KRAS/NF-kB/YY1/miR-489 Signaling Axis Controls Pancreatic ... · pancreatic cancer initiation and progression are questions that still remain largely unanswered. Most importantly,

Molecular and Cellular Pathobiology

KRAS/NF-kB/YY1/miR-489 Signaling AxisControls Pancreatic Cancer MetastasisPeng Yuan1,2, Xiao-Hong He1,2, Ye-Fei Rong3, Jing Cao4, Yong Li5, Yun-Ping Hu6,Yingbin Liu6, Dangsheng Li7,Wenhui Lou3, and Mo-Fang Liu1,2,4

Abstract

KRAS activation occurring in more than 90% of pancreaticductal adenocarcinomas (PDAC) drives progression and metas-tasis, but the underlying mechanisms involved in these processesare still poorly understood.Here,we showhowKRAS acts throughinflammatory NF-kB signaling to activate the transcription factorYY1, which represses expression of the tumor suppressor genemiR-489. In PDAC cells, repression ofmiR-489 by KRAS signaling

inhibited migration and metastasis by targeting the extracellularmatrix factors ADAM9 and MMP7. miR-489 downregulationelevated levels of ADAM9 and MMP7, thereby enhancingthe migration and metastasis of PDAC cells. Together, ourresults establish a pivotal mechanism of PDAC metastasis andsuggest miR-489 as a candidate therapeutic target for their attack.Cancer Res; 77(1); 100–11. �2016 AACR.

IntroductionPancreatic cancer (PDAC) is one of the most lethal malignant

tumors,with a 5-year survival rate less than 8% from2005 to 2011in United States (1). To date, lack of effective screening tool todetect asymptomatic premalignant or early-stage cancer results inthe diagnosis of majority patients at their advanced stages. Morethan 90% of the patients with PDAC show distal metastasis atadvanced stage, which is the major cause of mortality (2). Thus,effective therapeutic interventions to stop PDAC metastasis areurgently needed. However, our ability to design effective thera-peutic interventions aiming to stop PDAC metastasis is limitedbecause of our poor understanding of the molecular mechanismsunderlying pancreatic cancer metastasis (3).

One prominent feature of PDAC is the high frequency(>90%) of KRAS mutations that generate oncogenic forms of

KRAS in pancreatic tumors, the most common of which isKRASG12D (4). Mounting evidence indicates that such onco-genic mutations play critical roles in both the initiation and theprogression of pancreatic cancer via persistent activation ofKRAS signaling pathways (5). Sustained KRAS signaling leadsto the activation of inflammatory signaling pathways that playcritical roles in regulating the initiation of pancreatic intrae-pithelial neoplasia (PanIN) and the progression of PDAC(6, 7). Of note, NF-kB signaling, a major pathway that connectsinflammation with cancers, is activated by KRAS signaling andhas been shown to promote pancreatic cancer progression inanimal models (8–10). However, the downstream targets ofNF-kB signaling that are directly involved in pancreatic cancerprogression and metastasis still need to be defined.

microRNAs (miRNA) are a class of small, noncoding RNAs thatnegatively regulate protein-coding genes at the posttranscription-al level and are involved in virtually all types of carcinogenesis(11). In particular, a number of miRNAs have been found to bedysregulated in PDAC and involved in PDAC carcinogenesis (12).Nevertheless, how these miRNAs are dysregulated in PDAC andhow they interact with key regulatory signaling molecules inpancreatic cancer initiation and progression are questions thatstill remain largely unanswered. Most importantly, despite thewell-established role of oncogenic KRAS in PDAC, how KRASsignaling engages miRNAs to drive pancreatic cancer metastasisremains unknown.

In our attempt to identify important miRNAs involved inpancreatic carcinogenesis, we identified KRAS signaling–repressed miR-489 as part of an important mechanism under-pinning oncogenic KRAS-induced PDAC migration and metasta-sis. Oncogenic KRAS signaling activates NF-kB, leading toenhanced expression of YY1, the transcription factor that directlysuppresses MIR489 transcription. Our results showed that miR-489 decreases the migration of PDAC cells in cell cultures andinhibits lung and liver metastatic colonization of PDAC cells inmice but contributes little to cell proliferation and anchorage-independent growth. Mechanistically, we identified 2 metallo-proteinase genes,ADAM9 andMMP7, as novel targets ofmiR-489that mediate its antimetastatic effect in these cells. Collectively,our findings not only provide new mechanistic insights into how

1Center for RNAResearch, State Key Laboratory ofMolecular Biology–Universityof Chinese Academy of Sciences, CAS Center for Excellence in Molecular CellScience, Shanghai, China. 2Shanghai Key Laboratory of Molecular Andrology,Institute of Biochemistry and Cell Biology, Shanghai Institutes for BiologicalSciences, Chinese Academy of Sciences, Shanghai, China. 3Department ofPancreatic Surgery, Zhong Shan Hospital, Shanghai, China. 4School of LifeScience and Technology, Shanghai Tech University, Shanghai, China. 5Depart-ment of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland,Ohio. 6Department of General Surgery, Xinhua Hospital Affiliated to ShanghaiJiao TongUniversity School ofMedicine, Shanghai, China. 7Shanghai InformationCenter for Life Sciences, Shanghai Institutes for Biological Sciences, ChineseAcademy of Sciences, Shanghai, China.

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

P. Yuan, X.-H. He, and Y.-F. Rong contributed equally to this article.

Corresponding Authors: Mo-Fang Liu, Institute of Biochemistry and CellBiology, Shanghai Institutes for Biological Sciences, the Chinese Academy ofSciences, 320 Yueyang Road, Shanghai 200031, China. Phone: 86-21-54921146; Fax: 86-21-54921011; E-mail: [email protected]; and Wenhui Lou,Department of Pancreatic Surgery, Zhong Shan Hospital, 180 Fenglin Road,Shanghai 200032, China. Phone: 86-18-616881868; Fax: 86-21-64043947;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-1898

�2016 American Association for Cancer Research.

