microRNA 17/20 inhibits cellular invasion and tumormetastasis in breast cancer by heterotypic signalingZuoren Yua, Nicole E. Willmartha,1, Jie Zhoua,1, Sanjay Katiyara,b, Min Wanga, Yang Liua, Peter A. McCuec,Andrew A. Quonga, Michael P. Lisantia, and Richard G. Pestella,b,2

Departments of aCancer Biology, bMedical Oncology, and cPathology, Thomas Jefferson University and Hospital, Kimmel Cancer Center, Philadelphia, PA 19107

Edited* by Hilary Koprowski, Thomas Jefferson University–Jefferson Medical College, Philadelphia, PA, and approved March 24, 2010 (received for reviewFebruary 18, 2010)

microRNAs are thought to regulate tumor progression and in-vasion via direct interaction with target genes within cells. Herethe microRNA17/20 cluster is shown to govern cellular migrationand invasion of nearby cells via heterotypic secreted signals. micro-RNA17/20 abundance is reduced in highly invasive breast cancer celllines and node-positive breast cancer specimens. Cell-conditionedmedium from microRNA17/20–overexpressing noninvasive breastcancer cell MCF7 was sufficient to inhibit MDA-MB-231 cell migra-tion and invasion through inhibiting secretion of a subset of cyto-kines, and suppressing plasminogen activation via inhibition ofthe secreted plasminogen activators (cytokeratin 8 and α-enolase).microRNA17/20 directly repressed IL-8 by targeting its 3′ UTR, andinhibited cytokeratin 8 via the cell cycle control protein cyclin D1. Atvariance with prior studies, these results demonstrated a uniquemechanism of how the alteredmicroRNA17/20 expression regulatescellular secretion and tumor microenvironment to control migrationand invasion of neighboring cells in breast cancer. These findingsnot only reveal an antiinvasive function of miR-17/20 in breast can-cer, but also identify a heterotypic secreted signal that mediatesthe microRNA regulation of tumor metastasis.

cell secretion | plasminogen | cytokeratin 8 | α-enolase

MicroRNAs (miRNAs) are 21- to 22-nucleotide molecules thatregulate cellular phenotype through altering the stability or

translational efficiency of targeted mRNAs (1). Important associa-tions between miRNA gene expression and human cancer have im-plicated miRNA in human tumorigenesis (2–4). miRNA encodinggenes are frequently located at fragile sites, common breakpoints, orminimal regions of amplification (5). The human miR-17/20 cluster,located on chromosome 13q31, undergoes loss of heterozygosityin several different cancers, including breast cancer. In the humanB cell line P493-6, miR-17/20 inhibits cell growth via suppression ofE2F1 expression (6). Inmice, transgenicmice overexpressingmiR-17alone showed overall tissue growth retardation, smaller organs, andgreatly reduced hematopoietic cell lineages (7). Analysis of miRNAsregulated in cyclin D1 transgenic mammary tumors and reciprocallyregulated in cyclin D1–knockout mice identified the miR-17/20cluster as an important regulatory switch in mammary tumor growth.miR-17/20 inhibited breast cancer tumor growth through repressionof cyclin D1 expression via the 3′ UTR binding site (8). Recent evi-dence implicates miRNA in breast cancer metastasis by inhibitingtarget genes. miR-335 inhibited human breast cancer cell metastasisvia repression of SOX4, a regulator of progenitor cell developmentand migration (9). miR-10b promotes cell migration in vitro andinitiates breast tumor invasion in vivo by targeting gene HOXD10(10). miR-200 family prevented epithelial to mesenchymal transitionof breast cancer cells by repressing ZEB1 and SIP1 (11). However,the role of the miR-17/20 cluster in regulation of breast cancer me-tastasis remains unknown.Tumor cell migration and invasion is in part dependent upon

plasminogen activity (12), which is regulated by urokinase-typeplasminogen activator (uPA), plasminogen activator inhibitor(PAI)–1, PAI-2, and binding to the secreted proteins cytokeratin8 (CK8) (13) and α-enolase (α-ENO) (14). At variance with

prior studies in which miRNA regulate migration and invasion viagenes within the tumor epithelial cells (15), we demonstrate thatthe miR-17/20 cluster mediates breast cancer cell migration andinvasion via a heterotypic secreted signal. miRNA expressionwithin breast cancer epithelial cells conveys an antimetastaticphenotype that can be transmitted to other cell types.

