MiR-126 Acts as a Tumor Suppressor in Pancreatic Cancer

Angiogenesis, Metastasis, and the Cellular Microenvironment

MiR-126 Acts as a Tumor Suppressor in Pancreatic CancerCells via the Regulation of ADAM9

Shin Hamada1, Kennichi Satoh1,2, Wataru Fujibuchi3, Morihisa Hirota1, Atsushi Kanno1, Jun Unno1,Atsushi Masamune1, Kazuhiro Kikuta1, Kiyoshi Kume1, and Tooru Shimosegawa1

AbstractThe epithelial-mesenchymal transition (EMT) is a critical step for pancreatic cancer cells as an entry ofmetastatic

disease. Wide variety of cytokines and signaling pathways are involved in this complex process while the entirepicture is still cryptic. Recently, miRNA was found to regulate cellular function including EMT by targetingmultiple mRNAs. We conducted comprehensive analysis of miRNA expression profiles in invasive ductaladenocarcinoma (IDA), intraductal papillary mucinous adenoma, intraductal papillary mucinous carcinoma, andhuman pancreatic cancer cell line to elucidate essential miRNAswhich regulate invasive growth of pancreatic cancercells. Along with higher expression of miR-21 which has been shown to be highly expressed in IDA, reducedexpression of miR-126 in IDA and pancreatic cancer cell line was detected. The miR-126 was found to targetADAM9 (disintegrin and metalloproteinase domain-containing protein 9) which is highly expressed in pancreaticcancer. The direct interaction between miR-126 and ADAM9 mRNA was confirmed by 30 untranslated regionassay. Reexpression of miR-126 and siRNA-based knockdown of ADAM9 in pancreatic cancer cells resulted inreduced cellular migration, invasion, and induction of epithelial marker E-cadherin. We showed for the first timethat the miR-126/ADAM9 axis plays essential role in the inhibition of invasive growth of pancreatic cancer cells.Mol Cancer Res; 10(1); 3–10. �2011 AACR.

Introduction

Early invasion to surrounding tissue or rapid dis-semination to distant organ is characteristic feature ofpancreatic cancer. Only 20% of patients are eligible forsurgical resection and the prognosis is unfavorable even inthose patients (1). Epithelial-mesenchymal transition(EMT) plays important role during invasion and metas-tasis which requires involvement of multiple cytokinesand signaling pathways (2). Targeting the EMT-inducingmachinery in pancreatic cancer cells could be a noveltherapeutic strategy.In addition to cytokines and signaling molecules, a novel

paradigm has been brought into the cancer research field.

Now it is recognized that noncoding RNA includingmiRNA regulates various biologic processes including EMT(3). miRNA is a small RNA consisting of 20 to 23 nucleotidewhich targets 30 untranslated region (UTR) sequence ofmRNA for destabilization (4). One miRNA is able to targetmultiplemRNAswhich indicates thatmiRNA could orches-trate gene regulation of biologic processes. The role ofmiRNA in cancer progression has been extensively studiedon the basis of these evidences.Several miRNAs are differentially expressed in cancer cells

compared with normal cells. Their expression status deter-mines the biologic behavior of cancer cells. The miR-21 ishighly expressed in pancreatic cancer cells than in the normalpancreatic ductal cells (5). Furthermore, transient overex-pression of miR-21 attenuates the antiproliferative effect ofgemcitabine. The miR-145 is downregulated in breastcancer tissue compared with normal tissue (6). Reexpressionof miR-145 in breast cancer cell lines results in decreasedcellular migration and invasion.In this study, we carried out comprehensive analysis

of miRNA expression profiles in invasive ductal adeno-carcinoma (IDA), intraductal papillary mucinous ade-noma (IPMA), intraductal papillary mucinous carcinoma(IPMC), and human pancreatic cancer cell line to elucidateindispensable miRNAs which are differentially expressed inIDA and pancreatic cancer cell line. The miR-126 wassignificantly downregulated commonly in IDA and pan-creatic cancer cell line and its direct interaction with novel

