miRNA-520f Reverses Epithelial-to-Mesenchymal Transition ... · miRNA. miR-520f expression was...

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Molecular and Cellular Pathobiology miRNA-520f Reverses Epithelial-to-Mesenchymal Transition by Targeting ADAM9 and TGFBR2 Jasmijn G.M. van Kampen 1 , Onno van Hooij 1 , Cornelius F. Jansen 1 , Frank P. Smit 2 , Paula I. van Noort 3 , Iman Schultz 3 , Roel Q.J. Schaapveld 3 , Jack A. Schalken 1 , and Gerald W. Verhaegh 1 Abstract Reversing epithelial-to-mesenchymal transition (EMT) in cancer cells has been widely considered as an approach to combat cancer progression and therapeutic resistance, but a limited number of broadly comprehensive investigations of miRNAs involved in this process have been conducted. In this study, we screened a library of 1120 miRNA for their ability to transcriptionally activate the E-cadherin gene CDH1 in a pro- moter reporter assay as a measure of EMT reversal. By this approach, we dened miR-520f as a novel EMT-reversing miRNA. miR-520f expression was sufcient to restore endog- enous levels of E-cadherin in cancer cell lines exhibiting strong or intermediate mesenchymal phenotypes. In parallel, miR- 520f inhibited invasive behavior in multiple cancer cell systems and reduced metastasis in an experimental mouse model of lung metastasis. Mechanistically, miR-520f inhibited tumor cell invasion by directly targeting ADAM9, the TGFb receptor TGFBR2 and the EMT inducers ZEB1, ZEB2, and the snail transcriptional repressor SNAI2, each crucial factors in medi- ating EMT. Collectively, our results show that miR-520f exerts anti-invasive and antimetastatic effects in vitro and in vivo, warranting further study in clinical settings. Cancer Res; 77(8); 200817. Ó2017 AACR. Introduction Cancer is a leading cause of death across the world, with the most common type of cancer being carcinoma, which originates from epithelial tissues. Despite major advances in our under- standing of the molecular and genetic basis of cancer, metastasis still causes 90% of all cancer-related deaths, and remains one of most complex and challenging problems of contemporary oncol- ogy. Thus, there is an urgent need for the development of new therapeutic approaches to treat and prevent metastatic disease. A potential strategy to achieve this goal is to target epithelial-to- mesenchymal transition (EMT). EMT plays a key role in tumor cell invasion and metastasis (1). Within the normal epithelium, cells interact with neighboring cells and the basement membrane to provide tissue integrity, a paracellular barrier, and cell polarity. During carcinoma progres- sion, cells lose their epithelial characteristics and gain a more mesenchymal phenotype. This so-called EMT is associated with the ability of cells to migrate and invade into the surrounding tissue. These processes are the rst steps toward metastatic spread (2). In addition, cells that have undergone EMT share many characteristics with stem cells, such as increased drug resistance, complicating systemic therapy for metastatic disease (3, 4). EMT in tumor cells is the result of transcriptional reprogram- ming in the cell (5). In particular, transcriptional repression of CDH1 (encoding the cell-cell adhesion protein E-cadherin) has been shown to trigger EMT. Several transcription factors such as ZEB1, ZEB2, SNAI1, and SNAI2 are known to repress CDH1. EMT is a reversible process, and silencing the CDH1 repressors results in mesenchymal-to-epithelial transition (MET), restoring the non- invasive epithelial phenotype (69). Reversing EMT may, there- fore, be an effective approach to combat advanced tumors in various types of cancer. miRNA are small noncoding RNA molecules that posttran- scriptionally regulate gene expression by controlling the transla- tion and stability of mRNAs (10). Individual miRNAs can regulate up to hundreds of mRNAs and a single mRNA may be targeted by several miRNAs (11). Although initially discovered for their role in differentiation, they have now been shown to play critical roles in a wide range of cellular processes, such as proliferation and apoptosis (12). Furthermore, deregulation of miRNAs plays an important role in cancer (13). Over expression of oncogenic miRNAs leads to repression of tumor-suppressor genes, and the loss of tumor-suppressive miRNAs enhances the expression of oncogenes. Because miRNAs function as master regulators of gene expression, miRNA-based therapy is an attractive approach for cancer therapy. Recently, it was found that downregulation of miR-200 family members (including miR-141 and miR-200c) results in EMT (14). Ectopic expression of these miRNAs in mesenchymal tumor cells restored their epithelial phenotype. This transition was charac- terized by reactivation of E-cadherin expression, restoration of cellcell adhesion, inhibition of tumor cell invasion, and 1 Department of Urology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, the Netherlands. 2 MDxHealth B.V., Nijme- gen, the Netherlands. 3 InteRNA Technologies B.V., Utrecht, the Netherlands. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: G.W. Verhaegh, Radboud University Medical Center, Geert Grooteplein-Zuid 28, PO Box 9101, 6500 HB Nijmegen, the Netherlands. Phone: 31-24-3610510; Fax: 31-24-3635121; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-16-2609 Ó2017 American Association for Cancer Research. 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Transcript of miRNA-520f Reverses Epithelial-to-Mesenchymal Transition ... · miRNA. miR-520f expression was...