Results and DiscussionReduced miR-17/20 Abundance in Metastatic Breast Cancer. To testthe possibility that miR-17/20 is related to human breast cancer me-tastasis, humanbreast cancer specimensandhumanbreast cancer celllines were analyzed. miR-17/20 expression in lymph node–positivehuman mammary gland primary tumors and lymph node–negativehuman mammary gland primary tumors were examined with quan-titative real-time PCR (Fig.1A) and confirmed byNorthern blot. ThemiR-17/20 abundance in each tumor sample was normalized to thatof the matching normal mammary gland sample from the same pa-tient. Comparison between human mammary gland primary tumorswith lymph nodes positive and negative indicated a significant inversecorrelation between the miR-17–5p expression level and nodalmetastatic status of the breast cancer (Fig.1A). A further analysis ofmiR-17/20 abundance in three highly invasive human breast cancercell lines (MDA-MB-231, Hs578T, and SKBR3) and four non-invasive human breast cancer cell lines (BT474, MCF7, MDA-MB-468, and T-47D) were compared by miRNA Northern blot hybrid-ization (Fig.1B). The quantitative analysis showed an overall higherexpression of bothmiR-17–5p andmiR-20a in the noninvasive breastcancer cell lines than that in highly invasive lines (Fig. S1).

miR-17/20 Conditioned Medium Suppressed Breast Cancer CellMigration and Invasion. To determine whether miR-17/20 re-duces cellular migration and invasion directly, MDA-MB-231and MCF-7 cells were examined. Both cell lines were trans-duced with the miR-17/20 cluster or vector control. The over-expression of miR-17 and miR-20a was shown in Fig. S2. Cellmigration assays were performed on the miR-17/20 transducedcells. No significant difference was observed between the miR-17/20 overexpressing MDA-MB-231 cells and control cells.The miR-17/20 conditioned cell medium was assayed for migra-

tion and invasion. Surprisingly, we found that the miR-17/20 condi-tionedmedium fromMCF7cells reduced the invasionofMDA-MB-231 breast cancer cells in 3D collagen invasion assays (Fig. 1C) andreduced cell migration of MDA-MB-231 in Boyden chamber assays(Fig. 1D). Wound healing assays demonstrated the addition of

Author contributions: Z.Y., M.P.L. and R.G.P. designed research; Z.Y., N.E.W., J.Z., S.K.,M.W., Y.L., and A.A.Q. performed research; P.A.M. contributed new reagents/analytictools; Z.Y., N.E.W., J.Z., and S.K. analyzed data; and Z.Y. and R.G.P. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1N.E.W. and J.Z. contributed equally to this work.2To whom correspondence should be addressed. E-mail: richard.pestell@jefferson.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1002080107/-/DCSupplemental.

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conditioned medium from miR-17/20 transduced MCF7 breastcancer cells reduced theability ofMDA-MB-231cells tomigrate intothe wound (Fig. 1E). We fractioned the miR-17/20 conditionedmedium using a P10 column (protein cut off at 10 kDa molecularweight), and performed the cell migration assays. Both the highmolecular weight fraction (proteins >10 kDa) and low molecularweight fraction (proteins <10 kDa) inhibited breast cancer cell mi-gration, although the former had more inhibition than the latter,indicatingmultiple factors in themiR-17/20 conditionedmediumareinvolved in the inhibitionof cellmigration and invasion.ThemiR-17/20 conditionedmediumdidnot change the self-expression pattern ofmiR-17/20 in cells (Fig. S3). These observations suggest miR-17/20regulates cellular secretion, alters cellular microenvironment, andregulates cell migration and invasion via a heterotypic signal.

miR-17/20 Reduces Cell Secretion. To identify the secreted factorswithin the medium regulated by miR-17/20 transduction, MALDI-

