AMP-activated protein kinase promotes epithelial-mesenchymal transition in cancer cells by Twist1

49 Pages·2016·4.04 MB·English

©2018.PublishedbyTheCompanyofBiologistsLtd|JournalofCellScience(2018)131,jcs208314.doi:10.1242/jcs.208314
RESEARCHARTICLE
AMP-activated protein kinase promotes epithelial-mesenchymal
transition in cancer cells through Twist1 upregulation
MeeraSaxena*,§,SaiA.Balaji§,NehaDeshpande§,SanthalakshmiRanganathan,DivyaMohanPillai,Sravanth
KumarHindupur‡andAnnapoorniRangarajan¶
ABSTRACT Indeed, EMT is considered to be an important contributor in the
invasion and metastasis of several types of cancer (Thiery et al.,
Thedevelopmentalprogrammeofepithelial-mesenchymaltransition
2009).
(EMT), involving loss of epithelial and acquisition of mesenchymal
During EMT, epithelial cells shed their intercellular contacts,
properties, plays an important role in the invasion-metastasis
cell-extracellular matrix interactions and apico-basal polarity, and
cascade of cancer cells. In the present study, we show that
attainmoremesenchymalpropertiessuchasincreasedexpressionof
activation of AMP-activated protein kinase (AMPK) using A769662
mesenchymal markers, motility and invasive capabilities (Polyak
ledtoaconcomitantinductionofEMTinmultiplecancercelltypes,as
andWeinberg,2009).EMTcanbestimulatedbyaplethoraofcues
observed by enhanced expression of mesenchymal markers,
suchashypoxia,signalingthroughTGFβ,receptortyrosinekinases,
decrease in epithelial markers, and increase in migration and
NotchandWnt (PolyakandWeinberg,2009).Inresponsetothese
invasion. In contrast, inhibition or depletion of AMPK led to a
cues,severaltranscriptionfactorssuchasTwist1,Snail(alsoknown
reversal of EMT. Importantly, AMPK activity was found to be
asSNAI1),Slug(alsoknownasSNAI2),FOXC2,Zeb1andZeb2
necessary for the induction of EMT by physiological cues such as
hypoxia and TGFβ treatment. Furthermore, AMPK activation (Polyak and Weinberg, 2009) are upregulated, which serve as
important downstream mediators of EMT. Of these, Twist1 is
increased the expression and nuclear localization of Twist1, an
consideredasamasterregulatorofEMT(Yangetal.,2004).Several
EMT transcription factor. Depletion of Twist1 impaired AMPK-
mechanisms of regulation of EMT have been elucidated in recent
inducedEMTphenotypes,suggestingthatAMPKmightmediateits
years(PolyakandWeinberg,2009).However,adiscreethierarchy
effectsonEMT,atleastinpart,throughTwist1upregulation.Inhibition
or a central orchestrator linking these various mechanisms in the
or depletion of AMPK also attenuated metastasis. Thus, our data
EMTinteractomehasyettobeelaborated.
underscoreacentralroleforAMPK inthe inductionofEMTandin
AMP-activated protein kinase (AMPK) is an evolutionarily
metastasis,suggestingthatstrategiestargetingAMPKmightprovide
conserved stress-sensing kinase that becomes activated under a
novelapproachestocurbcancerspread.
variety of stresses, including hypoxia, ischemia and nutrient
KEYWORDS:AMPKsignaling,EMT,Twist deprivation,leadingtoachangeintheATP:AMPratio(Steinberg
and Kemp, 2009). It is a heterotrimeric serine/threonine protein
INTRODUCTION kinaseconsistingofacatalyticαsubunit(α1orα2),ascaffoldingβ
Metastasis,thespreadoftumorcellsfromaprimarysitetoadistant subunit(β1orβ2)andanucleotide-bindingγsubunit(γ1,γ2orγ3)
organ,isaccountableformorethan90%ofcancer-relatedmortality (Hardieetal.,2012).Besidesitspertinentroleintheregulationof
(ChafferandWeinberg,2011).Thisprocessinvolveslocalinvasion energy homeostasis, AMPK has been implicated in several
of cells from a primary tumor into the surrounding tissue, physiological processes including cell growth (Xiang et al.,
intravasation into the nearby microvasculature, migration through 2004), cell division (Jonesetal.,2005), maintenance ofepithelial
thebloodandlymphaticvessels,extravasationatadistantorgansite, cellpolarity(Zhangetal.,2006)andautophagy(Liangetal.,2007).
and their proliferation to form a secondary macroscopic tumor Apartfromthese,AMPKactivityhasalsobeenassociatedwithcell
(ChafferandWeinberg,2011).Itisbelievedthattheevolutionarily migrationinnormalphysiology(Nakanoetal.,2010).Forinstance,
conserved developmental program of epithelial-mesenchymal AMPK activation was shown to be important for migration of
transition (EMT) bestows cancer cells with properties that enable human umbilical vein endothelial cells (HUVECs) (Nagata et al.,
themtoinitiatetheinvasion-metastasiscascade(Yangetal.,2006). 2003)andtransendotheliallymphocytemigration(Martinellietal.,
2009).Inanotherstudy,inhibitionofAMPKwasshowntosuppress
TGFβ-mediatedapoptosisandEMTofnormalmurinehepatocytes
DepartmentofMolecularReproduction,DevelopmentandGenetics,Indian
InstituteofScience,Bangalore560012,Karnataka,India. (Wang et al., 2010). A few reports have also associated AMPK e
*Presentaddress:DepartmentofBiomedicine,UniversityofBasel,Mattenstrasse signalingwithcancercellmigrationandinvasion(Chenetal.,2011; c
28,4058Basel,Switzerland.‡Presentaddress:Biozentrum,UniversityofBasel, n
Klingelbergstrasse50/70,4056Basel,Switzerland. Chiuetal.,2009;Kimetal.,2011).Anotherrecentstudyshowed e
§Theseauthorscontributedequallytothiswork that indirect activation of AMPK by mitochondrial dysfunction ci
S
¶Authorforcorrespondence([email protected]) induced EMT in lung cancer cells (He et al., 2016). In contrast, ll
some studies have reported an inhibitory role for AMPK in e
C
S.A.B.,0000-0002-6539-9660;N.D.,0000-0002-3740-2324;S.R.,0000-0001- EMT of breast, prostate and lung cancer cells, mainly using
f
5531-2173;S.K.H.,0000-0002-8078-4006;A.R.,0000-0003-0972-2184 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and o
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttribution metformin as pharmacological activators of AMPK (Banerjee al
License(http://creativecommons.org/licenses/by/3.0),whichpermitsunrestricteduse, etal.,2016;Chouetal.,2014;Cufíetal.,2010;Hanetal.,2015;Lin n
distributionandreproductioninanymediumprovidedthattheoriginalworkisproperlyattributed. r
et al., 2015; Qu et al., 2014). Thus, there are conflicting reports u
o
Received11July2017;Accepted20June2018 abouttheroleofAMPKsignaling inEMTandcancer metastasis, J
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RESEARCHARTICLE JournalofCellScience(2018)131,jcs208314.doi:10.1242/jcs.208314
suggestingthatAMPKactivationmighthavecell-type-andcontext- themesenchymalmarkersVimandN-cadatproteinlevelsinthese
specific effects. This might, in turn, depend on the upstream epithelialcelltypes(datanotshown).Thus,thissetofdatarevealed
activatorsof AMPK used,some ofwhichcould additionallyhave thatAMPKactivationintheepithelialcancercellssufficestobring
AMPK-independenteffects.Thisincongruityneedstoberesolved about a partial EMT involving the downmodulation of epithelial
before AMPK-targeted therapeutics are widely used for cancer markers.
