fig. 4A) as compared with cells expres-sing cont-miR. miR-23btransfected cells also had low colonyformation ability, as the number of foci in miR-23bexpressingcells was decreased when compared with cont-miRtrans-fected cells (Fig. 4B; Supplementary Fig. S2). Less absorbancewas observed at 560 nm with miR-23btransfected cells thancont-miR in the migration assay (Fig. 4C), and miR-23b over-expression also signicantly reduced the invasiveness of can-cer cells (Fig. 4D). FACS analysis revealed that reexpression ofmiR-23b leads to a signicant increase (22%27%) in thenumber of cells in the G0G1 phase of the cell cycle, whereasthe S-phase population decreased from 19% to 20% to 4%,suggesting that miR-23b causes a G0G1 arrest in miR-23btransfected cells compared with controls (cont-miR; Fig. 4E).FACS analysis for apoptosis was conducted using Annexin-V/FITC/7-aminoactinomycin D (7-AAD) dye. The percentageof total apoptotic cells (early apoptotic apoptotic) wassignicantly increased (13%22%) in response to miR-23boverexpression compared with cont-miR (3%4%) with acorresponding 13% to 20% decrease in the viable cell popula-tion (Fig. 4F). Functional assays were also conducted in andro-gen-dependent LNCaP cells, and the results were consistent(Supplementary Fig. S3). These results indicate a tumor sup-pressor role for miR-23b in prostate cancer.

    miR-23b directly represses proto-oncogene Src kinaseand its downstream genes involved in proliferation/survival/invasion and migration pathways in prostatecarcinoma

    We investigated whether Src kinase is a direct functionaltarget of miR-23b in prostate cancer. Complimentary sites in

    30UTR of Src kinase for miR-23b are given in SupplementaryTable S7. We chose Src kinase because it has been reported tobe a center stage molecule in cancer with pleiotropic effectsdue to the multiple signaling pathways engaged by Src kinase(Fig. 5A). In prostate cancer, Src kinase has been reported to beinvolved in proliferation, migration, and invasion. Our resultsshowed that Src kinase is overexpressed in prostate cancer celllines compared with RWPE1 cells (Fig. 5B). Transient trans-fection of humanprostate cancer cells with Src 30UTRplasmidsalong with miR-23b led to a signicant decrease in relativeluciferase units when comparedwith SrcMut30UTR vector andcont-miR or SrcMut30UTR vector andmiR-23b (Fig. 5C). Theseresults indicate that Src kinase is a direct target of miR-23b inprostate cancer.

    We next determined whether overexpression of miR-23bregulates Src kinase at translational level and alters down-stream signaling events. Transient transfection of prostatecancer cells with miR-23b signicantly downregulated totalSrc and active Src (pSrcY416) protein expression (Fig. 5D).Western blot analysis showed reduced levels of downstreammolecules that are involved in proliferation, migration, andinvasion (Fig. 5E) in cells with suppressed Src expressionfollowing miR-23b overexpression. Akt is an important down-stream gene of Src. We found complimentary miR-23b bindingsequence in its 30UTR (Supplementary Table S7). Our resultsshowed that miR-23b can also directly target Akt (Supplemen-tary Fig. S4). This further compliments our results thatmiR-23bexerts its effects in prostate cancer, at least partly, through SrcAkt pathway axis. We do not rule out the involvement of othertarget genes ofmiR-23b action in prostate cancer, although ourresults do indicate that SrcAkt axis pathway is partly involved.

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    -1,000

    19 Pairs of samples1333

    LowLowHigh

    >401440

  • miR-23b downregulates EMT markers and suppressescellmigration and invasion independent of proliferation

    To further show that the effect of miR-23b on cell migrationand invasion is an event independent of cell proliferation, weexamined the effect of overexpression of miR-23b on EMTmarkers. A decrease in Vimentin and Slug (mesenchymalmarkers) and an increase in E-cadherin (epithelial marker) wasobserved inmiR-23btransfected cells comparedwith the cont-miR (Fig. 5F andG).Theprocess of EMT is involved in epithelial-derived tumors causing them to become invasive and meta-

    static. These results show that miR-23b suppressed EMT mar-kers along with the other migratory/invasive genes such as Srcand focal adhesion kinase (FAK) and thus conrm that effect ofmiR-23b on invasion/migration is independent of proliferation.

