In Vitro and In Vivo Multiple Myeloma Cell Growthclincancerres.aacrjournals.org/content/clincanres/19/8/2096.full.pdf · Targeting miR-21 Inhibits In Vitro and In Vivo Multiple Myeloma

Cancer Therapy: Preclinical

Targeting miR-21 Inhibits In Vitro and In Vivo MultipleMyeloma Cell Growth

Emanuela Leone1, Eugenio Morelli1, Maria T. Di Martino1, Nicola Amodio1, Umberto Foresta1,Annamaria Gull�a1, Marco Rossi1, Antonino Neri2, Antonio Giordano3,6, Nikhil C. Munshi4,5,Kenneth C. Anderson4, Pierosandro Tagliaferri1, and Pierfrancesco Tassone1,6

AbstractPurpose:Deregulated expression ofmiRNAs plays a role in the pathogenesis and progression ofmultiple

myeloma. Among upregulated miRNAs, miR-21 has oncogenic potential and therefore represents an

attractive target for the treatment of multiple myeloma.

Experimental Design: Here, we investigated the in vitro and in vivo anti-multiple myeloma activity of

miR-21 inhibitors.

Results: Either transient-enforced expression or lentivirus-based constitutive expression of miR-21

inhibitors triggered significant growth inhibition of primary patient multiple myeloma cells or

interleukin-6–dependent/independent multiple myeloma cell lines and overcame the protective

activity of human bone marrow stromal cells. Conversely, transfection of miR-21 mimics significantly

increased proliferation of multiple myeloma cells, showing its tumor-promoting potential in multiple

myeloma. Importantly, upregulation of miR-21 canonical validated targets (PTEN, Rho-B, and BTG2),

together with functional impairment of both AKT and extracellular signal–regulated kinase signaling,

were achieved by transfection of miR-21 inhibitors into multiple myeloma cells. In vivo delivery of

miR-21 inhibitors in severe combined immunodeficient mice bearing human multiple myeloma

xenografts expressing miR-21induced significant antitumor activity. Upregulation of PTEN and

downregulation of p-AKT were observed in retrieved xenografts following treatment with miR-21

inhibitors.

Conclusion:Our findings show the first evidence that in vivo antagonism of miR-21 exerts anti-multiple

myeloma activity, providing the rationale for clinical development of miR-21 inhibitors in this still

incurable disease. Clin Cancer Res; 19(8); 2096–106. �2013 AACR.

IntroductionMultiple myeloma is a genetically complex hematologic

malignancy characterized by abnormal infiltration of clonalplasma cells in the bone marrow (1, 2). Despite novelinsights into the pathobiology of the disease and the

availability of new research platforms and therapeutics(3–6), innovative treatment strategies are urgently needed.A major area of investigation is the human bone marrowmicroenvironment (hBMM), which plays an essential rolepromoting growth, survival, and drug resistance inmultiplemyeloma (7). Moreover, within the hBMM, tumor cellsprogressively accumulate genetic aberrations leading torelapsed and refractory disease (8). Currently, there is clearevidence that changes in gene copy number, chromosomaltranslocations, mutations, as well as transcriptional andepigenetic events during the evolutionary process of mul-tiplemyeloma (9, 10) result inprofoundderegulationof themiRNA network (11, 12), which in turn leads to aberranttranslation of mRNAs and cell signaling.

miRNAs are small, noncoding RNAs of 19 to 25 nucleo-tides, which regulate gene expression by inducing degrada-tion or translation inhibition of target mRNAs primarilythrough base pairing to partially or fully complementarysites in the 30-untranslated region (30-UTR; ref. 13).Aberrant expression of miRNAs occurs widely in humancancers, including both solid tumors and hematologicmalignancies (11, 12, 14). Deregulated miRNAs mayact as oncogenes (Onco-miRNA) or tumor-suppressors

Authors' Affiliations: 1Medical Oncology Unit, Department of Experimen-tal and Clinical Medicine, Magna Graecia University and T. CampanellaCancer Center, Catanzaro; 2Department of Medical Sciences University ofMilan, Hematology 1, IRCCS Policlinico Foundation, Milan; 3HumanPathology and Oncology Department, University of Siena, Siena, Italy;4Department of Medical Oncology, Dana-Farber Cancer Institute; 5BostonVeterans Administration Healthcare System, West Roxbury, Boston, Mas-sachusetts; and 6Sbarro Institute for Cancer Research and MolecularMedicine, Center for Biotechnology, College of Science and Technology,Temple University, Philadelphia, Pennsylvania

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

E. Leone and E. Morelli contributed equally to this work.

Corresponding Author: Pierfrancesco Tassone, Magna Graecia Univer-sity, Viale Europa, 88100 Catanzaro, Italy. Phone: 39-0961-3697029; Fax:39-0961-3697341; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-3325

�2013 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 19(8) April 15, 20132096

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Published OnlineFirst February 27, 2013; DOI: 10.1158/1078-0432.CCR-12-3325