TOFMSanalysiswas conductedofmiR-17/20 transducedMCF7cellmedium.Mediumderived fromvector transducedMCF7cells servedas control. The proteins recovered from the gel were subjected to in-gel tryptic digestion. Sequential MS/MS peptide sequencing of theexcised bands at approximately 40 to 50 kDa identified the proteinsα-ENO andCK8 (Fig. 2A and Figs. S4 and S5). CK8 and α-ENO aresecreted by human breast cancer cells (13, 16). Western blot con-firmed the reduction in α-ENO and CK8 protein abundance in miR-17/20 conditioned MCF7 cell medium (Fig. 2B). Several other pro-tein bands showing different abundance in the miR-17/20–over-expressing MCF7 cell medium were analyzed by MS as well;unfortunately, the identities of these proteins were not validated yet.A human cytokine antibody array was used to examine further the

miR-17/20 conditioned medium. Subtractive analysis identifiedsecreted cytokines (CXCL1, IL-10, IL-8, and NT4) which were de-creased in the miR-17/20 conditioned medium (Fig. 2C). This assaywas repeated two times with independent medium samples. Only

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Fig. 1. miR-17/20–conditioned medium inhibits breast cancer cell migration and invasiveness. (A) Quantitative real-time PCR analysis of miR-17–5p abun-dance in human mammary gland primary tumors with lymph nodes positive (n = 10) or negative (n = 10). The matching normal breast tissue sample from thesame patient was used for normalization. **P < 0.01. (B) Northern blot of miR-17–5p and miR-20a in highly invasive (MDA-MB-231, Hs578T, and SKBR3) andweakly invasive (BT474, MCF7, MDA-MB-468, and T-47D) human breast cancer cell lines as indicated. tRNAs served as loading control. (C) Three-dimensionalcollagen gel invasion assays. MDA-MB-231 cells were assayed for cellular invasiveness, and shown by fluorescence. Cells were cultured with conditionedmedium from MCF7 cells either expressing a control vector (Ctrl CM) or transduced with the miR-17/20 cluster (miRNA CM). The relative distance of cellinvasion was quantitated. CM, conditioned medium. (D) Boyden chamber assays. MDA-MB-231 cells were incubated either with control or miR-17/20–conditioned medium from MCF7 cells. Migrated cells were stained with crystal violet, and counted for quantitative analysis. (E) Wound healing assays. Cellculture plates attached with MDA-MB-231 cells were wounded and incubated with control or miR-17/20–conditioned medium. The wound healing wasdetermined at the time points as indicated. Data are presented as mean ± SEM (n = 3). **P < 0.01.

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those spots on the array showing decreased level in the miR-17/20conditioned medium of both experiments were chosen to follow up.A bioinformatic screening analysis using miRNA TargetScan

and Gene Ontology annotation tools identified four cell-secretedfactors that are predicted targets of miR-17/20 and are known tobe involved in the regulation of cell migration (VEGFA, NTN1,IL-8, and LAMA3; Fig. 2D). Quantitative analyses by ELISAconfirmed that IL-8 and CXCL1 were reduced in miR-17/20–transduced breast cancer cell medium (Fig. 2 E and F).To determine whether the decreased abundance of those cel-

lular secreted factors in miR-17/20 conditioned medium governscell migration and invasion, add-back experiments were con-ducted using transwell migration (Fig. 3A) and 3D invasion assays(Fig. 3B). IL-8, CK8, or CXCL1 either partially or fully revertedthe anti-migratory and anti-invasive phenotypes of miR-17/20conditioned medium.

