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Tumor and Stem Cell Biology

miR-214 and miR-148b Targeting InhibitsDissemination of Melanoma and Breast CancerFrancesca Orso1,2,3, Lorena Quirico1,2, Federico Virga1,2, Elisa Penna1,2, Daniela Dettori1,2,Daniela Cimino1,2,3, Roberto Coppo1,2, Elena Grassi1,2, Angela Rita Elia2, Davide Brusa4,5,Silvia Deaglio4,5, Maria Felice Brizzi6, Michael B. Stadler7, Paolo Provero1,2,8,Michele Caselle3,9, and Daniela Taverna1,2,3

Abstract

miR-214 andmiR-148b have been proposed to antagonize theeffects of each other in enabling or blocking metastasis, respec-tively. In this study, we provide evidence deepening their role andinterrelationship in the process of metastatic dissemination.Depleting miR-214 or elevating miR-148b blocked the dissemi-nation of melanoma or breast cancer cells, an effect that could beaccentuated by dual alteration. Mechanistic investigations indi-cated that dual alteration suppressed passage of malignant cellsthrough the blood vessel endothelium by reducing expression ofthe cell adhesion molecules ITGA5 and ALCAM. Notably, trans-endothelial migration in vitro and extravasation in vivo impaired

by singly alternating miR-214 or miR-148b could be overriddenby overexpression of ITGA5 or ALCAM in the same tumor cells. Inclinical specimens of primary breast cancer or metastatic mela-noma, we found a positive correlation between miR-214 andITGA5 or ALCAM along with an inverse correlation of miR-214and miR-148b in the same specimens. Our findings define anantagonistic relationship of miR-214 andmiR-148b in determin-ing the dissemination of cancer cells via tumor–endothelial cellinteractions, with possible implications for microRNA-mediatedtherapeutic interventions aimed at blocking cancer extravasation.Cancer Res; 76(17); 5151–62. �2016 AACR.

IntroductionDissemination of primary tumor cells and the consequent

formation of metastasis at distant organs are the main cause ofcancer-related mortality. The currently available treatments (sur-gery, radio-, chemo-, and targeted therapy) mainly control pri-mary tumors, but they only exert a mild effect on metastases,mostly because of resistancemechanisms activatedby tumor cells.Therefore, it is crucial to identify key regulators of metastaticdissemination and to develop new targeted therapy approaches.

MicroRNAs (miRNAs) are small noncoding, single-strandedRNAs that act as negative regulators of gene expression. Increasing

evidence shows that deregulation of miRNAs is a crucial eventin tumor progression. Several miRNAs, including miR-137,miR-221/222,miR-182,miR-34a, andmiR-214, have been foundto be involved in melanoma malignancy by regulating key genessuch as c-KIT, MITF, FOXO3, ITGB3, CCND1, p27Kip1, TFAP2,and ALCAM (1–3). Instead, let-7, miR-9, miR-10b, miR-21, miR-31, miR-146, miR-148b, miR-155, miR-200, and miR-221/222are the main players in breast cancer dissemination (4–6). There-fore, it is essential to identify new miRNAs and understand howthese small noncoding RNAs control the various steps of tumormalignancy and how they interact with one another to attemptmiRNA-targeted therapies that can affect the molecular pathwaysinvolved in cancer progression. Several clinical trials based on theuse of miRNA modulators in tumorigenesis have already beenestablished (7).

We previously demonstrated that miR-214 promotes mela-noma metastasis dissemination by increasing migration, inva-sion, extravasation, and survival of melanoma cells via a novelpathway involving the metastasis suppressors, TFAP2 transcrip-tion modulators (3). On the other hand, we found that miR-148b opposes breast cancer progression, acting directly on theintegrin signaling players ITGA5, ROCK1, and PIK3CA/p110a(6). More recently, we showed that miR-214 downregulatesmiR-148b in tumor cells via TFAP2C with the consequentupregulation of miR-148b direct targets (2). In order to explorethe relevance of miR-214 and miR-148b for miRNA-basedtherapeutic interventions in melanoma and breast cancer, weanalyzed the dissemination of miR-214-depleted andmiR148b-overexpressing cells in mice, investigated how thesesmall-RNAs influence cell metastatic traits and looked for themain molecular players involved in this process. We demon-strated that single or combined modulations of miR-214

1Molecular Biotechnology Center (MBC), University of Torino, Torino,Italy. 2Department of Molecular Biotechnology and Health Sciences,University of Torino, Torino, Italy. 3Center for Complex Systems inMolecular Biology and Medicine, University of Torino, Torino, Italy.4Department of Medical Sciences, University of Torino, Torino, Italy.5Immunogenetics Unit, Human Genetics Foundation, Torino, Italy.6Department of Medical Sciences, University of Torino, Torino, Italy.7Friederich Miescher Institute and Swiss Institute of Bioinformatics,Basel, Switzerland. 8Center for Translational Genomics and Bioinfor-matics, San Raffaele Scientific Institute, Milan, Italy. 9Department ofPhysics University of Torino, Torino, Italy.

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

F. Orso and L. Quirico contributed equally to this article.

Corresponding Author: Daniela Taverna, MBC and Department of MolecularBiotechnology and Health Sciences, University of Torino, Via Nizza, 52, 10126Torino, Italy. Phone: 39-011-670-6497; Fax: 39-011-670-6432; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-15-1322

�2016 American Association for Cancer Research.