treatment. Next, we investigated the effects of AMPK activation and
In light of these conflicting reports, we investigated the role of inhibition on cancer cells that had already undergone EMT to
AMPKinregulatingEMTinmultiplecancertypes.Inthepresent variousextents,suchasA549,MDA-MB-231andMDA-MB-435S
study, we show that AMPK activation with a direct, allosteric cells.ActivationofAMPKinthepartiallyepithelial-mesenchymal-
activatorA769662sufficestoinduceafunctionalEMTinmultiple transitionedA549cells,bearinganE-cadlowandVimlowphenotype,
cancercells,whereasitsinhibitionordepletionleadstoreversalof ledtoanincreaseinthemRNAlevelsofmesenchymalmarkersand
EMT. Importantly, we show that AMPK is required for EMT EMTtranscriptionfactors(Fig.1C).Immunofluorescenceanalysis
inductionbyupstreamstimulirelevanttocancerprogression,such also revealed a reduction in E-cad and increase in Vim protein
ashypoxiaandTGFβ.Furthermore,weshowthatAMPKmediates expression(Fig.1D).Reductionintheexpressionoftheepithelial
itseffects,atleastinpart,throughupregulationofTwist1,amajor markers E-cad and ZO-1, and increase in the expression of the
regulatorofEMT,andincreasingitsnuclearlocalization. mesenchymal markers Vim and N-cad, was also detected using
immunoblotting (Fig. S1F). Thus, AMPK activation further
RESULTS promoted the EMT phenotype of A549 cells. In contrast,
ToexploreapossibleroleforAMPKinEMT,weinitiatedourstudy inhibition of AMPK using 10μM Compound C, a potent,
byinvestigating theeffectsofAMPKactivation andinhibitionon reversible, and ATP-competitive inhibitor of AMPK (Zhou et al.,
the expression of markers associated with EMT in a variety of 2001), led to a reduction in Vim and N-cad expression at both
cancer cell lines including breast cancer cell lines MCF7, T47D, mRNA and protein levels, and an increase in E-cad and ZO-1
BT474 and MDA-MB-231, melanoma cell line MDA-MB-435S proteinlevels(Fig.1C,DandFig.S1F).
andlungadenocarcinomacelllineA549.First,wedeterminedthe In MDA-MB-231 cells bearing an E-cad− and Vimhigh
endogenousexpressionofepithelialmarkerE-cadherin(E-cad)and phenotype, AMPK activation led to a further increase in the
mesenchymal markers vimentin (Vim) and N-cadherin (N-cad) in mRNA expression of mesenchymal markers Vim and N-cad and
thesecelllines(Fig.S1A).ThecancercelllinesBT474,MCF7and EMT transcription factors Snai1, Slug and Zeb1, whereas AMPK
T47D exhibiting an E-cad+ and Vim− phenotype represent inhibitionledtoareduction intheirtranscript levels(Fig.1E).At
epithelial cell types. A549 exhibited an E-cadlow and Vimlow proteinlevels,weobservedasignificantincreaseinVimexpression
phenotype, reminiscent of a partially epithelial-mesenchymal- upon AMPK activation and a moderate decrease upon AMPK
transitioned cell line, whereas MDA-MB-231 and MDA-MB- inhibition(Fig.1FandFig.S1G).Asreportedpreviously(Sommers
435ScellsexhibitedanE-cad−andVimhighphenotype,representing etal.,1991),wefailedtodetecttheexpressionofE-cadinMDA-
mesenchymalcelltypes.Thepatternofexpressionofthesemarkers MB-231cells(Fig.S1A),andAMPKinhibitionalsodidnotleadto
matchedthatreportedintheliterature(Lombaertsetal.,2006). itsre-expressioninthesecells(datanotshown).However,AMPK
To begin our study, we investigated whether AMPK activation activationledtoadecreaseintheexpressionofZO-1,whereasits
wouldpromotetheinductionofEMTinepithelial-typecancercell inhibitionledtoincreasedZO-1inMDA-MB-231cells(Fig.S1G).
lines. Most previous studies have used AICAR or metformin for Similarresultswereobtainedwithanothermesenchymalcellline,
activating AMPK; however, these have additional AMPK- MDA-MB-435S(Fig.S1H).
independent effects as well (Foretz et al., 2010; Kalender et al., BecausepharmacologicalinhibitionofAMPK(withCompound
2010). To overcome this problem, we used A769662 (Cool et al., C)mighthaveoff-targeteffects(Liuetal.,2014),weadditionally
2006), which directly activates the heterotrimeric form of AMPK undertookRNAi-mediateddepletionofAMPK.Todoso,weused
bothallostericallyandbyinhibitingitsdephosphorylation(Sanders BT474 and A549 cells stablyexpressing AMPKα2 shRNA under
etal.,2007).WeconfirmedtreatmentwithA769662byperforming constitutive and inducible promoters, respectively, each showing
immunoblotanalysestodetectthelevelsofphosphorylatedacetyl- ∼50%and∼70%knockdownofAMPK(Fig.S1IandJ).Depletion
CoAcarboxylase(ACC),abonafidesubstrateofAMPK(Witters ofAMPKα2intheepithelialBT474cellsledtoafurtherincreasein
et al., 1991). An increase in the phosphorylation of ACC is the expression of epithelial markers E-cad and ZO-1 (Fig. S1I),
indicativeofAMPKactivation(Fig.S1B).TreatmentofT47Dand althoughwefailedtoseeanyexpressionofVim(datanotshown).In
MCF7cellswithA769662ledtoanincreaseinthetranscriptlevels the partially epithelial-mesenchymal-transitioned A549 cells,
of mesenchymal markers N-cad and Vim and EMT transcription AMPK knockdown led to an increase in the expression of E-cad
factorsSnai1,SlugandZeb1(Fig.1A).Moreover,sincedecreasein and ZO-1 while reducing the levels of Vim (Fig. S1J; clone #1).