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(TS-miRNAs; refs. 14–16), with the former generally upre-gulated and the latter downregulated in cancer cells.Because miRNAs regulate several mRNAs relevant in cancerpromotion, targeting of deregulatedmiRNAs in cancer cellsis emerging as a novel promising therapeutic approach in avariety of malignancies (17–19), including multiple mye-loma (20–25). Among those miRNAs involved in tumor-igenesis,miR-21plays a key role in tumorprogression and issignificantly upregulated in several human cancers (26). Ithas been recently shown that the Epstein–Barr virus (EBV)–encoded EBNA2, which is needed for the transformingcapacity of B cells in vitro, significantly increase miR-21expression (27). Interestingly, a genetically engineeredmouse model showed that constitutive tissue-specific over-expression of miR-21 resulted in a pre-B-cell lymphoma(28). Conversely, inhibition of miR-21 induces antiproli-ferative and apoptotic effects as well as enhances sensitivityto antitumor agents including gemcitabine, docetaxel,temozolomide, and 5-fluorouracil (29–31). These findingsstrongly support the notion thatmiR-21 represents a poten-tial therapeutic target in human cancer. In the last few years,there is increasing evidence for the role of miR-21 inpathogenesis of plasma cell dyscrasias. Upregulation ofmiR-21 has been found in both monoclonal gammopathyof undetermined significance and multiple myeloma (32);in the latter, miR-21 is regulated by interleukin (IL)-6through Stat3-pathway activation (33), and a Stat3/miR-21 positive feedback loop has been shown (34). Moreover,miR-21 induces resistance to multiple myeloma cell apo-ptosis triggered by dexamethasone, doxorubicin, or borte-zomib (35). Importantly, adhesion of multiple myelomacells to humanbonemarrow stromal cells (hBMSC) triggersupregulation of miR-21 expression, suggesting its role inbone marrow–mediated growth, survival, and drug resis-tance (35). This multiple myeloma cell growth and survivalpromoting activity of miR-21 suggests that it represents anattractive novel therapeutic target.

In this report, we characterized the anti-multiple myelo-ma activity and the molecular events triggered by miR-21inhibition in patient multiple myeloma cells and multiplemyeloma cell lines, which were maintained even whentumor cells were adherent to patient BMSCs in vitro. Wethen showed the in vivo cytotoxicity and mechanisms ofaction of miR-21 inhibitors in a murine xenograft model ofhuman multiple myeloma, providing the framework for itsclinical development.

Materials and MethodsReagents and cell culture

Multiple myeloma cell lines were cultured in RPMI-1640(Gibco, Life Technologies) supplemented with 10% FBS(Lonza Group Ltd.) and 1% penicillin/streptomycin(Gibco, Life Technologies). The IL-6–dependent multiplemyeloma cell line INA-6 (kindly provided from Dr. RenateBurger, University of Erlangen-Nuernberg, Erlangen, Ger-many) was cultured in the presence of rhIL-6 (R&D Sys-tems), as previously reported (36). Following informedconsent approved by our University Hospital Ethical Com-mitee, primary patient multiple myeloma (ppMM) cellswere isolated from bone marrow aspirates by Ficoll-Hypa-que density gradient sedimentation followed by antibody-mediated positive selection using anti-CD138 magnetic-activated cell separation microbeads (Miltenyi Biotech).Purity of immunoselected cells were assessed by flow-cyto-metric analysis using a phycoerythrin-conjugated CD38monoclonal antibody (mAb; CD38-PE; Imgenex) by stan-dard procedures (37, 38). hBMSCs were obtained by long-term culture of bone marrow mononuclear cells (39). Forcoculture, 1 � 105 ppMM cells were seeded on 2 � 104

hBMSCs for 24 to 48 hours in 96-well plates. RNA samplesof normal healthy bonemarrow-derived plasma cells (nPC)were purchased (AllCells).

Overexpression and inhibition of miR-21 in multiplemyeloma cells

Pre-miR-21 miRNA precursor molecules and miR-21inhibitors were purchased from Ambion (Applied Bio-systems) and were used to enforce or to antagonize mir-21 expression, respectively, at a final concentration of 100nmol/L. Pre-miR precursor-negative control and anti-miRmiRNA inhibitor-negative control were obtained fromAmbion (Applied Biosystems). A total of 1 � 106 cellswere transfected using Neon Transfection System (Invi-trogen), (1 pulse at 1,050 V, 30 milliseconds), and thetransfection efficiency evaluated by flow-cytometric anal-ysis relative to a FAM dye–labeled anti-miR–negativecontrol reached 85% to 90%. The same conditions wereapplied for transfection of multiple myeloma cells with10 mg of the p3x FLAG-PTEN (kindly provided byProf. Giuseppe Viglietto, Magna Graecia University, Cat-anzaro, Italy) or with the same amount of the empty p3xFLAG-CMV-7.1 vector. When cotransfected, we used 100nmol/L of synthetic miR-21 or miR-NC together with 10mg of p3x FLAG–PTEN or the same amount of empty p3xFLAG-CMV-7.1 vector.

