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Early events in celladhesion and polarityduring epithelial-mesenchymal transition

Ruby Yun-Ju Huang1,2, Parry Guilford3

and Jean Paul Thiery2,4,5,*1Department of Obstetrics and Gynaecology, NationalUniversity Hospital, 119074, Singapore2Cancer Science Institute of Singapore, NationalUniversity of Singapore, 119074, Singapore3Cancer Genetics Laboratory, University of Otago,Dunedin 9016, New Zealand4Department of Biochemistry, National University ofSingapore, 8 Medical Drive, Singapore 117597,Singapore5Institute of Molecular and Cell Biology, A*STAR,138763, Singapore

*Author for correspondence (jpthiery@imcb.a-star.edu.sg)

Journal of Cell Science 125, 4417–4422

� 2012. Published by The Company of Biologists Ltd

doi: 10.1242/jcs.099697

During evolution from primitive species, a

mechanism, termed epithelial-mesenchymal

transition (EMT), was established to create

mesenchymal cells from a pre-existing

epithelial cell layer. However, the direct

and full conversion from an epithelial to a

mesenchymal state is not observed in all

species (Yin et al., 2009). Intermediate-

staged cells harbor the greatest plasticity

because they are able to either reverse

to an epithelial state or progress to a

mesenchymal phenotype. Epithelial cells

are characterized by well-developed,

intercellular contacts, whereas mesenchymal

cells seldom form intercellular contacts.

Therefore, to better characterize the

hallmarks of intermediate-stage cells, it is

important to understand how intercellular

contacts change during early EMT. This

Cell Science at a Glance article focuses

on adhesion and polarity, which are

restructured during EMT in vitro. The

molecular anatomy of junctional complexes

and the dynamics of some of their

components during EMT will be described

in intermediate stages that lead to a fully

converted mesenchymal state.

Molecular anatomy of junctionalcomplexesA prototypic monolayered epithelium is

characterized by apical, lateral and basal

plasma membrane domains. The lateral

domain contains morphologically distinct,

well-demarcated intercellular adhesive

structures, including tight junctions (TJs),

adherens junctions (AJs) and desmosomes,

that contribute to the establishment of apical-

basal polarity (Nelson, 2009). Below, we

describe the dynamics involved in the

formation of these junctional complexes

during the establishment of apical-basal

polarity.

Nectins comprise a family of cell adhesion

proteins that contain three immunoglobulin

(Ig) domains. Nectins are involved in nascent

contact between epithelial cells (Takai and

Nakanishi, 2003) through Ca2+-independent

heterophilic binding (e.g. nectin-1 binds to

nectin-3), which forms the scaffold upon

which E-cadherin-based adhesive structures

assemble and recruit other adherens

components. Through their PDZ-binding

(See poster insert)

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domain, nectins associate with afadin, a largecytoplasmic protein that binds directly to

actin microfilaments (Takai and Nakanishi,2003). E-cadherin, a prototypic, Ca2+-dependent, adhesion molecule that containsfive Ig domains, subsequently accumulates

at these nectin scaffolds and forms trans-homophilic interactions through its N-terminus, and cis-interactions between the

first and second Ig domains (Harrison et al.,2011). The armadillo repeat-containingproteins b-catenin and p120-catenin interact

with the cytoplasmic domain of E-cadherinthrough their C-terminal and juxtamembranesegments, respectively. b-catenin binds to a-catenin, which, in turn, binds to other

proteins ensuring the connection to actinmicrofilaments (Nishimura and Takeichi,2009). In conjunction with afadin, proteins

that connect to actin microfilaments enforcenascent junctional stability. However, such astabilizing effect is still debated, because

actin polymerization is inhibited bymonomeric a-catenin (the form that bindscytoplasmic b-catenin), but not dimeric a-

catenin (Drees et al., 2005; Yamada et al.,2005).