theproteinlevels ofepithelialmarkersanddisruptionofadherens ImmunofluorescenceanalysisfurtherrevealedanincreaseinE-cad
e
junctions are hallmarks of EMT (Huang et al., 2012), we also expressionandadecreaseinVimexpressionuponAMPKdepletion c
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investigatedtheeffectsofAMPKactivationontheproteinlevelsof inthesecells(Fig.S1K).SimilarresultswereobtainedinA549cells e
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the epithelial markers epithelial cadherin (E-cad; also known as whereAMPKwasstablyknockeddownusinganothershRNAoligo c
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CDH1)andzonaoccludensprotein1(ZO-1,alsoknownasTJP1). sequence(Fig.S1L;clone#4).Similarly,mesenchymalMDA-MB- l
ImmunocytochemicalanalysisrevealedasignificantreductioninE- 231 cells that stably express AMPKα2 shRNA (Hindupur et al., el
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cad levels upon AMPK activation for 48h in these cell lines 2014) and showed ∼80% knockdown (Fig. S1M) revealed a
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(Fig.1B).ReducedexpressionofE-cadandZO-1wasalsodetected reduction in the levels of Vim and N-cad (Fig. S1M,N). These o
usinganimmunoblottingapproach(Fig.S1B).Similarresultswere resultswerevalidatedfurtherusinganadditional,inducibleshRNA al
obtained with AMPK activation in BT474 cells (Fig. S1C,D,E). targeting AMPKα2 (Fig. S1O; clone #4) in MDA-MB-231 cells. n
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However, in spite of the changes at transcript levels (Fig. 1A and Similar results were obtained upon AMPK knockdown in yet u
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Fig.S1C),AMPKactivationfor48hdidnotleadtothedetectionof anothermesenchymalcelltype,MDA-MB-435S(Fig.S1P).Thus, J
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Fig.1.AMPKactivitymodulatesEMTmarkerexpression.(A-F)CancercelllinesT47D,MCF7(A,B),A549(C,D)andMDA-MB-231(E,F)wereculturedinthepresence C
of100μMAMPKactivator(A769662),10μMAMPKinhibitor(CompoundC)orDMSO(vehiclecontrol)asspecifiedfor48h.Thereafter,cellswereharvestedand f
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subjectedtoqRT-PCRanalysisforthespecifiedtranscripts.GraphsinpanelsA,CandErepresentfoldchangeingeneexpressionnormalizedtoβ2M;errorbarsrepresent
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s.e.m.,n=3.Paralleldisheswerefixedusing4%paraformaldehydetoundertakeimmunocytochemistryanalysisforthespecifiedproteins(B,D,F).Photomicrographs a
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showrepresentativefluorescentimagestakenusinganOlympus1X71microscopeat20×magnificationandprocessedusingImageJsoftware.E-cadisstainedinred;
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Vimisstainedingreen;thenucleusisstainedinblue(Hoechst33342).GraphsinpanelsB,DandFrepresentrelativefluorescenceintensitymeasurementsnormalized u
tonumberofnuclei.Errorbarsrepresents.e.m.,n=3.Scalebars:20μm.*P<0.05;**P<0.01;***P<0.001;ns,notsignificant.A7,A769662;CC,CompoundC. o
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thesedatarevealedthatincancercellsthathaveundergoneEMTto (Thieryetal.,2009;Yangetal.,2008).Intriguingly,hypoxiaisalso
varying extent, AMPK activation further promoted, whereas its awell-known trigger for AMPK activation (Mungai et al., 2011).
inactivation or depletion reversed the morphological changes Based on our data above, we hypothesized that hypoxia-induced
associated with EMT, suggesting that AMPK plays a key role in EMT might be mediated by AMPK activation. To test this, we
regulatingtheprocessofEMT. exposed epithelial BT474 cells stablyexpressingeitherscrambled
Attainment of motility and invasive capabilities are hallmark orAMPKα2shRNAtothehypoxiamimeticCoCl .Increaseinthe
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features of a functional EMT (Polyak and Weinberg, 2009). To levelsofHif1αontreatmentwithCoCl revealedthegenerationofa
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assesswhetherAMPKcanmodulatethemotilityandmigrationof hypoxic environment (Fig. 3A). Consistent with a previous study
cancer cells, we undertook a scratch assay with BT474, A549, (Mungai et al., 2011), treatment with CoCl also triggered an
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MDA-MB-231 and MDA-MB-435S cells treated with either increase in AMPK activity, as revealed by elevated pACC levels
AMPK activator A769662 or its inhibitor Compound C. In (Fig.3A).Comparedwithuntreatedcontrolcells,adecreaseinthe
keeping with a failure to induce mesenchymal marker expression expression of E-cad and ZO-1 on CoCl treatment revealed the
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inepithelialcelllines,AMPKactivationfailedtoaltermigrationof induction of EMT in scrambled-shRNA-expressing BT474 cells.