Authors' Affiliations: 1Division of Gastroenterology, Tohoku UniversityGraduateSchool ofMedicine; 2DivisionofCancer StemCell,MiyagiCancerCenter Research Institute, Miyagi; and 3Computational Biology ResearchCenter, National Institute of Advanced Industrial Science and Technology,Koto-ku, Tokyo, Japan

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

Corresponding Author: Kennichi Satoh, Division of Cancer Stem Cell,Miyagi Cancer Center Research Institute, 47-1 Nodayama, Shiote, Mede-shima, Natori city, Miyagi, 981-1293, Japan. Phone: 81-22-384-3151; Fax:81-22-381-1195; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-11-0272

�2011 American Association for Cancer Research.

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target gene ADAM9 was identified by 30-UTR assay. ThemiR-126/ADAM9 axis was confirmed to regulate cellularmigration and invasion of pancreatic cancer cells for thefirst time which would delineate novel therapeutic target ofadvanced pancreatic cancer.

Materials and Methods

Cell line and cell cultureThe human pancreatic cancer cell lines ASPC-1,MiaPaca-

2, BxPC3, Panc-1, and KLM-1 were obtained from Amer-ican Type Culture Collection or Cell Resource Center forBiomedical Research Institute of Development, Aging andCancer (Sendai, Japan) and maintained as described previ-ously (7, 8).

Tissue samples and microdissectionNormal pancreatic tissue samples were obtained from

patients who underwent surgical resection of organ withpancreatic tissue due to the non-pancreatic disease in ourinstitute. Tissue samples of IDA, IPMA, and IPMC wereobtained from patients who underwent surgical resection inour institute. Tissue samples of IDAwere also obtained frompatients who received fine-needle aspiration (FNA) biopsyfor diagnosis. Histologic diagnosis of each sample wasconfirmed by pathologists who were not informed aboutcurrent research. For microdissection, tissue samples wereimmediately embedded in Tissue-Tek O.C.T. compoundmedium (Sakura), frozen in liquid nitrogen, and stored at�80�C. The frozen section in 8 mm thick was made usingcryostat (Jung CM3000; Leica). Five IDA samples, 6 IPMAsamples, and 6 IPMC samples were subjected to laser-captured microdissection using a Leica CIR MIC system(Leica Microsystems). Sample collection and usage wereconducted under written informed consent obtained fromeach patient before surgery.

RNA extractionTotal cellular RNA was extracted from microdissected or

bulk sample or pancreatic cancer cell line using the mirVanamiRNA Isolation Kit (Applied Biosystems).

Microarray and statistical analysisWe used the Agilent Human miRNA microarray v2.0

(Agilent Technologies) to compare the expression profiles ofmiRNA. Each array contains 723 human miRNA and 76viral miRNA. One hundred nanogram of total RNA fromeach sample was labeled by cyanine 3-cytidine bisphosphateusing miRNA Complete Labeling Reagent and Hyb kit(Agilent Technologies), then array slide was hybridized withlabeled miRNA for 20 hours. Hybridized array slides werescanned by Agilent Microarray Scanner (Agilent Technol-ogies), and fluorescent signal of each spotwas corrected usingAgilent Feature Extraction v.9.1.3.1 (Agilent Technologies).The array data were deposited in the Gene ExpressionOmnibus database (GSE29542).The significantly upregulated or downregulated miR-

NAs in IDA sample or pancreatic cancer cell line com-

pared with IPMA or IPMC sample were elucidated bystatistical analysis of array data. The raw data of eachsample were normalized by robust multichip averagemethod. These normalized data were adjusted to the linearmodel, and each miRNA was ranked using Bayesian t test.Differentially expressed miRNAs were determined at d(false discovery rate) ¼ 0.01. Normalization, linear modelfitting, and elucidation of differentially expressed miRNAswere conducted using AgiMiRNA package (9) in Biocon-ductor-2.7 on R version 2.12.0.

