CORRECTION

ERM proteins in cancer progression

Jarama Clucas and Ferran Valderrama

There was an error published in J. Cell Sci. 127, 267-275.

On page 268 of this article, the official name of NcK-interacting kinase (NIK) was incorrectly given as MAP3K14. The correct proteinsymbol is MAP4K4.

The correct sentence should read:

It has been proposed that the conserved threonine residues of these proteins can be phosphorylated in a tissue and cell-dependent manner

through, for example, Rho-associated kinase (ROCK) (Hebert et al., 2008; Matsui et al., 1998), protein kinase Ca (PRKCA, hereafterreferred to as PKCa) (Ng et al., 2001), NcK-interacting kinase (MAP4K4, hereafter referred to as NIK) (Baumgartner et al., 2006)lymphocyte-orientated kinase (STK10; hereafter referred to as LOK) (Belkina et al., 2009; Viswanatha et al., 2012) and others (see Table

1), which indicates that ERM proteins are involved in a wide variety of signalling pathways (Fig. 2).

We apologise to the readers for any confusion that this error might have caused.

Journal of Cell Science JCS170027.3d 25/2/15 08:11:00The Charlesworth Group, Wakefield +44(0)1924 369598 - Rev 9.0.225/W (Oct 13 2006)

� 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 000, 000–000 doi:10.1242/jcs.170027

1

Jour

nal o

f Cel

l Sci

ence

COMMENTARY ARTICLE SERIES: CELL BIOLOGY AND DISEASE

ERM proteins in cancer progression

Jarama Clucas and Ferran Valderrama*

ABSTRACT

Members of the ezrin–radixin–moesin (ERM) family of proteins are

involved in multiple aspects of cell migration by acting both as

crosslinkers between the membrane, receptors and the actin

cytoskeleton, and as regulators of signalling molecules that are

implicated in cell adhesion, cell polarity and migration. Increasing

evidence suggests that the regulation of cell signalling and the

cytoskeleton by ERM proteins is crucial during cancer progression.

Thus, both their expression levels and subcellular localisation would

affect tumour progression. High expression of ERM proteins has

been shown in a variety of cancers. Mislocalisation of ERM proteins

reduces the ability of cells to form cell–cell contacts and, therefore,

promotes an invasive phenotype. Similarly, mislocalisation of ERM

proteins impairs the formation of receptor complexes and alters the

transmission of signals in response to growth factors, thereby

facilitating tumour progression. In this Commentary, we address the

structure, function and regulation of ERM proteins under normal

physiological conditions as well as in cancer progression, with

particular emphasis on cancers of epithelial origin, such as those

from breast, lung and prostate. We also discuss any recent

developments that have added to the understanding of the

underlying molecular mechanisms and signalling pathways these

proteins are involved in during cancer progression.

KEY WORDS: ERM, Cancer progression, Cell signalling,

Cytoskeleton

IntroductionEzrin, radixin and moesin (ERM) are three highly homologous

protein members of the FERM (4.1-band ERM) superfamily

(Sato et al., 1992). Fundamentally, they are essential for linking

the actin cytoskeleton to the cell membrane and are key

organisers of specialised membrane domains, such as apical

microvilli (Lan et al., 2006; Takeuchi et al., 1994), lamellipodia

and filopodia (Baumgartner et al., 2006; Lamb et al., 1997). Their

function as cytoskeletal linkers places them at the centre of an

elaborate regulatory network of many cellular processes, whether

under normal and controlled conditions, such as migration,

growth and adhesion, or in pathological scenarios, such as cancer

cell invasion and metastasis. Importantly, several studies support

a role for ERM proteins in many fundamental signal transduction

pathways to influence cell adhesion (Pujuguet et al., 2003;

Takeuchi et al., 1994), migration (Crepaldi et al., 1997; Naba

et al., 2008; Ng et al., 2001; Valderrama et al., 2012) and

morphogenesis (Bretscher et al., 2002; Crepaldi et al., 1997;

Gautreau et al., 2000; Hsu et al., 2012) in response toextracellular cues. There is also evidence that the ERM proteinsare important for cell–cell and cell–matrix contacts – potentially

through interactions with cadherin complexes and integrins (Jungand McCarty, 2012; Pujuguet et al., 2003) – as well as for thereorganisation of the cytoskeleton (Belkina et al., 2009;Bretscher, 1983; Gautreau et al., 1999); yet, the molecular

mechanisms in which they participate within pathologicalprocesses such as cancer still remain ill-defined. In thefollowing sections, we provide an overview of the structure and

regulation of ERM proteins, and subsequently focus our attentionon recent advances that help to elucidate the role of these proteinsin cancer progression.