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Molecular and Cellular Pathobiology

miRNA-520f Reverses Epithelial-to-MesenchymalTransition by Targeting ADAM9 and TGFBR2Jasmijn G.M. van Kampen1, Onno van Hooij1, Cornelius F. Jansen1, Frank P. Smit2,Paula I. van Noort3, Iman Schultz3, Roel Q.J. Schaapveld3, Jack A. Schalken1, andGerald W.Verhaegh1

Abstract

Reversing epithelial-to-mesenchymal transition (EMT) incancer cells has been widely considered as an approach tocombat cancer progression and therapeutic resistance, but alimited number of broadly comprehensive investigations ofmiRNAs involved in this process have been conducted. In thisstudy, we screened a library of 1120 miRNA for their ability totranscriptionally activate the E-cadherin gene CDH1 in a pro-moter reporter assay as a measure of EMT reversal. By thisapproach, we defined miR-520f as a novel EMT-reversingmiRNA. miR-520f expression was sufficient to restore endog-enous levels of E-cadherin in cancer cell lines exhibiting strong

or intermediate mesenchymal phenotypes. In parallel, miR-520f inhibited invasive behavior in multiple cancer cell systemsand reduced metastasis in an experimental mouse model oflung metastasis. Mechanistically, miR-520f inhibited tumor cellinvasion by directly targeting ADAM9, the TGFb receptorTGFBR2 and the EMT inducers ZEB1, ZEB2, and the snailtranscriptional repressor SNAI2, each crucial factors in medi-ating EMT. Collectively, our results show that miR-520f exertsanti-invasive and antimetastatic effects in vitro and in vivo,warranting further study in clinical settings. Cancer Res; 77(8);2008–17. �2017 AACR.

IntroductionCancer is a leading cause of death across the world, with the

most common type of cancer being carcinoma, which originatesfrom epithelial tissues. Despite major advances in our under-standing of the molecular and genetic basis of cancer, metastasisstill causes 90% of all cancer-related deaths, and remains one ofmost complex and challenging problems of contemporary oncol-ogy. Thus, there is an urgent need for the development of newtherapeutic approaches to treat and prevent metastatic disease. Apotential strategy to achieve this goal is to target epithelial-to-mesenchymal transition (EMT).

EMT plays a key role in tumor cell invasion and metastasis (1).Within the normal epithelium, cells interact with neighboringcells and the basement membrane to provide tissue integrity, aparacellular barrier, and cell polarity. During carcinoma progres-sion, cells lose their epithelial characteristics and gain a moremesenchymal phenotype. This so-called EMT is associated withthe ability of cells to migrate and invade into the surroundingtissue. These processes are the first steps toward metastatic spread

(2). In addition, cells that have undergone EMT share manycharacteristics with stem cells, such as increased drug resistance,complicating systemic therapy for metastatic disease (3, 4).

EMT in tumor cells is the result of transcriptional reprogram-ming in the cell (5). In particular, transcriptional repression ofCDH1 (encoding the cell-cell adhesion protein E-cadherin) hasbeen shown to trigger EMT. Several transcription factors such asZEB1, ZEB2, SNAI1, and SNAI2 are known to repress CDH1. EMTis a reversible process, and silencing theCDH1 repressors results inmesenchymal-to-epithelial transition (MET), restoring the non-invasive epithelial phenotype (6–9). Reversing EMT may, there-fore, be an effective approach to combat advanced tumors invarious types of cancer.

miRNA are small noncoding RNA molecules that posttran-scriptionally regulate gene expression by controlling the transla-tion and stability ofmRNAs (10). IndividualmiRNAs can regulateup to hundreds of mRNAs and a single mRNAmay be targeted byseveral miRNAs (11). Although initially discovered for their rolein differentiation, they have now been shown to play critical rolesin a wide range of cellular processes, such as proliferation andapoptosis (12). Furthermore, deregulation of miRNAs plays animportant role in cancer (13). Over expression of oncogenicmiRNAs leads to repression of tumor-suppressor genes, and theloss of tumor-suppressive miRNAs enhances the expression ofoncogenes. BecausemiRNAs function asmaster regulators of geneexpression, miRNA-based therapy is an attractive approach forcancer therapy.

Recently, it was found that downregulation of miR-200 familymembers (includingmiR-141 andmiR-200c) results in EMT (14).Ectopic expression of these miRNAs in mesenchymal tumor cellsrestored their epithelial phenotype. This transition was charac-terized by reactivation of E-cadherin expression, restorationof cell–cell adhesion, inhibition of tumor cell invasion, and

1Department of Urology, Radboud Institute forMolecular Life Sciences, Radbouduniversity medical center, Nijmegen, the Netherlands. 2MDxHealth B.V., Nijme-gen, the Netherlands. 3InteRNA Technologies B.V., Utrecht, the Netherlands.

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

Corresponding Author: G.W. Verhaegh, Radboud University Medical Center,Geert Grooteplein-Zuid 28, PO Box 9101, 6500 HB Nijmegen, the Netherlands.Phone: 31-24-3610510; Fax: 31-24-3635121; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-16-2609

�2017 American Association for Cancer Research.

CancerResearch

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increased sensitivity to chemotherapeutic agents. Transcriptionfactors ZEB1 and ZEB2 were identified as the major targets ofEMT-regulatingmiR-200 familymembers. The other repressors ofCDH1, however, were not targeted by themiRNAs of themiR-200family. Identification of novel miRNAs that target the whole or abroader repertoire ofCDH1 repressors andother EMT regulators iscritical to formulating a universal miRNA-based anti-EMT or anti-metastatic therapy.

In this study, we aimed to identify novel miRNAs that are ableto reverse EMT and inhibit metastasis. Because repression ofCDH1 is a key molecular event in EMT, we screened miRNAs fortheir ability to re-activate CDH1 transcription. Positive miRNAswere further validated by analyzing their effect on tumor cellinvasion and metastasis. In addition, we identified potentialtarget genes of themost promisingmiRNA,miR-520f, to elucidatethe mechanism of action.