These nascent cadherin-based adhesions,often called ‘punctates’, progressively

mature to form AJs (Adams et al., 1996).This first requires the active assembly ofactin filaments within cadherin-rich

punctates, which involves nucleation ofactin polymerization by the actin-likeprotein complex ARP2/3 and actinin-4

(Tang and Brieher, 2012). Mature AJsprovide a scaffold for the assembly of acircumferential acto-myosin contractile beltat the apex of epithelial cells to ensure

stable interactions with neighboring cellsthat reside as epithelial sheets (Shapiroand Weis, 2009). EPLIN (also known

as LIMA1), an actin-filament-bundlingprotein, establishes such connections infully developed adherens junctions,

forming the zonula adherens at the apex ofthe lateral domain of polarized epithelialcells (Taguchi et al., 2011). Therefore, actin

dynamics have a profound effect on AJstability.

AJ formation and stability are controlledby several protein complexes, in particular

GTPases and their corresponding G-proteinexchange factors (GEFs). For example,TARA (also known as TRIOBP), an actin-

binding adaptor protein, activates the GEFtriple functional domain (TRIO), which inturn promotes actin polymerization through

Rac activation (Yano et al., 2011). The Rac,CDC42 GTPases and ARP2/3 also inducelocalized actin polymerization to initiate

nascent junctions in the vicinity oflamellipodia by forming complexes with

the Abi-WAVE complex [the Abelsoninteractor proteins 1 and 2 in complex witha member of the Wislott-Aldrich syndromeprotein (WASP) family]. Abi also binds

Diaphanous, a member of the formin family,which competes for the same binding site asWAVE. This Abi-Diaphanous complex

polymerizes linear actin filaments andstabilizes junctions. Both the Abi-Diaphanous and Abi-WAVE complexes

form in the vicinity of E-cadherin–b-catenin complexes (Ryu et al., 2009).SHROOM3, a PDZ domain-containingadaptor protein, binds and activates Rho-

associated protein kinase (ROCK), whichstimulates apical constriction of the actin beltthrough phosphorylation of the myosin light

chain (Nishimura and Takeichi, 2008).AJ assembly also requires actomyosincontractility (Miyake et al., 2006), with

Myosin IIA having a role in punctate E-cadherin clustering into AJs, and Myosin IIBproviding stability for the actin belt (Smutny

et al., 2011). During epithelial assembly,epithelial cells must sense and transducemechanical forces. These tensile forces thatare exerted on cadherin complexes result in

the unfolding of a-catenin to reveal crypticvinculin-binding sites, which nucleatepolymerization of new actin microfilaments

(Gomez et al., 2011; Yonemura et al., 2010).

Desmosomes are well-described adhesivejunctions that are characteristic to epithelial

cells. The cytoplasmic domains of desmocolins(DC1 and DC2) and desmogleins (DG1, DG2and DG3) bind to plakoglobin, plakophilins(PKP1, PKP2 and PKP3) and desmoplakins

(DP1 and DP2) (Franke, 2009; Thomasonet al., 2010). Desmoplakins form electron-dense plaques that firmly connect to the

thick network of intermediate cytokeratinfilaments (Thomason et al., 2010). Manydesmosomal proteins (plakoglobin, PKP2 and

desmogleins) associate with AJ proteins, suchas E-cadherin (Hinck et al., 1994; Lewis et al.,1997), b-catenin (Chen et al., 2002) and p120-

catenin (Kanno et al., 2008) to mutuallystrengthen cadherin- and desmosome-basedcell adhesions. Like AJs, desmosomesinitially form as immature Ca2+-dependent

structures (Thomason et al., 2010), andmature into hyper-adhesive desmosomes.Evidence suggests that AJs and desmosomes

cooperate during junctional assembly; thisstrengthening and maturation ultimatelycontributes to epithelial cell integrity (Huen

et al., 2002; Vasioukhin et al., 2001).