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(inhibition) and miR-148b (overexpression) significantlyinhibit metastatization when tumor cells cross the vessel endo-thelium by decreasing the expression of ITGA5 and ALCAM,two adhesion molecules respectively involved in tumor-Extra-Cellular Matrix (ECM) and cell–cell interactions.

Materials and MethodsCell culture

MA-2 and MC-1 cells were kindly provided by L. Xu andR.O. Hynes (8) and maintained as described in refs. 2 and 3.HumanHBL-100were fromATCCand 4175-TGL and SK-MEL-28were kindly provided, respectively, by J. Massagu�e (9) andL. Poliseno and maintained in standard conditions. Humanumbilical vein endothelial cells (HUVEC) were kindly providedby L. Primo (with GFP) or generated by M.F. Brizzi and main-tained as described in refs. 2 and 3. All used cell lines wereauthenticated in the last 6 months by BMR Genomics, using theCELL ID System (Promega).

Reagents and antibodiesmiR precursors and inhibitors. Pre-miRmiRNA Precursor NegativeControl #1, Pre-miRmiRNA Precursor Hsa-miR-214 (PM12124),Pre-miR miRNA Precursor hsa-miR-148b (PM10264), Anti-miRmiRNA InhibitorNegativeControl #1, Anti-miRmiRNA InhibitorHsa-miR-214 (AM12124), Anti-miR miRNA Inhibitor hsa-miR-148b (AM10264) (all from Applied Biosystems). TaqManMicro-RNA assays for miRNA detection: Hsa-miR-214 ID 002306,Hsa-miR-148b ID 000471, U6 snRNA ID001973, U44 snRNAID001904 (all from Applied Biosystems). Primary antibodies:anti-PIK3CA #4255 (Cell Signaling Technology), anti-GFP ab290(Abcam), anti-ITGA5 pAb RM10 kindly provided by G.Tarone, anti-CD166/ALCAM mAb MOG/07 (Novocastra Lab-oratories), anti-ROCK-1 pAb H-85, anti-hsp90 mAb F-8, anti-GAPDH pAb V-18, anti-ACTIN I-19 pAb (all from Santa CruzBiotechnology), anti-tubulin mAb B5-1-2 (Sigma), anti-CD31pAb (Becton Dickinson). Secondary antibodies: HRP-conju-gated goat anti-mouse IgG, goat anti-rabbit IgG, donkey anti-goat IgG (all from Santa Cruz Biotechnology), goat anti-ratIgG Alexa-Fluor-568 and goat anti-rat IgG Alexa-Fluor-488(Molecular Probes, Invitrogen Life Technologies). siRNAs:si-ITGA5 (Hs_ITGA5_5 siRNA), si-ALCAM (Hs_ALCAM_5siRNA), and All Stars Negative Control siRNA were purchasedfrom Qiagen.

Sponge design and recombinant vector preparationsmiR-214 sponges were described in ref. 3. miR-148b-specific

sponge sequences containing eight miRNA binding sites inter-rupted by 15-nts spacers were designed to be perfectly comple-mentary to themiR-148b seed region, with a bulge position 9–12to prevent undesired cleavage of the sponge RNA. Sponges weresynthesized by DNA 2.0, cloned into pJ241 plasmids, excisedusing flanking HindIII sites, blunted and subcloned into bluntedBamHI and SalI sites, downstreamof EGFP into pLenti CMV-GFP-Puro (658-5) vector (Addgene), giving rise to pLenti148-spon-geA/B. Nucleotides (in Supplementary Table S1) were verified bysequencing. pLemiR-empty, pLemiR-214, and pLemiR-148bexpression vectors were described in refs. 3 and 6. The samemiR-148b expression cassette was also subcloned in a pLenti4/V5 expression vector (kindly provided by C.M. DiPersio, AlbanyMedical College, Albany, NY).

Transient transfections, vectors, and generation of stablecell lines

To obtain transient anti-miR or pre-miR or siRNA expression,cells were plated at 50% confluency and immediately transfectedusing HiPerFect Transfection Reagent (Qiagen), with 100 nmol/Lanti-miR, 75 nmol/L pre-miR, or 100 nmol/L siRNA. For transientcDNA overexpression, cells were plated at 90% confluency andtransfected 24 hours later using Lipofectamine2000 reagent (Invi-trogen Life Technologies). For ITGA5 or ALCAM overexepressionpEGFP-N3-ITGA5 (6) or pLVX-ALCAM (2) expression vectorswere used. All stable cell lines were generated via lentiviralinfection. Sponge vectors were obtained as described above. ThepLKO.1-shALCAM lentiviral vector was from Open Biosystems(RHS3979). Lentiviruses were produced by calcium phosphatetransfection of 20-mg vector plasmid together with 15-mg pack-aging (pCMVdR8.74) and 6-mg envelope (pMD2.G-VSVG) plas-mids in 293T cells, according to Trono's lab protocol (http://tronolab.epfl.ch), and supernatants were harvested 48 hours aftertransfection.

Protein or RNA isolation, immunoblotting, qRT-PCRs formiRNA detection, and proliferation assays

Total protein or RNA extracts, immunoblotting, qRT-PCRs, andproliferation assays were performed as described in refs. 2 and 3.