Materials and MethodsCell culture

The PANC-1 cell line was purchased from the ATCC and T24cells were received from the University of Colorado HealthSciences Center (Department of Pathology) in 1998. Both celllines were authenticated in 2016 using the PowerPlex 21 system(Promega) by Eurofins Genomics (Germany). The tumorcell lines and culture conditions are described in the Supplemen-tary Materials and Methods and in Supplementary Table S1. Celllines were frequently tested for Mycoplasma infection, using aMycoplasma-specific PCR, and cells were propagated for no morethan 6 months or 30 passages after resuscitation from stocks.

Generation of the lentiviral library encoding miRNAsHuman miRNAs were selected from both the public miRNA

repository (www.mirbase.org) and proprietary small RNA deep-sequencing data (15). ThemiRNA sequences were amplified fromhuman genomic DNA, with the amplicons containing the full-length pre-miRNA hairpin and a flanking sequence on both sides.The primers for the amplicons were complemented with a 50

GCGC overhang and a restriction site for directional cloning intothe pCDH-CMV-MCS-EF1-puro vector (SBI, System Biosciences).Correct cloning was verified by DNA sequence analysis (seeSupplementary Materials and Methods and SupplementaryTable S2). VSV-G pseudotyped lentiviral particles were packaged,amplified, and provided by SBI (SBI, System Biosciences). Viralparticles were stored at �80�C.

Screening MET-inducing miRNAsT24-pEcad-luc/Rluc (Supplementary Table S1) cells were

infected with lentivirus (1.0 mL of undiluted lentiviral particles,average multiplicity of infection ¼ 64) in complete mediumcontaining 2 mg/mL polybrene (Sigma) in a 96-well format, andselected with 1 mg/mL puromycin (Sigma). Six days after infec-tion, firefly and Renilla luciferase activities were measured usingthe dual-luciferase reporter assay system (Promega), on a Victor3multilabel counter (PerkinElmer). The Fluc/Rluc ratiowas used asa measure for reporter activation.

Creation of doxycycline-inducible miRNA expression systemsThe miR-520f precursor sequence was amplified using

DNA from pCDH-miR-520f infected T24 cells as a template andpCDH-specific primers (Supplementary Table S3). The miR-520f

precursor was cloned into the NheI/EagI site of the pmRi-ZsGreen1–inducible Vector (Clontech, Supplementary Fig.S1A). T24 cells stably transfected with the pTet-On AdvancedVector (Clontech, Supplementary Fig. S1A) were obtained byclonal selection in G418-containing medium (600 mg/mL,Gibco). Inducible reverse tetracyclin-responsive transcriptionalactivator (rtTA) expression was confirmed by a luciferase reporterassay in T24-Tet-On clones that were transiently transfectedwith the pTRE-Tight-luc vector (Supplementary Fig. S1B). A stableT24-Tet-On clone was then transfected with the pmRi-ZsGreen1-miR-520f vector, and clonally selected in medium containingpuromycin (1 mg/mL, Sigma). Transfected clones displayed doxy-cycline-inducible miRNA expression at 1 mg/mL doxycycline(Supplementary Fig. S1CandS1D). Transfectionswere performedusing X-tremeGENE 9 transfection reagent, according to themanufacturer's instructions (Roche).

Transfection of miRNA mimics and siRNAsmiRNA mimics and siRNAs (Supplementary Table S4) were

transfected using Lipofectamine RNAiMAX Reagent (Invitrogen)according to the manufacturer's protocol.

Total RNA isolationTotal RNA was isolated using TRIzol, according to the manu-

facturer's instructions (Invitrogen).Concentration andpurity of theRNA was determined on a Nanodrop-1000 spectrophotometer(Thermo Scientific). For microarray analysis, RNA was furtherpurified using an RNeasy micro kit (QIAGEN). RNA integrity wasdeterminedon anAgilent Bioanalyzer 2100 (Agilent technologies).

Real-time and stem loop RT-PCRFor gene expression analysis, 2 mg DNase-I–treated total RNA

was used to generate cDNA, using random hexamer primers(Roche) and SuperScript II Reverse Transcriptase (Invitrogen).For miRNA analysis, a stem loop (SL) RT-PCR was performed; forthis, 100 ng total RNA was reverse transcribed using 0.375 pmolmiR–specific SL-RT primer. Real-time-PCR analysis was per-formed using LightCycler 480 SYBR Green I Master mix (Roche).RNA not subjected to reverse transcriptase was used as a controlfor nonspecific PCR amplification. All primers and amplificationconditions are listed in Supplementary Table S5. (SL) RT-PCRwasperformed on a LightCycler LC480 instrument (Roche). Expres-sion levels of B2M andGAPDH (mRNA) or RNU6-1 (U6; miRNA)were used for normalization. Relative gene expression levels werecalculated according to the mathematical model for relativequantification in real-time PCR described by Pfaffl (16).

Western blot analysisTo assess protein levels of E-cadherin and ADAM9, we per-

formed SDS-PAGE andWestern blot analysis on T24 and PANC-1protein extracts. A more detailed description can be found in theSupplementary Materials and Methods. Mouse anti–E-cadherin(HECD-1, Takara) and rabbit anti-ADAM9 (#2099, Cell SignalingTechnologies) were used to detect E-cadherin and ADAM9 respec-tively. Mouse anti–b-actin (AC-15, Sigma) staining was used fornormalization.