miR-17/20 Inhibits Plasminogen Activation. Tumor cell migration andinvasion is in part dependent upon plasminogen activity (12). CK8andα-ENObindandactivateplasminogen topromote cellmigrationand invasion (13, 14, 17). The reduction of CK8 and α-ENO abun-dance in the miR-17/20 conditioned medium led to a 40% to 50%reduction in plasminogen activity (Fig. 4A). As neither CK8 norα-ENO contains a miR-17/20 binding site within their 3′ UTRs, weconsidered the effect may be indirect, and examined the validatedtargets of miR-17/20. Cyclin D1 is one of the downstream targets ofmiR-17/20 in MCF7 cells via the 3′ UTR binding (8). Cyclin D1regulates expression of target genes through binding to target genepromoters in the context of local chromatin (18). To investigate themechanism by which miR-17/20 reduced CK8 abundance, we con-sidered the effect ofmiR-17/20onCK8maybe indirect via cyclinD1.MCF7 cells were treated with cyclin D1 siRNA, the conditionedmedium was collected, and functional assays were conducted. CK8abundance was reduced in the medium from cyclin D1 siRNAtreated MCF7 cells (Fig. 4B). α-ENO abundance did not showa change in cyclin D1 siRNA conditioned medium. Plasminogenactivity was reduced in cyclin D1 siRNA–treated cell-conditionedmedium (Fig. 4C), consistent with the reduction in CK8 abundancein themedium. These findings suggest the repression of cyclin D1 bymiR-17/20 (8) reduces CK8 level in the conditioned medium, whichresults in the reduction of plasminogen activation.Cyclin D1 and CK8 abundance were determined in breast cancer

tumor samples as well as matched normal tissue from the same pa-tient, as shown in Fig. 4D. Compared with normal tissues, the cyclinD1 and CK8 expression was increased and correlated positively inthe breast tumor samples.To determine the relative importance of plasminogen activity on

cell migration, PAI-1 was added to the empty vector–transducedMCF7 cell–conditioned medium. PAI-1 inhibited MDA-MB-231cell migration as miR-17/20 conditioned medium did (Fig. S6).

miR-17/20 Inhibits IL-8 Through 3′ UTR Binding. IL-8 abundance wasdecreased in the miR17/20 conditioned medium. A highly con-served sequence complementary to the “seed sequence”ofmiR-17–5p and miR-20a was identified within the IL-8 3′ UTR (Fig. 5A).miR-17/20 repressed IL-8 luciferase reporter gene activity, andmutation of the miR-17/20 binding site of the IL-8 3′UTR reducedthe repression (Fig. 5 B and C).The “fibroblastic system” is a broadly distributed cell surface–

associated regulator of cellular migration and invasion. The accu-mulation of plasminogen and its activators uPA and tissue-typeplasminogen activator at the cell surface augments their catalyticactivities. Plasmin, generated from plasminogen, plays an importantrole in degradation of extracellular matrix, facilitating cell migrationand invasion (19). α-ENO and CK8 bind to plasminogen to regulateplasminogen activation. α-ENO is a glycolytic enzyme that is a key 2-phospho-D-glycerate hydrolase in the cytoplasm of prokaryotes andeukaryotes. Plasminogen binds α-ENO via its C-terminal lysine res-idue (20), enhancing its activation and protecting activated plasminagainst inhibition by α2-antiplasmin. α-ENO expression enhancesplasminogen activation and promotes mammalian cell migration.Clinical evidence suggests increased plasminogen activity correlateswith breast cancer invasiveness. The level of plasminogen activator,such as uPA, correlates with poor overall survival in breast cancer(12). In metastatic breast cancers, miR-17/20 expression is reduced.Reduced miR-17/20 expression increases α-ENO secretion andincreases IL-8 and cyclinD1 abundance. Increased cyclinD1 inducesCK8 secretion, which in turn activates plasminogen and promotescancer cell migration and invasion, as increased α-ENO does. Thus,miR-17/20 regulates the migration and invasion of neighboring cellsvia heterotypic secreted signals as shown in Fig. 5D.

