MicroRNA-200a Promotes Anoikis Resistance and Metastasis ... · Anoikis is an important barrier to...

Human Cancer Biology

MicroRNA-200a Promotes Anoikis Resistance andMetastasis by Targeting YAP1 in Human Breast Cancer

San-Jian Yu1, Jing-Ying Hu2, Xia-Ying Kuang1, Jian-Min Luo1, Yi-Feng Hou1, Gen-Hong Di1, Jiong Wu1,Zhen-Zhou Shen1, Hou-Yan Song2, and Zhi-Ming Shao1

AbstractPurpose:Theprocess ofmetastases involves the dissociation of cells from theprimary tumor, penetration

into the basement membrane, invasion, and exiting from the vasculature to seed and colonize distant

tissues. miR-200a is involved in this multistep metastatic cascade. This study aimed to test the hypothesis

that miR-200a promotes metastasis through increased anoikis resistance in breast cancer.

Experimental Design: Breast cancer cells transfected withmimic or inhibitor formiR-200a were assayed

for anoikis in vitro. miR-200a expression was assessed by quantitative real-time PCR (qRT-PCR). Luciferase

assays, colony formation assays, and animal studies were conducted to identify the targets of miR-200a and

the mechanism by which it promotes anoikis resistance.

Results: We found that overexpression of miR-200a promotes whereas inhibition of miR-200a sup-

presses anoikis resistance in breast cancer cells. We identified Yes-associated protein 1 (YAP1) as a novel

target ofmiR-200a.Our data showed that targeting of YAP1 bymiR-200a resulted in decreased expression of

proapoptotic proteins, which leads to anoikis resistance. Overexpression ofmiR-200a protected tumor cells

fromanoikis andpromotedmetastases in vivo. Furthermore, knockdownof YAP1phenocopied the effects of

miR-200a overexpression, whereas restoration of YAP1 in miR-200a overexpressed breast cancer cells

reversed the effects ofmiR-200a on anoikis andmetastasis. Remarkably, we found that YAP1 expressionwas

inversely correlatedwithmiR-200a expression in breast cancer clinical specimens, andmiR-200a expression

was associated with distant metastasis in patients with breast cancer.

Conclusions: Our data suggest that miR-200a functions as anoikis suppressor and contributes to

metastasis in breast cancer. Clin Cancer Res; 19(6); 1389–99. �2013 AACR.

IntroductionBreast cancer is the second-leading cause of cancer-related

death in women. Exciting progress has been made in thesystemic therapy and earlier diagnosis of breast cancer (1).However, distant metastases remain a major obstacle in thesuccessful treatment of breast cancer, and themechanismofbreast cancer metastasis is still not completely understood(2, 3).Emerging evidence indicates that the expression of several

microRNAs (miRNAs), including miR-10b, miR-335 and

miR-31, is altered in the metastatic cascade (4–6). ThemiR-200 family has multiple functions in regulation of cancerstem cells, chemosensitivity, and apoptosis (7–10). It wasrecently reported that themiR-200a suppress the epithelial–mesenchymal transition (EMT), the initiating step ofmetas-tasis (11–14). Interestingly, miR-200 family promotesbreast cancer metastasis to lung in an isogenic model(15, 16).miRNAs are known to target themetastatic cascadeandenhancebothpro- andantimetastatic signals, especiallyin the early and late steps of tumor metastasis (4, 6, 17).

Anoikis is an important barrier to distant metastasis.Resistance to anoikis enhances the survival of cancer cellsduring systemic circulation, thereby facilitating secondarytumor formation in distant organs (14, 15). miR-200a, amember of the miR-200 family, involves multiple steps ofmetastasis, such as suppressing EMT and promoting thelung metastasis. However, the function of miR-200a on theregulation of anoikis remains unknown.

Yes-associated protein 1 (YAP1), a key node of the Hipposignaling pathway, has been implicated as a tumor sup-pressor in breast (18). Binding of Tyr-357–phosphorylatedYAP1 with the PYmotif allows p73 to escape Itch-mediatedubiquitination (19–21). Loss of function of YAP1 wasreported to protect cells from anoikis (18). In this study,we identified a new role of miR-200a in anoikis in breast

Authors' Affiliations: 1Breast Cancer Institute, Department of BreastSurgery, Cancer Hospital/Cancer Institute, Shanghai Medical College,Institutes of Biomedical Sciences, and 2Department ofMolecular Genetics,Shanghai Medical College and Key Laboratory of Molecular Medicine,Ministry of Education, Fudan University, Shanghai, PR China

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

Corresponding Author: Zhi-Ming Shao, Breast Cancer Institute, Depart-ment of Breast Surgery, Cancer Hospital/Cancer Institute, Shanghai Med-ical College, Institutes of Biomedical Sciences, FudanUniversity, Shanghai200032, PR China. Phone: 86-13611709888; Fax: 011-86-2164174774;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-1959

�2013 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 1389

on November 17, 2020. © 2013 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst January 22, 2013; DOI: 10.1158/1078-0432.CCR-12-1959

http://clincancerres.aacrjournals.org/

cancer and confirmed that YAP1 is a novel target of miR-200a.miR-200a–induced anoikis resistance was phenocop-ied by knockdown of YAP1. Furthermore, ectopic expres-sion of miR-200a promoted both anoikis resistance andmetastasis of human breast cancer cells in animal models.In summary, we combine experimental and clinical studiesto establish a role for miR-200a in anoikis resistance. Weshow that by targeting YAP1,miR-200a allows cells to evadeanoikis and embark on a metastasis spread.

Materials and MethodsCell lines and breast tumor specimens

Breast cancer cell lines obtained from American TypeCulture Collection (ATCC) were grown according tothe ATCC-recommended culture conditions. Bcap37 cellswere obtained from the Cell Bank of China andmaintainedin Dulbecco’s Modified Eagle’s Medium (DMEM) contain-ing 10% FBS and 1% antibiotics (Invitrogen). All primaryhuman breast cancers specimens were obtained frompatients undergoing surgery for breast cancer during2003 to 2005 at the Shanghai Cancer Center and stored inliquid nitrogen until analysis. All patients provided writteninformed consent. The study was approved by the EthicsCommittee of the Cancer Hospital, Fudan University(Shanghai, PR China).

siRNA,miRNA, plasmid construction, transfection, andluciferase assays

Specific siRNAs and negative controls (scrambled siRNA)were purchased from Genepharma, and miR-200a mimics,inhibitors, and negative controls were obtained from Ribo-bio. Cells cultured in 6-well plates were transfected with100pmol/well siRNAandmiRNAmimic orwith 400pmol/well antagomiRNA using the Hilymax Transfection Agent(Dojindo) according to the manufacturer’s protocol. Thenegative controls (miRmimics and antagomiRNA) consist-ing of random sequences had no detectable effects inhuman cell lines or tissues. The sequences of the siRNAs,miRNAs, and negative controls are listed in SupplementaryTable S1.