Translational RelevanceOncogenic miRNAs are an emerging target for the

treatment of human cancer. We investigated the thera-peutic potential of miR-21 inhibitors in humanmultiplemyeloma. The translational relevance of our study reliesin the strong anti-multiple myeloma activity of miR-21inhibitors, which is not abrogated by exogenous inter-leukin-6 or cell adherence to human bone marrow stro-ma, taking into account the dependency of multiplemyeloma from the bone marrow milieu. Moreover, weprovide evidence of a successful in vivo treatment withmiR-21 inhibitors inamurine xenograftmodelofhumanmultiple myeloma, offering a framework for clinicaldevelopment ofmiR-21 inhibitors inmultiplemyeloma.Notably, this therapeutic activity is dependentuponmiR-21 expression, which may represent a predictive bio-marker tomiR-21 inhibitors in this still incurabledisease.

Antitumor Activity of mir-21 Inhibitors in Multiple Myeloma

www.aacrjournals.org Clin Cancer Res; 19(8) April 15, 2013 2097




Cell proliferation assaysCell growth was evaluated by Trypan blue exclusion

cell count and bromodeoxyuridine (BrdUrd) prolifera-tion assay. Electroporated cells were incubated for 4hours in 6-well plates; after harvesting, they were platedin 24-well plates for Trypan blue exclusion cell count andin 96-well plates for BrdUrd proliferation assay. Cellswere counted at 24-hour intervals. BrdUrd uptake wasmeasured every 24 hours by the DELFIA cell proliferationassay, and luminescence was detected using a Victor 4plate reader (PerkinElmer). Each sample was run at leastin triplicate.

Survival assayCell survival was evaluated by MTT assay in 96-well

plates. In brief, transfected cells were seeded at a densityof 1� 104 cells per well in 100 mL of culture medium. Every24 hours, 10 mL of 5 mg/mL MTT (Sigma) reagent wereadded to each well, and cells were further incubated for 4hours at 37�C. Then medium was removed and 100 mL ofdimethyl sulfoxide (DMSO) were added to each well todissolve the formazan. The optical density (OD) was eval-uated at wavelength of 560 nm.Wells without cells (DMSOalone)were used as blank, and experimentswere repeated atleast 3 times. Data represent the mean � SD of 3 indepen-dent experiments.

Colony formation assayClonogenity was evaluated by a colony formation assay

in methylcellulose-based medium (Methocult H4100,StemCell Technologies), following manufacturer’s instruc-tions. A total of 2 � 102 cells/mL electroporated cells wereseeded in 24-well plates and incubated for 3 weeks; colonyformationwas then evaluated by counting colonies ofmorethan 100 cells. The experiments were repeated at least 3times. Data represent the mean � SD of 3 independentexperiments.

Quantification of IL-6 productionThe concentration of IL-6 in culture medium was mea-

sured by ELISA (Quantikine Rat IL-6; R&D Systems).hBMSCs were electroporated; following incubation periodsof 24 to 48hours, aliquots of culturemediumwere collectedand subjected to a specific ELISA for IL-6.

Reverse transcription and quantitative real-time PCRTotal RNA-containing miRNAs and mRNAs were

extracted from cells with Trizol Reagent (Invitrogen),according to the manufacturer’s instructions. All RNAextractions were carried out in a sterile laminar flow hoodusing RNase/DNase–free laboratory ware. The integrity oftotal RNA was verified by nanodrop (Celbio NanodropSpectrophotometer nd-1000). The single-tube TaqManmiRNA assay (Applied Biosystems) was used to detect andquantify mature miR-21, using ViiA7 RT reader (AppliedBiosystems); the protocol was conducted for 40 cycles at95�C for 3 minutes, 95�C for 15 seconds, and 60�C for 30seconds. miR-21 expression was normalized on RNU44

and then expressed as fold change ð2DDCtÞ. For mRNAdosage studies, oligo-dT–primed cDNA was obtainedthrough the High Capacity cDNA Reverse Transcription Kit(Applied Biosystems) and then used as a template to quan-tify PTEN (Hs01026371_m1), Rho-B (Hs1085292_m1),and BTG2 (Hs1105077_m1) levels by TaqMan assay(Applied Biosystems); normalization was conductedwith glyceraldehyde-3-phosphate dehydrogenase (GAPDH;Hs03929097_g1). Comparative real-time PCR (RT-PCR)was conducted in triplicate, includingno-template controls.Relative expression was calculated using the comparativecross-threshold (Ct) method.