    Depletion of Src by RNA interference mimics miR-23breconstitution in prostate cancer

    Phenocopy experiments were also carried out by siRNAinhibition of Src (Fig. 6; Supplementary Fig. S5). We used avalidated siRNA that resulted in 80% to 90% Src gene

    Figure 4. Transient transfection of miR-23b inhibits prostate cancer cell proliferation, colony formation, migration, invasion, and induces apoptosis and cell-cycle arrest. A, proliferation of PC3 and DU145 cells after miR-23b transfection was signicantly reduced compared with cont-miR. B, miR-23boverexpression signicantly inhibits colony-forming ability of prostate cancer cells. C,migration assays of PC3 and DU145 cells transfected withmiR-23b. D,invasion assay shows a signicant decrease in the number of invading PC3 and DU145 cells transfected with miR-23b. E, cell-cycle analysis showing anincrease in the G0G1 phase of PC3 and DU145 cells overexpressing miR-23b. F, apoptosis assay showing induction of apoptosis by miR-23b. , P < 0.05,SD.

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  • knockdown at themRNAand protein levels (Fig. 6A) for furtherexperiments. Our results showed that siRNA inhibition of Srcincreased G0G1 cell-cycle population (16%21%), whereasthere was a decrease of 11% to 12% in S-phase cell population(Fig. 6B and C). Approximately 15% to 18% of the cells were inthe apoptotic fraction in Src siRNAtransfected cells com-pared with 4% in nonspecic control (Fig. 6D and E). Theseresults suggest that inhibition of Src (that in turn depletes thedownstream Akt axis) by miR-23b overexpression is partlyresponsible for the observed phenotype in prostate cancercells, and siRNA depletion of Src mimics the effect of miR-23bOverexpression. We further carried out experiments with

    antimiR-23b and anti-miR-Neg-control to determine whetherthe protein expressions of the all the downregulated genes (bymiR-23b) are rescued. For this experiment, we chose DU145cell line as it expresses higher levels of miR-23b than PC3.Indeed the protein expression of all genes was rescued by anti-miR-23b (Fig. 6F).

    Intratumoral delivery of miR-23b suppressestumorigenicity in vivo

    We also conducted in vivo growth suppression experimentsto determine the tumor-suppressive effect of miR-23b afterlocal administration in established tumors. Tumor growth was

    Figure 5. miR-23b directly targets Src kinase and regulates downstream pathway genes and EMT. A, Src is a center stage molecule involved in variouspathways. B, Src kinase protein expression is high in prostate cancer cell lines than in nonmalignant RWPE1 cells. C, luciferase assays showingdecreased reporter activity after cotransfection of either wild-type Src-30UTR or its mutated 30UTR with miR-23b in PC3 and DU145 cells. Mut Src30UTR,mutated 30UTR sequence. D, Western blot analysis showing miR-23b represses Src kinase translationally. E, Western blot analysis showing decreasedexpression of Src kinase downstream genes. F and G, Western blotting and immunouorescence showing miR-23b downregulates EMT markers.

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  • signicantly suppressed by miR-23b over the course of exper-iment compared with the cont-miR. Average tumor volume incont-miR was 850 mm3 compared with the average tumorvolumeof 38mm3 inmice that receivedmiR-23b (Fig. 7A) at thetermination of the experiment.We also checked the expressionof miR-23b or Src kinase in harvested tumors. Our resultsshowed that miR-23b expression was signicantly high with acorresponding signicant decrease in the target Src kinasegene expression in miR-23btreated tumors compared withcontrols (Fig. 7B and C). These results conrm the tumorsuppressor effect of miR-23b in a prostate xenograft model.