CancerResearch

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on September 8, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 28, 2016; DOI: 10.1158/0008-5472.CAN-16-1898

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oncogenic KRAS-induced inflammatory signaling promotesPDAC metastasis but also indicate that miR-489 is a robustinhibitor of metastasis and a potential therapeutic target fortreating PDAC.

Materials and MethodsCell lines

The human PDAC cell lines BxPC-3 and PANC-1 cells wereobtained from ATCC (May, 2011) and cultured according to theirguidelines. All the cell lines were mycoplasma-free and recentlyauthenticated by cellular morphology and the STR analysis at Ji-Ying Inc. (January 2014 and May 2016) according to the guide-lines from ATCC (13). The 2 cell lines stably expressing luciferase(PANC-1-luc and BxPC-3-luc) were generated from their parentalcell lines as previously reported (14).

Antibodies and reagentsThe antibodies used in this study included: anti-KRAS (60309-

1; Proteintech), anti-PCNA (#2586; Cell Signaling Technology),anti-YY1 (ab38422; Abcam), anti-ETS-1 (sc-350; Santa Cruz Bio-technology), anti-p65 (10745-1-AP, Proteintech), anti-ADAM9(ab186833, Abcam), anti-MMP7 (10374-2-AP, Proteintech),anti-b-actin (A3854; Sigma-Aldrich), anti-mouse secondary anti-body (A9044, Sigma-Aldrich), and anti-rabbit secondary anti-body (A9169, Sigma-Aldrich). These antibodies were dilutedaccording to themanufacturers' instructions. The NF-kB signalinginhibitor BAY 11-7082 (S2913; Selleck) was first diluted in cancercell culture medium and then used as in previous studies withsome modifications (i.e., incubation at 5 mmol/L for 24 hours;ref. 15). The sequences of chemically synthesized DNA and RNAoligonucleotides are listed in Supplementary Table S1.

Cell proliferation and soft agar colony formation assaysThe assays were performed as described previously (16). In

brief, cancer cells were subjected to transfection for 24 hours. Forcell proliferation assay, 3,000 viable cells were seeded into eachwell of 96-well plates. Cell growth was determined byMTT assaysand verified by counting the cells excluding trypan blue. For softagar colony formation assay, 5,000 viable cancer cells were triplyseeded in1.5mLof tissue culturemediumwith1%glutamine andantibiotics, and 0.4% soft agar was layered onto 0.8% solidifiedagar in tissue culturemedium in6-well plates. After incubation for15 days, the colony foci were stained with 0.005% crystal violetand counted using a dissecting microscope. Experiments werecarried out in triplicate.

Wound-healing assayWound-healing assays were performed as previously described

(14). Cancer cells were transfected for 24 hours and starved inculture medium containing 0.2% FBS for 12 hours. The mono-layer of confluent cells was scratched using a 10-mL Pipetman tip.Cells were then photographed at different time points with anOlympus IX81 microscope. The relative wound areas were thenmeasured using ImageJ software (NIH, Bethesda, MD).

Transwell assayCancer cells were transfected for 24 hours, following by star-

vation for 12 hours. PANC-1 cells (2 � 104) or BxPC-3 cells (5 �104) were used in each 20% (in culture medium without FBS)Matrigel-precoated (356231BD; BD Biosciences) insert (8.0-mm

pore size inserts, 3422; Corning) in the Transwell assays accordingto the manufacturer's instructions. The nonmigrating cells weredetached, and themigrating cellswere stainedwith 40,6-diamidio-2-phenylindole (DAPI) and counted after 24 hours using afluorescence microscope. All fields were selected in a blindmanner.

Three-dimensional cell culture assayThe 3D cell culture assay was performed as recently described

(17). Transfected and viable cancer cells (1,000) in culture medi-um containing 2% Matrigel were seeded on the solidified 100%Matrigel-precoated chamber (177402; Nunc). The sphere mor-phology was observed with a microscope after 19 days.

Xenograft assays in miceNOD/SCID mice were purchased from SLAC Corporation and

were housed under standard housing conditions at the animalfacilities in the Institute of Biochemistry and Cell Biology, Shang-hai Institutes for Biological Sciences, Chinese Academy ofSciences. The lung and liver metastasis assay was performed asdescribed previously (14, 18). PANC-1-luc cells (4 � 104) orBxPC-3-luc cells (1� 105) in 100 mL Dulbecco PBS (D-PBS) wereinjected into6- to 8-weekNOD/SCIDmice through the tail vein (n¼ 5–6), and the luciferase activity in lungs was analyzed 50 dayslater. PANC-1-luc cells (1 � 105) orBxPC-3-luc cells (5 � 105) in50 mL D-PBS were injected into the spleens of 6- to 8-week-oldNOD/SCIDmice (n¼ 4–5), and the tumor numbers in livers wereanalyzed 60 days later. For bioluminescence imaging, mice weregiven luciferin 5 minutes before imaging and were then anesthe-tized (3% isoflurane). Luminescence imaging was performed andanalyzed using the Xenogen IVIS Imaging System (Xenogen, asubsidiary of Caliper Life Sciences).

Statistical analysesAll results are presented as the mean � SD. The Student t test

was performed to judge the significance of differences betweentreated groups and their paired controls. P values indicate the levelof statistical significance (�, P < 0.05; ��, P < 0.01; ��, P < 0.001).The fold changes of the relative miRNA or mRNA levels wereperformed using Mev-HCL software algorithm analyses (19).