BT474 cells (Fig. 2A and Fig. S2A). In the partially epithelial- Interestingly, hypoxia-induced EMT was inhibited upon AMPK
mesenchymal-transitionedA549cells,A769662treatmentledtoa knockdowninthesecells(Fig.3A).Immunoblotanalysisrevealed
marginal increase in migration whereas Compound C treatment similarresultsforpartialepithelial-mesenchymal-transitionedA549
resulted in a significant decrease in the motility (Fig. 2B and cells stably expressing AMPKα2 shRNA and incubated under
Fig. S2B). Interestingly, mesenchymalMDA-MB-231 and MDA- hypoxic conditions (3% oxygen) in a tri-gas incubator (Fig. 3B;
MB-435S cells showed a significant increase in migration when clone #4). Similarly, in mesenchymal MDA-MB-435S cells, an
treatedwithA769662andasignificantinhibitionofmotilitywith increaseintheexpressionofVimandN-cadin3%oxygenrevealed
CompoundCtreatment(Fig.2C,Fig.S2C,Fig.2DandFig.S2D). the induction of EMT under hypoxia, which was inhibited in the
Tofurtherconfirmthesedata,weadditionallyundertookyetanother presence of the AMPK inhibitor Compound C (Fig. 3C). Similar
cell migration assay with MDA-MB-435S cells involving resultswereobtainedupontreatmentwithCoCl inMDA-MB-231
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quantitative real-time impedance measurement using an ECIS cells stably expressing AMPKα2 shRNA (Fig. S3A) or in the
(electriccell-substrateimpedancesensing)-basedtechnique(Hong presenceofCompoundC(Fig.S3B).ThesedatarevealthatAMPK
et al., 2011a). In corroboration with the scratch assay results, we mightberequiredforhypoxia-inducedEMT.
foundthatalthoughA769662treatmentledtoamarkedincreasein Besides hypoxia, another potent inducer of EMT is TGFβ
migration of MDA-MB-435S cells (as revealed by an increase in (Polyak and Weinberg, 2009; Yang and Weinberg, 2008). It has
impedance),CompoundCtreatmentledtoadecreaseinmigration previously been shown that treatment of murine hepatocytes and
(asrevealedbyadecreaseinimpedance)comparedwiththecontrol AML12 cells with TGFβ leads to activation of AMPK signaling
cells(Fig.S2E).Further,toconfirmtheresultswithCompoundC, (Wangetal.,2010).Thus,wespeculatedthatAMPKactivitymight
weemployedRNAi-mediatedknockdownofAMPKα2.Consistent alsoberequiredforTGFβ-inducedEMTofcancercells.Toexplore
withpharmacological inhibitionofAMPK,A549,MDA-MB-231 this, MDA-MB-435S cells were treated with TGFβ for 48h. An
and MDA-MB-435S cells stably expressing AMPKα2 shRNA or increase in the protein levels of pSmad2 (Fig. 3D) confirmed the
transfectedwithsiRNAtargetingAMPKα2alsoshoweddecreased activationofTGFβsignalinginthesecells.Inkeepingwiththedata
migrationcomparedwithscrambledshRNAcontrolcells(Fig.2E, of Wang et al., we also saw an increase in AMPK activity upon
Fig.S2F,Fig.2F,Fig.S2G,Fig.2GandFig.S2H).Knockdownof TGFβactivation,asrevealedbyanincreaseinthelevelsofpACC
AMPK failed to change the migration in BT474 cells (data not (Fig.3D).Furthermore,increasedlevelsofVimandN-cadrevealed
shown). The effect of AMPK inhibition/knockdown on the theinductionofEMT(Fig.3D).Intriguingly,TGFβ-inducedEMT
migratory properties of mesenchymal cells (Fig. 2F, Fig. S2G, wassignificantly inhibitedin thepresenceoftheAMPKinhibitor
Fig. 2G and Fig. S2H) was more dramatic compared with the CompoundC(Fig.3D).SimilarresultswereobtaineduponTGFβ
partially epithelial-mesenchymal-transitioned A549 cells (Fig. 2E treatmentinthepresenceofCompoundCinA549(Fig.S3C)and
andFig.S2F). MDA-MB-231 cells (Fig. S3D). To further corroborate the data
Todetermine theeffectsofAMPKactivation andinhibitionon obtainedwithpharmacologicalinhibitionofAMPK,weaddressed
theinvasivepotentialofcancercells,weperformedinvitroBoyden theroleofAMPKinTGFβ-inducedEMTbysubjectingMDA-MB-
chamber invasion assays. Since mesenchymal cells showed 231cellsstablyexpressingeitherscrambledorAMPKα2shRNAto
maximal effect on AMPK modulation, we undertook invasion TGFβtreatmentfor48h.AnincreaseinthelevelsofVimandN-cad
assayswiththesecelltypes.Comparedwithvehicle-treatedcontrol revealed the induction of EMT marker expression. Depletion of
cells, we found that whereas A769662 treatment caused a AMPK prevented TGFβ-mediated induction of EMT markers at
significant increase in the invasiveness of MDA-MB-231 and protein levels (Fig. S3E). Together, these data reveal that AMPK
MDA-MB-435S cells, Compound C treatment decreased their mightberequiredforTGFβ-mediatedinductionofEMT.
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invasive potential (Fig. 2H,I). Furthermore, knockdown of RassignalinghasalsobeenshowntopromoteEMT(Mukherjee, c
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AMPKα2inMDA-MB-231andMDA-MB-435Scellsalsoledto 2010) and increase AMPK activity (Rios et al., 2013). To test e
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amarkeddecreaseintheirinvasivepotentialcomparedwithcontrol whether AMPK is also important for Ras-induced EMT, we c
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cells (Fig. 2J, Fig. S2I and Fig. 2K). These results collectively inhibited AMPK in HMLER breast cancer cells (Elenbaas et al., l
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demonstrated that whereas AMPK activation increased the 2001)usingCompoundC.AreductioninthelevelsofpAMPKand e
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migratoryandinvasivecapabilityofcancercells,therebybringing pACCrevealedtheeffectsofCompoundC(Fig.S3F).Weobserved
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aboutafunctionalEMT,itsinhibitionorknockdownreversedEMT, that inhibition of AMPK led to a decrease in the expression of o
suggesting that AMPK plays a critical role in the morphogenetic mesenchymalmarkerssuchasVimandN-cadwhileincreasingthe al
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processesinvolvedwithEMT. expression of the epithelial marker E-cad in HMLER cells
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Next, we investigated the role of AMPK in the context of (Fig.S3F).Thus,collectivelytheseresultsindicatedacriticalrole u
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physiological stimuli such as hypoxia, a known trigger of EMT forAMPKintheinductionofEMTbyvariousupstreamstimuli. J
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Because our study highlighted the role of AMPK in EMT were detected in the lungs of mice injected with scrambled e
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induction, and EMT is considered to be a prerequisite for shRNA cells, no metastatic growth was detected in mice
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metastasis (Polyak and Weinberg, 2009), we next investigated injected with AMPKα2 knockdown cells (Fig. 4A, Fig. S4A). o
the requirement of AMPK function for metastasis in vivo. To Similar results were obtained with MDA-MB-435S cells stably al
do so, we subjected MDA-MB-231 cells stably expressing either expressing scrambled or AMPKα2 shRNA (Fig. 4B, Fig. S4B). n
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scrambled- or AMPKα2-shRNA to the experimental tail vein An MTT assay revealed no significant change in cell u
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metastasis assay. While several microscopic metastatic colonies proliferation when AMPK was depleted (Fig. S4C), together J
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Fig.2.AMPKinducesafunctionalEMT.(A)Scratchassaywasperformed Importantly,inhibitionofAMPKpreventedtheinductionofTwist1
withBT474cellsaftertreatmentwith100µMAMPKactivator(A769662;A7)or expression by CoCl or TGFβ treatment (Fig. 5F,G). Taken
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DMSO(vehiclecontrol).Thegraphrepresentstimekineticsofwound together, these data suggested that AMPK might mediate its
confluencepercentage,calculatedbyIncuCyteZOOMsoftware.(B)Scratch
effectsonEMTthroughupregulationofTwist1.