    DiscussionIn this study, we found miR-23b to be signicantly silenced/

    downregulated in human prostate tumor samples when com-

    pared with adjacent normal samples. The downregulation ofmiR-23b expression was also observed in prostate cancer celllines when compared with a nonmalignant cell line. This isconsistent with a previous miRNA proling analysis of 9prostate carcinoma samples and various cell lines that showeddownregulation of miR-23b (24). The suppression of miR-23bexpression in tumors and cancer cell lines suggests a tumorsuppressor role in prostate cancer. However, neither thefunctional role nor the diagnostic or prognostic implicationsof miR-23b in prostate cancer have been previously dened.

    DNA methylationmediated downregulation of miRNAs byproximal CpG islands has been described by a number ofgroups (15, 25), and identication of other targets for meth-ylation may clarify the specic molecular events involvedin prostate cancer progression, enabling the prevention,

    Figure 6. Depletion of Src kinase bysiRNA mimics miR-23boverexpression. A, Src protein levelswere signicantly attenuated with 50nmol/L Src-siRNA duplex (Si)compared with a nonsilencing siRNAduplex (Con). P, PC3; D, DU145. BandC, cell-cycle analysis showing anincrease in the G0G1 phase of PC3and Du145 cells transfected withsiRNA. D and E, apoptosis assayshowing induction of apoptosis afterSrc knockdown by siRNA in PC3 andDU145 cells. , P < 0.05. F, Westernblot analysis showing the proteinexpressions of Src, and itsdownstream genes are rescued byantimiR-23b transfection.

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  • diagnosis, and treatment of prostate cancer to be approachedat a molecular level. Here, we show that miR-23b is frequentlysilenced through tumor-specic DNA methylation in prostatecancer tissues and cell lines and themethylation can be used asa diagnostic marker to distinguish malignant from normalcases. miRNAs possess several features that make them attrac-tive candidates as new prognostic biomarkers and powerfultools for the early diagnosis of cancer (26). In this study, wefound that miR-23b was predictive of OS and RFS. Multipleregression analysis also showed that miR-23b is an indepen-dent predictor of biochemical recurrence. miR-23b expressionalso distinguished malignant from normal tissues, indicatingthe diagnostic signicance of miR-23b in prostate cancer.

    An obstacle to understanding miRNA function has been therelative lack of experimentally validated targets. We validatedSrc kinase to be a direct target of miR-23b in prostate cancer.Src kinase requires phosphorylation within a segment of thekinase domain termed the activation loop for full catalyticactivity and this auto-phosphorylation site is tyrosine416 (27).Src kinase has been reported to be activated in prostate cancer(28, 29, 30). We showed that miR-23b inhibits Src kinase atprotein levels by directly binding to its 30UTR complimentarysequences. Src is involved in multiple signaling pathwaysincluding Ras/Raf/ERK1/2, PI3K/AKT, b-catenin/c-Myc/cyclin D1, and FAK/p130CAS/MMP-9, and increased Src activ-ity correlates with more aggressive phenotypes (28, 29, 31, 32).We observed that inhibition of Src by miR-23b overexpressionreduced signaling via theMEK/ERK pathway. A previous studyby Chang and colleagues (28) has shown that Src inhibition bysmall-molecule inhibitors induced apoptosis and cell-cyclearrest at the G0G1 phase of the cell cycle in prostate cancercell lines. Our results revealed that inhibition of Src bymiR-23boverexpression induced apoptosis andG0G1 arrest in prostatecancer cells. This effect on the cell cycle and apoptosis