Protein extraction and Western blot analysisTotal proteins were extractedwith a lysis buffer (Tris–HCl

15 mmol/L pH 7.5, NaCl 120 mmol/L, KCl 25 mmol/L,Tryton X-100 0.5%) and addition ofHalt Protease InhibitorSingle-Use Cocktail 1� (Thermo Scientific). After lysis in icefor 30 minutes, the solution was disrupted by gentle pipet-ting followed by 3� 10 sonication cycles; the lysate wascentrifuged at 12,000 rpm for 20 minutes and the super-natant was collected. For Western blot analysis, 60 mg oflysate were separated by electrophoresis on Mini ProteanTGX precast gels (4%–20%) and electro-transferred onto anitrocellulose membrane (Invitrogen). The membraneswere blocked for 1 hour in 5% milk at room temperatureand then incubated overnight at 4�C in milk 5% with thefollowing antibodies: PTEN (A2B1; Santa Cruz Biotechnol-ogy); phospho-Akt (Ser473) rabbit mAb (Cell Signaling);AKT (pan; 11E7) rabbit mAb (Cell Signaling); phospho-p44/42 mitogen-activated protein kinase (MAPK; Erk1/2;Thr202/Tyr204) rabbit mAb (Cell Signaling); p44/42MAPK (Erk1/2) rabbit mAb (Cell Signaling); g-tubulinAntibody (C-20) goat polyclonal; GAPDH–horseradishperoxidase rabbit polyclonal immunoglobulin G (IgG;Santa Cruz Biotechnology). Membranes were washed 3times in PBST and then incubated with a secondary anti-body conjugated with horseradish peroxidase in 0.5%milkfor 2 hours at room temperature. After 3 washes withPBSTween, chemiluminescence was detected using PierceECL Western Blotting Substrate (cat. 32109; Pierce). Inten-sity of the bands was analyzed using Quantity One analyz-ing system (Bio-Rad).

Virus generation and infection of multiple myelomacells

Multiple myeloma cells stably expressing miR-21inhibitor were transduced by the lentiviral vector miR-Zip-21 anti-miR-21 construct (System Biosciences). Thesupernatant was collected 48 hours posttransfection andwas centrifuged (3,000 � g for 15 minutes at 4�C) toremove cell debris; it was then passed through a 0.45-mmfilter and used for 2 rounds of transduction of U-266 andMM.1S cells (1 � 106) in the presence of 8 mg/mLpolybrene (Sigma-Aldrich). Multiple myeloma cellsunderwent 3 rounds of infection (8 hours each round),and transduced cells were selected in medium containing1 mg/mL puromycin.

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Clin Cancer Res; 19(8) April 15, 2013 Clinical Cancer Research2098




Animals and in vivomodel of humanmultiplemyelomaMale CB-17 severe combined immunodeficient (SCID)

mice (6- to 8-weeks old; Harlan Laboratories, Inc.) werehoused and monitored in our Animal Research Facility.All experimental procedures and protocols had beenapproved by the Institutional Ethical Committee (MagnaGraecia University) and conducted according to protocolsapproved by the National Directorate of VeterinaryServices (Italy). In accordance with institutional guide-lines, mice were sacrificed when tumors reached 2 cm indiameter or in the event of paralysis or major compro-mise, to prevent unnecessary suffering. Animal experi-mental procedures have been carried out as in previousreports (40–42). Briefly, mice were subcutaneously inoc-ulated with 1 � 106 OPM-2 cells, and treatment startedwhen palpable tumors became detectable, approximately3 weeks following injection of multiple myeloma cells.Each dose contained 20 mg of NLE-formulated (MaxSup-pressor In Vivo RNA-LANCEr II) synthetic oligo, whichequals 1 mg/kg per mouse, as previously described (21).Treatments were conducted intratumorally every 2 daysfor a total of 8 injections.

Statistical analysisEach experiment was carried out at least 3 times and all

values are reported as means� SD. Comparisons betweengroups were made with Student t test, whereas statisticalsignificance of differences among multiple groups wasdetermined by GraphPad software (www.graphpad.com).Graphs were obtained using SigmaPlot version 12.0. Pvalue of less than 0.05 was accepted as statisticallysignificant.

ResultsmiR-21 expression in multiple myeloma cell lines andprimary patient multiple myeloma cells

By RT-PCR, we evaluated the miR-21 expression in INA-6, MM.1S, NCI-H929, RPMI-8226, OPM-2, U-266, andKMS-26 multiple myeloma cell lines as compared withnPCs. Among these cell lines, we found variable miR-21expression: KMS-26, U-266, and OPM-2 showed the high-est, whereas other cells expressed very low levels of miR-21 (Fig. 1A). Notably, we foundmore than 3-fold increasein miR-21 expression when INA-6 cells, which are IL-6–dependent, were cultured adherent to hBMSCs (Fig. 1B).Consistent with data achieved in cell lines, miR-21 levelswere upregulated in ppMM cells as compared with nPCs;moreover, ppMM cells further increased miR-21 expres-sion (>4.18-fold) when cells were cultured adherent tohBMSCs (Fig. 1C), showing that miR-21 in multiplemyeloma is indeed upregulated by the bone marrowmilieu.