The coding domain sequence of human YAP1mRNAwascloned into NotI/BamHI sites of the pQCXIH vector (Clon-tech). The 30-untranslated region (UTR) of human YAP1mRNA was cloned into the XhoI/NotI sites of the psi-CHECK2 Luciferase vector (Promega). The In-FusionAdvantage PCR Cloning Kit (Clontech) was used in theplasmid cloning. For luciferase assays, HEK293T cellswere plated in 6-well plates and transfected with 4 mg ofthe psiCHECK-YAP1 30-UTR reporter vector per well and100 pmol of the miR-200a mimic or 400 pmol antagomiR-200a or a negative control. Site-specific mutants of the 30-UTR reporters for YAP1 were also tested. The clone and sitemutant primer sequences are listed in SupplementaryTable S1.

Protein extraction and Western blot analysisCells were lysed in SDS cell lysis buffer supplemented

with Complete Protease Inhibitor Cocktail Tablets (RocheDiagnostics) for 30 minutes on ice. The homogenates werecentrifuged at 13,000 rpm for 10 minutes at 4�C. Super-natants were collected, and protein concentrations weredetermined by the Bradford Assay (BioRad). The proteinswere then resolved on SDS-PAGE and transferred to poly-vinylidene fluoride membranes (Millipore). The mem-branes were probed with the following primary antibodies:p73, cleaved caspase-3, andBim(1:1,000, Epitomics);Noxa(1:500; AbD Serotec); YAP1 (1:1,000), Bax (1:1,000), andglyceraldehyde-3-phosphate dehydrogenase (GAPDH;1:2,000; Proteintech). After further washes, the membraneswere incubated with the appropriate horseradish peroxi-dase–conjugated secondary antibodies (1:5,000; Protein-tech), and blots were developed using ECL (Millipore).Quantity One software (BioRad) was used to quantify YAP1and GAPDH expression levels.

RNA extraction and quantification of mature miRNAsThe tissues were homogenized with Polytron PT100.

Total RNA was extracted using the TRIzol reagent (Invitro-gen) or the miRVana miRNA Isolation Kit (Ambion). Theprimers for miR-200a and U6 detection assays werepurchased from Ribobio. Total RNAs, isolated from celllines or tissues, were reverse-transcribed using a specificstem-loop RT primer (50 nmol/L) and the ReverTraAceqPCR RT Kit (Toyobo). The RT conditions consistedof 15 minutes at 42�C followed by 5 minutes at 98�C.Levels of mature miRNAs were quantified by quantitativereal-time PCR (qRT-PCR) using the SYBR Green Real-timePCR Master Mix (Toyobo). Quantitative PCR was carriedout using an Applied Biosystems 7900HT real-time PCRsystem, and data were collected and analyzed using ABISDS version 2.3. To calculate relative concentrations,miR-200a and U6 CT values were obtained for all sam-ples. The mRNA level of GAPDH was used as an internalcontrol for gene-specific mRNA analysis. The normalizedexpression of each sample was designated as CT andobtained by dividing the CT value of miR-200a by theU6 CT of the same sample. The relative amount of miRNAor mRNA in each sample was calculated using the

Translational RelevanceDistant metastasis remains a major obstacle to suc-

cessful breast cancer treatment. Anoikis (detachment-induced apoptosis) has been suggested to act as a barrierto metastasis. Here, we report that miR-200a, function-ing as an anoikis suppressor, promotes anoikis resistanceby targeting Yes-associated protein 1 (YAP1) and subse-quently leads to distant metastasis in breast cancer. Ourstudy also provides evidence that overexpression ofmiR-200a might affect the prognosis of patients with breastcancer. Therefore, understanding the mechanism andfunction of miR-200a as a prometastatic miRNA mayprovide new therapeutic opportunities for breast cancertreatment.

Yu et al.

Clin Cancer Res; 19(6) March 15, 2013 Clinical Cancer Research1390




comparative CT method. The results are presented as foldchange of expression in cells or cancer tissues.

Anoikis assayAnoikis was induced by polyHEMA (Sigma) culture. A

solution containing 20 mg/mL polyHEMA in 95% ethanolwas made, and 0.95 ml/mm2 of this solution was pipettedinto 35-mm wells. The plates were allowed to sit, partiallycovered, until the ethanol evaporated and the poly-HEMAhad solidified. Periodically, the plates were rocked by handto help ensure even coating. Before use, the wells werewashed twice with PBS and once with DMEM to removeresidual ethanol. In all, 105 cells of each line, suspended in 2mL DMEM with 10% FBS, were incubated in the poly-HEMA-coatedwells for 48 hours in a humidified (37�C, 5%CO2) incubator. After incubation, the cells were used forfurther analyses.

Apoptosis and colony formation assays of cells treatedin suspension cultureFollowing induction of anoikis for 48 hours, caspase-3

activity was measured using the Caspase-Glo 3/7 Assay Kit(Promega) according to the manufacturer’s instructions.Apoptosis was detected by fluorescein isothiocyanate(FITC)-Annexin V/propidium iodide (PI) double staining(Invitrogen). Afterwashing twicewithPBS, cells were labeledwith FITC-Annexin V and PI for 10 minutes in the dark atroom temperature, and cellular fluorescence was measuredusing a FACScan flow cytometer (Becton Dickinson). Eachexperiment was carried out in triplicate. After suspensionculture, cells were seeded into normal plates at a density of2,000 cells/100-mm2 dishes. For colony formation assays,plates were incubated for 2 weeks, and the resulting colonieswere stained with crystal violet and counted.

Animal studiesMDA-MB-231 cells were transfected with synthetic hsa-

mir-200a, YAP1 siRNA, or scrambled RNA and then labeledwith 1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocya-nine perchlorate (DiI; Invitrogen). Labeled cells were resus-pended in culturemediumat a concentration of 107 cell/mLand injected into the center of the yolk sac of transgenic fli:EGFP zebrafish (25 embryos/group and 100 cells/embryo).One and 3 days after injection, DiI-labeled MDA-MB-231cells were detected in GFP-labeled vessels using a fluores-cence microscope, and surviving cells were counted in thetail vein (22). Cell-Injected zebrafish were examined withOlympus BX61/SZX12 microscopes and photographedwith a DP70 digital camera. Images were processed usingAdobe Photoshop software. The data were derived from 3replicated experiments.Four- to 6-week-old athymic female BALB/c nu/nu mice

were provided by the Shanghai Institute of Materia Medicaat theChinese Academyof Science.MDA-MB-231 cells wereinfected with pMR-miR-200a and pMR-miR-Ctrl (Geneco-poeia). YAP1-reintroduced MDA-MD-231 cells were alsoused in the experiments. GFP-labeled MCF-7 cells weretransfected with antagomiR-200a and antagomiR-Ctrl.