Real-time reverse transcriptase PCRThe expression level of hsa-miR-126 in each normal

pancreas, FNA sample, or cancer cell line sample wasquantified by real-time reverse transcriptase PCR (RT-PCR)using TaqManMicroRNA Assays (Applied Biosystems) andStepOnePlus real-time PCR system (Applied Biosystems).The expression level of hsa-miR-126 in each sample wasnormalized by expression level of U6.

Immunohistochemistry and in situ hybridizationNormal pancreatic tissue or IDA tissue collected at the

time of surgery were fixed in 10% paraformaldehydeovernight and embedded in paraffin wax. Immunohis-tochemistry of ADAM9 was conducted as described pre-viously using ADAM9 antibody (2099 Cell SignalingTechnology, Inc.) and the Histofine Kit (Nichirei; ref. 10).In situ hybridization of hsa-miR-126 was conducted usingmiRCURY LNA microRNA ISH Optimization Kit[formalin-fixed, paraffin-embedded (FFPE); EXIQON]and DIG-labeled hsa-miR-126 detection probe (EXI-QON) at final concentration of 50 nmol/L. The protein-ase-K treatment, hybridization of probe, and stringentwashing were conducted according to the manufacturer'sprotocol. The hybridized DIG-labeled probe was detectedusing Sheep anti-DIG-AP (11 093 274 910, RocheApplied Science) and NBT/BCIP ready-to-use tablets(Roche Applied Science).

Expression of miRNA in pancreatic cancer cell lineTransient expression of hsa-miR-126 in Panc-1 and

ASPC-1 cells was conducted using the Pre-miR miRNAPrecursor (Applied Biosystems) at final concentration of200 nmol/L. Control treatment was conducted using thePre-miR miRNA Precursor Molecules Negative Control(Applied Biosystems). Transfection of the miRNA pre-cursor was conducted using Lipofectamine 2000(Invitrogen).

RNA interferenceThe transient knockdown of ADAM9 was conducted

by using the ON-TARGET plus SMARTpool siRNAagainst human ADAM9 (Dharmacon) at the final concen-tration of 100 nmol/L. As a negative control, the ON-TARGET plus Non-targeting pool siRNA (Dharmacon)was used at the same concentration. The siRNA transfectionwas conducted by using Lipofectamine 2000 TransfectionReagent (Invitrogen).

Hamada et al.

Mol Cancer Res; 10(1) January 2012 Molecular Cancer Research4




Cell growth assayPanc-1 and ASPC-1 cells were transfected with hsa-

miR-126 precursor or negative control precursor,ADAM9 siRNA or negative control siRNA and incubat-ed for 24 hours. Then, 6,000 cells per well were plated in96-well plate in triplicate. The bromodeoxyuridine

(BrdUrd) incorporation assay was conducted 72 hoursafter.

Migration assay and invasion assayCellular migration and invasion were assessed by two-

chamber assay using the 8-mm pore, 24-well BD

Figure 1. A, laser-capturedmicrodissection of tumor epithelium. B, cluster analysis ofmiRNAexpression profiles. Left, the normalization of rawdata. Right, theheat map of miRNA expression profile. C, Venn diagram of differentially expressed miRNAs.

miR-126/ADAM9 Regulates Invasive Growth of Pancreatic Cancer

www.aacrjournals.org Mol Cancer Res; 10(1) January 2012 5




FALCON Cell Culture Insert and BD BioCoat MatrigelInvasion Chamber (BD Biosciences), respectively. Panc-1and ASPC-1 cells were transfected with hsa-miR-126precursor or negative control precursor, ADAM9 siRNAor negative control siRNA and cultured for 72 hours.Then, 10,000 cells per chamber for Panc-1, 50,000 cellsper chamber for ASPC-1 were plated, and cellular migra-tion and invasion were assessed 24 hours after plating.Migrated or invaded cells were counted in random 5 high-power fields.

Scratch assayScratch assay was conducted as described previously (2).