Transfected miR-21 inhibitors exert anti-multiplemyeloma effects in vitro

To study the anti-multiple myeloma activity of miR-21inhibition, we transfected tumor cells with miR-21 inhibi-tors. By Trypan blue exclusion cell count and BrdUrdproliferation assay, we found significantly decreased cellgrowth of multiple myeloma cell lines highly expressingmiR-21 levels (KMS-26, U-266, and OPM-2; Fig. 2A and B).In these cells, we also observed reduction of cell survival byMTT assay (Fig. 2C). Conversely, miR-21 inhibitors did notaffect cell proliferation or survival of multiple myeloma cell

Figure 1. miR-21 expression in patient with multiple myeloma cells and cell lines. A, qRT-PCR analysis ofmiR-21 expression using total RNA from 7 multiplemyeloma cell lines and bonemarrow–derived plasma cells (nPCs) from healthy donors. RawCt values were normalized toRNU44 housekeeping snoRNA andexpressed as DDCt values relative to miR-21 levels in nPCs. Values represent mean � SE of 3 different experiments. B, qRT-PCR of miR-21 expressionin INA-6 cells cultured in the presence or absence of IL-6 (2.5 ng/mL) or cocultured with hBMSCs and then immunopurified by immunomagneticsorting with anti-CD138 beads. Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as DDCt values calculated using thecomparative cross-threshold method. miR-21 expression levels in INA-6 cells cultured in the absence of IL-6 were set as an internal reference. Valuesrepresentmean�SEof 3 different experiments. C, qRT-PCRofmiR-21 expression in nPCs (n¼ 3) and ppMM (n¼ 3) cells before and 6 hours after seeding onhBMSCs, and then immunopurified by immunomagnetic sorting with anti-CD138 beads. Raw Ct values were normalized to RNU44 housekeeping snoRNAand expressed as DDCt values calculated using the comparative cross-threshold method. miR-21 expression levels in nPCs were used as an internalreference. Data are the average of 3 independent experiments carried out in triplicate. P values were obtained using two-tailed t test.






lines with low miR-21 expression (NCI-H929, MM.1S, andRPMI-8226; Supplementary Fig. S2).

Lentiviral transducedmiR-21 inhibitors affectmultiplemyeloma cell proliferation and clonogenicity

We next evaluated the effects of constitutive inhibition ofmiR-21. U-266 cells (expressing highmiR-21) orMM.1S cells(expressing low miR-21) were transduced with a lentiviralvector carrying a miR-21 inhibitory sequence or with alentiviral empty vector. By BrdUrd proliferation assay, wefound significantly decreased cell growth of U-266 cellstransduced with miR-21 inhibitors as compared with con-

trols (Fig. 3B). In contrast, no significant effects on cellproliferation were observed in MM.1S cells transduced withmiR-21 inhibitors (Fig. 3A). Constitutive expression of miR-21 inhibitors in U-266 cells also significantly inhibited col-ony formation inmethylcellulose cultures (75% reduction incolonies; Fig. 3B), whereas clonogenicity of MM.1S cells wasnot affected (Fig. 3A). Taken together, these results showthat miR-21 inhibition obtained either by transfection ortransduction of miR-21 inhibitors exerts antiproliferativeactivity in multiple myeloma cells. The growth-inhibitoryactivity strongly relies on high basal miR-21 expression, asmultiple myeloma cells with low miR-21 were not affected.

Figure 2. Effects of ectopic expression of miR-21 inhibitors in multiple myeloma cell lines. A, Trypan blue exclusion growth curves were generated fromKMS-26, U-266, and OPM-2 cells transfected with miR-21 inhibitors or scrambled controls (miR-NC inhibitors). B, BrdUrd proliferation assay wasconducted inKMS-26,U-266, andOPM-2cells 48hours after transfectionwithmiR-21 inhibitors or scrambledcontrols. C,MTTsurvival assaywas conductedin KMS-26, U-266, and OPM-2 cells 48 hours after transfection with miR-21 inhibitors or scrambled controls. Averaged values � SD of 3 independentexperiments are plotted. P values were obtained using two-tailed t test.

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miR-21 inhibition modulates the expression andactivity of several signaling moleculesmiR-21 is known as a growth promoting and antiapop-

totic factor in several human cancers through the targetingof multiple tumor suppressor genes (26). Among validatedtargets of miR-21, PTEN, Rho-B, and BTG2 are the moststudied and involved in cell-cycle progression and/or apo-ptosis regulation (43–45). We therefore evaluated the

effects of miR-21 inhibition on the expression of thesetargets and found that these genes were upregulated atmRNA level after transfection of miR-21 inhibitors, ascompared with control cells transfected with scrambledsequences (miR-NC inhibitors; Fig. 3C). Moreover, byWestern blot analysis, we found a marked upregulation ofPTEN protein expression 24 to 48 hours after cell transfec-tion with miR-21 inhibitors (Fig. 3D). Notably, we found