Those cells were resuspended in PBS at a concentration of107 cells/mL, and animals were injectedwith 100 mL of eachcell line. In all experiments, the cells were introduced via tailvein injection. The animals were sacrificed and autopsied 3weeks after injection. Metastasis was assessed by macro-scopic observation of the major organs for secondarytumors and confirmed by histology. All animal experimentswere carried out with approval from the Shanghai MedicalExperimental Animal Care Commission.

Statistical analysisDatawere shown asmean� SEMunless otherwise noted.

Statistical significance was analyzed by the unpaired Stu-dent t test, and P < 0.05 was considered to be statisticallysignificant.

ResultsmiR-200a is a determinant of anoikis in breast cancer

miR-200a expressionwas evaluated in 7 breast cancer celllines by qRT-PCR. Three cell lines (MCF-7, MDA-MB-468,and HBL100) had high levels of miR-200a, whereas theremaining cell lines showed significantly lower expression(Fig. 1A) consistent with the previous report (23). Toexamine the role of miR-200a in anoikis, MDA-MB-231(i.e., low miR-200a expression) and MCF-7 (i.e., high miR-200a expression) cells were transfected with a miR-200amimic and antagomiR-200a, respectively. The transfectedcells were transferred into polyHEMA-coated plates for 48hours incubation, which prevents cell attachment andinduces anoikis (Fig. 1B and C and Supplementary Fig.S1). First, Annexin V/PI staining showed that MCF-7 antag-omiR-200a cells were sensitive to suspension-induced apo-ptosis (Fig. 1B and C, P¼ 0.003). In contrast, theMDA-MB-231 miR-200a mimic cells were resistant to suspension-induced apoptosis (Fig. 1B and C, P < 0.01). Second,caspase-3 cleavage was observed in MCF-7-antagomiR-200a cells when they were brought into suspension. Incontrast, cleavage of caspase-3 was suppressed in MDA-MB-231 cells transfected with miR-200a mimic (Fig. 1D).Third, following 48 hours of suspension culture, the cellswere replated and cultured in normal conditions, and thecolonies were scored after an additional 2 weeks of culture.Transfection of miR-200a mimic increased colony forma-tion in MDA-MB-231 cells (Fig. 1E, P < 0.01). However,silencing miR-200a with antagomiR-200a reduced colonyformation inMCF-7 cells (Fig. 1E, P < 0.01). Together, theseresults show a critical role for miR-200a in the suppressionof anoikis in breast cancer cells.

YAP1 is negatively regulated by miR-200aTo identify the miR-200a targets, we conducted an in silico

search using Targetscan, MiRanda, and Diana (24–27).Cytoscape was used to find an intersection between genesthat regulate the apoptotic processes (GO: 0042981) andpredicted target genes of miR-200a (Fig. 2A; refs. 28, 29).Among the potential targets which are listed in Supplemen-tary Table S2, 5 candidate genes (TGFB2, TIAM1, FOXA1,

MicroRNA-200a Promotes Anoikis Resistance by Targeting YAP1

www.aacrjournals.org Clin Cancer Res; 19(6) March 15, 2013 1391




15

10

5

0

−5

−10

25

20

15

10

5

0

300

250

200

150

100

50

0

MCF-7

miR-Ctrl

miR-Ctrl

miR-Ctrl

miR-Ctrl

miR-200a mimic

miR-200a mimic

miR-200a mimic

miR-200a mimic

miR-Ctrl

MCF-7

Cleaved caspase-3

1.0 1.3 0.5 2.1

18 kDa

36 kDaGAPDH

MDA-MB-231

MCF-7

MDA-MB-231

MCF-7

Colo

ny n

um

bers

MDA-MB-231

miR-200a mimic

AdherentA B

C

E

D

AntagomiR-Ctrl

AntagomiR-Ctrl

AntagomiR-Ctrl

AntagomiR-Ctrl

AntagomiR-200a

AntagomiR-200a

AntagomiR-200a

AntagomiR-200a

miR

-Ctrl

miR

-200a mim

ic

AntagomiR

-Ctrl

AntagomiR

-200a

AntagomiR-Ctrl AntagomiR-200a

Suspension

Adherent

Apopto

sis

(%

)

Suspension

MDA-MB-231

HBL100

MCF-7

MCF-7

MDA-M

B-468

MDA-M

B-231

MDA-M

B-231

MCF-7

MDA-M

B-231

ZR-75-1

ZR-75-30

Bcap37

Re

lative

miR

-20

0a

exp

ressio

n

Figure1. miR-200a induces anoikis resistance in breast cancer cells. A,miR-200a andU6expression in 7 breast cancer cell lineswas determined by qRT-PCR.Relative expression was calculated by obtaining normalized CT values, such that the CT value of miR-200a was normalized to the U6 CT value fromthe same sample. The resulting value was then transformed by applying 2�DCT . Mean � SD of normalized CT from 3 independent experiments. B, apoptosiswas evaluated after culturing MDA-MB-231 and MCF-7 cells in adherent and suspension and staining with Annexin V at 48 hours. The flow cytometricprofile depicts Annexin V/FITC staining on the x-axis and PI staining on the y-axis. C, the number represents the percentage of early apoptotic cells in eachcondition. Mean � SEM of apoptotic cells from 3 independent experiments. D, after suspension culture, the transfected cells were lysed for Westernblotting. The protein levels of cleaved caspase-3were normalized toGAPDH. E, forty-eight hours after the induction of anoikis, transfectedMDA-MB-231 andMCF-7 cells were used for the colony formation assay. Colonies were stained with crystal violet after 2 weeks in culture (left). Mean � SEM of coloniesfrom 3 independent experiments was shown in the right. ��, P < 0.01.

Yu et al.