assaywasperformedwithA549cellsaftertreatmentwith100µMAMPK
TogaugetheroleofTwist1downstreamofAMPKintheprocess
activator(A769662),10µMAMPKinhibitor(CompoundC)orDMSO(vehicle
control).Thegraphrepresentstimekineticsofwoundconfluencepercentage, ofEMT,weinvestigatedtheeffectsofTwist1depletioninAMPK-
calculatedbyIncuCyteZOOMsoftware.Errorbarsrepresents.e.m.,n=2.(C,D) mediated EMT response of cancer cells. For this, we generated
ScratchassaywasperformedwithMDA-MB-231(C)andMDA-MB-435S(D) MDA-MB-231cellsstablyexpressingTwist1shRNA(Manietal.,
cellsaftertreatmentwith100µMAMPKactivator(A769662),10µMAMPK 2008). These cells showed ∼90% reduction of Twist1 both at
inhibitor(CompoundC)orDMSO(vehiclecontrol).Graphsrepresenttime
transcriptlevel(Fig.5H)andproteinlevel(Fig.S5B,C)compared
kineticsofwoundconfluencepercentage,calculatedbyIncuCyteZOOM
withcontrolshGFPcells.Consistentwiththeliterature(Manietal.,
software.Errorbarsrepresents.e.m.,n=3and2,respectively.(E)Scratch
assaywasperformedwithA549cellsstablyexpressinginducibleshRNA 2008), Twist1 knockdown led to a reduction in the expression of
againstAMPKα2(clone#4)culturedwithandwithoutdoxycycline.Thegraph EMTmarkerssuchasVim,N-cad,fibronectin1(FN1;alsoknown
representstimekineticsofwoundconfluencepercentage,calculatedby as CIG), Snail and Slug (Fig. 5I). As shown previously, AMPK
IncuCyteZOOMsoftware.Errorbarsrepresents.e.m.,n=2.(F)Scratchassay activationwithA769662ledtoincreasedEMTmarkerexpressionin
wasperformedwithMDA-MB-231cellsstablyexpressingscrambledshRNAor
thecontrolshGFPcells;however,AMPKactivationfailedtodoso
AMPKα2shRNA.Thegraphrepresentstimekineticsofwoundconfluence
in the Twist1 knockdown cells (Fig. 5I). Similar results were
percentage,calculatedbyIncuCyteZOOMsoftware.Errorbarsrepresent
obtainedinMDA-MB-435ScellsexpressingTwist1siRNAoligos
s.e.m.,n=2.(G)ScratchassaywasperformedwithMDA-MB-435Scells
transfectedwithcontrolsiRNAorAMPKα2siRNA.Graphsrepresentthe (Fig. S5D,E), indicating that AMPK might mediate its effects on
distancemigratedbetweenthecelledgesby48h,calculatedbyImageJ EMT,atleastinpart,throughTwist1.
software.Errorbarsrepresents.e.m.,n=3.(H,I)Boydenchamberinvasion Furthermore, to determine whether AMPK requires Twist1 for
assaywasperformedfor24hwithMDA-MB-231(H)andMDA-MB-435Scells mediatingfunctionalchangesassociatedwithEMT,weinvestigated
(I)treatedwith100µMAMPKactivator(A769662),10µMAMPKinhibitor
the effects of AMPK activation in the migration and invasion of
(CompoundC)orDMSO(vehiclecontrol).Graphsrepresentnumberofcells
Twist1knockdowncells.Asseenbefore,AMPKactivationledtoan
invadedperfield(9-10fieldswerecountedperexperiment).Errorbars
represents.e.m.,n=3.(J,K)Boydenchamberinvasionassaywasperformedfor increase in the migration and invasive potential of control shGFP
24hwithMDA-MB-231cellsstablyexpressingscrambledshRNAorAMPKα2 cells (Fig. 5J,K and Fig. S5F); however, AMPK activation in
shRNA;n=2(J)orMDA-MB-435ScellstransfectedwithcontrolsiRNAor shTwist1cellsfailedtodoso(Fig.5J,KandFig.S5F).