Figure 3. Activity of miR-21 inhibitors in multiple myeloma cell lines. BrdUrd proliferation assay and colony formation assay of MM.1S (A) and U-266 (B) cellstransduced with a lentivirus carrying either the empty vector or miR-21 inhibitory sequences. Averaged values � SD of 3 independent experimentsare plotted. P values were obtained using two-tailed t test. C, qRT-PCR of PTEN, BTG2, and Rho-B expression was conducted in U-266 cells 48 hours aftertransfection with either miR-21 inhibitors or scrambled controls (miR-NC inhibitors). The results are shown as average mRNA expression afternormalization with GAPDH and DDCt calculations. Data represent the average � SD of 3 independent experiments. D, Western blot analysis of PTEN24 to 48 hours after transfection ofU-266 cells withmiR-21 inhibitors or scrambled controls. E, levels of total AKT, total ERK, p-AKT, and p-ERK 24 to 48 hoursafter transfection of U-266 cells with either miR-21 inhibitors or scrambled controls. Relative protein level values are derived from densitometric scan.






that both p-AKT and p-extracellular signal–regulated kinase(ERK) were reduced by miR-21 inhibitors, whereas totalAKT and ERK were unaffected (Fig. 3E).

Enforced expression of miR-21 mimics enhancesproliferation of multiple myeloma cells

To establish the oncogenic role of miR-21 in our model,we evaluated the effects of transiently enforced expressionof synthetic miR-21 mimics in tumor cells. Specifically, we

examined whether miR-21 mimics exerted a growth-pro-moting activity in MM.1S (low miR-21) or in U-266 (highmiR-21) cells. We found that miR-21 mimics enhancedgrowth of MM.1S (Fig. 4B) cells, whereas no effects wereobserved in U-266 cells (Fig. 4A). We then evaluatedwhether miR-21 overexpression downregulated canonicaltargets. As shown in Fig. 4C, quantitative RT-PCR (qRT-PCR) analyses showed a decrease in PTEN, BTG2, andRho-BmRNA expression (78%, 62%, and 42%, respectively).

Figure 4. Effects of transient enforced expression of miR-21 mimics in multiple myeloma cell lines. Growth curves and BrdUrd uptake (48-hour time point) ofU-266 (A) and MM.1S (B) cells transfected with either miR-21 mimics or scrambled controls (miR-NC). Enforced expression of miR-21 mimics was alsotriggered in MM.1S cells overexpressing a miRNA insensitive PTEN construct (mi-PTEN). C, qRT-PCR of PTEN, BTG2, and Rho-B expression levels wasdone 48 hours after transfection of MM.1S cells with either miR-21 mimics or scrambled controls. The results shown are average mRNA expression afternormalization with GAPDH and DDCt calculations. D, Western blot analysis of PTEN 48 hours after transfection of MM.1S cells with either miR-21mimics or scrambled controls. Relative protein level values are derived from densitometric scan. E, qRT-PCR of PTEN expression levels in MM.1S cellscotransfected with either mi-PTEN or an empty vector and miR-21 mimics or scrambled controls (48-hour time point). The results shown are average mRNAexpression after normalization with GAPDH and DDCt calculations. Data represent the average � SD of 3 independent experiments.

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Moreover,Western blot analysis showed that levels of PTENproteinwere reduced inMM.1S cells overexpressingmiR-21compared with controls (Fig. 4D). To investigate if PTENdownregulation mediates the growth promoting activity ofmiR-21, we transfected MM.1S cells with an expressionvector encoding PTEN gene lacking regulatory 30-UTRsequence. We showed that transfected cells indeed over-expressed PTEN gene, which was not downregulated bymiR-21 (Fig. 4E). Importantly, the PTEN rescue completelyabrogated the miR-21 growth-promoting activity (Fig. 4B).We conclude that the proliferative effect of miR-21 isdependent onPTENsuppression inmultiplemyeloma cells.

miR-21 inhibitors antagonize the hBMSCs protectiverole on multiple myeloma cellsIt is well known that the human bone marrow milieu

strongly support survival and proliferation of multiplemyeloma cells. Because miR-21 expression in multiplemyeloma cells was significantly enhanced by adherence ofcells to hBMSCs (Fig. 1B and C), we next evaluated whethermiR-21 inhibition could overcome the supportive effects ofthe human bone marrow milieu. To this aim, we evaluatedthe antitumor activity of miR-21 inhibition in a contextclosely resembling the intramedullary stage of multiplemyeloma. Specifically, we cultured IL-6–dependent multi-ple myeloma cell line INA-6 adherent to hBMSCs andenforced the expression ofmiR-21 inhibitors in one or bothcell types. As shown in Fig. 6A, miR-21 inhibition affectedviability of multiple myeloma cells to a similar extent asdoes hBMSCs deprivation. miR-21 inhibition was alsoobserved in INA-6 cells cultured in an IL-6–enriched culturemedium(Fig. 5A).Wewonderedwhether this effectwas due

to miR-21 inhibition directly in INA-6 cells, in hBMSCs, orin both. Therefore, we investigated the effects of miR-21inhibition in INA-6 cells cocultured with nontransfectedhBMSCs and found that the antitumor effect was similar tothat obtained whenmiR-21 inhibition was induced in bothcell types (Fig. 5B). In contrast, no effects were observedwhen miR-21 was inhibited only in hBMSCs adherent toINA-6 cells (Fig. 5B). Furthermore, viability and IL-6 secre-tion in culture medium were not affected by miR-21 inhi-bition in hBMSCs (data not shown). Finally, ppMM cellswere cultured adherent to hBMSCs and then exposed tomiR-21 inhibitors, which were able to overcome the sup-portive effect of human bone marrow milieu (Fig. 5C). Onthese findings, we conclude thatmiR-21 inhibitors abrogatethe supporting activity of hBMSConmultiplemyeloma celllines and ppMM cells.