    prompted us to study the effect on Src downstream targetgenes AKT, BAD, and STAT3, a Src target and key transcrip-tional factor for c-Myc and cyclin D1 (33) that are involved inthe G0G1 phase of cell cycle. We found that all these geneswere downregulated at the protein level. Inhibition of Src hasbeen found to decrease the invasion and migration of prostatecancer cells (29) through selective inhibition of Src substrates,such as FAK. Our results indicate that miR-23b inhibitedprostate cell migration and invasion and also downregulatedc-Jun and FAK. Overexpression of miR-23b caused a decline inthe mesenchymal markers Vimentin and Snail, whereas therewas an increase in the expression of E-cadherin, an epithelialmarker. These results suggest that miR-23b suppresses migra-tion and invasion independent of cell proliferation and that ithas an anti-metastatic effect on prostate cancer cells. Todetermine whether Src inhibition is, at least partly, responsiblefor the changes observed after miR-23b overexpression, wecarried out phenocopy experiments after inhibiting Src bysiRNA. Our results showed that inhibition of Src was respon-sible for G0G1 cell-cycle arrest, whereas there was a decreasein S-phase cell population. Induction of apoptosis was alsoobserved in Src siRNAtransfected cells compared with non-specic control. These results were similar to that of miR-23boverexpression assays. We further determined that the proteinexpressions of the genes that were downregulated by miR-23bwere rescued by antimiR-23b. These results strongly indicatethat the tumor-suppressive effect of miR-23b is, at least partly,mediated by SrcAkt axis inhibition in prostate cancer.

    The antiproliferative effects of miR-23b observed in thisstudy were conrmed in prostate tumor xenograft models. Inconclusion, our study shows that miR-23b has an importanttumor suppressor role both in vitro and in vivo.

    Accumulating evidence indicates thatmodulation ofmiRNAalso represents an attractive strategy for therapeutic gene

    1,000900800700600500400300200100

    0

    cont-miR

    cont-miRA

    B C

    miR-23b

    miR-23b

    cont-miR miR-23b

    Src

    GAPDH

    C-1 C-2 23B-1 23B-2

    24 27 30

    8,0007,0006,0005,0004,0003,0002,0001,000

    0

    33 36 39 42 45

    Tum

    or

    volu

    me

    (mm3

    )

    Rel

    ative

    miR

    -23b

    exp

    ress

    ion

    Figure 7. miR-23b inhibit tumorgrowth in vivo. ,P < 0.05. A, tumorvolume following intratumoralinjection of cont-miR or miR-23bprecursor into established tumors., P < 0.05. B, average expressionof miR-23b in excised tumors. D,depletion of Src kinase bymiR-23bin excised tumors. Protein wasextracted from 2 tumors thatreceived cont-miR (C-1-2) or miR-23b precursor (23B-1-2).

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  • silencing. miRNAs potentially inuence cellular behaviorthrough the regulation of extensive gene expression networks.Therapeutic modulation of a single miRNA may thereforeaffect many pathways simultaneously to achieve clinical ben-et. Our study is the rst report showing that miR-23b hasdiagnostic/prognostic signicance and inhibits the SrcAktaxis genes, a center stage pathway that regulates proliferation,survival, and migration/invasion in cancer. It also highlightsthe therapeutic potential of miR-23b in the treatment ofprostate cancer.

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

    Authors' ContributionsConception and design: S. Majid, A.A. Dar, Y. Tanaka, R. DahiyaDevelopment ofmethodology: S.Majid, A.A. Dar, Y. Tanaka, G. Deng, R. DahiyaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Majid, A.A. Dar, S. Arora, V. Shahryari, G. Deng

    Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Majid, A.A. Dar, S. Saini, S. Arora, S. Yamamura, Y.Tanaka, R. DahiyaWriting, review, and/or revision of the manuscript: S. Majid, R. DahiyaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S. Majid, S. Saini, S. Arora, M.S. Zaman,I. Chang, R. DahiyaStudy supervision: S. Majid, S. Arora, R. Dahiya

    AcknowledgmentsThe authors thank Dr. Roger Erickson for his support and assistance with the

    preparation of the manuscript.

    Grant SupportThis researchwas supported by theNational Center for Research Resources of

    the NIH through grant numbers RO1CA 138642, RO1CA130860, RO1CA160079,and T32DK007790, and VA Merit Review and VA Program Project.