Materials and MethodsHuman Breast Tumor Samples. Human breast cancer specimens and matchingnormal breast tissue samples (frozen tissues) were provided by Peter McCue

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Fig. 2. miR-17/20 decreased cellular secretion. (A) Proteomic analysis of themiR-17/20 or control conditioned medium from MCF7 cells. SDS/PAGE–MSanalysis of two bands that were differentially expressed in the miR-17/20–conditioned medium identified two proteins, Y1 and Y2. Y1 corresponds toα-ENO, and Y2 corresponds to CK8. (B) Western blot analysis of control or miR-17/20–conditioned medium for proteins as indicated. The abundance of α-ENOand CK8 decreased in themiR-17/20–conditionedmedium. (C) Human cytokineantibody array analysis of the conditionedmedium from either control or miR-17/20–transduced MCF7 cells indicated altered abundance of cytokines CXCL1,IL-10, IL-8, and NT-4. (D) Bioinformatic analysis demonstrated an overlap be-tween predicted targets of miR-17/20 (n = 959) and known cellular secretedfactors implicated in cellular migration (IL-8, VEGFA, LAMA3, and NTN1). Theoverlap between the predicted targets of miR-17/20 and the cytokines secretedin the miR-17/20–conditioned medium identified IL-8 as a target. ELISAs forIL-8 (E) or CXCL1 (F) confirmed their decreased abundance in the miR-17/20–conditioned medium. Data are presented as mean ± SEM (n = 5).

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(Philadelphia, PA). All procedures were approved by the institutional reviewboard of Thomas Jefferson University.

Vectors and Oligonucleotides. pMSCVpuro vector was used to express miR-17/20 cluster as described previously (6, 8). WT and mutated IL-8 3′ UTRsequences were inserted into the XbaI–FseI site immediately downstream ofthe stop codon in the pGL3 control firefly luciferase reporter vector.

All DNA oligonucleotides were synthesized by Integrated DNA Technol-ogies. The primer sequences, siRNA sequences, and miRNA probe sequencesfor Northern blot are available upon request.

Cell Culture, Retrovirus Infection, siRNA/Plasmid Transfection, and LuciferaseReporter Assay. All cell types used in this study were cultured in DMEMcontaining penicillin and streptomycin (each 100 mg/L) and supplementedwith 10% FBS. Retroviral production and infectionmethodswere as described(21). The cellular transfection with plasmid DNA and luciferase reporter as-say were described earlier (8).

miR-17/20–Conditioned Medium Preparation. MCF-7 cells were transduced withthe pMSCVpuro vector expressingmiR-17/20 cluster or vector control as describedpreviously (6, 8). The same number of miR-17/20–overexpressing MCF7 cells andcontrol cells were plated at equal seeding densities. After attachment to theplates, cells were washed twice with PBS solution and overlaid with phenol red–free DMEM without serum and allowed to grow for another 24 h before col-lecting the medium (supernatant) from the cultures. The collected medium wascentrifuged at 2,000 × g for 10 min and filtered through a 0.2-μmmembrane toremove cellular debris and intact cells to yield conditioned medium.

MALDI MS.MALDI mass spectra were recorded with a PerSeptive Voyager-DESTRMALDI time-of-flightmass spectrometer operated in the reflectionmode.The measured peptide masses were used for database searching with Pro-Found algorithm.

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Fig. 4. miR-17/20 suppresses CK8 secretion and thereby plasminogen ac-tivity through cyclin D1. (A) Plasminogen activity in control and miR-17/20–conditioned MCF7 cell medium. Data are mean ± SEM (n = 4), **P < 0.01.CM, conditioned medium; Pg, plasminogen; PA, plasminogen activator; Pm,plasmin; VLK-pNA, Val-Leu-Lys-4-nitroanilide; NA, nitrophenolate anion. (B)Western blot of the cyclin D1 siRNA conditioned medium and cell lysatesdemonstrated reduction of cyclin D1 in MCF7 cells and decrease of CK8 se-cretion. Cyclin D1 siRNA did not affect α-ENO secretion. (C) Plasminogenactivation was assessed in the conditioned medium from cyclin D1–deficientor control cells. Data are mean ± SEM (n = 3). **P < 0.01. (D) Western blotshowed the cyclin D1 and CK8 abundance in breast cancer tumor samplesand matching normal tissues. T, tumor sample; N, normal tissue sample.

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Cellular Transwell Migration Assay. Cellular transwell migration assay wasperformed as described previously (22).

Cellular 3D Invasion Assay. The3DinvasionassaywasadaptedfromVialetal. (23).