Cell invasion assayInvasion assays were performed using BioCoat Matrigel

Invasion Chambers (Corning), according to the manufacturer'sinstructions. Before the invasion assay, T24-imiR-520f cells were

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incubated in thepresence of doxycycline (1mg/mL) for 2days, andtransduced cells were puromycin-selected and passaged one time.MiRNA mimic or siRNA transfected cells were collected by tryp-sinization 72 hours after transfection.We seeded 40,000 cells intothe invasion chambers in serum-free medium. The invasionchambers were placed in a 24-well plate containing 500 mLmedium with 10% FCS as a chemo-attractant. As a control,40,000 cells were seeded in a 24-well plate. After 48 hours ofincubation, cells in the inner compartment of the invasion cham-ber were removed by aspiration and cleaning with cotton swabs.Invasive cells were quantified by incubating the bottom of theinvasion chamber in CellTiter-Glo (CTG) reagent (Promega), andthen analyzing luminescence on a Victor3 multilabel counter(PerkinElmer). The percentage of invasive cells was calculated asthe CTG activity of the invasive cells, normalized for the CTGactivity of the input control (i.e., cells grown in a 24-well plate).

Experimental metastasis assayAnimal experiments were approved by the ethical committee

on animal research of the Radboud university medical center(Nijmegen, NL). For this experiment, we used 24 female, 6- to8-week-old NOD-SCID immunodeficient mice (Charles Riverlaboratories) that were divided into four groups of 6 mice.Groups 1 and 2 were given doxycycline-containing (0.2 mg/mL) drinking water during the entire experiment, whereasgroups 3 and 4 received doxycycline-free water. Groups 1 and3 were injected with 0.8 � 106 (200 mL volume) T24-imiR-520fcells and groups 2 and 4 with 0.8 � 106 (200 mL volume) T24-Tet-On cells, all via the lateral tail vein (17). Mice were mon-itored daily, and when the first mice started to suffer fromserious tumor burden, all animals were sacrificed. The numberand size of metastases were determined macroscopically (i.e.,by visual examination through a dissection microscope) andmicroscopically on hematoxylin and eosin (H&E)–stained sec-tions (4-mm) of Tissue-Tek–embedded, frozen mouse lungs.Lung tumor burden in the H&E-stained sections was deter-mined by scanning the sections using a Pannoramic 250 FlashII digital slide scanner and analyzing the percentage of tumorarea using Pannoramic viewer (3DHISTECH). Twenty 20-mmsections from Tissue-Tek–embedded frozen mouse lungs wereused for TRIzol RNA isolation.

GeneChip Human Exon ST arrayGene expression was examined by Affymetrix GeneChip

Human Exon 1.0 ST arrays (Affymetrix). RNA was processedusing the GeneChip WT PLUS Reagent Kit (Affymetrix). Thearrays were further processed using the GeneChip Hybridiza-tion, Wash, and Stain Kit (Affymetrix). All kits were usedaccording to the manufacturer's protocols. GeneChips werescanned with a GeneChip Scanner (Affymetrix), generating CELfiles for each array. Gene-expression values were derived fromthe CEL file using the model-based Robust Multiarray Average(RMA) algorithm as implemented in Partek software (PartekGenomics Suite 6.6). RMA performs normalization, back-ground correction and data summarization. Differentiallyexpressed genes between conditions were calculated usingANOVA. The data discussed in this publication have beendeposited in NCBI's Gene Expression Omnibus (18) and areaccessible through GEO Series accession number GSE92988(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE92988).

30-UTR reporter assaysThe ADAM9 and TGFBR2 30-UTR (untranslated region)

regions and mutant variants were cloned into the psiCHECK-2 reporter vector (Promega). Mutant variants of the ADAM930-UTR were created using SOEing PCR (Supplementary Fig. S2;Supplementary Table S6). A more detailed description can befound in the Supplementary Data. For the 30-UTR reporterassays, 5,000 T24 cells per well were seeded in a 96-well plate.One day after seeding, cells were transfected with 100 ngpsiCHECK-2-30-UTR reporter DNA (using X-tremeGENE 9 DNAtransfection reagent; Roche), and 20 nmol/L miRNA mimic.The luciferase activities were measured 48 hours posttransfec-tion using the dual-luciferase reporter assay system (Promega).firefly and Renilla luciferase signals were measured on a Victor3multilabel reader (PerkinElmer).

Statistical analysesThe data are presented as means � SEM from at least three

independent experiments. Two-tailed t testswere performedusingGraphPad Prism (GraphPad Software, Inc.). Clinical data,ADAM9 and TGFBR2 expression of muscle-invasive bladderurothelial carcinoma and pancreatic adenocarcinoma patientsincluded in The Cancer Genome Atlas (TCGA) database weredownloaded from cBioPortal (19). Kaplan–Meier analyses wereperformed using GraphPad Prism (Graphpad Software, Inc.). AP value of <0.05 was considered statistically significant.

ResultsIdentification of MET-inducing miRNAs

To identify miRNAs that can reverse EMT, we developed ascreening model to test their ability to induce transcriptionalactivation of CDH1. We cloned the core element of the CDH1promoter that is responsible for cell type–specific expression (20)in an expression vector todrivefirefly luciferase expressionwith anHSV-Tk promoter–driven Renilla luciferase cassette as an internalcontrol (Fig. 1A; Supplementary Fig. S3A). This reporter constructwas stably transfected into the bladder cancer cell line T24 (T24-pEcad-luc/Rluc). T24 has a mesenchymal phenotype, as deter-mined by the CDH1/VIM ratio (Supplementary Fig. S3B), whichhas been shown to correlate well with EMT status (21). Because ofits low endogenousCDH1 expression, T24 is a suitablemodel cellline for studying re-activation of CDH1 and reversal of EMT.