Study approvalPDAC specimens and paired normal adjacent tissues were

collected during surgery from Zhong Shan Hospital, which isaffiliated with Fudan University (Shanghai, China) with writteninformed consent frompatients. Sampleswere immediately snap-frozen and stored at �80�C. The specimen collection wasapproved by the Medical Ethical Committee of the hospital. Allanimal experimentswere performedunder protocols approvedbythe Institute of Biochemistry andCell Biology, Shanghai Institutesfor Biological Sciences, Chinese Academy of Sciences and inaccordance with the Guide for the Care and Use of LaboratoryAnimals (NIH publication nos. 80–23, revised 1996).

ResultsDownregulation of miR-489 is crucial for oncogenic KRAS topromote cell migration in human PDAC cells

To gain new insight into the role of miRNAs in oncogenicKRAS-initiated pancreatic tumorigenesis, we compared theexpression of 53 miRNAs that are dysregulated during PDAC

MIR489 Is a Key Regulator for PDAC Metastasis

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progression in the KrasG12Dtransgenic animal model and inKRASG12D-transfected human pancreatic ductal epithelial cells(20, 21) between 2 human PDAC cell lines, namely, wild-typeKRAS-containing BxPC-3 and KRASG12D-containing PANC-1 cells(Supplementary Fig. S1A, left). Our qRT-PCR assays showed that17miRNAs were differentially expressed between the 2 PDAC celllines (Supplementary Fig. S1B, left column), including 3miRNAs(miR-489,miR-889, andmiR-574) enriched in BxPC-3 cells and 4miRNAs (miR-125b,miR-491,miR-21, andmiR-23b) enriched inPANC-1 cells (by >3.0-fold). To examine the effect of oncogenicKRAS signaling on miRNA expression in these PDAC cells, weknocked down KRAS in PANC-1 cells and ectopically expressedKRASG12D in BxPC-3 cells, respectively (Supplementary Fig. S1C).miR-21 was the most markedly upregulated by KRASG12D over-expression in BxPC-3 cells (Supplementary Fig. S1B, middlecolumn) and downregulated by KRAS knockdown in PANC-1cells (right column), consistent with previous studies reporting animportant role for miR-21 in oncogenic KRAS–induced cell pro-liferation (22, 23). Interestingly, miR-489, among all the testedmiRNAs, showed the greatest reduction in KRASG12D-transfectedBxPC-3 cells (Supplementary Fig. S1B, middle column) and thegreatest increase in KRAS siRNA–transfected PANC-1 cells (rightcolumn).

The strong negative correlation between KRAS signaling andmiR-489 prompted us to examine whether downregulation ofmiR-489 is involved in KRAS signaling–mediated pancreatictumorigenesis. As expected, ectopic expression of KRASG12D inBxPC-3 cells substantially increased cell proliferation (Fig. 1A),anchorage-independent growth (Fig. 1B), and cell migrationin vitro (Fig. 1C and D). Intriguingly, restoration of miR-489expression in these cells by transfection of themiR-489 expressionvector pSIF-miR-489 barely affected cell proliferation and softagar colony formation (Fig. 1A and B; Supplementary Fig. S1D)but rescued the KRASG12Dexpression–induced cell migration in adosage-dependent manner (Fig. 1C and D). Conversely, knock-down of KRAS in PANC-1 cells markedly decreased cell prolifer-ation (Fig. 1E), anchorage-independent growth (Fig. 1F), and cellmigration in vitro (Fig. 1G and H), whereas concurrent suppres-sion of miR-489 by anti-miR-489 showed no significant effect oncell proliferation (Fig. 1E and F; Supplementary Fig. S1E) butsignificantly reversed the impact of KRAS knockdown on cellmigration in these KRAS-mutant cells (Fig. 1G and H). Theseresults together suggest that downregulation of miR-489 contri-butes little to KRAS signaling–driven cell proliferation but is animportant mechanism underlying KRAS signaling–driven cellmigration of PDAC cells.

miR-489 is downregulated by the KRAS/NF-kB/YY1 axis inPDAC cells

We next asked how KRAS signaling downregulates miR-489 inPDAC cells. To this end, we first searched for potential transcrip-tion factor–binding sites inMIR489 promoter using the PROMOand TransFac programs (24, 25). Interestingly, ETS1 and YY1, 2transcription factors that have been shown to control miRNAexpression in cancer cells (26, 27), were predicted as candidatetranscription factors forMIR489 (Fig. 2A, top). To explorewhetherETS1 or YY1 directly regulates MIR489, we first performed achromatin immunoprecipitation (ChIP) assay in PANC-1 cells.We found that the PMIR489(promoter of MIR489) fragment waseffectively enriched by anti-YY1, but not by anti-ETS1, comparedwith the IgG control (Fig. 2B).Moreover, RNAi knockdownofYY1

strongly enhanced miR-489 expression in these cells, whereasknockdown of ETS1 had little effect (Fig. 2C). We further foundthat overexpression of YY1, but not ETS1, potently decreasedmiR-489 expression in PANC-1 cells (Fig. 2D). These results stronglysuggest that YY1 is a repressor forMIR489. To further substantiatethis conclusion, we constructed 2 luciferase reporters under con-trol of either the wild-type humanMIR489 promoter or a mutantversion with 2 putative YY1-binding sites deleted (termed the WTPMIR489 reporter and the mut PMIR489 reporter, respectively; Fig.2A, bottom). As expected, the activity of the WT PMIR489 reporterwas potently enhanced by knockdown of YY1 but reduced by YY1overexpression inPANC-1 cells (Fig. 2E). ThemutPMIR489 reporterdisplayed about 4-fold activity in PANC-1 cells compared withWT, and it was not affected by either YY1 knockdown or over-expression (Fig. 2E). Collectively, these results support the notionthat YY1 is a direct transcriptional repressor for MIR489.