YAP1, and ITSN1) were selected. We conducted colonyformation assay to screen the genes. YAP1 attracted ourattention because itmostly promoted colony formation aftersuspension culture. (Supplementary Fig. S3). YAP1 expres-sion was also reported lost in human breast tumors (18).To confirm YAP1 is a target of miR-200a, 30-UTR of

the YAP1 gene was cloned into the luciferase constructpsiCHECK2 (Fig. 2B). Luciferase assay revealed that miR-200a significantly reduced the activity of the luciferasereporter gene fused to the YAP1 30-UTR by 52.5% �

4.9% compared with miR-Ctrl in HEK293T cells. Intrigu-ingly, miR-200a did not decrease the luciferase activity of amutant construct that contained substitutions at 3 nucleo-tides within the miR-200a–binding site (Fig. 2C). In con-trast, when thewild-type reporterwas cotransfectedwith theantagomiR-200a, the relative luciferase activity of thereporter was significantly enhanced (P < 0.01; Fig. 2D).These results suggested that YAP1 was a direct target ofmiR-200a. Furthermore, we found that transfection with themiR-200a mimic caused a 60% reduction of YAP1 protein

Diana

Targetscan GO: 0042981

Regulation of

apoptotic process

miRanda

A B

C D

SV40 pro TK proRenilla luciferase Firefly luciferase3′ UTR polyA SV40 polyA497 3420

Hsa-miR-200a 3′

YAPI 3′ UTR BS 5′

YAP1

2.5

2.0

1.5

1.0

0.5

0.0

2.0

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Lucife

rase a

ctivity (

FLU

/RLU

)

Lucife

rase a

ctivity (

FLU

/RLU

)GAPDH

70 kDa

miR

-Ctrl

miR

-200

a m

imic

miR-Ctrl

****

miR-200a mimic

Antag

omiR

-Ctrl

Antag

omiR

-200

a

AntagomiR-CtrlAntagomiR-200a

1.0 0.4 1.0 1.5

36 kDa

YAP1

GAPDH

70 kDa

36 kDa

YAPI 3′ UTR Mutant BS 5′

YAP1 3′ UTR

YAP1 MT3′ U

TR

YAP1 3′ UTR

YAP1 MT3′ U

TR

8

657 1238

Figure 2. miR-200a targets YAP1 through an interaction with the YAP1 30-UTR. A, the intersection between genes that regulate the apoptotic processes(GO: 0042981) and Targetscan, MiRanda, and Diana predicted target genes is defined as the overlapping fraction of all 4 databases. B, schematicdiagrams of human YAP1 30-UTR luciferase constructs with wild-type and mutant (YAP1-30-UTR) miR-200a target sequences. C, HEK293T cells weretransfected with the luciferase constructs shown in B and 100 pmol miR-200a and harvested 48 hours later. Firefly luciferase activity is normalized to Renillaluciferase activity. Values are expressed as the mean � SD of 3 replicate, independent experiments. MDA-MB-231 cells were transfected with anmiR-200amimic or control oligonucleotide and subjected toWestern blotting (top). D, HEK293T cells were transfectedwith the luciferase constructs shown inB and 400 pmol antagomiR-200a and harvested 48 hours later. Firefly luciferase activity is normalized to Renilla luciferase activity. Values are expressedas the mean � SD of 3 replicate, independent experiments. MCF-7 cells were transfected with the antagomiR-200a and control oligonucleotide for48 hours and then subjected to Western blotting (top). The protein levels of YAP1 were normalized to GAPDH. ��, P < 0.01.






expression in MDA-MB-231 cells (Fig. 2C). In contrast,suppression of miR-200a with antagomiR-200a increasedYAP1 protein expression in MCF-7 cells (Fig. 2D). Thesedata indicate that miR-200a directly targets the 30-UTR ofYAP1 to repress the expression of YAP1 protein.

miR-200a suppresses anoikis by targeting YAP1We next investigated the correlation between miR-200a

and YAP1 expression in breast cancer cell lines. Four among7 cell lines expressed high levels of miR-200a presentedundetectable or low-levels of YAP1. Two cell lines (MDA-MB-231 and ZR-75-30) that expressed low levels of miR-200a expressed high levels of YAP1 (Fig. 3A). To furtherexplore the effect of YAP1 on suspension-induced apopto-sis, MDA-MB-231 and ZR-75-30 cells were transfected withYAP1 siRNA followed treated with 48 hours of suspensionculture, then replated, and cultured under normal condi-tions. Both cell lines exhibited increased colony formation(Supplementary Fig. S1A and S1B).

Because YAP1 is a key modulator of p73, which regulatesproapoptotic genes, we examined caspase-3 cleavage andcaspase-3/7 activity in YAP1 siRNA–transfected cells. Thedata showed knockdownof YAP1 inhibited caspase-3 cleav-age in both MDA-MB-231 and ZR-75-30 cells (Fig. 3B) andthe caspase-3/7 activity also repressed by YAP1 siRNA inthose cells. Furthermore, we examined whether the expres-sion of Noxa, Bax, and Bim, 3 pro-apoptotic proteins, wasaffected bymiR-200a in breast cancer cell lines (19, 30–32).Western blotting revealed that basal levels of p73, Noxa,Bax, andBimwere reduced following transfectionwithmiR-200a and YAP1 siRNA in MDA-MB-231 and ZR-75-30 cellstreated in suspension culture (Fig. 3D). These data sug-gested that miR-200a targeting of YAP1 suppresses theproapoptotic protein expression and allows the cells toevade anoikis.

Transfection of miR-200a promoted in vivo breastcancer cell metastasis in zebrafish and mouse models

To analyze the in vivo effect of miR-200a on tumor cellmetastasis, transgenic fli:EGFP zebrafish embryoswithGFP-labeled blood vessels were prepared as an animal model.The transparent nature of zebrafish embryos allows visual-ization of tumor cells dissemination in living fish. Immuneprivilege of zebrafish embryos facilitates implantation ofmammalian tumor cells (22, 33). MDA-MB-231 cells weretransfectedwithmiR-200amimic or YAP1 siRNA, andMCF-7 cells were transfected with antagomiR-200a or antago-miR-Ctrl. After 48 hours, the transfected cells were labeledwithDiI and injected into the abdomen of zebrafish embry-os. After injection, cells that seeded in the blood vessels ofthe zebrafishwere detected, and zebrafish embryos contain-ing metastatic cells were counted at 24 hours and 72 hourseparately. (Fig. 4A and Supplementary Table S3). Cellstransfected with the YAP1 siRNA or miR-200a mimic wereseeding in nearly 75% injected zebrafish embryos, respec-tively. However, control MDA-MB-231 cells were observedin 40%. The metastatic cells in the tail vessels were alsocounted (Fig. 4B). Approximately 40% of miR-200a mimic

or YAP1 siRNA–transfected cells were able to seed in the tailvein, but only 20% of the control transfected cells could doso (Supplementary Table S4). In addition, MCF-7 antag-omiR-200a–transfected MCF-7 cells showed an obviousdecrease of survival cells in zebrafish tail vein comparedwith antagomiR-Ctrl–transfected cells (Fig. 4A and B).