We performed a functional screen, using a lentiviral-basedmiRNA expression library containing 1120 human miRNA pre-cursors in our model cell line T24-pEcad-luc/Rluc (Fig. 1A). ThemiR-200 family members miR-141 and miR-200c were consid-ered positive controls because they are known to re-activateCDH1expression by targeting ZEB1 and ZEB2. Re-activation ofCDH1 byeach miRNA was quantified by calculating the firefly luciferase/Renilla luciferase (FLuc/RLuc) ratio, where miRNAs with a z-score> 2.0 were considered positive. We identified 29 positive miRNAsin our screen, including miR-141 and miR-200c (Fig. 1B). ThesemiRNAs were further validated by studying their effects on theendogenous expression ofCDH1 in wild-type T24 cells. Of the 29positive miRNAs from the screen, 10 miRNAs also inducedendogenous CDH1 expression (Fig. 1C). MiR-520f showed thehighest induction of endogenousCDH1 expression (Fig. 1C). Theidentity of miR-520f was confirmed by Sanger DNA sequenceanalysis of the proviral DNA of transduced cells. MiR-520f isa primate-specific miRNA that lies in a miRNA cluster on

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chromosome 19 (C19MC). It is not expressed in most normal ormalignant human tissues, with the exception of testicular germcell tumors and some thymomas (data from TCGA and own data,not shown).

miR-520f reverses EMT and reduces tumor cell invasion in vitroTo confirm that miR-520f is able to reverse EMT, we analyzed

the expression of CDH1 and several EMT-related genes as well asE-cadherin protein levels in T24 cells transfected with miRNAmimics. Our results obtained with the miR-520f mimics con-firmed the upregulation of CDH1 that we found using lentiviralbased miR-520f expression (Fig. 2A). Western blot analysisshowed that protein levels of E-cadherin are also elevated bymiR-520f (Fig. 2B). Moreover, we were able to confirm theseresults in the pancreatic cancer cell line PANC-1 (Fig. 2A and B),which is characterized as an intermediate mesenchymal cell linebased on its CDH1/VIM ratio (Supplementary Fig. S3B). In bothcell lines, miR-520f mimic transfection markedly increased miR-520f levels (Supplementary Fig. S1E). In T24, upregulation ofCDH1 was accompanied by downregulation of SNAI2, while inPANC-1, it was accompanied by downregulation of ZEB1 andZEB2 (Fig. 2A). In T24, SNAI1 was upregulated (Fig. 2A), yetexpression of SNAI1 remains very low in T24 cells (data notshown). Furthermore, miR-520f significantly downregulated themesenchymalmarkers CDH2 and VIM in PANC-1, but not in T24(Fig. 2A).

To further show EMT reversal, we looked at the morphology ofour cell line models using phase-contrast microscopy. T24 cellstransfectedwithmiR-520f gained amore epithelial-likemorphol-ogy (Supplementary Fig. S1F). PANC-1 cells, which have a typicalcobblestone and epithelial-like morphology, maintained theirmorphology after transfection with miR-520f (SupplementaryFig. S1F). In addition, we observed an increased membranousE-cadherin staining in miR-520f transfected PANC-1 cells (Sup-plementary Fig. S4).

Because miR-520f is able to re-activate CDH1 and down reg-ulates several EMT-inducing factors, we hypothesized that miR-520f may reduce tumor cell invasion. To test this, we infected T24cells with the miR-520f-precursor-containing lentivirus and stud-ied their invasion capacity using Matrigel-coated cell invasionchambers. Tumor cell invasion was inhibited up to 65% by miR-520f (Fig. 2C). To validate these findings, we also tested theinvasion capacity of T24 cells with doxycycline-inducible miR-520f expression (T24-imiR-520f) and PANC-1 cells transfectedwith miR-520f mimics. MiR-520f inhibited invasion over 40% inthe induced T24-imiR-520f cells and about 55% in mimic trans-fected PANC-1 cells (Fig. 2D and E).

Identification of miR-520f target genesTo further study the role of miR-520f in EMT and tumor cell

invasion, we identified potential target genes of miR-520f bymicroarray analysis of miR-520f–transfected PANC-1 cells.Genes that were downregulated and contained miR-520f bind-ing sites, as assessed using in silico prediction tools, wereselected as candidate targets and validated by qPCR analysis.Using this approach, ADAM9 (encoding a disintegrin andmetalloprotease domain-containing protein 9) and TGFBR2(encoding Transforming growth factor beta receptor II) wereidentified as potential targets of miR-520f (Fig. 3A and B).Western blot analysis showed that ADAM9 was also down-regulated at the protein level (Fig. 3C).

To determine whether ADAM9 and TGFBR2 are direct targetsof miR-520f, we performed 30-UTR luciferase reporter assays.We used psiCHECK-2 reporter constructs containing either thewild-type or mutated ADAM9 30-UTR or TGFBR2 30-UTR frag-ments (Supplementary Fig. S5A). The mutants contained sev-eral nucleotide substitutions in the predicted miR-520f–bind-ing sites, which should disrupt miRNA binding (Fig. 3D). InT24 cells transfected with the ADAM9 30-UTR wild-type con-struct, which contains two miR-520f–binding sites, luciferase

Figure 1.