AMPKα2siRNA;n=3(K).Graphsrepresentnumberofcellsinvadedperfield Because Twist1 is a transcription factor and its nuclear
(9-10fieldswerecountedperexperiment).Errorbarsrepresents.e.m.Forall
localization is critical for its function, we investigated nuclear
scratch/migrationassayexperimentsabove,cellswerepretreatedwith10µg/ml
localizationofTwist1underAMPK-activatedconditions.Asseen
MitomycinCfor2hbeforethescratch/woundwasmade.Forallmigrationassay
experimentsperformedusingIncuCyteZOOM,10×magnification(widemode) in the immunofluorescence images (Fig. 5C), in addition to
wasused.*P<0.05;**P<0.01;***P<0.001.A7,A769662;CC,CompoundC; increased levels of Twist1, we also noted increased nuclear
Ctrl,control;Scr,scrambled;sh,shRNA. localization of Twist1 in the AMPK-activated condition. Because
AMPKactivation ledtoasignificantincreaseinTwist1inMDA-
MB-231 cells (Fig. 5A and Fig. S5A), we performed subcellular
suggesting that AMPK functions might be required for the fractionation experiments with the same cells. Cell fractionation
metastasis of these cancer cells. experiments also showed elevated nuclear Twist1 upon AMPK
Next, we investigated the mechanisms by which AMPK might activation(Fig.6A).Furthermore,immunofluorescenceimagingof
regulateEMT.InductionofEMTisassociatedwiththeupregulation MDA-MB-231andMCF7cellstransientlytransfectedwithaGFP-
ofseveraltranscriptionfactorssuchasTwist1,Snail,SlugandZeb1 taggedTwist1constructalsoshowedincreasednuclearlocalization
(YangandWeinberg,2008).Ofthese,Twist1hasbeenconsidered of Twist1 in the A769662-treated condition (Fig. 6B), suggesting
to be a major regulator of EMT, invasion and metastasis (Wang AMPK-mediatedpost-translationalmodificationsofTwist1leading
et al., 2016; Yang et al., 2004). Therefore, we analyzed the to its nuclear localization. Because phosphorylation by mitogen-
expression of Twist1 under conditions of AMPK activation. We activated protein kinases (MAPKs), extracellular signal-regulated
noticed an AMPK-mediated increase in Twist1 transcripts in a kinases(ERKs)andJunN-terminalkinases(JNKs)hasbeenshown
varietyofcelllinestested,includingMCF7,BT474,A549,MDA- to increase serine phosphorylation and stability of Twist1 (Hong
MB-231 and MDA-MB-435S cells (Fig. 5A). Notably, the et al., 2011b), we investigated the effect of AMPK activation on
epithelial-mesenchymal-transitioned cells showed a greater Twist1stability.Incycloheximidechaseexperiments,inhibitionof
increase in Twist1 transcript levels upon AMPK activation AMPKledtoadecreaseinTwist1stability(Fig.6C).Moreover,we
compared with the non-invasive cells (Fig. 5A). A western blot observed increased Twist1 stability in the presence of AMPK
analysisrevealedelevatedTwist1proteinlevelsinthepresenceof activator A769662 (data not shown). Taken together, our data
e
A769662 (Fig. 5B and Fig. S5A). An immunocytochemical revealed that AMPK activation leads to an increase in the c
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analysisfurtherconfirmedelevatedTwist1levels(Fig.5C). expression, stability and nuclear localization of Twist1 protein, e
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To validate whether Twist1 regulation is through AMPK whichultimatelycouldpromoteEMT. c
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activation, we next investigated the effects of AMPK inhibition Inconclusion,ourstudyuncoversacentralroleforAMPKinthe l
l
on Twist1 expression in the epithelial-mesenchymal-transitioned positiveregulationofEMTincancercells,mediated,atleastinpart, e
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cancer cells lines A549, MDA-MB-231 and MDA-MB-435S. throughTwist1upregulation.
f
Indeed,inthesecelllines,inhibitionofAMPKwithCompoundC o
led to a significant decrease in the levels of Twist1 transcripts DISCUSSION al
n
(Fig. 5D). Consistent with this, MDA-MB-231 and MDA-MB- EMTissuggestedtobeaninitialandimportantstepintheinvasion-
r
435S cells stably expressing AMPKα2 shRNA also showed less metastasis cascade (Thiery et al., 2009). Therefore, an increased u
o
expression of Twist1 compared with scrambled cells (Fig. 5E). understanding of the molecular players involved in this process is J
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RESEARCHARTICLE JournalofCellScience(2018)131,jcs208314.doi:10.1242/jcs.208314
Fig.3.AMPKisnecessaryforEMTinductionby
differentupstreamstimuli.(A)BT474cellsstably
expressingscrambledshRNAorAMPKα2shRNA
wereculturedinthepresenceof150µMCoCl for
2
48h.Thereafter,cellswereharvestedand
immunoblotanalysiswasundertaken.Thegraph
representsdensitometricquantificationofthe
specifiedproteinsnormalizedtoα-tubulin;error
barsrepresents.e.m.;n=2.(B)A549cellsstably
expressinginducibleshRNAagainstAMPKα2
(clone#4)wereculturedwithandwithout
doxycyclineinambientoxygen(20%O )orin
2
hypoxic(3%O )conditionsinatri-gasincubator
2
for48h.Thereafter,cellswereharvestedand
subjectedtoimmunoblotanalysis.Thegraph
representsdensitometricquantificationofthe
specifiedproteinsnormalizedtoα-tubulin;error
barsrepresents.e.m.;n=2.(C)MDA-MB-435S
cellswereculturedinambientoxygen(20%O )or
2
inhypoxic(3%O )conditionsinatri-gasincubator
2
inthepresenceof10µMAMPKinhibitor
(CompoundC)for24h.Thereafter,cellswere
harvestedandsubjectedtoimmunoblotanalysis.
Thegraphrepresentsdensitometricquantification
ofthespecifiedproteinsnormalizedtoα-tubulin;
errorbarsrepresents.e.m.;n=4.(D)MDA-MB-
435Scellswereculturedwith5ng/mlTGFβinthe
presenceorabsenceof10µMAMPKinhibitor
(CompoundC)for24h.Thereafter,immunoblot
analysiswasundertakenforthespecifiedproteins.
Thegraphrepresentsdensitometricquantification
ofthespecifiedproteinsnormalizedtoα-tubulin;
errorbarsrepresents.e.m.;n=4.*P<0.05;
**P<0.01;***P<0.001.α-Tub,α-tubulin;CC,
CompoundC;Doxy,doxycycline;ns,not
significant;Scr,scrambled;sh,shRNA.
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RESEARCHARTICLE JournalofCellScience(2018)131,jcs208314.doi:10.1242/jcs.208314
Fig.4.AMPKknockdownimpairsmetastasisofbreastcancercells.(A,B)MDA-MB-231andMDA-MB-435ScellsstablyexpressingscrambledshRNA
orAMPKα2shRNAwereinjectedthroughthetailveinofnudemiceandscoredforlungmetastasis.Photomicrographsshowhistologicsectioningof
representativelungmetastasesoriginatingfromMDA-MB-231(A)andMDA-MB-435S(B)cells(magnification×20;hematoxylinandeosinstaining).Scalebars:
30μm.GraphsrepresentincidenceoflungmetastasesfoundinmiceinexperimentsAandB;n=5miceforeachgroup.Scr,scrambled;sh,shRNA.
likely to lead to a better understanding of cancer metastasis and dissolutionofadherensjunctionsfromthecellmembrane(thefirst
identify newer targets for cancer treatment. Although several hallmarkofanactiveEMT),thuspushingthecellstowardsinitiating
signaling pathways and physiological conditions have been anEMT.Furthermore,eventhoughAMPKactivationupregulated
identified as activators of EMT, and a plethora of transcription the transcript levels of mesenchymal markers (Vim and N-cad) in
factorshavebeenidentifiedasdownstreameffectorsofEMT,ithas these cells, their protein levels are not changed (data not shown),
remainedunclearwhetherthesevariousinducersofEMTfunction and consistent with this, their migratory properties also did not
independently, work in conjunction, or converge on a common change.Hence,thesecellsseemtoonly‘partially’undergoanEMT.