To further examine the effects of miR-200a on metastasisin a mouse model, the metastatic potential of stably miR-200a and scramble-transfected MDA-MB-231 cells wereinjected into tail vein in BALB/c nude mice. Intravenousinjection of pMR-miR-200a–transfectedMDA-MB-231 cellslabeled withGFP inducedmarked lung colony formation invivo, whereas MDA-MB-231 cells transfected with the con-trol vector induced fewer lung metastases (Fig. 4C, Supple-mentary Fig. S1C). Statistic results showed a significantdifference between the number of lung colonies derivedfrom miR-200a–overexpressing MDA-MB-231 cells andcontrol cells (Fig. 4D). In addition, we introduced a con-struct expressing YAP1 into miR-200a stably expressing cellline ofMDA-MB-231. The restoration of YAP1 expression inthe stable cell line was confirmed by Western blotting(Supplementary Fig. S4C). The tail vein injection studyshowed that restoration of YAP1 could significantly reversethe metastasis promotion imposed by miR-200a (Fig. 4CandD). Collectively, the data suggest that overexpression ofmiR-200a or silencing of YAP1 may promote breast cancercells evasion of anoikis in the circulation, thereby facilitat-ing cancer metastasis.

Inverse correlation between YAP1 and miR-200aexpression in breast cancer patients

To address whether the expression of YAP1 is associatedwithmiR-200a inpatientswith breast cancer,miR-200a andYAP1 expression were examined in specimens from 16patients with breast cancer by qRT-PCR and Western blot-ting, respectively. Our investigation showed that the expres-sion ofmiR-200awas inversely correlated with that of YAP1in 16 patients such that YAP1 was expressed at a lower levelin specimens overexpressing miR-200a (Fig. 5A and B).Moreover, analysis of 40 human breast cancer specimensby qRT-PCR showed a significant negative correlationbetween YAP1 and miR-200a expression levels by Pearsoncorrelation analysis (R ¼ �0.562, P ¼ 0.000159, n ¼40); Fig. 5C). These data provides further evidence of afunctional link between miR-200a and YAP1 in breastcancer.

miR-200a is associated with breast cancer metastasisTo further understand the relationship between

miR-200a and breast cancer metastasis, we conductedmiR-200a expression analyses on a series of breast tumorsamples (n ¼ 110, Cancer Hosipital Fudan UniversityCollection). The relationship between themiR-200a expres-sion levels and clinicopathologic parameters of breast can-cer was summarized in Table 1. The results showed that nosignificant correlationbetween themiR200a expression andage, histologic type, size, stage, estrogen receptor (ER),progesterone receptor (PR), and Her2. However, we found

Yu et al.





that miR-200a expression was upregulated by more than7-fold in lymph node–positive specimens compared withlymph node–negative specimens. Moreover, in metasta-sis-positive patients, the miR-200a expression was 10-

fold compared with metastasis-negative patients (Table1). Thus, these findings suggested that high miR-200alevels were associated with metastatic behavior in breastcancer.

15

10

5

0

−5

−10

miR-200a

r = −0.801P = 0.03n = 7

YAP1

YAP1

Cleaved caspase-3

70 kDa

0.9

1.0 1.00.5 0.4 0.20.3

1.0 1.00.3 0.3 0.10.2

1.0 1.00.1 0.0 0.10.0

1.0 1.00.2 0.6 0.80.1

1.0 1.00.7 0.5 0.60.3

0.5 1.7 0.9 1.5 1.1 1.0

1 1

0.9 1 0.4 1

ZR-75-30 ZR-75-30

10.5

36 kDa

70 kDa

70 kDa

36 kDa

24 kDa

22 kDa

16 kDa

20 kDa

36 kDa

20 kDa

36 kDa

GAPDH

GAPDH

Cleaved caspase-3

GAPDH

GAPDH

MDA-MB-231 MDA-MB-231

MDA-MB-231 ZR-75-30

Adherent

A B

C D

Suspension

Adherent

ScrambleYAP1 siRNA

YAP1

P73

Noxa

Bim

Bax

Rela

tive

caspase-3

/7 a

ctivity (

RLU

)

10,0009,0008,0007,0006,0005,0004,0003,0002,0001,000

0

600,000

500,000

400,000

300,000

200,000

100,000

Suspension

****

Fold

change

MDA-M

B-468

MDA-M

B-231

MCF-7

HBL100

Bcap37

YAP1 siR

NA

YAP1 siR

NA

miR

-200a mim

ic

miR

-200a mim

icYA

P1 siR

NA

Scram

ble

Scram

ble

Scram

bleYA

P1 siR

NA

Scram

ble

ZR-75-1

ZR-75-30

MDA-M

B-231

ZR-75-30

MDA-M

B-231

ZR-75-30

Figure 3. miR-200a attenuates anoikis induced apoptosis in breast cancer cells. A, relative expression levels of YAP1 and miR-200a in human breast cancercell lines and Western blot analysis of YAP1 protein expression. Pearson correlation of the YAP1 and miR-200a expression levels in cell lines (n ¼ 7,R¼�0.803, P¼ 0.03). B, after adherent or suspension culture, the transfected cells were lysed for Western blotting. The protein levels of cleaved caspase-3were normalized to GAPDH. The data were derived from 3 replicated experiments. C, forty-eight hours after the induction of anoikis, lysates werecollected fromMDA-MB-231andZR-75-30cells transfectedwithYAP1siRNA toanalyze the activationof caspase-3/7 using theCaspaseGLOassay system.Values are expressed as the mean � SD of 3 replicate, independent experiments. D, cellular lysates of the miR-200a mimic, YAP1 siRNA, and controloligonucleotide transfectedMDA-MB-231 and ZR-75-30 cells were used forWestern blotting to detect YAP1, p73, Noxa, Bim, BAX, andGAPDH expression.The data were derived from 3 replicated experiments. ��, P < 0.01.