Briefly, Panc-1 or ASPC-1 was transfected with indicatedmiRNA precursor or siRNA at final concentrations of 200or 100 nmol/L, respectively. Cells were grown until con-

fluent state and then themonolayer of the cells was scratchedusing sterile tips. Cellular migration toward the scratchedarea was assessed 24 hours after.

Western blottingWestern blot analysis was done as described previously

(2). The primary antibodies used in this study were asfollows: a-tubulin (T5168; Sigma-Aldrich), E-cadherin(610181; BD), N-cadherin (610920; BD), and ADAM9(2099; Cell Signaling Technology, Inc.). As a secondaryantibody, horseradish peroxidase (HRP)-conjugated anti-mouse antibody (GE Healthcare) or HRP-conjugatedanti-rabbit antibody (7074; Cell Signaling Technology)was used. Reactive bands were detected using ECLplus Western blotting detection reagents (GE Healthcare).The specific bands were subjected to densitometry analysis

Figure 2. A, BrdUrd incorporationassay after miR-126 expression(n ¼ 3). B, attenuated cellularmigration and invasion aftermiR-126 expression (n ¼ 3).C, attenuated cellular migrationtoward the scratched area aftermiR-126 expression. D, E-cadherininduction and N-cadherinreduction after miR-126expression. Bottom, densitometricanalysis (n ¼ 3). E, schematic viewof evolutionally conserved 30-UTRsequence of ADAM9 mRNA.Bottom, possible hybridization ofmiR-126 andADAM9 30-UTR. HPF,high-power field.

Hamada et al.





using Image J software (http://rsbweb.nih.gov/ij/index.html).

30-UTR assayPotential target site of hsa-miR-126 in the 30-UTR

sequence of ADAM9 mRNA was identified using TargetS-can (http://www.targetscan.org/). The DNA sequencewhich corresponds to this 30-UTR sequence of ADAM9mRNA was subcloned into the pmirGLO vector (Promega;pMG-A9). The DNA sequence of ADAM9 30-UTR inwhich the seed sequence for the hsa-miR-126 binding wasmutated was also subcloned into the pmirGLO vector(Promega; pMG-A9M). Panc-1 and ASPC-1 cells weretransfected with hsa-miR-126 precursor or negative controlprecursor and incubated for 24 hours. Then, 100,000 cellsper well were plated into 12-well plates and transfected with100 ng per well of pMG-A9 or pMG-A9M reporter vector.Twenty-four hours after transfection, luciferase assay wasconducted using Dual-Luciferase Reporter Assay Kit(Promega).

Statistical analysisStatistical analysis was conducted using Excel (Microsoft).

The difference between 2 groups was analyzed by Mann–

Whitney U test. The differences between multiple groupswere analyzed by ANOVA with Fisher least significantdifference method. The value of P < 0.05 was consideredas statistically significant.

Results

Comprehensive analysis of the differentially expressedmiRNAs in IDA samplesMicrodissection was done as shown in Fig. 1A. Each

sample's miRNA expression profile including pancreaticcancer cell line was assessed by microarray. By compar-ing the miRNA expression profiles of IDA samples orcancer cell lines versus IPMA or IPMC samples by clusteranalysis after the normalization (Fig. 1B), differentiallyexpressed miRNAs were elucidated (Fig. 1C). ThosemiRNAs which were differentially expressed commonlyin IDA samples or cancer cell lines compared with theIPMA or IPMC samples are summarized in Supplemen-tary Tables S1 and S2. The miR-21 was upregulated inIDA and cancer cell lines and previous reports havesuggested its correlation with gemcitabine resistance orpatients' outcome (5, 11). In addition, the tumor sup-pressor miR-145 was downregulated in IDA and cancer

Figure 3. A, decreased expression of ADAM9 after miR-126 expression. Right, densitometric analysis (n ¼ 3). B, in situ hybridization of miR-126 (i–iv) andimmunohistochemistry (IHC) of ADAM9 (v–viii); i, ii, v, vi, normal pancreas; iii, iv, vii, viii, pancreatic cancer. Black bar shows 50 mm. C, comparison of miR-126expression in normal pancreas tissues (n ¼ 5) and pancreatic cancer tissues (n ¼ 20) by quantitative real-time RT-PCR. ��, P < 0.01.






cell lines (12). These results indicate the validity ofcurrent experimental design. Among those differentiallyexpressed miRNAs, we chose miR-126, as recent reportssuggested the tumor suppressive role in other type ofcancer (13, 14).