We next examined whether KRAS signaling represses MIR489via YY1 in PDAC cells. The level of YY1 protein was significantlylower in wild-type KRAS-containing BxPC-3 cells than inKRASG12D-containing PANC-1 cells (Fig. 2F, left, lane 1 vs. lane4). Interestingly, YY1mRNA and protein weremarkedly increasedby ectopic expression of KRASG12D in BxPC-3 cells (Fig. 2F, left,lane 6) and significantly reduced by KRAS knockdown in PANC-1cells (Fig. 2F, left, lane 3), indicating that oncogenic KRAS acti-vates YY1 expression in PDAC cells. Then, we searched and foundthat NF-kB family member was predicted to be transcriptionfactor in YY1 promoter, which is consistent with the study inmyoblasts (28). As NF-kB is a well-documented downstreameffector of KRAS signaling (8, 10), we examined whether theYY1/miR-489 axis is regulated by KRAS/NF-kB signaling inhuman PDAC cells. Both KRAS knockdown and treatment withan NF-kB inhibitor, BAY 11-7082, significantly elevated miR-489expression, with a concomitant reduction of YY1 expression inPANC-1 cells, whereas the elevation of miR-489 expression byKRAS knockdown or BAY 11-7082 treatment was completelysuppressed by ectopic expression of Flag-YY1 in these cells (Fig.2G). These results suggest that the YY1/miR-489 axis is controlledby KRAS/NF-kB signaling in PDAC cells. To further substantiatethis conclusion, we examined the effect of KRAS knockdown orBAY 11-7082 treatment on the activity of the PMIR489reporter inPANC-1 cells. In a similar manner, both KRAS knockdown andtreatmentwithNF-kB inhibitor significantly enhanced the activityof the WT PMIR489reporter in PANC-1 cells, and their effects werecompletely abrogated by ectopic expression of Flag-YY1 (Fig. 2H).In sharp contrast, KRAS knockdown or BAY 11-7082 treatmentbarely affected the activity of the mut PMIR489reporter (Fig. 2H).Taken together, these results indicate that the MIR489 gene isrepressed by the novel KRAS/NF-kB/YY1 axis in human PDACcells.

miR-489 inhibits the migration and metastasis of PDAC cellsWenext examined the role ofmiR-489 in PDAC tumorigenesis.

Modulation of miR-489 expression in PDAC cells, includingtransfection of pSIF-miR-489 in PANC-1 cells (which have lowerendogenousmiR-489 expression) or anti-miR-489 in BxPC-3 cells(which show a high level of endogenous miR-489 expression;Supplementary Fig. S1A, right), barely altered cell proliferationand anchorage-independent growth (Supplementary Fig. S2).Western blot analyses confirmed that the proliferation markerproliferating cell nuclear antigen (PCNA)was little altered inpSIF-miR-489–transfected PANC-1or anti-miR-489–transfected BxPC-

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Figure 1.

Downregulation of miR-489 is crucial for oncogenic KRAS to promote cell migration in human PDAC cells. A–D, Restoration of miR-489 expression overrodethe effect of ectopicKRASG12D onwound-healing (C) and Transwell cell migration (D) but only slightly altered oncogenic KRAS signaling–promoting cell proliferation(A) and anchorage-independent growth (B) in BxPC-3 cells. The cells were transfected with pCMV-myc-G12D-KRAS (G12D-KRAS) or cotransfected withG12D-KRAS and the indicated amounts of pSIF-miR-489, with pCMV-myc and pSIF-H1 vectors serving as negative controls. A, Cell proliferation assays. Top, MTTassays; bottom, Western blot analyses of KRAS and PCNA proteins. B, Soft agar colony formation assays. Top, the number of soft agar foci per field; bottom,representative images (scale bar, 100 mm). C, Wound-healing assays. Top, quantitative results of wound closure 16 hours after wounding; bottom, representativeimages at 0 and 16 hours after wounding (scale bar, 100 mm). D, Transwell migration assays. Top, number of migratory cells per field 24 hours after thecells were plated; bottom, representative images (scale bar, 40 mm). E–H, Inhibition of miR-489 by anti-miR-489 overrode the effect of KRAS knockdown onwound-healing (G) and Transwell cell migration (H) but only slightly alteredKRAS knockdown–reduced cell proliferation (E) and anchorage-independent growth (F)in PANC-1 cells. The cells were transfected with KRAS siRNA (KRAS-siR) or cotransfected with KRAS-siR and anti-miR-489, with scrambled RNA (scr siR)and control RNA (ctrl RNA) serving as negative controls. Themean� SD of three separate experiments was plotted. Statistics: Student t test, � , P < 0.05; �� , P < 0.01;�� , P < 0.001. n.s., not significant.






3 cells (Supplementary Fig. S2, middle). These results togetherdemonstrate that miR-489 does not affect the proliferation ofPDAC cells. We further examined the effect of miR-489 on PDACcell migration and metastasis. In vitro studies showed that miR-489 overexpression in PANC-1 cells significantly reduced cellmigration in both wound-healing (Fig. 3A, top) and Transwellmigration assays (Fig. 3B, top) and markedly suppressed theability of these cells to spread out and form colonies in a 3-dimensional (3D) cell culture assay (Fig. 3C, top). To determinethe function of miR-489 on PDAC metastasis in vivo, we infectedfirefly luciferase–labeled PANC-1 (PANC-1-luc) cells with pSIF-miR-489 pseudovirus and performed tail vein xenografts. Weobserved strong luciferase foci in the lungs of mice injected with

control pSIF-H1 pseudovirus–infected cells but a dramatic reduc-tion in the luciferase signal in the lungs ofmice injected withmiR-489–overexpressing cells (Fig. 3D, top), suggesting that miR-489inhibits the lungmetastasis of PDAC cells.Hematoxylin and eosin(H&E) staining of the lung sections confirmed that miR-489–overexpressing cells colonized the lung much less efficiently thanthe control populations (Fig. 3E, top).We next injected pSIF-miR-489 pseudovirus–infected PANC-1-luc cells into mouse spleensand examined livermetastasis. Comparedwith controls, miR-489overexpression substantially reduced the liver metastatic coloni-zation of PDAC cells (Fig. 3F, top) and resulted in fewermetastaticliver nodules (Fig. 3G, top). In a reciprocal experiment, inhibitionof miR-489 by anti-miR-489 in BxPC-3-luc (firefly luciferase–

