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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 ([email protected])
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
Journal of Cell Science 125 (19)4418
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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
Journal of Cell Science 125 (19) 4419
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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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