Wound Healing Assay. MDA-MB231 cells were counted and plated in equalnumbers in12-well tissuecultureplatestoachieve90%confluence.Thereafter,a vertical wound was created using a 0.1-μL pipette tip. The wounded wellswere covered with either miR-17/20–conditioned medium or control mediumfromMCF7 cells. Imageswere collectedwithaCCDcameraatplanned intervalsand were digitized and stored using Metamorph 3.5 software (24). Imageswere captured at designated times to assess wound closure.

Cytokine Array Analysis. Human cytokine antibody arrays were obtained fromRaybiotech. The hybridizationwas performed following the instructions fromthe user manual. The intensities of signals were quantified using AlphaIm-ager software.

ELISA. The human IL-8 ELISA kit and human CXCL1/GROα ELISA kit werepurchased from R&D Systems. The reagent preparation and assay procedurefollowed the manufacturer’s instructions.

Plasminogen Activation Assay. To assay the plasminogen activator activity inconditionedmedium,abiochemical reactionwasperformed in96-well plateat37 °C for 1 to 2 h followed by absorbance readings at 405 and 570 nm. Thereaction includes 150 μL of 20 mM Hepes buffer (pH 7.4), 5 μL of 10 mM Val-Leu-Lys-4-nitroanilide (Sigma), 0.1Uof humanplasminogen (MPBiomedicals),and 20 μL of conditioned medium. The absorbance value for each well wascorrected by subtracting the reading at 570 nm from the reading at 405 nm.

miRNA Northern Blot Analysis. Northern blot analysis of miRNAs was per-formed as previously described (8).

miRNA Quantitative Real-Time PCR Analysis. miRNA specific primer sets werepurchased from Ambion. SYBR Green Master Mix was from Applied Bio-systems. The 7900 HT Sequence Detection system (Applied Biosystems) wasused for PCR. 5s rRNA was used for normalization.

Western Blot Analysis. Cell-conditioned medium was concentrated using a 3KMWCO Amicon centrifugal filter (Millipore). The following antibodies wereused for Western blotting: anti–cyclin D1 was from Neomarker; and anti-CK8(sc-8020), anti-α-enolase (sc-100812), and anti–β-tubulin (sc-9104) were fromSanta Cruz Biotechnology.

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Fig. 5. miR-17/20 inhibits IL-8 expression through 3′UTR interaction. (A) Schematic representation of thehuman IL-8 3′ UTR showing the highly conserved miR-17/20 binding site (in italics and boxed) between spe-cies. The “seed” sequence of miR-17/20 (nt 2–10) is in-dicated in blue. (B) The pGL3 reporter vectors carryingthe wide type or mutated IL-8 3′ UTR are indicated. (C)Luciferase reporter assay showed the suppression ofluciferase activity by IL-8 3′ UTR. In the miR-17/20–overexpressing MCF7 cells, the luciferase activity wassuppressed more than that in control cells. But for themutated construct in which the miR-17/20 binding sitewas mutated, the luciferase activity was significantlyrescued compared with the WT construct in the miR-17/20 transduced cells. Values are equal to mean ± SEM (n= 6). **P < 0.01. (D) Schematic representation of thehypothetical molecular mechanisms by which miR-17/20regulates breast cancer cellular migration and invasion.Pg, plasminogen; PA, plasminogen activator; IL-8R, IL-8receptor; CXCR2, CXCL1 receptor.

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Statistical Analysis. Data are presented as mean ± SEM. Student t test wasused for analysis, and P values <0.05 were considered significant.

ACKNOWLEDGMENTS. We thank Dr. Davidson for discussion, and Atenssa L.Cheek for the preparation of this manuscript. This work was supported inpart by National Institutes of Health (NIH) Grants R01CA70896, R01CA75503,

R01CA107382 (to R.G.P.), and R01CA120876 (to M.P.L). The Kimmel CancerCenter was supported by the NIH Cancer Center Core Grant P30CA56036 (toR.G.P.). This project is funded in part from the Dr. Ralph and Marian C. FalkMedical Research Trust and a grant from Pennsylvania Department of Health(R.G.P.). The Department specifically disclaims responsibility for an analysis,interpretations or conclusions.

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