Migration, invasion, and transendothelial migrationTranswell assays

To measure migration and matrigel invasion, 7.5� 104 MA-2or MC-1, 3 � 104 HBL-100, and 5 � 104 4175-TGL or SK-MEL-28 were seeded in serum-free medium in the upper chambers ofcell culture transwells with 8.0-mm pore size membrane (BDBiosciences), precoated or not with 4 mg/well growth factor-reduced matrigel (BD Biosciences) or in BioCoat MatrigelInvasion Chambers (Becton Dickinson). The lower chamberswere filled with complete growth medium. After 18 hours, themigrated cells on the lower side of the membrane were fixed in2.5% glutaraldehyde, stained with 0.1% crystal violet andphotographed using an Olympus IX70 microscope. For trans-endothelial migration assay, 105 HUVECs were seeded incomplete medium in the upper part of Transwell inserts with5.0-mm pore size membrane (Costar, Corning Inc.) coated byfibronectin at 5 mg/cm2 or 0.1% gelatin, and grown till con-fluency. Then, 5 � 104 cells labeled with CellTracker OrangeCMRA or Green CMFDA (Molecular Probes, Invitrogen LifeTechnologies) were seeded onto the HUVECs monolayer.Twenty hours later, HUVECs and nontransmigrated cells wereremoved and the red or green fluorescent cells that migrated onthe lower side of the membrane were fixed in 4% paraformald-heyde and photographed using Zeiss Axiovert200M micro-scope. Migration, invasion, and transendothelial migrationwere evaluated by measuring the area occupied by migratedcells using the ImageJ software (http://rsbweb.nih.gov/ij/).

ALCAM localization imagingA total of 2 � 105 HUVEC-GFP were seeded on coverslips

coated with fibronectin at 5 mg/cm2, and grown till confluency.Then, 5 � 104 ALCAM-overexpressing MA-2 cells (stably trans-duced with pLVX-ALCAM expressing vector) were seeded on theHUVEC-GFP monolayer. Twenty-four hours later, the cocul-tures were fixed in cold methanol and immunostained forALCAM protein. Briefly, samples were blocked with 5% BSA,

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incubated with anti-CD166/ALCAM mAb MOG/07 (Novocas-tra Laboratories, 1:100 dilution) for 2 hours, then with anti-mouse IgG Alexa-Fluor-568 for 45 minutes and finallymounted on microscope slides for analyses and photos, per-formed with Confocal Leica SP5 microscope.

In vivo tumor growth and metastasis assaysAll experiments performed with live animals complied with

ethical care. For tumor growth, 5 � 106 MC-1 cells (in PBS)were subcutaneously injected into the flanks of 8- to 12-week-old NOD/SCID/IL2R_null (NSG) immunocompromised mice,animals dissected 4 weeks later and proteins extracted fromtumors. For experimental metastasis assays, 5 � 105 MA-2 orMC-1 or SK-MEL-28 or 3 � 105 4175-TGL cells were injectedinto the tail vein of 8- to 12-week-old SCID or NOD/SCID/IL2R_null (NSG) immunocompromised mice and the animalswere dissected 7 or 4 weeks later, respectively. Green or redfluorescent lung metastases were evaluated and photographedin fresh lungs in toto using a Leica MZ16F fluorescence stereo-microscope. The number of metastases was measured onphotographs using the ImageJ software (http://rsbweb.nih.gov/ij/). Micrometastases were evaluated on paraffin-embed-ded and haematoxylin and eosin (H&E)-stained slides, scannedwith Panoramic Desk (3DHistech, Euroclone).

In vivo extravasation assayA total of 1.5 � 106 MC-1 or MA-2 or SK-MEL-28 cells,

previously labeled with CellTracker Orange CMRA (MolecularProbes, Invitrogen Life Technologies), were injected into the tailvein of 4- to 6-week-old female NSG mice (Charles River Labo-ratories). Two or 48 hours later, mice were sacrificed, and 4%paraformaldehyde was injected into the trachea. Lungs weredissected and photographed in toto using a Leica MZ16F fluores-cence stereomicroscope and red fluorescence was quantified 48hours following injections using the ImageJ software (http://rsbweb.nih.gov/ij/). Lungs were embedded in OCT (Killik,BioOptica), frozen, cryostat-cut in 6-mm-thick sections. Localiza-tion of tumor cells, inside/outside the vessels, was evaluated onsections at a Zeiss AxioObserver microscope with the ApoTomeModule (3), following blood vessels staining with an anti-CD31primary antibody in immunofluorescence.

Human tumor correlation analysesNormalized expression values for mRNA and miRNA in breast

cancer were downloaded from the European Genome-phenomeArchive (EGAS00000000122, EGAD00010000434, andEGAD00010000438; refs. 10, 11), and 1302 samples were used.Instead, for melanomas, expression values were downloadedfrom The Cancer Genome Atlas (TCGA, https://tcga-data.nci.nih.gov/tcga/) and 352 samples were used. All analyses wereperformed with R using the packages stats (lm) and ggplot2(12). R: A language and environment for statistical computing.R Foundation for Statistical Computing, Vienna, Austria (URLhttps://www.R-project.org/).