In vivo delivery ofmiR-21 inhibitors exert anti-multiplemyeloma activity against human multiple myelomaxenografts

Finally, we studied the in vivo antitumor potential ofmiR-21 inhibitor oligonucleotides in nonobese diabetic/severe combined immunodeficient (NOD/SCID) micebearing human multiple myeloma xenografts. On thebasis of in vitro findings, we induced OPM-2 xenograftsinto a cohort of 12 mice. When tumors became palpable,mice were randomized into 2 groups and treated intra-tumorally with miR-21 inhibitors or scrambled inhibitors(miR-NC inhibitors). As shown in Fig. 6A, repeatedinjection of miR-21 inhibitors (1 mg/kg; 8 injections, 2days apart), significantly reduced growth of multiplemyeloma xenografts. Importantly, we next evaluated if

Figure 5. miR-21 inhibition antagonizes prosurvival effect of bone marrowmilieu. A, MTT assay of INA-6 cells cultured adherent to hBMSCs, in IL-6–enrichedculture medium, or in IL-6–free culture medium. The assay was conducted 48 hours after transfection of cocultured cells with miR-21 inhibitors or scrambledcontrols (miR-NC inhibitors). B, MTT assay of INA-6 cells cultured with hBMSCs was conducted 48 hours after either INA-6 cells or hBMSCs wereindependently transfected with miR-21 inhibitors or scrambled controls. C, MTT assay conducted in ppMM cells cultured in the presence or absence ofhBMSCs. The assay was conducted 48 hours after transfection with miR-21 inhibitors or scrambled controls. Averaged values � SD of 3 independentexperiments are plotted including. P values were obtained using two-tailed t test.






the antiproliferative activity of miR-21 inhibitors wasrelated to modulation of miR-21 canonical targets. Con-sistent with our in vitro experiments, we found upregula-tion of PTEN both at mRNA and protein levels inretrieved tumors from animals treated with miR-21 inhi-bitors (Fig. 6B and C). Moreover, inhibition of miR-21significantly reduced the phosphorylation of AKT, adownstream target of PTEN and key mediator of tumorcell survival (Fig. 6C), suggesting that the in vivo anti-multiple myeloma activity of miR-21 inhibitors is relatedto PTEN upregulation and AKT activation impairmentwithin tumors.

DiscussionIn this report, we show that antagonism of miR-21 by

oligonucleotide inhibitors exerts antitumor activity invitro and in vivo against human multiple myeloma xeno-grafts in SCID/NOD mice. To our knowledge, this is thefirst evidence of a successful in vivo treatment with miR-21inhibitors in a murine xenograft model of human mul-tiple myeloma, which has important potential clinicalapplications. We show that efficacy of strategies based onmiR-21 inhibition is dependent upon miR-21 expressionlevels in multiple myeloma cells. Indeed, in multiplemyeloma cells expressing high miR-21 levels (KMS-26,U-226, and OPM-2), miR-21 inhibitors reduce cell pro-liferation, survival, and clonogenicity. In contrast, noanti-multiple myeloma effects were observed in cells withlow endogenous miR-21 expression. These data suggestthatmiR-21 expression is a potential biomarker predictiveof therapeutic response to miR-21 inhibitors to be vali-dated in future clinical trials.

The oncogenic role exerted by miR-21 in multiplemyeloma pathogenesis is predicted upon its upregulatedlevels even at early stages of disease. This notion isfurther supported by the role of miR-21 in IL-6/Stat3signaling, a central pathway for multiple myeloma cellgrowth and drug resistance (33, 46, 47). In this report,we show increased proliferation in multiple myelomacells transfected with miR-21 mimics, further supportingthis view. Consistent with prior reports (35), we foundthat miR-21 expression in multiple myeloma cells isupregulated by adherence to hBMSCs. Importantly, weshow that the antitumor properties of miR-21 inhibitorswere not attenuated either by exogenous IL-6 or byadherence of ppMM cells or multiple myeloma cell linesto hBMSCs. These findings strongly suggest the clinicalpotential of targeting miR-21. Specifically, the humanbone marrow milieu induces resistance to conventionaltherapies including melphalan, doxorubicin, and dexa-methasone (7); in contrast, upregulation of miR-21 pro-moted by the bone marrow suggests that targeting thismiRNA may be even more active against multiple mye-loma cells in this milieu.