ERM structure and regulationERM protein structureERM proteins have been highly conserved through evolution,

presenting more than 75% amino acid identity within thecommon FERM domain and F-actin-binding site that are sharedbetween all members of this superfamily (Gary and Bretscher,

1995). ERM proteins consists of a FERM domain (,300 aminoacids) situated at the N-terminus, a C-terminal region (,100amino acids), and an a-helical region (,200 amino acids) thatlinks the C-terminus and the FERM domain (Fig. 1) (Algrain

et al., 1993; Gary and Bretscher, 1995; Turunen et al., 1994). TheFERM domain comprises three lobes that are arranged as acloverleaf – F1, F2 and F3 – and mediates the link between the

ERM proteins and specific membrane-bound proteins localised inactin-rich regions. The C-terminal domain, also referred to as theC-terminal ERM-associated domain (C-ERMAD), contains the

F-actin-binding site, which mediates the link between the actincytoskeleton and the plasma membrane (Berryman et al., 1993;Pearson et al., 2000).

ERM activationThe C-terminal domain of ERM proteins is capable of binding to

the FERM domain of the same molecule, through self-association, to form a monomer or, with another ERMmolecule, to form a homo- or heterodimer, as observed for

ezrin and moesin (Fig. 1) (Gary and Bretscher, 1995). Thisintramolecular interaction leads to a closed conformation orhead–tail interaction, and the consequent masking of both themembrane and actin binding sites, resulting in inactivation of

the proteins (Bretscher et al., 2002; Gary and Bretscher, 1995).The ERM proteins will remain in this inactive conformationuntil the FERM domain binds to phosphatidylinositol (4,5)-

bisphosphate [PtdIns(4,5)P2] (Bretscher et al., 2002; Fievet et al.,2004; Niggli et al., 1995; Pearson et al., 2000) – which is locatedat the cell membrane – and the conserved threonine residues –

T567, T564 and T558, for ezrin, radixin and moesin, respectively– become phosphorylated (Fig. 2) (Fievet et al., 2004; Matsuiet al., 1998; Nakamura et al., 1995; Niggli and Rossy, 2008). This

activation results in directly linking the actin cytoskeleton to the

Division of Biomedical Sciences, St George’s University of London, CranmerTerrace, London SW17 0RE, UK.

*Author for correspondence (fvalderr@sgul.ac.uk)

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 267–275 doi:10.1242/jcs.133108

267

Jour

nal o

f Cel

l Sci

ence

plasma membrane through the positively charged F3 lobe of theFERM domain, which has been proposed to have a high affinityfor negatively charged phospholipids within the plasma

membrane (Pearson et al., 2000). Alternatively, ERM can bindto the PDZ domains (protein–protein recognition modules) ofother scaffolding proteins, such as ERM-binding phosphoprotein

50 (EBP50; also known as Slc9a3r1) or Na+/H+ exchangeregulatory factor 2 (NHERF2; also known as Slc9a3r2) (Arpinet al., 2011; Garbett and Bretscher, 2012; Gautreau et al., 1999;

Harris and Lim, 2001; Reczek et al., 1997). As their role insignalling has become more apparent, further studies have nowdemonstrated that all three proteins can bind to several

transmembrane receptors, including several receptor kinases(RTKs), epidermal growth factor receptor (EGFR), hepatocyte

growth factor receptor (HGFR), platelet-derived growth factorreceptor (PDGFR) (Berryman et al., 1993; Crepaldi et al., 1997;Krieg and Hunter, 1992; Matsui et al., 1998; Naba et al., 2008;Orian-Rousseau et al., 2007) and to co-receptors, such as CD44,

CD43, ICAM1 and ICAM2 (Fig. 2) (Matsui et al., 1998; Orian-Rousseau et al., 2007; Takeuchi et al., 1994).