Figure 2.

miR-489 is downregulated by the KRAS/NF-kB/YY1 axis in PDAC cells. A, Top, schematic representation of predicted YY1- and ETS1-binding sites within thehuman MIR489 promoter. Bottom, construction of the wild-type MIR489 promoter reporter (WT PMIR489 reporter) and the YY1 binding site–deleted mutantreporter (mut PMIR489 reporter). B, Top, YY1- and ETS1-binding sites within the humanMIR489 promoter. Bottom, ChIP analyses of YY1 or ETS1 binding to theMIR489promoter using antibodies against YY1 or ETS1, respectively. C and D, Effects of YY1 or ETS1 knockdown (C) or overexpression (D) on miR-489 expressionthrough qRT-PCR analyses of miR-489 expression (left) and Western blot analyses of YY1 or ETS1 protein expression (right) in PANC-1 cells. E, Modulation ofthe PMIR489 promoter activity by YY1 knockdown (top) or overexpression (bottom) in PANC-1 cells. F, Regulation of KRAS signaling on YY1 expression inPANC-1 and BxPC-3 cells. Top, qRT-PCR analyses of YY1 mRNA; bottom, Western blot analyses of YY1 protein. G, Effect of the KRAS/NF-kB/YY1 axis on miR-489expression in PANC-1 cells. Top, qRT-PCR analyses of miR-489; bottom, Western blot analyses of p65 and YY1 protein. H, Modulation of PMIR489 promoteractivity by the KRAS/NF-kB/YY1 axis in PANC-1 cells. The mean � SD of three separate experiments was plotted. Statistics: Student t test, � , P < 0.05; �� , P < 0.01;�� , P < 0.001. n.s., not significant.

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Figure 3.

miR-489 inhibits the migration andmetastasis of PDAC cells. A–C,miR-489 inhibited the migration of PDAC cells in vitro. PANC-1 and BxPC-3 cells were transfectedwith pSIF-miR-489 or the control vector pSIF-H1 (top) and anti-miR-489 or the control RNA (ctrl RNA; bottom), and the assays were performed 24 hoursposttransfection.A,Wound-healing assays. Left, representative images at 0 and 16 hours after wounding (scale bars, 100 mm); right, quantification of wound closure16 hours after wounding. B, Transwell migration assays. Left, representative images (scale bars, 40 mm); right, quantification of migrating cell numbers24 hours after the cellswere plated.C, 3D cell culture assays,with images showing spheres on day 19 after the cellswere grown in 3D cell culture (scale bars, 300 mm).The inset shows an amplified view of individual pSIF-H1–transfected PANC-1 cells (top) or anti-miR-489–transfected BxPC-3 cells (bottom) invading the surroundingMatrigel (scale bars, 50 mm). D and E, miR-489 inhibited lung metastatic colonization of PDAC cells in NOD/SCID mice. D, Bioluminescence imaging ofNOD/SCID mice on day 50 after tail vein injection of pSIF-miR-489 pseudovirus–infected PANC-1-luc cells (top) or anti-miR-489–transfected BxPC-3-luc cells(bottom). Left, representative images; right, bioluminescence quantification of lung metastasis (mean� SD, n¼ 5 mice in each group). E, H&E staining of the lungs(left; scale bars, 2 mm) and the number of metastatic nodules (right) in mice on day 50 after tail vein injection of the indicated PDAC cells. Data on theright are presented as mean � SD (n ¼ 5 mice in each group). F and G, miR-489 inhibited liver metastatic colonization of PDAC cells in NOD/SCID mice. F,Bioluminescence imaging of NOD/SCID mice on day 60 after splenic injection of pSIF-miR-489 pseudovirus–infected PANC-1-luc cells (top) or anti-miR-489–transfected BxPC-3-luc cells (bottom). Left, representative images; right, bioluminescence quantification of liver metastasis (mean� SD, n¼ 4mice in each group).G, Brightfield imaging of the livers (left; scale bars, 5 mm) and the number of visible liver metastases (right) in mice on day 60 after splenic injection of theindicated PDACcells. Data on the right arepresented asmean�SD (n¼4mice in eachgroup). All data aremean�SD. Statistics: Student t test, � ,P<0.05; �� ,P<0.01;�� , P < 0.001.






labeled BxPC-3) cells, which have a higher endogenous miR-489expression (Supplementary Fig. S1A, right), led to a significantincrease in cell migration in wound healing (Fig. 3A, bottom)and Transwell migration (Fig. 3B, bottom), cell invasion in 3Dcell culture (Fig. 3C, bottom), and lung and liver metastasis(Fig. 3D–G, bottom). These results together indicate that miR-489 inhibits the migration and metastasis of PDAC cells,supporting that miR-489 is a critical regulator of PDACmetastasis.