TJ assembly commences after theformation of AJs and desmosomes. TJs

prevent the passage of fluid between theluminal compartment and the underlying

stroma. Despite the identification of morethan 40 proteins involved in TJs, their

precise molecular composition remainsunclear (Anderson and Van Itallie, 2009).Notably, the paracellular transport of ions

or small metabolites occurs through TJsvia anastomosing fibrils, designated ‘TJ

strands’, which initiate from homo- andheterotypic interactions between the

tetraspan proteins claudins (Furuse, 2010);for instance, claudin-3 binds to claudin-1and claudin-2 (Furuse and Moriwaki, 2009).

The cytoplasmic domains of claudins andoccludins associate with the zonula

occludens proteins [ZO1, ZO2 and ZO3(also known as TJP1, TJP2 and TJP3,respectively)], which contain three PDZ

domains. Occludins and other tetraspanproteins, such as tricecullin, are recruited

for TJ maturation following claudinactivity. ZO proteins interact with other

PDZ-containing proteins, such as multi-PDZ-domain protein 1 (MUPP1, alsoknown as MPDZ) and PALS1-associated

to tight junction protein (PATJ, also knownas INADL), and assemble into scaffolds that

are connected to actin microfilaments(Furuse, 2010; Furuse and Tsukita, 2006).

Also closely associated with TJs arethe transmembrane junctional adhesionmolecules (JAMs; JAM1, JAM2 and

JAM3) that contain two Ig domains(Fukuhara et al., 2002), and the coxsackie

virus and adenovirus receptor (CXADR).These proteins interact with ZO1 andafadin. Nectin adhesion complexes also

contribute to the recruitment of claudins,occludins and JAMs.

The highly polarized epithelial cellsreside above a layer of basementmembrane, comprising a complex network

of extracellular matrix (ECM). Epithelialcells connect to the basement membrane

through integrins that are located on thebasal epithelial surface, and bind to peptide

motifs found on collagens, laminins andother ECM components (Hay, 1993;LeBleu et al., 2007; Timpl and Brown,

1996). Hemi-desmosomes are specializedadhesive structures that are assembled by

clustering of the a6b4 integrin (Margadantet al., 2008). The cytoplasmic domain of the

b4 integrin subunit subsequently recruitsan array of proteins, including plectin,which is connected to cytokeratin

filaments (Margadant et al., 2008). Otherintegrin heterodimers cluster into focal

complexes, focal adhesions or fibrillaradhesions that are linked to the actin

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microfilament network. This so-called

‘integrin adhesome’ also includes numerouskinases, phosphatases and adaptor proteins(Geiger and Yamada, 2011) and senses

mechanical forces through Talin andp130CAS (also known as BCAR1) (del Rioet al., 2009; Sawada et al., 2006).

Apical-basal polarity is progressively

established during junctional maturation. In

vitro studies indicate that the basal side is firstdetermined when cells attach and spread to

the substratum. As the adhesion junctionalcomplexes mature, the lateral cell domain isincreasingly segregated from the apicaldomain, with mature TJs defining the

boundary between the apical and lateraldomains. Apical-basal cell polarity iscontrolled by three evolutionarily conserved

protein complexes in Drosophila: (1) theCrumbs complex, comprising Crumbs3,PALS1 (known as Stardust) and Patj; (2) the

PAR complex, comprising PAR6, PAR3 andatypical protein kinase C (aPKC); and (3) theScribble complex, consisting of Scribble,

Lethal giant larvae (Lgl) and Discs large(Dlg) (St Johnston and Ahringer, 2010). Inimmature cell-cell contacts, PAR6 is bound toaPKC, independent of PAR3, which is found

in nascent AJs associated with afadin (Martin-Belmonte and Perez-Moreno, 2012). Duringmaturation, PAR3 relocates into the Crumbs

complex and is recruited to TJs (Nelson,2009; St Johnston and Ahringer, 2010).