C

D

pMR-miR-CtrlpMR-miR-200a

GFP

H&E

pMR-miR-200a

pQCXIH-YAP1

pMR-miR-200a

pQCXIH

Num

ber

of m

icro

meta

sta

ses

per

section

0

5

10

15

20

25

30

pMR-miR-Ctrl

pMR-miR200a+pQCXIHpMR-miR-200a

pMR-miR200a+pQCXIH-YAP1

MDA-MB-231MDA-MB-231

**

**

A0 hour 24 hour 72 hour

miR-200a

miR-Ctrl

YAP1 siRNA

Scramble

0 hour

AntagomiR-200a

AntagomiR-Ctrl

B

MDA-M

B-231

MDA-M

B-231

MCF-7

miR-Ctrl

Scramble

AntagomiR-Ctrl

miR-200a mimic

YAP1 siRNA

AntagomiR-200a

*

**

*

Cell

num

bers

in tail

vein

0

10

20

30

40

50

60

Figure 4. Detection of metastatic breast cancer cells in in vivo animal models. A, MDA-MB-231 cells were transfected with miR-200a mimic, miR-Ctrl, YAP1 siRNA, orscramble RNA. MCF-7 cells were transfected with antagomiR-200a and antagomiR-Ctrl. All transfected cells were labeled with DiI and injected into the center of the yolksac of transgenic fli:EGFP zebrafish embryos. Twenty-four and 72hours after injection,DiI-labeled MDA-MB-231 and MCF-7 cells were detected in GFP-labeled vesselsusing a fluorescence microscope. The data were derived from 3 replicatedexperiments. B, the number of metastatic cells in zebrafish tail vessels at 72 hours areexpressed as the mean � SD of 3 replicate, independent experiments. C,representative GFP and hematoxylin and eosin (H&E)-stained lung sections frommiceintravenously injected with MDA-MB-231 cells. The arrowhead points to the tumorfocus formed in the lung. The number of metastases in the lung were counted andanalyzed. D, lung colony numbers were macroscopically counted using dissectingmicroscopes. �, P < 0.05; ��, P < 0.01.

Yu et al.





DiscussionIn this study, we show that miR-200a promotes breast

cancer metastasis by targeting YAP1. We also proved thatupregulation of miR-200a is associated with anoikis resis-tance. Our data show that miR-200a could reduce theexpression of YAP1 by directly binding to the 30-UTR ofYAP1. As a result, caspase-3, p73, and its downstream pro-apoptotic proteins were indirectly inhibited by miR-200a.Silencing YAP1 largely phenocopied miR-200a–inducedanoikis resistance. In addition, an inverse correlationbetween miR-200a and YAP1 expression was observed inbreast cancer cell lines and tumor specimens.The miR-200 family is known to suppress EMT, the

initiating step of metastasis, by targeting Zeb1 and Zeb2(13). To verify whether miR-200a influences EMT of breastcancer cells, we examined the expression of 4 EMT markers(E-caherin, N-cadherin, vimentin, and fibronectin) by qRT-PCR inMDA-MB-231 andMCF-7 cell lines. With miR-200amimic transfection, MDA-MB-231 cells showed lowervimentin expression and higher E-cadherin expression thancontrol group. In contrast, we observed elevated vimentinand decreased E-cadherin in antagomiR-200a–transfectedMCF-7 cells (Supplementary Fig. S2). These results, togetherwith those previous studies, indicate that overexpressionmiR-200a may suppress metastasis in breast cancer (12,13, 23, 34). However, some evidence showed that over-expression of miR-200 in mouse breast cancer cells pro-moted lung metastasis (15, 16). Previous identified miR-200a targets were inadequate to explain miR-200a prome-tastatic phenotypes. It is impliedmiR-200amay be involved

inmultiple steps of metastasis. A recent article reported thatmiR-200s promote 4TO7mouse carcinoma cells metastaticcolonization by directly targeting Sec23a, which mediatessecretion of metastasis-suppressive proteins (16, 35). How-ever, relatively little is known about the role of miR-200a inthe translocation of tumor cells. The function of miR-200ain anoikis is still need to be elucidated. Herein, we identify anovel role for miR-200a in imbuing human breast cancercells with anoikis resistance. In vivo mouse model showedthat antagomiR-200a–transfected MCF-7 cells failed toform lung metastasis. Considering tumor metastasisinclude several steps, several regulators involve in the phe-notype of metastatic potential (14, 36). The impact of miR-200a expression on the metastasis of breast cancer seems tovary according to histopathologic character and stage ofcancer. Our results provide an explanation for how miR-200a inhibits EMT but promotes lung metastasis. At theinitial stage, low levels of miR-200a promote invasion andintravasation. However, once tumor cells enter into thecirculation, high miR-200a expression is required for effi-cient survival and further colonization of secondary organs.Thus, miR-200a may exert its prometastatic effects at mul-tiple steps in the metastatic cascade.

The proper execution of apoptosis is a fundamentalsafeguard against tumorigenesis and metastasis. YAP1 is awell-defined proapoptotic transcriptional factor (19). Theinhibition of YAP1 expression greatly reduces detachment-induced apoptosis. Nuclear translocation of YAP1 wasinhibited by phosphorylation at the Ser127 site (37).Cotranscriptional function of YAP1 was related to thisposttranslational modification (38). Our study has shown

Figure 5. miR-200a expression isinversely correlated with YAP1expression in breast cancer tissue.A, Western blot analyses of YAP1expression in 16 breast cell lines (top2 panels). B, ranks of YAP1 andmiR-200a expression. C, Pearsoncorrelation of the YAP1 and miR-200a expression levels in patientswith breast cancer (n ¼ 40; R ¼�0.5621, P ¼ 0.00158).

YAP11.0

T1

25

20

15

10

5

0

0 2 4 6 8 10 12 14 16 18 20

ΔC T V

alu

e Y

AP

1/G

AP

DH

Rela

tive

expre

ssio

n (

Ra

nks)

ΔC T Value miR-200a/U6

miR-200a

A C

B18

16

14

12

10

8

6

4

2

0

YAP1

T2 T3 T4 T5 T6 T7 T8

T1 T2 T3 T4 T5 T6 T7 T8

T9 T10 T11 T12 T13 T14 T15 T16

T9 T10 T11 T12 T13 T14 T15 T16

2.1 0.5 0.0 1.2 0.5 0.7 0.1

0.1 0.2 0.9 0.8 0.6 1.7 3.2 0.0

GAPDH

YAP1

GAPDHr = −0.562

P < 0.01n = 40






that miR-200a directly targets the 30-UTR of YAP1 to repressthe expression of YAP1. These findings suggested that miR-200a negatively regulates YAP1 and not only provides anunderlying mechanism for the aberrant expression of YAP1in breast cancer but also shows the importance of posttran-scriptional regulation of YAP1.

Resistance to anoikis, which is a critical step in themetastatic cascade, allows cells to survive within the circu-latory and lymphatic systems (35, 39). Our findings show

that miR-200a promotes tumor cell survival in the circula-tory system through conferring anoikis resistance, indicat-ing thatmiR-200amay play roles in the prognosis of cancer.In ovarian cancer, overexpression of the miR-200a corre-lated with a poor prognosis (40). Patients with high expres-sion ofmiR-200a had a shorter median overall survival timethan in those lacking significant miR-200a expression (27.5vs. 61.0 months, respectively; ref. 41). The present resultsindicated that miR-200a expression correlates with lymphnodemetastasis anddistantmetastasis, which are significantprognosis factors in patients with breast cancer. However,the correlation was not observed betweenmiR-200a expres-sion and traditional pathologic markers, such as histologictype, hormone receptor status, and tumor–node–metastasis(TNM) stage. One of the reason may be a relatively limitednumber of patients was used. The other could be the com-plex function of miR-200a in cancer progression.