ADAM9 and MMP7 are novel targets of miR-489To dissect the molecular mechanism of the inhibitory effect of

miR-489 on PDAC metastasis, we used miRNA target predictiontools (29) to search for potential target genes of miR-489. Wefound that ADAM9 and MMP7, 2 metalloproteinase genes thathave been implicated as being crucial to PDAC progression (30,31), were predicted to be targets of miR-489 (Fig. 4A, top). Toexperimentally test whether miR-489 targets ADAM9 andMMP7,we constructed luciferase reporters by cloning the wild-type 30-untranslated regions (UTR) ofADAM9 andMMP7 or theirmutantversions (with deletion of the 7-bp sequence complementary tothe 50 sequence of miR-489) downstream of the firefly luciferasecDNA in the pmirGLO vector (Fig. 4A, bottom). We found thatcotransfection of pSIF-miR-489 into 293T cells substantially

decreased the luciferase activity of the wild-type reporters butbarely affected that of the mutant reporters (Fig. 4B), suggestingthat ADAM9 and MMP7 are targets of miR-489. To further cor-roborate this conclusion,we transfectedpSIF-miR-489 into PANC-1 cells and found that both ADAM9 and MMP7 proteins weregreatly reduced in pSIF-miR-489–transfected cells (Fig. 4C, left,lane 3). qRT-PCR analyses showed that their mRNA levels werealso significantly reduced in PANC-1 cells transfected with pSIF-miR-489 (Fig. 4C, right). In contrast, inhibition of miR-489 byanti-miR-489 in BxPC-3 cells led to enhanced ADAM9 andMMP7expression (Fig. 4D). These results together confirmedADAM9andMMP7 as authentic targets of miR-489. In addition, depleting YY1or KRAS proteins in PANC-1 cells significantly reduced ADAM9and MMP7 expression, whereas inhibition of miR-489 by anti-miR-489 effectively increased their expression in these cells (Sup-plementary Fig. S3). This result is consistentwithour abovefindingthat the KRAS/NF-kB/YY1 axis is required for repressing MIR489(Fig. 2), further supporting the notion that ADAM9 andMMP7 aredirect targets of miR-489 in human PDAC cells.

The miR-489:ADAM9/MMP7 regulatory axis is functionallyimportant for migration and metastasis of PDAC cells

As miR-489 inhibits the migration and metastasis of PDACcells (Fig. 3), we then examined whether miR-489 exerts its

Figure 4.

ADAM9 and MMP7 are novel targets of miR-489. A, ADAM9 and MMP7 were predicted to be miR-489 targets. Top, predicted miR-489 regulatory elements (seedsequences in upper case) in ADAM9 and MMP7 30UTRs. Bottom, sequences of the wild-type (pmirGLO-ADAM9-30UTR, pmirGLO-MMP7-30UTR) and themutated (pmirGLO-ADAM9-30UTR mut, pmirGLO-MMP7-30UTR mut) 30UTR luciferase reporters. B, Luciferase reporter assays for the ADAM9-30UTR (top) andMMP7-30UTR (bottom) reporters in 293T cells, indicating that miR-489 targets ADAM9 and MMP7. C and D, ADAM9 and MMP7 were repressed by miR-489 inPDAC cells. C, Effect of miR-489 overexpression on ADAM9 and MMP7 expression in PANC-1 cells. D, Effect of miR-489 inhibition on ADAM9 and MMP7expression in BxPC-3 cells. Left, Western blot analyses of ADAM9 and MMP7 protein expression. Right, qRT-PCR analyses of ADAM9 and MMP7 expression. Themean � SD of three separate experiments was plotted. Statistics: Student t test, �� , P < 0.01; �� , P < 0.001. n.s., not significant.

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inhibitory effects on PDAC metastasis by targeting ADAM9and MMP7. Intriguingly, either ADAM9 or MMP7 knockdownby shRNA in PANC-1 cells barely altered cell proliferation andanchorage-independent growth (Supplementary Fig. S4A andS4B) but robustly decreased cell migration in wound-healing,Transwell migration, and 3D cell culture migration assays(Supplementary Fig. S4C–S4E) and significantly suppressedboth lung and liver metastases in NOD/SCID mice (Supple-mentary Fig. S4F–S4I). Interestingly, we found that eitherADAM9 inhibitor SI-27 (32, 33) or MMP7 inhibitor sulfur-

2-(4-chlorine-3-trifluoromethyl phenyl)-sulfonamido-4-phe-nylbutyric acid (SCTPSPA; ref. 34) substantially suppressedthe lung metastasis of PANC-1-luc cells (Supplementary Fig.S4J and S4K). These results indicate that inhibition of ADAM9and MMP7 recapitulates the inhibitory effect of miR-489 onthe migration and metastasis of PDAC cells. To verify thefunctional role of the miR-489:ADAM9/MMP7 regulatory axisin PDAC migration and metastasis, we constructed miR-489–resistant expression vectors (pCMV-myc-ADAM9 and pCMV-myc-MMP7, without their 30UTRs) for ectopic expression of

Figure 5.