Screening for EMT-reversing miRNAs.A, Schematic representation of theCDH1 promoter cloned in the reporterconstruct and transduction of T24-pEcad-luc/Rluc cells with the lentiviral-based miRNA expression library. B,Scatter plot of z-scores correspondingto the firefly/Renilla luciferase ratiosin T24-pEcad-luc/Rluc cellstransduced with different miRNAlentiviral particles. MOI, multiplicity ofinfection. C, Endogenous expression ofCDH1 in T24-pEcad-luc/Rluc cellstransduced with different miRNAlentiviral particles, relative to emptyvector transduced cells.

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reporter activity was decreased up to 60% by miR-520f, com-pared with control mimic transfected cells (Fig. 3E). Regulationby miR-520f of the mutant ADAM9 30-UTR constructs, whereonly one of the two predicted miR-520f–binding sites wasdisrupted, was reduced compared to the wild-type construct(Fig. 3E). When both binding sites in the ADAM9 30-UTR weredisrupted, the effect of miR-520f was completely abolished(Fig. 3E). In the TGFBR2 30-UTR wild-type construct, containingone miR-520f-binding site, miR-520f decreased luciferasereporter activity by 35%, compared with control mimic trans-fected cells (Fig. 3F). In the mutant construct, the effect of miR-520f was completely abrogated (Fig. 3F).

To assess the role of ADAM9 and TGFBR2 in tumor cellinvasion inhibition mediated by miR-520f, we transfectedPANC-1 cells with siRNAs targeting ADAM9 and TGFBR2.Western blot and qPCR analyses showed that ADAM9 wassuccessfully silenced by both siRNAs (Fig. 4A). Interestingly,tumor cell invasion of cells in which ADAM9 was silenced,was reduced (Fig. 4B). Similarly, knockdown of TGFBR2 usingsiRNAs also resulted in a reduction of tumor cell invasion(Fig. 4A and B). To assess the clinical relevance of ADAM9 andTGFBR2 targeting by miR-520f, we used TCGA data to exam-ine the overall survival of muscle-invasive bladder urothelialcarcinoma and pancreatic adenocarcinoma patients with highor low ADAM9 expression. High or low ADAM9 expressionwas defined as the top quartile or lower quartile, respectively,of the ADAM9 expression levels. High ADAM9 expression wasassociated with a significantly worse overall survival in both

cancer types (Fig. 4C). Furthermore, high TGFBR2 expression(defined as top 10% vs. lower 10% in TCGA gene-expressiondatasets) was associated with a significantly worse overallsurvival of pancreatic cancer, but not of bladder cancerpatients (Fig. 4D).

miR-520f inhibits metastasis formation in vivoWe next performed a pilot study to test the antimetastatic

activity of miR-520f in vivo, by injecting tumor cells with orwithout miR-520f expression in the tail vein of NOD-SCIDmice and monitoring the formation of lung metastases (Fig.5A). For this purpose, we used T24 cells with doxycycline-inducible miR-520f expression (see materials and methods). Tocontrol not only for the effect of miR-520f, but also for Tet-onactivity, we used T24 cells transfected with only the pTet-OnAdvanced Vector (T24-Tet-On) next to T24 cells transfectedwith both the pTet-On Advanced Vector and the pmRi-ZsGreen1-miR-520f vector (T24-imiR-520f). For both cell lines,we included a group that received doxycycline treatment andone that did not.

After RNA analysis, we found that tumors in mice that wereinjected with cells containing the pmRi-ZsGreen1-miR-520fvector, but that did not receive doxycycline, showed expressionof miR-520f, most likely due to leaky promoter activity (Sup-plementary Fig. S6A). Furthermore, it was found that doxycy-cline reduced metastasis formation (Supplementary Fig. S6B).Therefore, to determine the effect of miR-520f on tumormetastasis, we compared animals injected with T24-Tet-On

Figure 2.

miR-520f reverses the EMT phenotype. A, RT-qPCR analysis of EMT marker expression in T24 and PANC-1 cells transfected with 20 nmol/L miR-520f mimiccompared with control mimic transfected cells (N ¼ 3). B, Western blot analysis of E-cadherin levels in T24 and PANC-1 cells transfected with 20 nmol/LmiR-520f mimic compared with nontransfected (NT) and control mimic (NC1)–transfected cells. C, Invasion of T24 cells infected with lentivirus encodingthe miR-520f precursor compared with empty-vector (EV) infected cells (multiplicity of infection ¼ 30, N ¼ 3). D, Invasion of T24-imiR-520f cells stimulatedwith doxycycline (þDox) compared with nonstimulated (�Dox) cells (N ¼ 4). E, Invasion of PANC-1 cells transfected with 20 nmol/L miR-520f mimic comparedwith control mimic transfected cells (N ¼ 4); � , P < 0.05; ��, P < 0.01; ���, P < 0.001.

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cells that received no doxycycline (as a negative control), withanimals injected with T24-imiR-520f cells that also received nodoxycycline. The body weight of the animals in all groups wascomparable during the study (Supplementary Fig. S6C). Thetotal tumor area, as assessed on H&E sections, was reduced inthe animals injected with T24-imiR-520f cells (Fig. 5B and C).Gene expression analysis in tumor foci confirmed the highexpression levels of miR-520f in the T24-imiR-520f cells (Fig.5D). Furthermore, the miR-520f–positive tumors displayed atrend toward upregulation of CDH1 expression (Fig. 5E), asignificant downregulation of SNAI2 (Fig. 5F), and a trendtoward downregulation of ADAM9 expression (Fig. 5G).