    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 May 31, 2012; revised September 14, 2012; accepted October 9, 2012;published OnlineFirst October 16, 2012.

    References1. Siegel R, Ward E, Brawley O, Jemal A. Cancer statistics, 2011: the

    impact of eliminating socioeconomic and racial disparities on prema-ture cancer deaths. CA Cancer J Clin 2011;61:21236.

    2. Albertsen PC, Hanley JA, Fine J. 20-year outcomes following conser-vative management of clinically localized prostate cancer. JAMA2005;293:2095101.

    3. Mimeault M, Batra SK. Recent advances on multiple tumorigeniccascades involved in prostatic cancer progression and targetingtherapies. Carcinogenesis 2006;27:122.

    4. Miranda KC, Huynh T, Tay Y, Ang YS, Tam WL, Thomson AM, et al. Apattern-based method for the identication of MicroRNA binding sitesand their corresponding heteroduplexes. Cell 2006;126:120317.

    5. Shi XB, Tepper CG,White RW. MicroRNAs and prostate cancer. J CellMol Med 2008;12:145665.

    6. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, et al. AmicroRNA expression signature of human solid tumors denes cancergene targets. Proc Natl Acad Sci U S A 2006;103:225761.

    7. Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, et al.MicroRNA expression proles classify human cancers. Nature 2005;435:8348.

    8. Rosenfeld N, Aharonov R, Meiri E, Rosenwald S, Spector Y, ZepeniukM, et al. MicroRNAs accurately identify cancer tissue origin. NatBiotechnol 2008;26:4629.

    9. Heinzelmann J, Henning B, Sanjmyatav J, Posorski N, Steiner T,Wunderlich H, et al. Specic miRNA signatures are associated withmetastasis and poor prognosis in clear cell renal cell carcinoma.WorldJ Urol 2011;29:36773.

    10. Kosaka N, Iguchi H, Ochiya T. Circulating microRNA in body uid: anew potential biomarker for cancer diagnosis and prognosis. CancerSci 2010;101:208792.

    11. Chuang JC, Jones PA. Epigenetics and microRNAs. Pediatr Res2007;61:24R9R.

    12. Saito Y, LiangG,EggerG, FriedmanJM,Chuang JC,CoetzeeGA, et al.Specic activation ofmicroRNA-127with downregulation of the proto-oncogene BCL6 by chromatin-modifying drugs in human cancer cells.Cancer Cell 2006;9:43543.

    13. Lujambio A, Ropero S, Ballestar E, FragaMF, Cerrato C, Setien F, et al.Genetic unmasking of an epigenetically silenced microRNA in humancancer cells. Cancer Res 2007;67:14249.

    14. Lujambio A, Calin GA, Villanueva A, Ropero S, Sanchez-CespedesM, Blanco D, et al. A microRNA DNA methylation signature forhuman cancer metastasis. Proc Natl Acad Sci U S A 2008;105:1355661.

    15. ToyotaM, Suzuki H, Sasaki Y,MaruyamaR, Imai K, Shinomura Y, et al.Epigenetic silencing of microRNA-34b/c and B-cell translocation gene

    4 is associated with CpG island methylation in colorectal cancer.Cancer Res 2008;68:412332.

    16. Huang YW, Liu JC, Deatherage DE, Luo J, Mutch DG, Goodfellow PJ,et al. Epigenetic repression of microRNA-129-2 leads to overexpres-sion of SOX4 oncogene in endometrial cancer. Cancer Res 2009;69:903846.

    17. Esteller M. Epigenetics in cancer. N Engl J Med 2008;358:114859.18. Rodriguez-Paredes M, Esteller M. Cancer epigenetics reaches main-

    stream oncology. Nat Med 2011;17:3309.19. Lujambio A, Esteller M. How epigenetics can explain human metas-

    tasis: a new role for microRNAs. Cell Cycle 2009;8:37782.20. MedinaPP,NoldeM,SlackFJ.OncomiRaddiction in an in vivomodelof

    microRNA-21-induced pre-B-cell lymphoma. Nature 2010;467:8690.21. ObadS, dosSantosCO,Petri A,HeidenbladM,BroomO,RuseC, et al.