The dynamic changes of junctionalcomplexes during EMTEMT triggers the sequential destabilizationof cell-cell adhesive junctions and the

regulatory machinery that controls cellpolarity. This transforms epithelial cellsinto a plastic and motile state, with amesenchymal phenotype (Thiery and

Sleeman, 2006). The cartoon in theaccompanying poster depicts two bonafide epithelial cells that become

progressively separated mesenchymal-likecells through the gradual loosening of cell-cell contacts, which is comparable to

‘unzipping’. Separation starts with thesequential loss of TJ, AJ and desmosomeintegrity, transitioning cells from a

polarized epithelium to a post-EMTmesenchymal state. This transitioninvolves intermediate stages, which offersthe greatest potential for epithelial

plasticity and highlights one of theimportant hallmarks of EMT, reversibility.

EMT reversibility is closely tied to the

dynamic and efficient remodeling of celladhesion complexes (Yap et al., 2007).Adhesion proteins undergo constant

recycling into and out of junctions, whichis counter-intuitive, as these junctions are

assumed to be stable. For instance, afraction of the E-cadherin molecules atAJs in highly polarized cells have a half-residence time of only several minutes in

the junction (de Beco et al., 2009). As such,E-cadherin is in constant traffic between thecell surface and the recycling endosomes

(Bryant and Stow, 2004; de Beco et al.,2009; Hong et al., 2010; Le et al., 1999;Troyanovsky et al., 2006). However, in

cells with an intermediate phenotype, suchas A431 squamous carcinoma cells and E-cadherin-transfected Chinese hamster ovary(CHO) cells, E-cadherin turnover is not

only faster, but also appears to be governedby non-clathrin-mediated endocytosis(Hong et al., 2010). TJs are also constantly

remodeled whilst maintaining a barrierbetween epithelial cells (Shen et al., 2008;Steed et al., 2010). Thus, EMT is likely to

be dependent on these constitutivelyestablished dynamic changes at celljunctions.

Early intermediate state of EMT

The absence of well-developed TJs in in

vitro cell lines might be responsible for the

paucity of research emphasizing the role ofTJs in EMT (Thiery and Huang, 2005).The central role of TJ stability in EMT

regulation was discovered serendipitouslyin a luminescence-based interactomemapping for transforming growth factor-

beta (TGFb) signaling (Barrios-Rodiles etal., 2005; Ozdamar et al., 2005). Occludinwas found to bind directly to TGFb type Ireceptor (TGFBR1). TGFBR1 forms a

complex with PAR6 and occludin at TJsindependently from its ligand. Uponstimulation with TGFb, TGFBR2 is

recruited to the TJ complex andphosphorylates PAR6, which binds to theE3-ubiquitin ligase, SMURF1. Smurf1

then ubiquitinates RhoA leading to itsdegradation, which interferes with thecortical actin cytoskeleton and ultimatelycontributes to TJ disassembly (Ozdamar

et al., 2005). In mammary epithelial cells,activated EGFR-related protein 2 (ERBB2)directly interacts with the PAR6-aPKC

complex and disrupts the apical polarityof multi-acinar structures. This ERBB2-PAR6-aPKC association promotes

PAR3 dissociation from the PAR6-aPKCcomplex (Aranda et al., 2006). Therefore,the breakdown of TJs and disruption of the

PAR complex owing to the dissociation ofPAR3 are the key events in the earlyintermediate state of EMT.

Late intermediate state of EMT

The late intermediate state of EMT is

hallmarked by AJ destabilization. First, theE-cadherin complex is destabilized by post-

translational modifications, such as increasedphosphorylation, its internalization anddegradation of E-cadherin and b-catenin

(Bryant and Stow, 2004; D’Souza-Schorey,2005). Evidence for this comes from growth

factor or receptor tyrosine kinase (RTK)-mediated induction of EMT; examplesinclude hepatocyte growth factor (HGF) and

its receptor Met, epidermal growth factor(EGF) and EGFR, and fibroblast growth

factor (FGF) and FGFR (Thiery, 2002). InHGF-induced EMT, Met co-localizes with E-

cadherin and they are both co-endocytosed(Kamei et al., 1999) through the upregulationof endogenous ADP-ribosylation factor 6

(ARF6) GTPase (Palacios et al., 2001). Thisleads to the phosphorylation of E-cadherin on

two tyrosine residues and its subsequentubiquitination by the E3-ubiquitin ligase