It has been proposed that the conserved threonine residues of

these proteins can be phosphorylated in a tissue and cell-dependent manner through, for example, Rho-associated kinase(ROCK) (Hebert et al., 2008; Matsui et al., 1998), protein kinase

Ca (PRKCA, hereafter referred to as PKCa) (Ng et al., 2001),NcK-interacting kinase (MAP3K14, hereafter referred to as NIK)(Baumgartner et al., 2006) lymphocyte-orientated kinase (STK10;

hereafter referred to as LOK) (Belkina et al., 2009; Viswanathaet al., 2012) and others (see Table 1), which indicates that ERMproteins are involved in a wide variety of signalling pathways(Fig. 2). Ezrin has three tyrosine sites (Y145, Y353 and Y477)

that are phosphorylated through several kinases (Table 1), whichcan lead to the activation of different signalling pathways(Gautreau et al., 1999; Mak et al., 2012; Srivastava et al., 2005).

It is now accepted that the association between ERM proteinsand receptors at the plasma membrane is important for mediatingmany of these pathways. In particular, their association with

CD44, EGFR and HGFR has been suggested to have a role in themigration process that is associated with cancer cell invasion andmetastasis (Chen and Chen, 2006; Mak et al., 2012; Martin et al.,

2003; Zhu et al., 2012). However, there are conflicting data withregard to which kinases are responsible for not only the activationbut the phosphorylation and regulation of ERM proteins. Forexample, it has been suggested that ROCK phosphorylates ERM

proteins in vitro (Estecha et al., 2009; Hebert et al., 2008; Matsuiet al., 1998), but other studies suggest that ROCK does notphosphorylate ERM proteins in vivo, and that their activation

instead involves PtdInsP2 and Rho (Jeon et al., 2002; Matsuiet al., 1999); it has also been suggested that the Rho/ROCKpathway is not essential for this phosphorylation event

F-actin-binding domain

F3 FF3

F1 11

F2

N-terminal C-terminal

α-helical domain

Phosphatase

PtdIns(4,5)P2 +Kinase

nding

P

A

B

P

Fig. 1. Structure of ERM proteins, and their active and inactive states.(A) The N-terminal FERM domain consists of the three lobes F1, F2 and F3(blue), and is linked through an a-helical domain (orange) to the F-actin-binding domain (green) at the C-terminus. (B) After binding to of PtsdIns(4,5)P2 at the plasma membrane, specific kinases can phosphorylate theirconserved threonine residues (ezrin, T567; radixin, T564, moesin, T558,respectively), in turn allowing the protein to unfold so the F-actin domain isexposed and can link to the cytoskeleton. (Left) Upon dephoshorylation of theF-actin-binding domain, the FERM and F-actin binding domains interactintramolecularly and/or intermolecularly to form inactive monomers or dimers.

Cell survival

Extracellular space

Intracellular space

PtdIns(4,5)P2

P

F-actin

Cell migration and invasion

Cell adhesion

PI3K/Akt

Cell morphology

Transmembrane receptors (e.g. CD44, EGFR, ICAM1, ICAM2, CD43)

PKC EGF/HGF Rac1 RhoA

Rac1 E-cadherin RhoGTPases

Wnt/β-catenin

Cell proliferation

P

P

+ Kinase

Fig. 2. ERM activation and signallingpathways. ERM proteins bind toPtdInsP2 and are then recruited tospecific areas of the cell membranethrough their N-terminal FERM domain(left), which exposes the conservedthreonine residue of the F-actin domain.Specific kinases (see Table 1) can nowphosphorylate the conserved tyrosineresidue, after which the ERM proteincan function as the link between cellmembrane and cytoskeleton (middle).Once activated, ERM proteins can bindone of several transmembranereceptors and initiate a range ofsignalling transduction pathways, eitherdirectly as shown or indirectly throughother scaffolding proteins (right).

COMMENTARY Journal of Cell Science (2014) 127, 267–275 doi:10.1242/jcs.133108

268

Jour

nal o

f Cel

l Sci

ence

(Yokoyama et al., 2005). The serine/threonine-protein kinase

MST4 has been shown to phosphorylate ezrin (ten Klooster et al.,2009); however, knockdown of this kinase in the human placentalchoriocarcinoma cell line JEG-3 that is known to express MST4had no effect on the level of ezrin phosphorylation (Viswanatha

et al., 2012). The majority of relevant studies that used a varietyof established immortalised cell lines have implicated particularkinases; but once further work is carried out in vivo, it may

become apparent that activation and regulation of these proteinsis dependant not only on the abundance of a particular kinase,but also on its temporal–spatial expression in different cells,

tissues or organs. Although it would be beneficial to discusssuch discrepancies further, this is outside the scope of thisCommentary and we, therefore, focus our attention on the

molecular mechanisms and kinases that have been suggested tobe involved in cancer progression.