Statistical analysisThe results are shown as mean � SD or as mean � SEM, as

indicated, and a two-tailed Student t testwas used for comparison.�, P < 0.05; ��, P < 0.01; ��, P < 0.001 were considered to bestatistically significant. ns indicates a nonstatistically significant Pvalue.

ResultsmiR-214 depletion and miR-148b overexpression inhibitmelanoma and breast cancer metastasis formation in mice

High levels ofmiR-214 are found inmalignantmelanomas andbreast tumors and promote metastatization in mice by regulatinga complex network of players, in a negative or positive manner,including the downregulation of the antimetastatic miR-148b.Here, we evaluated the potential therapeutic value of miR-214depletion and miR-148b overexpression in tumor progression inmice.

Specific miR-214 or miR-148b sponges A or B were bio-infor-matically designed, cloned at the 3' end of a Green FluorescentProtein (GFP) expression cassette in lentivirus vectors as inSupplementary Table S1, Supplementary Fig. S1A, and ref. 3 andtested in MC-1 or MA-2 melanoma cells. Specifically, cellswere transfected with pLenti-214-spongeA/B or pLenti-148b-spongeA/B or pLenti-empty (control) vectors together withprecursors for miR-214 or miR-148b or controls (pre-miR-214or pre-miR-148bor pre-control), andGFP levelswere evaluated incells at the microscope (Supplementary Fig. S1B and S1C), byWestern Blot-WB- (Supplementary Fig. S1D and S1E), or FACSanalysis (Supplementary Fig. S1F and S1G). As shown, all spongeswere able to inhibit GFP expression, suggesting miR-214 or miR-148b binding to their complementary sequences.

MC-1 orMA-2melanoma or 4175-TGL breast cancer cells werethen transduced with lentiviruses expressing miR-214-sponges(pLenti-214-spongeA/B) or miR-148b (pLenti4/V5-148b, pLe-miR-148b) or with empty controls (pLenti-empty, pLenti4/V5-empty, pLemiR-empty). Alternatively, MA-2 or SK-MEL-28 mel-anoma or 4175-TGL breast cancer cells were transduced withlentiviruses expressing miR-148b-sponges (pLenti-148b-spon-geA/B) or miR-214 (pLemiR-214) or with empty controls(pLenti-empty, pLemiR-empty) to verify the mechanism in theopposite directions.miR-214 andmiR-148b levelswere evaluatedby qRT-PCR analyses as shown in Supplementary Figs. S2 to S5.Cells with the expected miR-214 or miR-148b modulations wereinjected in the tail vein of immunocompromised mice, andmetastasis dissemination was evaluated 4 to 7 weeks later bymeasuring the number/area of lung metastasis in H&E-stainedlung or liver sections or the fluorescent (green or red) tumor cellspresent in thewhole organs (Fig. 1A–C; Supplementary Figs. S6A–S6D and S7A–S7D). Single miR-214 downmodulation or miR-148b overexpression significantly blocked metastasis dissemina-tion forMC-1 orMA-2melanoma or 4175-TGL breast cancer cellscompared with controls. Relevantly, simultaneous depletion ofmiR-214 and increased levels of miR-148b further blocked tumorspreading of melanoma and breast tumor cells compared withsingle modulations, suggesting a combined action. In parallel,singlemiR-214overexpressionormiR-148bdownmodulation, orsimultaneous double targeting, favored melanoma or breastcancer cell dissemination compared with controls.

In conclusion, we can consider miR-214 and miR-148b aspromising targets for miRNA-based therapeutic interventions intumor progression.

miR-214 inhibition and miR-148b overexpression control-specific tumor cell metastatic traits

To understand which metastatic traits of melanoma (MA-2,MC-1, and SK-MEL-28) and normal or tumor breast (HBL-100and 4175-TGL) cells were affected by miR-214 and miR-148b

miR-214 and miR-148b Targeting in Tumor Progression

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Figure 1.

miR-214 depletion and miR-148b overexpression inhibitmetastasis formation in mice. Lung or liver colonyformation in immunodeficient mice, 7 (A) or 4 (B and C)weeks after tail-vein injection of MC-1 (A) or 4175-TGL(B and C) cells. Cells were transduced with controls(pLenti-empty, pLenti4/V5-empty) or miR-214-sponge(pLenti-214-spongeB) or miR-148b-overexpressing(pLenti4/V5-148b) vectors, single or in combination, asindicated in the panels. Representative pictures ofH&E-stained sections (A, B, a–d, bar, 500 mm; C, a–d, bar,100 mm) are shown. Graphs (bottom of each figure)represent quantitated results as mean � SEM H&E-stainedcolonies number (A) or as a percentage of metastatic/totalareas (B) in lungs or as mean metastases/field in liver,referring to the indicated number of mice (n). Twoindependent experiments were performed andrepresentative results are shown.