Effect of miR-126 reexpression in pancreatic cancer celllinesCellular proliferation after the transfection of miR-

126 precursor was not altered in Panc-1 and ASPC-1(Fig. 2A). However, cellular migration or invasion afterthe transfection of miR-126 precursor was attenuated inboth cell lines by up to 10% to 50% (Fig. 2B). Decreased

cellular migration in miR-126–transfected Panc-1 orASPC-1 was also confirmed by scratch assay (Fig. 2C).In addition, expression of E-cadherin in Panc-1 or ASPC-1 after the transfection of miR-126 precursor wasincreased whereas expression of N-cadherin was decreased(Fig. 2D). These effects of miR-126 were consideredto oppose EMT of cancer cells. The possible miR-126target genes by database analysis are summarized inSupplementary Table S3. ADAM9 was included in thosegenes and previous report has shown its frequent expres-sion in pancreatic cancer and correlation with poorprognosis (15). Potential seed sequence in ADAM930-UTR which could interact with miR-126 was

Figure 4. A, transient knockdown ofADAM9 by siRNA. B, BrdUrdincorporation assay after ADAM9knockdown (n ¼ 3). C, attenuatedcellular migration and invasionafter ADAM9knockdown (n¼3). D,attenuated cellular migrationtoward the scratched area afterADAM9 knockdown. E, E-cadherininduction and N-cadherinreduction after ADAM9knockdown. Bottom, densitometryanalysis (n ¼ 3). HPF, high-powerfield.

Hamada et al.





identified with evolutionary conservation (Fig. 2E).Among a large number of ADAM family members, thedatabase analysis identified only ADAM9 as a predictedmiR-126 target, based on the evolutionarily conserved 30-UTR seed sequence which defines the miRNA-targetingspecificity (16–18).

Expression of miR-126 attenuates ADAM9 expressionTo confirm that ADAM9 is a direct target of miR-126,

we assessed the expression levels of ADAM9 in miR-126–transfected Panc-1 or ASPC-1. The expression levels ofADAM9 in miR-126–transfected cells were decreased(Fig. 3A). Expression of miR-126 and ADAM9 in normalpancreas or pancreatic cancer tissue was assessed by in situhybridization and immunohistochemistry, respectively(Fig. 3B). Normal pancreas expresses miR-126 and lacksADAM9 expression. Expression of miR-126 in pancreaticcancer was diminished, whereas cancerous glands were pos-itive for ADAM9 staining. Quantitative real-time RT-PCRalso confirmed higher expression of miR-126 in normalpancreas tissues than in pancreatic cancer tissues (Fig. 3C).

ADAM9 knockdown attenuates cellular migration andinvasionAs a next step, we assessed the biologic role of

ADAM9 expression in Panc-1 and ASPC-1 by siRNA-basedknockdown experiment. Transient transfection of siRNAagainst ADAM9 efficiently repressed ADAM9 expression inPanc-1 and ASPC-1 (Fig. 4A). Knockdown of ADAM9 didnot alter cellular proliferation of Panc-1 or ASPC-1 (Fig.4B). Cellular migration and invasion of Panc-1 or ASPC-1was attenuated until 50% to 60% of control treatment(Fig. 4C) and decreased cellular migration in ADAM9siRNA–transfected Panc-1 or ASPC-1 was also confirmedby scratch assay (Fig. 4D). Along with these biologicchanges, E-cadherin expression was increased and N-cad-herin was decreased by ADAM9 knockdown (Fig. 4E). Onthe basis of these findings, we concluded that ADAM9regulates cellular migration and invasion in pancreatic cancercells as a target gene of miR-126.