ERM proteins in cancer progressionERM proteins have been mainly characterised in their rolein epithelial morphogenesis, adhesion and migration (Arpin et al.,2011; Fehon et al., 2010; Bretscher et al., 2002), three key events

that, when altered, can participate in the induction of a cancerphenotype. Therefore, it is not surprising that ERM proteins havebeen suggested to influence tumour progression. Many clinical

studies have linked high expression of ezrin with poor outcome inpatients that suffer from a wide variety of cancers, includingbreast, lung or prostate, and only little attention has been given toradixin and moesin. The mislocalisation of these proteins can

interfere with the formation of receptor complexes and leads tochanges in signalling pathways in response to growth factors,which could aid the progression of a tumour (Arpin et al., 2011)

(Fig. 3). It is becoming increasingly apparent that all three ERM

proteins can modulate the activity of the Ras superfamily of smallGTPases, including the Ras and Rho subgroups, by interactingwith their guanine exchange factors (GEFs) and guaninenucleotide dissociation inhibitors (GDIs), which are vital for

their activation (Sperka et al., 2011; Takahashi et al., 1997;Valderrama et al., 2012). Ras is a molecular switch that isimportant for RTK-dependent activation of the MAP kinase

pathway, such as through EGFR and HGFR (Easty et al., 2011;Orian-Rousseau et al., 2007; Ridley et al., 1995). ERM proteinshave been proposed to be responsible for a new step in Ras

activation in that they are recruited to F-actin and co-receptors,specifically to receptors involved in cell adhesion, such as b1integrin and CD44, at the plasma membrane in response to

activation of RTKs. In doing so, ERM proteins can form acomplex that acts as a scaffold and binds both GDP-bound Rasand the N-terminal region of the GEF son of sevenless homolog(SOS) (Sperka et al., 2011). Ezrin has specific binding

domains for CD44, Ras and the autoinhibitory Dbl-homology–pleckstrin-homology (DH–PH) domain of SOS butonly after ezrin is in a complex with F-actin and its co-receptor

can it bind to the DH–PH domain of SOS and expose theallosteric binding site that is specific for GDP binding of Ras(Sperka et al., 2011).

All three ERM proteins have been suggested to act as scaffoldsin Ras activation, and it is well known that oncogenic mutationsin Ras, or even in the RTKs EGFR and HGFR, can result in thedevelopment of a cancerous phenotype (Bonaccorsi et al., 2004;

Fehon et al., 2010; Ridley, 2001). Therefore, it is tempting tospeculate that ERM overexpression, which is commonly seen in avariety of cancers, is the cause for an increase in the activation of

Table 1. Kinases proposed to phosphorylate ERM proteins

Kinase(mode of phosphorylation) ERM protein Function of phosphorylated ERM protein

Phosphorylatedresidue References

Akt2 (directly) Ezrin NHE3 translocation and activation T567 (Shiue et al., 2005)CDK5 (directly) Ezrin Cell senescence T235

T567(Yang et al., 2003)

EGFR (indirectly) Ezrin Cell proliferation, lamellipodia formation T567, Y145, Y353 (Gandy et al., 2013;Krieg and Hunter, 1992)

HGFR (indirectly) Ezrin Cell scattering T477 (Naba et al., 2008)Lck (directly) Ezrin T-cell activation Y145 (Autero et al., 2003)LOK (unknown) Ezrin

MoesinCytoskeletal rearrangement in hematopoietic cellsmicrovilli formation in polarised epithelial cells

T567T588

(Belkina et al., 2009;Viswanatha et al., 2012)

MRCK (directly) Moesin Effector of Cdc42 in filopodia formation T588 (Nakamura et al., 2000)Mst4 (directly) Ezrin Brush border polarity T567 (ten Klooster et al., 2009)NIK (MAP3K14; directly) Ezrin

RadixinMoesin

Lamellipodia formation and cell morphology T567T564T588

(Baumgartner et al., 2006)

PKA (directly) Ezrin Gastric parietal cell activation Ser66 (Zhou et al., 2003)PKCa (PRKCA; directly) Ezrin, radixin,

moesinCell migration T567 (Ng et al., 2001)