HAKAI (also known as CBLL1) (Fujitaet al., 2002; Mukherjee et al., 2012). EGFalso induces EMT in non-transformed cells,

such as normal ovarian surface epithelium(Salamanca et al., 2004) and in various human

carcinoma cells that overexpress EGFR (Barret al., 2008). During the early stages of EGF-induced EMT, a rapid increase in tyrosine

phosphorylation of the E-cadherin–b-catenincomplex disrupts cell-cell adhesion and

causes cell dissociation (Fujii et al., 1996;Shibamoto et al., 1994), possibly through

caveolin-1-dependent endocytosis of E-cadherin (Lu et al., 2003). Aside fromRTKs, Src tyrosine kinase also has a crucial

role during this stage of EMT (Rodier et al.,1995). Tyrosine phosphorylation of its

substrates b-catenin and p120-catenindecreases their binding affinity to E-cadherin. In lysophosphatidic acid (LPA)-

induced ovarian cancer cell dispersal, the Src-related tyrosine kinase, Fyn, phosphorylates

b-catenin and p120-catenin, thus,destabilizing the b-catenin–p120-catenin–a-

catenin complex and resulting in thebreakdown of cell adhesions (Huang et al.,2008). However, Src can also induce

E-cadherin endocytosis-mediated AJdisassembly by tyrosine phosphorylation-

independent mechanisms. In MDCK cells,Src induces the depletion of T-cell lymphoma

and invasion metastasis 1 (TIAM1),a Rac-specific GEF, through TIAM1phosphorylation and proteasomal degradation

(Palovuori et al., 2003; Woodcock et al., 2009).Interestingly, E-cadherin-mediated cell

adhesion might also inhibit ligand-dependentRTK activation, such as that of EGFR (Perrais

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et al., 2007; Qian et al., 2004), which raises thequestion as to how cells that express E-

cadherin at AJs trigger EMT through growthfactor-mediated stimulation. One plausibleexplanation is that cells that respond to RTKsignals might have entered an early

intermediate state through a b-catenin-dependent mechanism.

Unlike in AJs, desmosome stability is

affected by serine phosphorylation bymembers of the protein kinase C (PKC)family (Volberg et al., 1992). Plakophilin

2, which regulates intercellular junctionassembly, is a crucial scaffold forPKCa (Bass-Zubek et al., 2008). PKCasignaling also regulates the dynamics of

desmoplakin through the Ca2+ ATPaseSERCA2 (also known as ATP2A2),which stabilizes desmosomes (Hobbs

et al., 2011). However, plakoglobin, whichis structurally related to b-catenin, can bephosphorylated at both tyrosines and

serines (Gaudry et al., 2001; Shibamotoet al., 1994). Desmosome disassemblyoften accompanies AJ disruptionduring late intermediate EMT, when TJs

are also weakened. This suggests aninterdependence between desmosomes andAJs, which is not surprising given the

fact that E-cadherin, plakophilin anddesmoplakin genes are direct targets oftranscriptional repressors, such as Zinc

finger E-box-binding homeobox 1 (ZEB1),which is a key driver of EMT (Vandewalleet al., 2005). Initial transcriptional changes,

such as upregulation of snail homologue 1and 2 (SNAI1 and SNAI2), frequently occurat this late intermediate state, and theiractivation is often transient and results in a

partial EMT (Carrozzino et al., 2005;Savagner et al., 1997). In an induciblesystem, the incomplete downregulation of

E-cadherin by SNAI1 in MDCK cellsresults in a partial EMT effect on TJs(Carrozzino et al., 2005). However, when

expression of SNAI1 and SNAI2 issustained, a full EMT phenotype can beinitiated. SNAI1-overexpressing mouse

EpH4 epithelial cells fully execute EMTby downregulating occludins and claudins-3, -4 and -7 (Ikenouchi et al., 2003),followed by the post-transcriptional loss of

ZO1 (Ohkubo and Ozawa, 2004).