DiscussionDespite continuous efforts in developing new therapeutic

strategies, metastatic disease remains the leading cause of deathfrom cancer. As EMT plays a fundamental role in metastasis,reversing this process may be a promising approach to targetmetastatic tumor cells. In this study, we identified miR-520f as anovel EMT reversing miRNA. We demonstrated that miR-520f isable to transcriptionally activate CDH1 expression in our screen-ing model. Furthermore, miR-520f elevated endogenous levels ofE-cadherin in cell lines with a strong and intermediate mesen-chymal phenotype.

Figure 3.

ADAM9 and TGFBR2 are direct targets of miR-520f. A, Microarray analysis showing genes >1.5-fold up- or downregulated in miR-520f mimic–transfectedPANC-1 cells compared with control mimic transfected cells. B,Gene-expression analysis ofADAM9 and TGFBR2 in T24 and PANC-1 cells transfected with 20 nmol/LmiR-520f mimic compared with control mimic-transfected cells (N ¼ 3). C, Western blot analysis of ADAM9 in T24 and PANC-1 cells transfected with 20 or5 nmol/L miR-520f mimic compared with nontransfected (NT) and control mimic (NC1) transfected cells. D, Sequences of the ADAM9 30-UTR (NM_003816.2)and TGFBR2 30-UTR (NM_001024847.2) constructs with miR-520f–binding sites and/or mutated miR-520f–binding sites. E, ADAM9 30-UTR reporter assay inT24 cells transfected with 100 ng wild-type or mutant vector and 20 nmol/L miR-520f or control mimics (N ¼ 3). F, TGFBR2 30-UTR reporter assay in T24 cellstransfected with 100 ng wild-type or mutant vector and 20 nmol/L miR-520f or control mimics (N ¼ 4); � , P < 0.05; �� , P < 0.01; ��� , P < 0.001.

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Because several transcription factors are known to repressCDH1, we hypothesized that miR-520f regulates these so calledEMT inducers, and studied their expression in our model celllines. Interestingly, miR-520f downregulated SNAI2 in T24cells, and ZEB1 and ZEB2 in PANC-1 cells. However, we assumethat these are not direct targets of miR-520f, as there are nopredicted binding sites of miR-520f in the 30-UTRs of SNAI2and ZEB1 (TargetScan.org 7.0). Moreover, downregulation ofthese repressors was mutually exclusive in our cell line models.This is in line with the fact that the EMT inducing transcriptionfactors are differentially expressed in different tumor types(22). Nevertheless, these results indicate that miR-520f canregulate different EMT inducers in multiple cell lines, and thatother targets of miR-520f are likely to regulate these repressors.Furthermore, we have shown that miR-520f inhibits tumor cellinvasion in multiple cell line models. Our data demonstratethat miR-520f reverses the EMT phenotype, which suggests thatmiR-520f has antimetastatic activity, making it an attractivetherapeutic drug for different cancers.

We identifiedADAM9 as a direct target ofmiR-520f. Our resultsshow that miR-520f can bind to at least two seed-complementarysites in the 30-UTR of ADAM9. Point mutations of several nucleo-tides in the miR-520f-binding sites were sufficient to abrogate theeffect ofmiR-520f, showing thatADAM9 is a direct target. Elevatedlevels of ADAM9 are observed in multiple cancers and have beencorrelated with cancer progression and metastases (23–27).

Furthermore, knockdown of ADAM9 has been shown to reducecellular migration and invasion (28–31). This indicates thatADAM9 plays an important role in the mechanism by whichmiR-520f inhibits invasion. Consistent with these data, we alsoshow that siRNA mediated knockdown of ADAM9 inhibitstumor cell invasion. Over expression of ADAM9 has beenshown to enhance growth factor-mediated disruption ofcell–cell contacts and internalization of E-cadherin (32). There-fore, targeting ADAM9 may inhibit tumor metastasis by regu-lating E-cadherin-mediated cell–cell adhesion and cellularmotility and invasion. This is supported by our finding thatsiRNA mediated knockdown of ADAM9 increased membra-nous E-cadherin staining (Supplementary Fig. S4). Further-more, our TCGA clinical data analysis shows that high ADAM9expression is associated with a significantly worse overall sur-vival in both bladder cancer and pancreatic cancer patients.

In addition to ADAM9, we also identified TGFBR2 as a directtarget of miR-520f. Our results show that miR-520f can bind to atleast one seed-complementary site in the 30-UTRof TGFBR2. Pointmutations of several nucleotides in the miR-520f–binding sitewere sufficient to abrogate the effect of miR-520f, showing thatTGFBR2 is also a direct target of miR-520f. TGFb signaling isknown to stimulate cancer progression through the induction ofEMT (33). TGFb receptor II plays an important role in the TGFbsignaling pathway and has been shown to drive cancer progres-sion (34, 35). As TGFb signaling is known to induce transcriptions

Figure 4.

ADAM9 and TGFBR2 are involved intumor cell invasion and overall survivalof cancer patients. A, Gene-expressionanalysis of ADAM9 and TGFBR2, andWestern blot analysis of ADAM9 inPANC-1 cells transfectedwith 20 nmol/LADAM9 or TGFBR2 siRNAs or siRNAcontrol (N ¼ 3); ���, P < 0.001. B,Invasionof PANC-1 cells transfectedwith20 nmol/L ADAM9 or TGFBR2 siRNAsor siRNA control (N ¼ 3); � , P < 0.05;��� ,P<0.001.C andD,Overall survival ofpancreatic adenocarcinoma andmuscle-invasive bladder cancer patientswith high and low expression of ADAM9(C) or TGFBR2 (D) mRNA (C,N¼ 88 andN ¼ 204, and D, N ¼ 52 and N ¼ 121,respectively).