The miR-489:ADAM9/MMP7 axis is functionally important for regulating migration and metastasis in PDAC cells. A–C, ADAM9 and MMP7 overexpression rescuedthe migration of PDAC cells in vitro. BxPC-3 cells were cotransfected with pSIF-miR-489 and pCMV-myc-ADAM9 (myc-ADAM9), pCMV-myc-MMP7 (myc-MMP7),or their control vector pCMV-myc, and the assayswere performed 24 hours posttransfection.A,Wound-healing assays. Left, representative images at 0 and 16 hoursafter wounding (scale bar, 100 mm); right, quantification of wound closure 16 hours after wounding. B, Transwell migration assays. Left, representativeimages (scale bar, 40 mm); right, quantification of migrating cell numbers 24 hours after the cells were plated. C, 3D cell culture assays, with images showingspheres on day 19 after the cells were grown in 3D cell culture (scale bar, 300 mm). The inset shows an amplified view of pCMV-myc-ADAM9- or pCMV-myc-MMP7–transfected BxPC-3 cells invading the surrounding Matrigel (scale bar, 150 mm). D and E, ADAM9 and MMP7 overexpression rescued lung metastaticcolonization of PDAC cells in NOD/SCID mice. D, Bioluminescence imaging of NOD/SCID mice on day 50 after tail vein injection of BxPC-3-luc cells cotransfectedwith pSIF-miR-489 and pCMV-myc-ADAM9, pCMV-myc-MMP7, or the control vector pCMV-myc. Left, representative images; right, bioluminescencequantification of lung metastasis (mean � SD; n ¼ 5 mice in each group). E, H&E staining of the lungs (left; scale bar, 2 mm) and the number of metastaticnodules in mice on day 50 after tail vein injection of the indicated PDAC cells. Data in the right are presented as mean � SD (n ¼ 5 mice in each group). F and G,ADAM9 and MMP7 overexpression rescued liver metastatic colonization of PDAC cells in NOD/SCID mice. F, Bioluminescence imaging of NOD/SCID mice onday 60 after splenic injection of BxPC-3-luc cells cotransfected with pSIF-miR-489 and pCMV-myc-ADAM9, pCMV-myc-MMP7, or the control vector pCMV-myc.Left, representative images; right, bioluminescence quantification of liver metastasis (mean � SD; n ¼ 4 mice in each group). G, Brightfield imaging of thelivers (left; scale bar, 5 mm) and the number of visible liver metastases (right) in mice on day 60 after splenic injection of the indicated PDAC cells. Data on theright are presented as mean � SD (n ¼ 4 mice in each group). All data are mean � SD. Statistics: Student t test, � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






Figure 6.

Comparison of miR-489 expression and YY1, ADAM9, andMMP7 expression in human PDAC specimens. A,miR-489 expression was further reduced in human PDACspecimens from metastatic patients, whereas expression of YY1, ADAM9, and MMP7 was enhanced. qRT-PCR analyses of miR-489 and YY1, ADAM9, andMMP7 mRNA expression in 30 primary tumor and paired adjacent normal tissue specimens from nonmetastatic or metastatic patients. B, Pearson correlationanalyses of miR-489 and YY1mRNA (left), ADAM9mRNA (middle), orMMP7mRNA (right) in 60 human PDAC specimens. C, Immunohistochemical staining of YY1,ADAM9, and MMP7 (brown) and in situ hybridization of miR-489 (blue) in human PDAC sections with wild-type KRAS (left, n ¼ 10) or mutant KRAS (right,n ¼ 10). Sections were counterstained with Mayer hematoxylin (blue) for immunohistochemical staining or nuclear fast red sodium salt (light red) for in situhybridization analyses. Four of 10 stained tumor specimens in each group are shown as representative images. Scale bars, 50 mm. D, Model of miR-489as a key regulatory node linkingoncogenicKRASmutations toPDACmetastasis. All data aremean�SD. Statistics: Student t test, � ,P<0.05; �� ,P<0.01; �� ,P<0.001.

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ADAM9 and MMP7 proteins. Cotransfection of these vectorsin PANC-1 cells barely altered cell proliferation and anchor-age-independent growth (Supplementary Fig. S5) but largelyrescued the impact of miR-489 on cell migration in vitroas well as lung and liver metastases in NOD/SCID mice(Fig. 5), supporting the notion that targeting ADAM9 andMMP7 is an important mechanism of miR-489–mediatedsuppression of cell migration and metastasis of PDAC cells.Collectively, these results indicate that the miR-489:ADAM9/MMP7 axis is of functional importance in regulating PDACmetastasis.

The expression of miR-489 and YY1, ADAM9, and MMP7mRNAs is correlated in human PDAC specimens

To test whether our above findings in PDAC cells are clin-ically relevant, we examined the miR-489 level as well as thelevels of YY1, ADAM9, and MMP7 mRNAs in 30 humanprimary PDAC tumors from nonmetastatic and metastaticpatients, respectively. We found that compared with pairedadjacent normal tissue, the miR-489 level was reduced inprimary tumors from nonmetastatic patients and furtherreduced in those from metastatic patients, whereas YY1,ADAM9, and MMP7 mRNAs were all significantly upregulatedin nonmetastatic primary tumors and further upregulated inmetastatic primary tumors (Fig. 6A). Importantly, we found asignificant inverse correlation between miR-489 and YY1mRNA levels (Pearson R ¼ �0.797, P < 0.001), ADAM9 mRNAlevels (Pearson R ¼ �0.804, P < 0.001), or MMP7 mRNA levels(Pearson R¼�0.818, P < 0.001; Fig. 6B). We further found thatPDAC tumors with wild-type KRAS showed greater miR-489expression and diminished YY1, ADAM9, and MMP7 immu-nohistochemical staining (Fig. 6C, left; n ¼ 10), whereastumors with mutant KRAS showed a significantly lower levelof miR-489 expression and more intense YY1, ADAM9, andMMP7 staining (Fig. 6C, right, n ¼ 10; Supplementary Fig. S6).Collectively, these results strongly suggest that the newly dis-covered YY1/miR-489:ADAM9/MMP7 regulatory axis is clini-cally relevant in human PDAC.

DiscussionKRAS mutations occur with a frequency of up to 90% in

pancreatic cancer and as early as the PanIN stage (4). MutantKRAS, alone or in conjunction with other mutant genes such asp53 or p16, drives initiation of pancreatic cancer by inducingacinar-to-ductal transformation andmaintaining progression tometastasis (5, 35). Moreover, both basic and clinical studieshave pointed out that many risk factors influence PDAC pro-gression by inducing inflammation, which accompanies cancerfrom initiation to metastasis (36).NF-kB signaling has beenrecognized as one of the most important pathways that linkinflammation to cancer, including PDAC, which confers a recip-rocal interaction between inflammation and tumor progression(37, 38). The upregulation of p65, the most common NF-kBfamily member, greatly induces the constitutive activation ofNF-kB signaling and promotes PDAC progression into metas-tasis, but the key downstream molecular events still remainlargely elusive (8, 10, 39). In the present study, we establishedmiR-489 as a key regulator of PDAC metastasis, acting down-stream of KRAS/NF-kB signaling (Fig. 6D). The importance ofmiR-489 in mediating PDAC metastasis was supported by

multiple lines of evidence, including in vitro cell migration andinvasion assays, in vivo xenograft experiments, andhumanPDACspecimens.