In summary, we combined experimental and clinicalstudies to establish a role formiR-200a in anoikis resistance.We showed that miR-200a promotes anoikis resistance andmetastasis via targeting YAP1.

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

Authors' ContributionsConception and design: S.-J. Yu, H.-Y. Song, Z.-M. ShaoDevelopment of methodology: S.-J. Yu, J.-Y. Hu, X.-Y. KuangAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): S.-J. Yu, J.-Y. Hu, X.-Y. Kuang, J.-M. LuoAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): S.-J. Yu, Y.-F.Hou,H.-Y. Song, Z.-M. ShaoWriting, review, and/or revision of the manuscript: S.-J. Yu, H.-Y. Song,Z.-M. ShaoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J.-Y. Hu, G.-H. Di, J.Wu, Z.-Z. Shen,Z.-M. ShaoStudy supervision: G.-H. Di, J. Wu, Z.-Z. Shen, Z.-M. Shao

AcknowledgmentsThe authors thank all the subjects of this study for their participation, Yin

Chen and her colleagues at Shanghai Institute of Materia Medica, ChineseAcademy of Science for their excellent works in animal experiments, andDongyin Guan and Dan Liu for their critical reading of the manuscript.

Grant SupportThis research is supported by grants from the National Natural Science

Foundation of China (30971143, 30972936, and 81001169), the ShanghaiUnited Developing Technology Project of Municipal Hospitals(SHDC12010116), the Key Clinical Program of the Ministry of Health(2010–2012), and the Shanghai Key laboratory of Breast Cancer(12DZ2260100).

The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked adver-tisement in accordancewith 18U.S.C. Section 1734 solely to indicate this fact.

Received June 15, 2012; revised January 3, 2013; accepted January 10,2013; published OnlineFirst January 22, 2013.

References1. Berry DA, Cronin KA, Plevritis SK, Fryback DG, Clarke L, ZelenM, et al.

Effect of screening and adjuvant therapy on mortality from breastcancer. N Engl J Med 2005;353:1784–92.

2. Eccles SA, Welch DR. Metastasis: recent discoveries and novel treat-ment strategies. Lancet 2007;369:1742–57.

3. Weigelt B, Peterse JL, van't Veer LJ. Breast cancer metastasis:markers and models. Nat Rev Cancer 2005;5:591–602.

4. Ma L, Teruya-Feldstein J, Weinberg RA. Tumour invasion and metas-tasis initiated by microRNA-10b in breast cancer. Nature 2007;449:682–8.

Table 1. The relationship between miR-200aexpression and clinicopathologic parameters inbreast cancer

Clinicopathologicparameters

Numberof cases

Median expressionof miR-200a(normalized CT)

a P

Age, y 0.676�40 11 8.135 � 4.353>40 99 9.880 � 3.706

Histologic type 0.887DCIS 19 10.24 � 3.947IDC 91 9.160 � 3.404

TNM stage 0.523I þ II 104 9.295 � 3.534III 6 11.05 � 2.894

Lymph node status <0.001Negative 45 11.15 � 2.835Positive 65 8.281 � 3.653

Tumor size 0.322T1 þ T2 97 9.430 � 3.491T3 þ T4 13 10.55 � 3.370

ER 0.713Negative 43 9.880 � 3.756Positive 67 9.141 � 3.335

PR 0.923Negative 47 9.880 � 3.063Positive 63 9.160 � 4.243

Her2 0.874Negative 91 9.160 � 3.580Positive 19 10.06 � 3.096

Distant metastasisb <0.001Absent 76 10.95 � 3.351Present 34 7.574 � 2.418

All cases 110 11.12 � 3.770

aNormalized CT ¼ CT miR-200a � CT U6.bIn this assay,metastasis defined as distantmetastasis aftersurgery.

Yu et al.





5. Tavazoie SF, AlarconC,Oskarsson T, PaduaD,WangQ, Bos PD, et al.Endogenous human microRNAs that suppress breast cancer metas-tasis. Nature 2008;451:147–52.

6. ValastyanS,Reinhardt F,BenaichN,CalogriasD,Sz�aszAM,WangZC,et al. A pleiotropically actingMicroRNA,miR-31, inhibits breast cancermetastasis. Cell 2009;137:1032–46.

7. Schickel R, Park S-M, Murmann AE, Peter ME. mir-200c regulatesinduction of apoptosis through CD95 by targeting FAP-1. Mol Cell2010;38:908–15.

8. CochraneDR, HoweEN, Spoelstra NS, Richer JK. Loss ofmiR-200c: amarker of aggressiveness and chemoresistance in female reproduc-tive cancers. J Oncol 2010;2010.

9. Iliopoulos D, Lindahl-Allen M, Polytarchou C, Hirsch HA, Tsichlis PN,Struhl K. Loss of miR-200 inhibition of Suz12 leads to Polycomb-mediated repression required for the formation and maintenance ofcancer stem cells. Mol Cell 2010;39:761–72.

10. Shimono Y, Zabala M, Cho RW, Lobo N, Dalerba P, Qian D, et al.Downregulation of miRNA-200c links breast cancer stem cells withnormal stem cells. Cell 2009;138:592–603.

11. Bracken CP, Gregory PA, Kolesnikoff N, Bert AG, Wang J, ShannonMF, et al. A Double-Negative feedback loop between ZEB1-SIP1 andthemicroRNA-200 family regulates epithelial-mesenchymal transition.Cancer Res 2008;68:7846–54.

12. Park S-M, Gaur AB, Lengyel E, Peter ME. The miR-200 family deter-mines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Gen Dev 2008;22:894–907.

13. KorpalM, Lee ES, HuG, Kang Y. ThemiR-200 family inhibits epithelial-mesenchymal transition andcancer cellmigration bydirect targeting ofE-cadherin transcriptional repressors ZEB1 and ZEB2. J Biol Chem2008;283:14910–4.

14. Chaffer CL, Weinberg RA. A perspective on cancer cell metastasis.Science 2011;331:1559–64.

15. Dykxhoorn DM, Wu Y, Xie H, Yu F, Lal A, Petrocca F, et al. miR-200enhances mouse breast cancer cell colonization to form distantmetastases. PLoS One 2009;4:e7181.