downstreammediator.OurstudyidentifiesacentralroleforAMPK InmetastableA549cells,whichalreadyseemtobeinapartialEMT
in mediating an EMT downstream of several physiological cues. state as evident by E-cadlow and Vimlow phenotype, AMPK
Even though the LKB1-AMPK axis was initially associated with activation significantly decreased the protein expression of E-cad
tumor-suppressive roles (Huang et al., 2008), recent studies have andZO-1andupregulatedmesenchymalmarkers(Fig.S6).Thus,
demonstrated pro-tumorigenic functions for AMPK, including metastable A549 cells, which seem to be ‘primed’ for EMT, are
inhibition of apoptosis, promoting anchorage-independent growth hence pushed further into EMT in response to AMPK activation.
and, more recently, cancer cell migration (Hindupur et al., 2014; Mesenchymal MDA-MB-231 cells do not express E-cad while
JeonandHay,2015;Kimetal.,2011;Ngetal.,2012;Wangetal., expressingZO-1atalowbasallevel.AMPKactivationwasableto
2010).Inourstudy,weprovideevidenceforadirectroleofAMPK furtherdownregulateZO-1expressioninthesemesenchymalcells,
in the induction of EMT in cancer cells, as well as in the andsignificantlyincreasedtheexpressionofmesenchymalmarkers
maintenance of the EMT phenotype. We further show that (Fig. S6) and their migratory and invasive potential. Hence, it
activation of AMPK suffices to bring about a functional EMT appearsthatalthoughAMPKactivationisabletodownregulatethe
e
characterized by increased migration and invasion potential. expression of epithelial markers across all states of EMT in c
n
Moreover, we show that AMPK activation is mandatory for EMT different cell types, additional cues might be required to also e
i
inductionmediatedbyupstreamphysiologicalcuessuchashypoxia increase the protein expression of mesenchymal markers in c
S
andTGFβ.Additionally,weshowthatAMPKmediatesitseffects epithelial cells (MCF7, T47D and BT474) to bring about a l
on EMT via upregulation of TWIST1 gene expression and its complete,functionalEMT.Incontrast,primedpartialepithelial- el
C
increasednuclearlocalization. mesenchymal-transitioned cells (A549) and mesenchymal cells
f
AMPK activation with A769662 affected the expression of (MDA-MB-231 and MDA-MB-435S) seem to already possess o
epithelialmarkersE-cadandZO-1todifferentextentsindifferent the requisite machinery to bring about a complete, functional al
n
cell lines promoting an EMT. In the epithelial breast cancer cells EMTinresponsetoAMPKactivation.
r
MCF7, T47D and BT474, AMPK activation decreased the Ontheotherhand,AMPKinhibitionordepletionincreasedthe u
o
expression of epithelial markers E-cad and ZO-1 and caused epithelialcharacteristicsofthecellswhiledecreasingmesenchymal J
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Fig.5.Seenextpageforlegend. C
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properties.IntheepithelialBT474cells,AMPKknockdownledtoa ZO-1inadditiontoreductioninmesenchymalmarkersVimandN- al
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further increase in epithelial markers (Fig. S6). In the partially cad.AMPKinhibitionordepletioninthemesenchymalMDA-MB-
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epithelial-mesenchymal-transitionedA549cells,AMPKinhibition 231 and MDA-MB-435S cell lines decreased the expression of u
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or knockdown led to an increase in epithelial markers E-cad and mesenchymalmarkersVimandN-cad(Fig.S6);however,itfailed J
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RESEARCHARTICLE JournalofCellScience(2018)131,jcs208314.doi:10.1242/jcs.208314
Fig.5.AMPKregulatesTwist1expression.(A)Variouscancercelllines cells(Banerjeeetal.,2016).AMPKsignalinghasbeenshownto
(MCF7,BT474,A549,MDA-MB-231andMDA-MB-435S)wereculturedinthe inhibitEMTin othercancer typessuch asmelanomas,lung and
presenceof100µMAMPKactivator(A769662)orDMSO(vehiclecontrol)for prostate cancer (Chou et al., 2014; Kim et al., 2012; Lin et al.,
48h.Thereafter,cellswereharvestedforRNAisolationandsubjectedto
2015).ThesediscrepanciescouldbeduetotheextentofAMPK
qPCRanalysisforTwist1geneexpression.Graphrepresentsfoldchanges
(normalizedtohousekeepinggeneβ2M)ofTwist1geneexpressioninthe activation by different pharmacological agents, or due to their
specifiedcelllines;errorbarsrepresents.e.m.,n=3.(B)MDA-MB-435Scells non-specificactivities.Metformin,abiguanideusedasafirst-line
wereculturedinthepresenceof100μMAMPKactivator(A769662)orDMSO anti-diabeticdrug,isanindirectmodulatorofAMPK.Itinhibits
(vehiclecontrol)for48h.Thereafter,cellswereharvestedandsubjectedto complex I of the mitochondrial respiratory chain, causing an
immunoblotanalysisforTwist1andα-tubulin.Thegraphrepresents increase in intracellular AMP levels, thereby activating AMPK
densitometricquantificationoftheTwist1proteinnormalizedtoα-tubulin;error
(Owen et al., 2000). However, AMPK-independent roles of
barsrepresents.e.m.,n=3.(C)MDA-MB-231cellswereculturedinthe
metforminhavebeenreported(Foretzetal.,2010;Kalenderetal.,
presenceof100µMAMPKactivator(A769662)orDMSO(vehiclecontrol)for
24h.Thereafter,thedishesweresubjectedtoimmunocytochemistryanalysis 2010) and its use as a bona fide AMPK activator needs further
forTwist1protein.Photomicrographsshowrepresentativefluorescentimages investigation.A769662andAICAR,bothanalogsofAMP,bind
takenat20×magnificationusinganOlympus1X71microscope;Hoechst directlytotheauto-inhibitorydomainofAMPKanddonotcause
33342wasusedfornuclearstaining,andwaspseudo-coloredgreenfor achangeintheATP:ADPratiooraltermitochondrialfunctionto
representation.Twist1isstainedinred.Scalebars:10μm.Thegraph
exerttheiraction.However,AICARmonophosphate(ZMP),the
representscytoplasmicandnuclearfluorescenceintensity(FI)measurements
activeformofAICARwithincells,isanaturalintermediateofthe
percell,n=30cellsquantifiedineachofthethreebiologicalreplicates;theerror
purine nucleotide synthesis pathway and is metabolized by
barrepresentss.e.m.(D)qPCRanalysisforTwist1geneexpressioninA549,
MDA-MB-231andMDA-MB-435Scellsculturedinthepresenceof10μM AICARtransformylase(Kimetal.,2016).Therefore,theturnover
AMPKinhibitor(CompoundC)orDMSO(vehiclecontrol)for48h.Thegraph of the active form of AICAR within cells is dependent on their
representsfoldchange(normalizedtoβ2M);errorbarsrepresents.e.m.,n=3. proliferation rate. Thus, these modulators of AMPK might
(E)qPCRanalysisforTwist1geneexpressioninMDA-MB-231andMDA-MB- activate it to different extents. Our results with A769662
435ScellsstablyexpressingscrambledshRNAorAMPKα2shRNA.The
showed that AMPK is a positive regulator of EMT in multiple
graphrepresentsfoldchange(normalizedtoβ2M);errorbarsrepresents.e.m.,
cancer cell lines. We have additionally corroborated our results
n=3.(F,G)MDA-MB-231andBT474cellswereculturedinthepresenceof
10µMAMPKinhibitor(CompoundC)with150µMCoCl (F)orTGFβ(G)for with genetic approaches using either siRNA- or inducible-
2
48h.Thereafter,cellswereharvestedandquantifiedforTwist1mRNAlevels shRNA-mediatedknockdown.