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modulations, we evaluated proliferation, migration, invasionthrough matrigel and transendothelial migration on a HUVECmonolayer in vitro. Cells were either transduced with lentivirusesfor the depletion (pLenti-214-spongeB, pLenti-148b-spongeB) oroverexpression (pLemiR-214, pLenti4/V5-148b) of miR-214 ormiR-148b or with empty controls (pLemiR-empty, pLenti-empty,pLenti4/V5-empty) or transiently transfected with miRNA pre-cursors or inhibitors (pre-miR-214, anti-miR-148b, pre/anti-con-trol). miR-214 and miR-148b modulations were evaluated byqRT-PCR assays as shown in Supplementary Figs. S2 to S5. Singleor double alterations of miR-214 and miR-148b did not signif-icantly affect proliferation compared with controls in any testedcell line (Fig. 2A and B; and Supplementary Fig. S8). Instead,modulations of miR-214 or miR-148b significantly affectedmigration, cell movement across a HUVEC monolayer (transen-dothelial migration) and invasion in matrigel compared withcontrols (Fig. 3A–F; Supplementary Fig. S9A–F). Precisely, in allassays, cell movement was impaired in miR-214-depleted andmiR-148b-overexpressing cells. Opposite results were observedwhen miR-214 was highly expressed in cells while miR-148bexpressionwas reduced. Relevantly, in transendothelialmigrationassays, but generally not in migration or invasion analyses,simultaneous miR-214 inhibition and miR-148b overexpression

almost always led to combined effects, like for in vivo metastasis(Fig. 1), suggesting a combinatorial, specific effect ofmiR-214 andmiR-148b at the level of tumor–endothelial cell interactions.

miR-214depletion andmiR-148boverexpression impair tumorcell extravasation in mice

The effect of single or combined sponge-induced miR-214depletion and miR-148b overexpression was investigated onin vivo cell extravasation of tumor cells. CMRA-labeled MC-1 orSK-MEL-28 cells stably expressing miR-214-sponge (pLenti-214-spongeB) or miR-148b (pLenti4/V5-148b) or control vectors(pLenti-empty or pLenti4/V5-empty) were injected in the tail veinof immunocompromised mice (Fig. 4A–L and graph, Supple-mentary Figs. S10 a–S10h and graph). Lodging to the lungvasculature was evaluated 2 hours after injection (Fig. 4A–D;Supplementary Fig. S10a–S10b), and no difference was observedamongmodified cells. Instead, a strongdecrease in early (48hoursafter injection) lung colonization was observed following singleor combined sponge-driven miR-214 downmodulation andmiR-148b overexpression compared with controls in MC-1 (Fig.4E–H)or SK-MEL-28 (Supplementary Fig. S10c–S10d) cells. Notethat cells were localized inside blood vessels or associated withthem at 2 hours, as shown in Supplementary Figs. S10e–S10f.Instead, cells were found in the lung parenchyma at 48 hours(Fig. 4I–L; Supplementary Fig. S10g–S10h). As for metastasisdissemination (Fig. 1) and transendothelial migration (Fig. 3),simultaneous miR-214 inhibition and miR-148b overexpressionled to combined effects, suggesting, once more, a combinatorial,specific effect of miR-214 and miR-148b at the level of tumor–endothelial cell interactions.

Depletion of miR-214 and overexpression of miR-148b affectthe adhesion molecules ITGA5 and ALCAM

To identify the molecular players involved in reduced cancerdissemination/extravasation by miR-214 depletion and miR-148b overexpression in tumor cells, expression of ITGA5 andALCAM, two validated miR-148b direct targets, known to behighly relevant for cancer cell dissemination, was analyzed incell cultures or in mouse subcutaneous tumors. Single or com-bined miR-214 depletion and miR-148b overexpression in mel-anoma (MA-2, MC-1, SK-MEL-28) and breast cancer (4175-TGL)cells were obtained following stable transduction of lentivirusvectors for the expression of miR-214 sponges (pLenti-214-spon-geB) or miR-148b (pLenti4/V5-148b) or empty controls (pLenti-empty, pLenti4/V5-empty). miR-214 and miR-148b alterationswere evaluated by qRT-PCR analyses as in Supplementary Figs. S2to S5. Important reduction of ITGA5 or ALCAM expressionwas observed for single or combined miRNA-modulations.Combined inhibitions of ITGA5 and ALCAM by dual miR-214/miR-148b interventions were rarely observed compared withsingle alterations (Fig. 5A–D; and Supplementary Fig. S11A–E).Considering the relevance of ITGA5 and ALCAM expressionimpairment for the inhibition of transendothelial migrationfollowing simultaneous miR-214/miR148b alterations, as pre-sented below (see the next paragraph), we can speculate that theeffects of combined miR-214/miR-148b changes occur specifical-ly when tumor cells get in contact with endothelial cells. Alter-natively, supplementary alterations of gene expression could beinvolved in the passage of tumor cells through the blood vessels.Opposite stable or transient modulations were obtained in someof the cells (MA-2, 4175-TGL) listed above and in HBL-100

Figure 2.

miR-214 depletion and miR-148b overexpression do not affect proliferation.A and B, proliferation of MC-1 (A) or 4175-TGL (B) cells stably transduced withcontrol (pLenti-empty, pLenti4/V5-empty) or miR-214-sponge (pLenti-214-spongeB) or miR-148b-overexpressing (pLenti4/V5-148b) vectors, single or incombination, as indicated in the graphs. Results are indicated as mean � SD ofthe proliferation ratio versus plated cells, measured by optical density at 0–72hours. At least two independent experiments (with triplicates) were performed,and representative results are shown.