16. Korpal M, Ell BJ, Buffa FM, Ibrahim T, Blanco MA, Celia-Terrassa T,et al. Direct targeting of Sec23a by miR-200s influences cancer cellsecretome and promotes metastatic colonization. Nat Med 2011;17:1101–8.

17. Hurst DR, Edmonds MD, Welch DR. Metastamir: the field of metas-tasis-regulatory microRNA Is spreading. Cancer Res 2009;69:7495–8.

18. Yuan M, Tomlinson V, Lara R, Holliday D, Chelala C, Harada T, et al.Yes-associated protein (YAP) functions as a tumor suppressor inbreast. Cell Death Differ 2008;15:1752–9.

19. Levy D, Adamovich Y, Reuven N, Shaul Y. Yap1 phosphorylation by c-Abl is a critical step in selective activation of proapoptotic genes inresponse to DNA damage. Mol Cell 2008;29:350–61.

20. Huang J, Wu S, Barrera J, Matthews K, Pan D. The hippo signalingpathway coordinately regulates cell proliferation and apoptosis byinactivating yorkie, the drosophila homolog of yap. Cell 2005;122:421–34.

21. Basu S, Totty NF, Irwin MS, Sudol M, Downward J. Akt phosphor-ylates the Yes-associated protein, YAP, to induce interaction with14-3-3 and attenuation of p73-mediated apoptosis. Mol Cell2003;11:11–23.

22. Rouhi P, Jensen LD, Cao Z, Hosaka K, Lanne T, Wahlberg E, et al.Hypoxia-induced metastasis model in embryonic zebrafish. Nat Pro-toc 2010;5:1911–8.

23. Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G,et al. The miR-200 family and miR-205 regulate epithelial to mes-enchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol2008;10:593–601.

24. Grimson A, Farh KK-H, JohnstonWK, Garrett-Engele P, Lim LP, BartelDP.MicroRNA targeting specificity inmammals: determinants beyondseed pairing. Mol Cell 2007;27:91–105.

25. John B, Enright AJ, Aravin A, Tuschl T, Sander C, Marks DS. HumanmicroRNA targets. PLoS Biol 2004;2:e363.

26. Betel D, Wilson M, Gabow A, Marks DS, Sander C. The microRNA.orgresource: targets and expression. Nucleic Acids Res 2008;36 Suppl 1:D149–53.

27. Maragkakis M, Reczko M, Simossis VA, Alexiou P, Papadopoulos GL,Dalamagas T, et al. DIANA-microT web server: elucidating microRNAfunctions through target prediction. Nucleic Acids Res 2009;37 Suppl2:W273–6.

28. Dimmer EC, Huntley RP, Alam-Faruque Y, Sawford T, O'Donovan C,MartinMJ, et al. TheUniProt-GOannotation database in 2011. NucleicAcids Res 2012;40(D1):D565–70.

29. Smoot ME, Ono K, Ruscheinski J, Wang P-L, Ideker T. Cytoscape 2.8:new features for data integration and network visualization. Bioinfor-matics 2011;27:431–2.

30. Levy D, Adamovich Y, Reuven N, Shaul Y. The Yes-associated protein1 stabilizes p73 by preventing Itch-mediated ubiquitination of p73. CellDeath Differ 2006;14:743–51.

31. Matallanas D, Romano D, Yee K, Meissl K, Kucerova L, Piazzolla D,et al. RASSF1A elicits apoptosis through an MST2 pathway directingproapoptotic transcription by the p73 tumor suppressor protein. MolCell 2007;27:962–75.

32. Strano S, Munarriz E, Rossi M, Castagnoli L, Shaul Y, Sacchi A, et al.Physical Interaction with Yes-associated protein enhances p73 tran-scriptional activity. J Biol Chem 2001;276:15164–73.

33. Eguiara A, Holgado O, Beloqui I, Abalde L, Sanchez Y, Callol C, et al.Xenografts in zebrafish embryos as a rapid functional assay for breastcancer stem-like cell identification. Cell Cycle 2011;10:3751–7.

34. Burk U, Schubert J, Wellner U, Schmalhofer O, Vincan E, Spaderna S,et al. A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells. EMBO Rep2008;9:582–9.

35. Simpson CD, Anyiwe K, Schimmer AD. Anoikis resistance and tumormetastasis. Cancer Lett 2008;272:177–85.

36. Hanahan D, Weinberg Robert A. Hallmarks of cancer: The next gen-eration. Cell 2011;144:646–74.

37. Oka T, Mazack V, Sudol M. Mst2 and Lats kinases regulate apoptoticfunction of Yes kinase-associated protein (YAP). J Biol Chem 2008;283:27534–46.

38. Aqeilan RI, Donati V, Palamarchuk A, Trapasso F, KaouM, Pekarsky Y,et al. WW domain–containing proteins, WWOX and YAP, compete forinteraction with ErbB-4 and modulate its transcriptional function.Cancer Res 2005;65:6764–72.

39. Chiarugi P, Giannoni E. Anoikis: a necessary death program foranchorage-dependent cells. Biochem Pharmacol 2008;76:1352–64.

40. Hu X, Macdonald DM, Huettner PC, Feng Z, El Naqa IM, Schwarz JK,et al. A miR-200 microRNA cluster as prognostic marker in advancedovarian cancer. Gynecol Oncol 2009;114:457–64.

41. Nam EJ, Yoon H, Kim SW, Kim H, Kim YT, Kim JH, et al. MicroRNAexpression profiles in serous ovarian carcinoma. Clin Cancer Res2008;14:2690–5.






2013;19:1389-1399. Published OnlineFirst January 22, 2013.Clin Cancer Res San-Jian Yu, Jing-Ying Hu, Xia-Ying Kuang, et al. Targeting YAP1 in Human Breast CancerMicroRNA-200a Promotes Anoikis Resistance and Metastasis by

Updated version

10.1158/1078-0432.CCR-12-1959doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2013/01/22/1078-0432.CCR-12-1959.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/19/6/1389.full#ref-list-1

This article cites 40 articles, 9 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/19/6/1389.full#related-urls

This article has been cited by 4 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/19/6/1389To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-12-1959

http://clincancerres.aacrjournals.org/content/suppl/2013/01/22/1078-0432.CCR-12-1959.DC1

http://clincancerres.aacrjournals.org/content/19/6/1389.full#ref-list-1

http://clincancerres.aacrjournals.org/content/19/6/1389.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/19/6/1389


MicroRNA-200a Promotes Anoikis Resistance and Metastasis ... · Anoikis is an important barrier to...

Documents

Transcript of MicroRNA-200a Promotes Anoikis Resistance and Metastasis ... · Anoikis is an important barrier to...