miR-126 regulates ADAM9 expression by directinteraction with ADAM9 30-UTRThe interaction of miR-126 and ADAM9 mRNA 30-

UTR was assessed by 30-UTR assay. The 30-UTR sequenceof wild-type ADAM9 mRNA (pMG-A9) or mutatedsequence (pMG-A9M) was subcloned into the pmirGLOvector (Fig. 5A). Reexpression of miR-126 decreased report-er activity of pMG-A9 in Panc-1 or ASPC-1 compared withthe control treatment (Fig. 5B). Mutation in the seedsequence (pMG-A9M) abrogated these effects which indi-cates the direct interaction of miR-126 and ADAM9 30-UTR.

Discussion

IPMA or IPMC manifests better prognosis than IDA asits noninvasive growth. Once the invasive growth of

IPMC occurs, the prognosis is as poor as IDA (19).Various cytokines and signaling pathways contribute tothis complex biologic process which requires mobilizationof cancer cells from the site of origin. However, thecontribution of miRNA in invasion of pancreatic cancerand metastasis is not yet fully understood. We tried toelucidate miRNAs whose differential expressions are indis-pensable in invasive growth of cancer cells by comparingthe miRNA expression profiles of IPMA, IPMC, IDA, andpancreatic cancer cell lines. This approach confirmed thedifferential expression of several miRNAs such as miR-21or miR-145 which are involved in the cancer cell invasionin previous studies (5, 6).In this study, we focused on miR-126 as its role in

pancreatic cancer was not yet clarified, whereas its expres-sions in endothelial cells and immune cells are retained intumor (20). Previous reports suggested that miR-126 actsas a tumor suppressor in lung, gastric, and breast cancerby targeting EGFL7, Crk, or SLC7A5 which yieldsgrowth advantages (13, 14, 21, 22). These evidencessuggested possible tumor suppressive role of miR-126 inpancreatic cancer. Transient expression of miR-126attenuated cellular migration or invasion, and the data-base analysis identified ADAM9 as a potential target ofmiR-126 based on the evolutionally conserved 30-UTRsequence. ADAM9 is uniformly expressed in pancreaticcancer cells, but its upstream regulator was not identified(15). Recent report identified that ADAM9 promotescancer cell invasion by modulating tumor–stromal cellinteraction (23), and our observation indicated that

Figure 5. A, wild-type seed sequence of ADAM9 30-UTR and mutatedseed sequence. Underlined sequence was modified to disrupthybridization with miR-126. B, cotransfection of miR-126 attenuatedreporter activity of pMG-A9 and mutated seed sequence canceled thiseffect. ��, P < 0.01 (n ¼ 3).






ADAM9 could also function in cell autonomous manner.Transient expression of miR-126 and siRNA-basedknockdown of ADAM9 revealed similar effects in pan-creatic cancer cells which confirmed the role of ADAM9as a downstream effector of miR-126.Present study provided a novel therapeutic target, miR-

126/ADAM9 axis in pancreatic cancer. Other differentiallyexpressed miRNAs identified in this study also could beuseful targets. Because the detection of miRNA in bloodsample is technically possible (24), an miRNA which pre-dicts that invasive pancreatic cancer could lead to the earlydetection of curable disease. As another approach, thesedifferentially expressed miRNAs could be applied to indi-vidualize pancreatic cancer patients before and during the

therapy. These approaches will be beneficial to developeffective therapy against pancreatic cancer. Further study isnecessary to dissect the complex regulation of pancreaticcancer progression by miRNA.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received June 14, 2011; revised October 11, 2011; accepted November 1, 2011;published OnlineFirst November 7, 2011.

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2012;10:3-10. Published OnlineFirst November 7, 2011.Mol Cancer Res Shin Hamada, Kennichi Satoh, Wataru Fujibuchi, et al. the Regulation of ADAM9MiR-126 Acts as a Tumor Suppressor in Pancreatic Cancer Cells via

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MiR-126 Acts as a Tumor Suppressor in Pancreatic Cancer