Figure 3.

miR-214 depletion and miR-148b overexpression inhibit transendothelial migration. Transwell migration assays were used to evaluate migration (through a porousmembrane) or transendothelial migration (through an HUVEC monolayer on top of a porous membrane) for MC-1 (A, B) or 4175-TGL (C and D) or SK-MEL-28(E and F) cells stably transduced with control (pLenti-empty, pLenti4/V5-empty) or miR-214-sponge (pLenti-214-spongeB) or miR-148b-overexpressing(pLenti4/V5-148b) vectors, single or in combination, as indicated in the panels. For migration, results are indicated as a ratio of mean� SEM of the area covered bymigrated versus plated tumor cells; for transendothelial migration, results are shown as mean � SEM of the area covered by tumor migrated cells. Atleast two independent experiments (with triplicates) were performed, and representative results are shown.

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normal breast cells to further evaluate thismechanism. Here, cellswere transduced with pLenti-148b-spongeA/B or pLemiR-214 orthe relative empty controls (pLenti-empty, pLemiR-empty) ortransiently transfected with anti-miR-148b or pre-miR-214 oranti/pre-control and modulations evaluated by qRT-PCR assaysas in Supplementary Fig. S2 to S5. Here, increased levels of ITGA5and ALCAM and their downstream players ROCK1 and PIK3CAwere observed (Supplementary Fig. S11A–E), thus reinforcing theimportance of this pathway in miR-214/miR-148b-driven tumordissemination.

Impairment of tumor dissemination bymiR-214 depletion andmiR-148b overexpression depends on ITGA5 and ALCAMexpression inhibition

In order to understand if reduction of ITGA5 or ALCAM wasessential for miR-214/miR-148b-driven extravasation inhibition,wemodulated ITGA5 or ALCAM levels in the presence ofmiR-214depletion and miR-148b overexpression.

First, ITGA5 or ALCAM expression was inhibited by RNAinterference in a transient (si-ITGA5 or si-ALCAM) or stable (sh-ALCAM) manner in MA-2 melanoma or 4175-TGL breastcancer cells and transendothelial migration or extravasationevaluated by comparison with empty (si-control, sh-control)cells. As shown in Supplementary Fig. S12, reduced levels ofITGA5 or ALCAM significantly impaired transendothelial

migration (Supplementary Fig. S12A and S12B) or lung extrav-asation, 48 hours after injection (tail vein) in immunocom-promised mice (Supplementary Fig. S12C and S12D) com-pared with controls. Modulations of ITGA5 or ALCAM wereevaluated by Western blot analysis (Supplementary Fig. S12).Tumor–endothelial cell contacts are shown in a confocal micro-scope image (Supplementary Fig. S12E) in which red (ALCAMstaining) MA-2 tumor cells were in contact with green (GFPexpression) HUVECs. All these results suggest the relevance ofITGA5 and ALCAM in the control of tumor cell transendothelialmigration or extravasation during tumor progression. At thispoint, ITGA5 or ALCAM were overexpressed in MC-1 or 4175-TGL cells previously transduced with pLenti-214-spongeB orpLenti4/V5-148b or pLenti-empty/pLenti4/V5-empty lentivirusvectors, in single or dual combinations, and cells used toevaluate transendothelial migration. Modulations of ITGA5 orALCAM were evaluated by Western blot analysis (Fig. 6A–F). Inall conditions, we observed increased transendothelial migra-tion, compared with controls, when ITGA5 or ALCAM wasoverexpressed in cells. Thus, suggesting that repression oftransendothelial migration, driven by miR-214-depletion andmiR-148b-upregulation, depends on the reduction of theseadhesion molecules in tumor cells. In fact, transendothelialmigration inhibition was rescued when ITGA5 or ALCAM levelswere increased.

Figure 4.

miR-214 depletion and miR-148boverexpression inhibit extravasation inmice. In vivo extravasation 2 hours (A–D) or48 hours (E–H) following tail-vein injectionsin immunodeficient mice of CMRA-labeledMC-1 cells transduced with control (pLenti-empty, pLenti4/V5-empty) or miR-214-sponge (pLenti-214-spongeB) or miR-148b-overexpressing (pLenti4/V5-148b) vectors,single or in combination, as indicated in thepanels. Representative pictures ofwhole redfluorescent lungs at 2 hours or 48 hoursafter injection (A–H) and representativefields of murine lung sections, 48 hoursafter injection (I–L), stained for CD31 andcounterstained with DAPI are shown; bar,800 mm. Results are indicated in the graphs(bottom) as mean � SEM of the number ofextravasated cells at 48 hours for n¼ 4miceper group. White arrows, extravasated cells.Two independent experiments wereperformed, and representative resultsare shown.






Taken together, our results prove that the negative targeting ofmiR-214 and/or the positive modulation of miR-148b impairsdissemination by acting on ITGA5 or ALCAM, two main playersfor tumor cell extravasation.

miR-214 expression correlates with ITGA5 and ALCAM levels,while it anticorrelates withmiR-148b, inmelanomametastasesand in primary breast tumors

Breast cancer (n ¼ 1302; refs. 10, 11) and melanomametastasis (TCGA, https://tcga-data.nci.nih.gov/tcga/; n ¼352) datasets were used to evaluate miR-214, miR-148b,ITGA5, and ALCAM expression and to look for possible corre-lations or anticorrelations with one another (Fig. 7A and B).Relevantly, we found that miR-214 and miR-148b anticorre-late in melanoma metastases (P ¼ 7.91e�05) and in primarybreast tumors (P ¼ 5.43e�08), while miR-214 significantlycorrelates with ITGA5 and ALCAM in both datasets. P valueswere the following: for melanoma metastases: ITGA5 (P ¼

3.09e�24) and ALCAM (P ¼ 2.69e�09); for primary breasttumors: ITGA5 (P ¼ 1.68e�23) and ALCAM (P ¼ 9.27e�07).These results are therefore in line with our above-presentedinvestigations, thus strengthening the link between miR-214,miR-148b, ITGA5, and ALCAM and its relevance in humantumor progression.