We investigated the molecular basis of miR-21 inhibitoranti-multiple myeloma activity. It is well known that tumorsuppressor genes, including PTEN, BTG2, and Rho-B, arevalidated targets ofmiR-21 (26).Here, we showed thatmiR-21 inhibition triggered upregulation of these genes inmultiple myeloma cell lines, whereas miR-21 mimics hadthe opposite effect. Modulation of PTEN expression andactivity may be of particular clinical relevance in multiplemyeloma, as PTENmutations are rare events in this disease(43) and PTEN downregulates AKT and ERK activity (48),

Figure6. miR-21 inhibition antagonizesmultiplemyeloma tumor growth in vivo. A, in vivo tumor growth ofOPM-2 xenografts intratumorally treatedwithmiR-21inhibitors or scrambled controls (miR-NC inhibitors). Palpable subcutaneous tumor xenografts were treated every 2 days with 20 mg of miR-21 inhibitorsfor a total of 8 injections (indicated by arrows). A separate control group of tumor-bearing animals was injected with scrambled controls. Tumors weremeasured with an electronic caliper every 2 days, and averaged tumor volume of each group � SD are shown. P values were obtained usingtwo-tailed t test. B, qRT-PCR of PTEN expression in lysates from retrieved OPM2 xenografts. The results shown are average mRNA expression levels afternormalization with GAPDH and DDCt calculations. Data represent the average � SD of 3 independent experiments. C, Western blot analysis of PTEN, totalAKT and p-AKT levels in lysates from a representative retrieved OPM-2 xenograft. Relative protein level values are derived from densitometric scan.

Leone et al.





thereby decreasing multiple myeloma cell proliferation,survival, and drug resistance (49). Importantly, upregula-tion of miR-21, p-AKT, and p-ERK in multiple myelomacells mediates hBMM-induced tumor cell growth and sur-vival (7), and a p-AKT/miR-21 positive feedback loop hasbeen already suggested (50). It is therefore of note thatmiR-21 inhibitors upregulate PTEN and decrease AKT and ERKphosphorylation inmultiplemyeloma cells in our study. Bytransfection of a PTEN expression vector not containing 30-miRNA target sequence, which could be not downregulatedby miR-21, we showed that PTEN suppression was indeedessential for miR-21–induced proliferative activity. More-over,whilemiR-21 inhibitors produced aprolonged growthinhibitory activitywithpERK impairment at 48hours, pAKTwas strongly suppressed only at 24 hours and almostrecovered at 48 hours. We interpretate these findings as arebound activation of pAKT, which does not translate ingrowth rescue.In conclusion, our results both further highlight the

oncogenic role ofmiR-21 inmultiplemyeloma and providethe framework for clinical development of miR-21 inhibi-tors to improve patient outcome in multiple myeloma.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

DisclaimerThe Editor-in-Chief of Clinical Cancer Research (K.C. Anderson) is an

author of this article. In keeping with the AACR’s Editorial Policy, the articlewas peer reviewed and a member of the AACR’s Publications Committeerendered the decision about acceptability.

Authors' ContributionsConception and design: P. Tagliaferri, P. TassoneAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): E. Leone, E. Morelli, N. Amodio, U. Foresta, A.Gull�a, A. NeriAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): E. Leone, E. Morelli, M.T. Di Martino,N. Amodio, A. Gull�a, A. GiordanoWriting, review, and/or revision of the manuscript: E. Leone, E. Morelli,N.C. Munshi, K.C. Anderson, P. Tagliaferri, P. TassoneAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M. RossiStudy supervision: P. Tagliaferri, P. Tassone

Grant SupportThis work has been supported by funds of Italian Association for Cancer

Research (AIRC), Principal Inverstigator: P. Tassone "Special ProgramMole-cular ClinicalOncology—5permille" n. 9980, 2010/15. KCA is anAmericanCancer Society Clinical Research Professor.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 24, 2012; revised January 30, 2013; accepted February20, 2013; published OnlineFirst February 27, 2013.

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Correction

Correction: Targeting miR21 Inhibits In Vitro andIn Vivo Multiple Myeloma Cell Growth

In this article (Clin Cancer Res 2013;19:2096–106), whichwas published in theApril 15, 2013, issueofClinical Cancer Research (1), a sectionof the grant supportwas mistakenly omitted. It should read as follows: "This work was supported inpart by NIH grant P50CA100707 SPORE in Multiple Myeloma (to K.C.Anderson), and by funding from the Italian Association for Cancer Research(AIRC), PI: PT. ‘Special Program Molecular Clinical Oncology—5 per mille’ n.9980, 2010/15. KCA is an American Cancer Society Clinical ResearchProfessor." The authors regret this error.

Reference1. Leone E, Morelli E, Di Martino MT, Amodio N, Foresta U, Gull�a A, et al. Targeting miR21 inhibits

in vitro and in vivo multiple myeloma cell growth. Clin Cancer Res 2013;19:2096–106.

Published online October 15, 2014.doi: 10.1158/1078-0432.CCR-14-2028�2014 American Association for Cancer Research.

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2013;19:2096-2106. Published OnlineFirst February 27, 2013.Clin Cancer Res Emanuela Leone, Eugenio Morelli, Maria T. Di Martino, et al. Growth

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