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factors ZEB1, ZEB2, and SNAI2, miR-520f might downregulatethese transcription factors through targeting TGFBR2 (36, 37).Furthermore, knockdown of TGFBR2 has been shown to inhibittumor cell invasion (38, 39). This is in line with our resultsshowing that siRNA mediated knockdown of TGFBR2 inhibitstumor cell invasion. Furthermore, high TGFBR2 expression isassociated with a significantly worse overall survival in pancreaticcancer patients.

The antimetastatic potential ofmiR-520fwas further supportedby our in vivo results. In our mouse metastasis model, miR-520freduced lung metastasis formation. The effect on metastasis,however, was not significant due to the large variation in theformation of metastases in the control group and the small groupsizes. Expression analysis of RNA derived from the mouse lungtumors showed that miR-520f expression was raised significantlyin tumor cells carrying the miR-520f expression vector. Further-more, we observed a trend toward CDH1 upregulation andADAM9 downregulation, and significant SNAI2 downregulationin miR-520f–expressing tumor cells, consistent with our in vitrodata. This suggests that targeting EMTwas successful and a feasibleapproach to inhibit metastasis. In this model, we were unable todetermine the effect of miR-520f on intravasation, because thetumor cells were injected directly into the circulation. However,miR-520f might also be effective in restraining cells from intra-

vasation by targeting EMT. It would be interesting to assess theeffect of miR-520f in an orthotropic cancer model.

Although miR-520f has not been linked to EMT or invasionbefore, it has been described to play a role in drug resistance. Laiand colleagues (40) reported that in PANC-1 cells, miR-520fenhanced sensitivity to gemcitabine. Furthermore, Harvey andcolleagues (41) found that miR-520f increased the sensitivity of acisplatin resistant neuroblastoma cell line to etoposide and cis-platin. This drug-sensitizing effect of miR-520f might be throughinhibition of EMT. EMT has been shown to give rise to cancer cellswith a stemcell-like phenotype, including resistance to therapy (3,4). Interestingly, Josson and colleagues (42) described that knock-down of ADAM9 in a prostate cancer cell line induced sensitivityto doxorubicin, cisplatin, taxotere, gemcitabine, and VP-16. Thisindicates that miR-520f might also enhance drug sensitivity bytargeting ADAM9. Taken together, these studies emphasize thetherapeutic potential of miR-520f in advanced cancers, as it notonly inhibits tumor cell invasion, but it also re-sensitizes resistanttumor cells to chemotherapy.

In conclusion, our study showed for thefirst time thatmiR-520fis able to reverse EMT and inhibit metastasis, underlining thetherapeutic potential of this miRNA. The effect of miR-520f canbe, at least partially, explained by targeting ADAM9 and TGFBR2,but other factors are likely to play a role aswell (Fig. 6).Our results

Figure 5.

miR-520f reduces metastasis formation in vivo. A, Schematic representation of the experimental set-up. NOD-SCID mice were injected with T24 tumor cellsvia the tail vein, and formation of lung metastatic foci was analyzed. B, H&E–stained lung sections of mice injected with tumor cells that do or do not expressmiR-520f. Dark purple areas indicate tumor tissue; light purple regions indicate normal mouse lung tissue. C, Bar graph representing the percentage of tumortissue in mouse lung sections. D–G, Relative expression of miR-520f (D), CDH1 (E), SNAI2 (F) and ADAM9 (G) in miR-520f-expressing lung tumors comparedwith control tumors; � , P < 0.05.

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warrant further research to explore the therapeutic potential ofmiR-520f.

Disclosure of Potential Conflicts of InterestR.Q.J. Schaapveld has ownership interest (including patents) in InteRNA

Technologies BV. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: J.G.M. van Kampen, R.Q.J. Schaapveld, J.A. Schalken,G.W. VerhaeghDevelopment of methodology: J.G.M. van Kampen, P.I. van Noort, R.Q.J.Schaapveld, G.W. VerhaeghAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.G.M. van Kampen, O. van Hooij, F.P. Smit, P.I. vanNoort, G.W. VerhaeghAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.G.M. van Kampen, O. van Hooij, C.F. Jansen,F.P. Smit, P.I. van Noort, I. Schultz, R.Q.J. Schaapveld, G.W. VerhaeghWriting, review, and/or revisionof themanuscript: J.G.M. vanKampen,O. vanHooij, P.I. vanNoort, I. Schultz, R.Q.J. Schaapveld, J.A. Schalken,G.W. Verhaegh

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.G.M. van Kampen, O. van Hooij, C.F. JansenStudy supervision: P.I. van Noort, I. Schultz, R.Q.J. Schaapveld, J.A. Schalken,G.W. Verhaegh

AcknowledgmentsPart of the results shown here are based upon data generated by the TCGA

Research Network: http://cancergenome.nih.gov/. We are grateful to JeroenMooren and Debby Smits of the Central Animal Laboratory Nijmegen for theirhelp and expertise on the animal work. Furthermore, we would like to thankDiede van Bladel, Joost Hendriks, and Kirsten van Niekerk for their help withexperiments.

Grant SupportThis work was supported by InteRNA Technologies BV and by the Dutch

Technology Foundation STW (project number: 12439).The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 6, 2016; revised January 25, 2017; accepted January 25,2017; published OnlineFirst February 16, 2017.

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