DiscussionWe previously demonstrated the prometastatic role of miR-

214 (3) and its link with the antimetastatic miR-148b (2). Here,we show that single or combined miR-214 inhibition and miR-148b overexpression in tumor cells strongly modulate metas-tasis formation by acting mainly during the passage across thevessel endothelium (transendothelial migration/extravasa-tion), a metastatic trait that involves two direct miR-148btargets, the adhesion receptors ITGA5 and ALCAM, in a cell–fibronectin and cell–cell dependent manner. Our data suggest

Figure 5.

miR-214 depletion and miR-148b overexpression affect ITGA5 and ALCAM expression. A–D, Western blot analysis of ITGA5 or ALCAM protein expression levelsin MC-1 (A), MC-1-SQ (B), 4175-TGL (C), SK-MEL-28 (D), transiently transfected with precursors for miR-148b (pre-miR-148b) or inhibitors for miR-214(anti-miR-214) or negative controls (pre-control or anti-control), single or in combination, or stably transduced with controls (pLenti-empty, pLenti4/V5-empty) ormiR-214-sponge (pLenti-214-spongeB) or miR-148b-overexpressing (pLenti4/V5-148b) vectors, single or in combination, as indicated. Protein modulationswere calculated relative to empty controls, normalized on loading controls (GAPDH or actin or hsp90 or tubulin) and expressed as percentages (%). Threeindependent experiments were performed, and representative ones are shown. SQ, subcutaneous tumors.

Orso et al.





Figure 6.

Transendothelial migration depends on ITGA5 and ALCAM expression in miR-214-depleted and/or miR-148b-overexpressing tumor cells. A–F, transendothelialmigration was evaluated in MC-1 (A, C, E) or 4175-TGL (B, D, F) cells stably transduced with miR-214-sponge (pLenti-214-spongeB) or miR-148b-overexpressing(pLenti4/V5-148b) vectors, single or in combination, and, in addition, transiently transfectedwith recombinant vectors for the overexpression of ITGA5 or ALCAM orempty controls, as indicated in the panels. ITGA5 and ALCAM levels were evaluated by Western blot analysis, 24 hours or 48 hours following transfections. Proteinmodulations were calculated relative to negative controls, normalized on loading controls (GAPDH or actin) and expressed as percentages (%). Transmigrationresults are indicated as mean � SEM of the area covered by tumor-migrated cells. At least two independent experiments (with triplicates) were performed,and representative results are shown.






that miR-214 and miR-148b are valuable candidates formiRNA-based targeted therapy.

miR-214 is highly expressed inmalignant cutaneous andocularmelanomas (3, 13, 14) as well as in breast, osteosarcoma, ovary,pancreas, prostate, and gastric cancers (15–20). In line with thesefindings, upregulation ofmiR-214 in various tumor cells increasesmetastasis formation (2, 3, 21–23).On the other hand,miR-148bis poorly expressed in melanomas and in breast, pancreatic, and

hepatocellular carcinomas (1, 6, 24, 25) and its modulation intumor cell lines reveals its antimetastatic function (6, 26–28).Here, by modulating miR-214 and miR-148b, respectively, in anegative and positive manner, we show that miR-214 and miR-148b are part of a miR-ON-miR regulatory axis where miR-214favors tumor dissemination following the downregulation ofmiR-148b and the consequent upregulation of miR-148b directtargets, ITGA5 and ALCAM as well as some of their downstream

Figure 7.

miR-214 anticorrelates with miR-148b,while it correlates with miR-148b targetsITGA5 and ALCAM, in melanomametastases or human primary breasttumors. The indicated datasets wereused to evaluate miR-214, miR-148b,ITGA5, and ALCAM expression inmelanoma metastases (A) and inprimary breast tumors (B). Statisticallysignificant negative or positivecorrelations are shown for miR-214 andmiR-148b (anticorrelations) and for miR-214 and ITGA5 or ALCAM (correlations).Correlations of normalized expressionare represented with a dot plotsuperimposing the regression line.The shaded area represents the 0.95standard error confidence interval ofthe model predictions. Statisticallysignificant R2 and P values and numberof samples (n) are indicated.

Orso et al.





players (8). Similarly, recent investigations underline the rele-vance ofmultiplemiR connections (miR-ON-miR) or ofmiRs andtranscription factors (TF) reciprocal regulations (miR-ON-TF-ON-miR). Examples are Lin28-let-7-miR-181 in megakaryocyte dif-ferentiation (29), miR-181b-FOS-miR-21 in glioma progression(30), miR-103/107-miR-200 in epithelial-to-mesenchymal tran-sition (31), miR-199a/miR-214-let-7b-miR-34a-miR-762-miR-1915 in breast cancer metastasis (32). All these lines of evidenceopen up the possibility to target multiple players of the samepathway and give hope for combined therapeutic interventions.In line with this hypothesis, it has recently been shown that thesimultaneous systemic delivery of two small non-coding RNAsacting as tumor suppressors, miR-34 and let-7, leads to reducednon–small lung cancer growth (33).