byqPCR.Thegraphsrepresentfoldchange(normalizedtoβ2M);errorbars Most importantly, our data revealed that TGFβ, hypoxia, and
represents.e.m.,n=3.(H)QuantificationofTwist1mRNAlevelsbyqPCRin oncogenic Ras-mediated EMT are significantly inhibited by
MDA-MB-231cellsstablyexpressingGFPshRNAorTwist1shRNA.The
depletion or inhibition of AMPK. This suggested that activation
graphrepresentsfoldchangeofTwist1geneexpression(normalizedtoβ2M);
ofAMPKcouldbeakeycentraleventintheEMTprogram.Recent
errorbarsrepresents.e.m.,n=3.(I)MDA-MB-231cellsstablyexpressingGFP
shRNAorTwist1shRNAwereculturedinthepresenceof100µMAMPK studies have identified critical functions of AMPK under
activator(A769662)orDMSO(vehiclecontrol)for48h.Thereafter,thecells pathological/physiological stresses associated with tumor
wereharvestedandqPCRanalysiswasundertakenforthespecified progression, including nutrient deprivation, hypoxia, and matrix
transcripts.Thegraphrepresentsrelativegeneexpressionofthespecified detachment (Hindupur et al., 2014; Steinberg and Kemp, 2009).
genes(normalizedtoβ2Mexpression);errorbarsrepresents.e.m.,n=4.
Our identification of a stress-response kinase as a necessary
(J,K)MDA-MB-231cellsstablyexpressingGFPshRNAorTwist1shRNAwere
component of the EMT program additionally supports the notion
culturedinthepresenceof100µMAMPKactivator(A769662)orDMSO
thatthephenomenonofEMT might be astressresponse thataids
(vehiclecontrol).(J)Thegraphrepresentsthedistancemigratedbetweenthe
celledgesinascratchassayin48h,calculatedbyImageJsoftware;errorbars cancer cell dissemination and metastasis. Furthermore, given that
represents.e.m.,n=3.(K)Thespecifiedcellstreatedsimilarlywerealso theearlierstudieshavelinkedtheattainmentofanEMTphenotype
subjectedtoBoydenchamberinvasionassay.Thegraphrepresentsthetotal withtheacquisitionofstemnessproperties(Manietal.,2008),itis
numberofcellsinvadedperfield;errorbarsrepresents.e.m.,n=4.*P<0.05; plausible that AMPK activity may determine the differentiation
**P<0.01;***P<0.001.α-Tub,α-tubulin;A7,A769662;CC,CompoundC;Scr,
statusofepithelialcancers.
scrambled;sh,shRNA.
EMT is largely effected by a set of transcription factors whose
expressionandfunctionareundertightcontrol.Twist1,considered
tobringbackE-cadexpression(datanotshown).Itisknownthatthe asamasterregulatorofEMTandmetastasis(Yangetal.,2004),is
E-cad promoter is hypermethylated in MDA-MB-435S cells, expressed in a variety of cancers (Ansieau et al., 2010). Several
completely silencing E-cad expression, whereas it is partially transcriptionalregulatorsofTwist1havebeenidentified,including
methylatedinMDA-MB-231cells,leadingtoaverylowexpression HIF1α,interleukin-6(IL-6),NF-κBandtheSmad-dependentTGFβ
of E-cad that cannot be detected by western blotting (Lombaerts pathway(Chengetal.,2008;Phametal.,2007;Tanetal.,2012).
etal.,2006).ItispossiblethatAMPKsignalingmaynotbeableto Our studyshowed thatTwist1 gene expressionistranscriptionally
alleviatesuchhypermethylation-inducedgenesilencing.Thesedata regulated by AMPK. Indeed, AMPK has been shown to regulate
e
suggestedthatAMPKisrequiredtomaintaintheEMTphenotype, gene expression by phosphorylating various transcription factors c
n
whereas its inhibition leads to reversal of EMT, also known as (CantóandAuwerx,2010),orviaitseffectsonchromatinstructure e
i
mesenchymal-to-epithelialtransition(MET)(Nietoetal.,2016). by modulating histone deacetylase activity or histones (Bungard c
S
Our data revealed that activation of AMPK using A769662 etal.,2010;McGeeetal.,2008).However,theprecisemechanism l
l
promotesEMT.Incontrast,metformin,alsoanAMPKactivator, of Twist1 transcriptional regulation by AMPK remains to be e
C
has been shown to inhibit the EMT phenotype of breast cancer elucidated.
f
cells (Qu et al., 2014). Another study showed that activation of Additionally, our study revealed that AMPK activation led to o
AMPKbyOSU-53,anewallostericactivatorofAMPK,reversed increased stability and nuclear localization of Twist1. Such a al
n
EMT(Chouetal.,2014).MetforminwasshowntoinhibitEMT multi-stepregulationofTwist1byAMPKsuggeststhatthecellular
r
by AMPK-mediated abrogation of Erk activity, leading to eventsdownstreamoftheAMPK-Twist1axisplayacriticalrolein u
o
downregulation of Snail and Slug in lung and breast cancer orchestrating the EMT program. Twist1 is also known to be J
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