    SilencingofmicroRNA families by seed-targeting tiny LNAs. NatGenet2011;43:3718.

    22. Majid S, Saini S, Dar AA, Hirata H, Shahryari V, Tanaka Y, et al.MicroRNA-205 inhibits Src-mediated oncogenic pathways in renalcancer. Cancer Res 2011;71:261121.

    23. Majid S, Dar AA, Saini S, Yamamura S, Hirata H, Tanaka Y, et al.MicroRNA-205-directed transcriptional activation of tumor suppres-sor genes in prostate cancer. Cancer 2010;116:563749.

    24. Porkka KP, Pfeiffer MJ, Waltering KK, Vessella RL, Tammela TL,Visakorpi T. MicroRNA expression proling in prostate cancer. CancerRes 2007;67:61305.

    25. Kozaki K, Imoto I, Mogi S, Omura K, Inazawa J. Exploration of tumor-suppressive microRNAs silenced by DNA hypermethylation in oralcancer. Cancer Res 2008;68:2094105.

    26. Schaefer A, JungM, Mollenkopf HJ, Wagner I, Stephan C, Jentzmik F,et al. Diagnostic and prognostic implications of microRNA proling inprostate carcinoma. Int J Cancer 2010;126:116676.

    27. Thomas SM, Brugge JS. Cellular functions regulated by Src familykinases. Annu Rev Cell Dev Biol 1997;13:513609.

    28. ChangYM,Bai L, LiuS, Yang JC,KungHJ, EvansCP.Src family kinaseoncogenic potential and pathways in prostate cancer as revealed byAZD0530. Oncogene 2008;27:636575.

    29. Nam S, Kim D, Cheng JQ, Zhang S, Lee JH, Buettner R, et al. Action ofthe Src family kinase inhibitor, dasatinib (BMS-354825), on humanprostate cancer cells. Cancer Res 2005;65:91859.

    30. Fizazi K. The role of Src in prostate cancer. Ann Oncol 2007;18:176573.

    31. Lee LF, Louie MC, Desai SJ, Yang J, Chen HW, Evans CP, et al.Interleukin-8 confers androgen-independent growth and migration ofLNCaP: differential effects of tyrosine kinases Src and FAK. Oncogene2004;23:2197205.

    miR-23b in Prostate Cancer

    www.aacrjournals.org Cancer Res; 72(24) December 15, 2012 6445

    on March 16, 2015. 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

    Published OnlineFirst October 16, 2012; DOI: 10.1158/0008-5472.CAN-12-2181

  • 32. Kotha A, Sekharam M, Cilenti L, Siddiquee K, Khaled A, Zervos AS,et al. Resveratrol inhibits Src and Stat3 signaling and induces theapoptosis of malignant cells containing activated Stat3 protein. MolCancer Ther 2006;5:6219.

    33. Prathapam T, Tegen S, Oskarsson T, Trumpp A, Martin GS. Acti-vated Src abrogates the Myc requirement for the G0/G1 transitionbut not for the G1/S transition. Proc Natl Acad Sci U S A 2006;103:2695700.

    Majid et al.

    Cancer Res; 72(24) December 15, 2012 Cancer Research6446

    on March 16, 2015. 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

    Published OnlineFirst October 16, 2012; DOI: 10.1158/0008-5472.CAN-12-2181

  • 2012;72:6435-6446. Published OnlineFirst October 16, 2012.Cancer Res

    Shahana Majid, Altaf A. Dar, Sharanjot Saini, et al.

    Prognostic Significance in Prostate CancerMethylation-Silenced Tumor Suppressor with Diagnostic and miR-23b Represses Proto-oncogene Src Kinase and Functions as

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