We propose that the presented miR-214-ON-miR-148b regu-latory axis controls tumor dissemination acting, in particular,when tumor cells cross the blood vessels endothelium, via themodulation of ITGA5 and ALCAM (8, 34–38). In fact, the silenc-ing of ALCAM or ITGA5 inhibits transendothelial migration invitro and extravasation in vivo. More relevantly, when miR-214 isdepleted and/or miR-148b overexpressed, transendothelialmigration suppression can be overcome by ITGA5 or ALCAMupregulation, thus suggesting that these two adhesion moleculesare essential modulators of tumor–endothelial cell interactions.These results are in line with the fact that ALCAM is a cell-to-celladhesion receptor known to play a major role in mediating theinteractions between endothelial and tumor or immune cellsduring transendothelial migration (39–41). Moreover, ALCAMwas found to be involved in cell movement (42) and in theconversion of the prometastatic pro-MMP-2 to its active form inmalignancy (43). On the other hand, modulations of integrinlevels were associated with various metastatic phenotypes (35,44–46). In particular, increased ITGA5 expression has beenobserved in metastatic melanoma cell lines compared with pri-mary ones (47), and survivin-dependent ITGA5 upregulation wasshown to enhance motility of melanoma cells (48). InMDAMB231 breast cancer cells, ITGA5 promoted lungmetastasisin both spontaneous and experimental lung metastasis models(35). In breast and ovarian cancer patients, ITGA5 expression wasshown to be predictive of metastasis and poor prognosis (6, 46,49). ITGA5 has also been linked to extravasation; in fact, it hasbeen shown that Cav1–Rho-GTPase-dependent control of cellextravasation depends on ITGA5 levels and inhibition of thisregulatory axis impairs extravasation and survival of metastaticcells (50). The fact that high levels of ITGA5 and ALCAM correlatewith miR-214 and that miR-214 expression anticorrelates withmiR-148b in human breast and melanoma, tumors, or metasta-ses, further strengthens the importance of these players and theirdirect functional connections in tumorigenesis.

With particular relevance for targeted therapy, the evidencethat single or combined miR-214 downregulation and miR-148b upregulation in tumor cells inhibit metastasis formation

in mice, gives hope for an miRNA-based therapy. Due to therelevance of ALCAM and ITGA5 in the pathway presented here,one could even speculate to target these two adhesion mole-cules with specific antibodies, in addition to miRNA targeting,to further affect metastasis formation. Considering that themain issue of the miRNA-based targeted therapy is the in vivodelivery, it is essential to identify safe, selective, and efficientcompound systemic deliveries. For this purpose, we are cur-rently developing new tools to administer miR-214 inhibitorsand miR-148b precursors to animals and test their efficacy onmetastasis formation.

In conclusion, our data demonstrate that the cascade of events,includingmiR-214,miR-148b, ALCAM, and ITGA5, is controllingmelanoma and breast cancer progression and it can be exploitedfor combinatorial therapeutic interventions.

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

Authors' ContributionsConception and design: F. Orso, L. Quirico, D. TavernaDevelopment of methodology: M.F. Brizzi, M.B. StadlerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Virga, E. Penna, D. Cimino, D. Brusa, S. DeaglioAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Orso, L. Quirico, F. Virga, D. Dettori, D. Cimino,E. Grassi, A.R. Elia, M.B. Stadler, P. Provero, M. CaselleWriting, review, and/or revision of the manuscript: F. Orso, L. Quirico,M.B. Stadler, D. TavernaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): F. VirgaStudy supervision: D. TavernaOther (animal care): R. Coppo

AcknowledgmentsWe are grateful to Lei Xu and Richard Hynes for the A375P cell line and its

metastatic variants, JoanMassagu�e for the 4175-TGL cells, LauraPoliseno for theSK-MEL-28 cells, Luca Primo for the GFP-HUVECs, C.M. DiPersio for thepLenti4/V5 expression vector, Jacek Krol for advice with the sponge constructs,Flavio Cristofani for his help with the immunocompromised mice, and MarcoForni for pictures of histological sections.

Grant SupportD. Taverna was supported by Compagnia di San Paolo, Torino, 2008.1054,

AIRC 2010, 2013 (IG2010-10104DT and IG2013-14201DT), FondazioneCassa di Risparmio Torino CRT (2014.1085DT). M. Caselle was supported byProgetto di ricerca di Ateneo 2012/SanPaolo Torino CASMATEN12 and F. Orsoby FIRB giovani 2008 (RBFR08F2FS-002 FO). E. Penna is an FIRC fellow (2012–2014).

The costs of publication of this articlewere defrayed inpart 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 14, 2015; revised May 20, 2016; accepted June 5, 2016;published OnlineFirst June 21, 2016.

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2016;76:5151-5162. Published OnlineFirst June 21, 2016.Cancer Res Francesca Orso, Lorena Quirico, Federico Virga, et al. Melanoma and Breast CancermiR-214 and miR-148b Targeting Inhibits Dissemination of

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