Transcriptional regulation of SNAI1 andSNAI2 at this stage also contributes to the

effect that EMT has on other polaritycomplex proteins, such as Crumbs3. InMDCK cells, expression of TGFbupregulates SNAI1 transcripts at around48 hours, which is followed bydownregulation of Crumbs3 transcription

after 96 hours, concomitant with the lossof E-cadherin (Whiteman et al., 2008).

Full mesenchymal state of EMT

The full mesenchymal state of EMT isachieved through the transcriptionalrepression of desmosomal, AJ and TJproteins (Moreno-Bueno et al., 2008).

Interestingly, b-catenin, p120-catenin andplakophilin 2 have nuclear localizationsignals (NLS) and can shuttle between

the nucleus, cytoplasm and cell membrane(Fagotto et al., 1998; Mertens et al., 2001;Roczniak-Ferguson and Reynolds, 2003).

Once displaced from junctional complexes,a fraction of these proteins is targeted tothe nucleus where they interact with

transcription factors to activate theirdownstream target genes, including EMTdrivers (Gottardi and Gumbiner, 2001).Nuclear b-catenin has been identified in

single migratory cells at the coloncarcinoma invasive front, which hasundergone full EMT (Brabletz et al.,

2005a; Brabletz et al., 2005b). It hasbeen suggested that nuclear b-catenincoactivates Wnt signaling with TCF4 and

lymphoid enhancer-binding factor 1(LEF1), and that formation of the b-catenin–TCF4 complex directly executesfull EMT through induction of the E-

cadherin transcriptional repressor ZEB1(Sanchez-Tillo et al., 2011). However,thus far, there is no direct evidence for

the involvement of nuclear p120-catenin orplakophilin 2 in the execution of EMT.

Conclusions and future perspectivesThe conversion of polarized epithelia into

mesenchymal-like cells follows an orderedsequence. Initially, EMT targets thePAR polarity complex and the closelyassociated TJs. During the intermediate

states, AJs and desmosomes weakenand fragment through post-translationalmodifications and transcriptional

repression. Concomitant with these ‘loss-of-function’ events, many ‘gain-of-function’ events occur, where vimentin-

containing filaments replace intermediatecytokeratin filaments in mesenchymal-likecells to induce motile and invasive

properties that potentially favor localinvasion and metastasis (Thiery, 2002).This plasticity is closely linked to thejunctional dynamics with which cell-cell

adhesions are formed and remodeled.

Epithelial and mesenchymal phenotypes

govern the sensitivity of targeted therapiesor chemotherapies in human cancers.Thus, it might be possible to exploit

reversibility of EMT for therapeutic

purposes (Arumugam et al., 2009; Buck

et al., 2007; Thomson et al., 2005; Yauch

et al., 2005). Drug resistance tends to either

result from or induce carcinoma clones thathave undergone EMT (Kajiyama et al.,

2007; Yang et al., 2006). Therefore,

reversing EMT could be a promising

strategy to increase carcinoma sensitivity

to drugs. For example, tyrosine kinase

inhibitors have been implicated in EMT

reversal in cancers (Choi et al., 2010;

Lorch et al., 2004; Nagai et al., 2011).

New genes that mediate EMT are

expected to be uncovered in the nearfuture. Therefore, elucidation of their

translational modifications and integration

into specific gene regulatory networks will

contribute towards the development of

EMT-based therapeutic interventions

(Chua et al., 2011). The EMT reversal

process is reminiscent of nascent

junctional maturation; consequently, further

exploration of the factors involved is

essential to shed light on the molecularbasis of EMT. It will be very exciting to see

whether these factors constitute valuable

targets for therapeutic EMT reversal.

FundingThis work was supported by the Agency

for Science, Technology and Research

(A*STAR) Biomedical Research Council,

Singapore; and the Health Research

Council of New Zealand International

Investment Opportunities Fund (IIOF)

[grant number 1010124640].

A high-resolution version of the poster is available for

downloading in the online version of this article at

jcs.biologists.org.

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