We found for the first time that the transcription factor YY1 actsdownstream of KRAS/NF-kB signaling to suppress the expressionof miR-489. YY1 is a ubiquitously distributed transcription factorthat directly binds to the promoter region of its target genes (40).Consistently, our results showed that YY1 inhibits miR-489expression by binding to 2 closely spaced sites that are locatednear the MIR489 transcription start site and that this negativeregulation greatly attenuates the expression of miR-489, thuspromoting PDAC metastasis. These findings enriched our under-standing of themechanismof KRAS signaling and YY1 function incancer development, especially in PDAC metastasis progression.Previous studies have shown that YY1 acts as an oncogene topromote cancer development by regulating the cell cycle, cellapoptosis, chemoresistance, inflammation, and cell invasion (40,41), which is in keeping with our finding of upregulation of YY1and YY1 downregulation of miR-489 to promote PDAC cellmetastasis. In addition, dysregulation of YY1 in PDAC and severalother types of cancers has been correlated with the poor prognosisof patients (41–44).

The important role of miRNAs in tumorigenesis is well estab-lished. An increasing body of evidence has demonstrated thatmiRNAs control nearly all aspects of tumorigenesis, includingcancer cell proliferation, apoptosis, and metastasis (11). Here,we uncovered the role of miR-489 as a critical suppressor ofPDAC metastasis, which is consistent with the global miRNAsprofiling data from a previous study showing a strong negativecorrelation ofmiR-489 expression in human PDAC tumors withliver metastasis (45). In addition, miR-489 has been found to bedownregulated in breast, ovarian, and non–small cell lungcancers (46–48). Of note, upregulation of miR-21 by oncogenicKRAS have been shown to promote pancreatic tumor growth(22). Interestingly, our findings show that oncogenic KRASsignaling employs miR-489 to specifically promote metastasis.More importantly, we found that miR-489 expression was wellcorrelated with the progression of PDAC, which is consistentwith the report that miR-489 may be a useful biomarker forPDAC clinicopathologic parameters (45). Moreover, in light ofthe role of miR-489 in PDAC metastasis, this miRNA is also apotential candidate for designing novel therapeutics targetingthe metastasis of PDAC.

We went further to show that miR-489 negatively regulatesPDACmetastasis by targeting 2 keymetastasismediators,ADAM9andMMP7. These genes encode metalloproteinases that remodelthe extracellularmatrix, thereby facilitating cancer cells in forminga local or distant metastasis and are highly expressed in manykinds of cancers, including PDAC (49, 50). Given the importantrole ofADAM9 andMMP7 in regulatingmetastasis, the regulationof these 2 importantmetalloproteinases by theKRAS/NF-kB/YY1/miR-489 axis may be critical to PDAC metastasis.

In summary, our study defined a regulatory axis centered onmiR-489 that plays an important role in oncogenic KRAS-drivenPDAC metastasis. Mechanistically, miR-489 targets 2 metallo-proteinase genes, ADAM9 and MMP7, to inhibit metastasis.Oncogenic KRAS signaling activates inflammatory NF-kB signal-ing to upregulate YY1, the transcriptional repressor of MIR489,thus attenuating the level of miR-489 and thereby promo-ting tumor progression and metastasis. This study not onlyidentified an miRNA-based mechanism underlying oncogenic






KRAS–associated inflammation that promotes PDAC metastasisbut also provided a potential candidate molecule with which todesign oligonucleotide drugs targeting PDAC metastasis.

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

Authors' ContributionsConception and design: P. Yuan, Y. Li, W. Lou, M.-F. LiuDevelopment ofmethodology: P. Yuan, X.-H. He, Y.-F. Rong,W. Lou,M.-F. LiuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Yuan, X.-H. He, Y.-F. Rong, J. Cao, Y. HuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. Yuan, X.-H. He, Y.-F. Rong, Y.-P. Hu, M.-F. LiuWriting, review, and/or revision of themanuscript: P. Yuan, Y. Li, Y. Liu, D. Li,W. Lou, M.-F. LiuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. LiuStudy supervision: W. Lou, M.-F. Liu

AcknowledgmentsWe thank Prof. Xinyuan Liu for pGL3-basic and pmirGLO vectors and

Prof. Ying Yu for help in animal luminescence imaging. The authors are gratefulto Dr. Yonggang Zheng for his critical reading of this article.

Grant SupportThis work was supported by grants from the National Natural Science

Foundation of China (31325008, 91640201, 91419307, 31300656, and31270840), Ministry of Science and Technology of China (2014CB943103,2014CB964802, and2012CB910803), Science and TechnologyCommission ofShanghaiMunicipality (13ZR1464300 and 16XD1404900), andChinese Acad-emy of Sciences (KJZD-EW-L01-2, 2013KIP202, and "Strategic Priority ResearchProgram" Grant XDB19010202).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 15, 2016; revisedOctober 15, 2016; acceptedOctober 19, 2016;published OnlineFirst October 28, 2016.

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2017;77:100-111. Published OnlineFirst October 28, 2016.Cancer Res Peng Yuan, Xiao-Hong He, Ye-Fei Rong, et al. Cancer Metastasis

B/YY1/miR-489 Signaling Axis Controls PancreaticκKRAS/NF-

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