PKCa (PRKCI; directly) Ezrin Intestinal epithelial cell differentiation T567 (Wald, 2008)PKCh (PRKCQ; directly) Moesin Intracellular signalling (pathway yet unknown) T558 (Pietromonaco et al., 1998;

Simons et al., 1998)ROCK (Rho-kinase; directly) Ezrin

RadixinMoesin

Effector of RhoAFas-mediated apoptosisCell migration and invasion

T567T564T588

(Hebert et al., 2008;Matsui et al., 1998)

Slik Drosophila

moesinCortical contraction and rigidity during mitosis dMoesin T559 (Carreno et al., 2008)

SLK Ezrin microvilli formation in polarised epithelial cells T567 (Viswanatha et al., 2012)Src (directly) Ezrin Cell spreading Y145

Y477(Mak et al., 2012)

COMMENTARY Journal of Cell Science (2014) 127, 267–275 doi:10.1242/jcs.133108

269

Jour

nal o

f Cel

l Sci

ence

Extracellular matrix

Signalling: -Polarity -Morphology -Adhesion -Survival etc...

Normal acini

Intraluminalproliferation

Carcinomain situ

Secretions

TF

BRCA1

Dsg3

PKC-induced cell migration

CD44

Directionof invasion

Ezrin

Radixin

X

Overexpressionof ezrin

Loss ofcell-cellcontacts

Cellsurvival

PI3K

Cytoskeletalreorganisation

and/orlamellipodiaformation

X

TF

Overexpressionof radixin?

Radixin-dependentVav-GEF activity

Vav

Rac1

Rac1 GTP X

GDP

TF

Moesin

CD44

Overexpressionof β-catenin

ROCK+ PKC

NF2 RhoA

RhoA GTP

GDP

β-catenin

β-catenin

EMT

X

A

C Cancer progression

B Normal physiological conditions

TGF-β

Fig. 3. Specific roles of ERMproteins in epithelial cancerprogression. (A) In epithelialcancers, tumours commonly developwithin the lumen of individual glands,such as acini in breast and prostate,in a process known as intraluminalproliferation. Cells that have acquiredinvasive ability break out of the basalcell layer and the basementmembrane of these glands, anddevelop a tumour in situ. (B) Undernormal physiological conditions, ERMproteins arrange the cytoskeleton ofepithelial cells, as well as induceseveral signalling pathways for cellsurvival, polarity and morphology tocreate an apical–basal polarity andform cell–cell contacts that constitutethe organised structures commonlyseen in glandular tissue.(C) Overexpression of ERM proteinspromotes cancer progression in arange of epithelial cancers, such asbreast, lung and prostate. Asepithelial cells undergo oncogenicmutations, they begin to lose cell–cellcontacts and change their polarity ormorphology, thereby developing amore mesenchymal phenotype thatfacilitates the proliferation of cells intothe lumen of the acini. Disruptingthese layers leads to their invasioninto surrounding tissue andsubsequent spreading to secondarysites around the body. The roles of thedifferent ERM proteins are illustrated.Ezrin overexpression promotessurvival of cancer cells, migrationand/or invasion of cancer cells tosurrounding tissue and disrupts cell-cell contacts. Ezrin is upregulated byoncogenic transcription factors suchas Myc and the downregulation oftumour suppressor factors. Thisresults in the disruption of cell–cellcontacts through interaction of ezrinwith Fes kinase, which promotesPKC- and CD44-induced cellmigration, and decreases b-cateninlevels as well as increases cellsurvival through activation of PI3K.Radixin induces Rac1-mediated cellmigration through inhibition of Vav.Moesin upregulation, through ROCK-and PKC-dependent phosphorylation,induces EMT and allows cells tobecome more motile and to changepolarity; it also promotes RhoA-induced cell migration. Moesin alsointeracts with CD44 and itsupregulation displaces the tumoursuppressor NF2, promoting CD44-mediated cell migration, as well asinduces the translocation of b-cateninto the nucleus.

COMMENTARY Journal of Cell Science (2014) 127, 267–275 doi:10.1242/jcs.133108

270

Jour

nal o

f Cel

l Sci

ence

these signalling pathways. Recently, the role of the three ERMproteins in tumour progression has become the focus of attention,

in order to determine the exact molecular roles these proteinshave during cancer progression. Below, we discuss the roles ofeach of the ERM proteins individually, and aim to present acomprehensive argument against functional redundancy between

these three homologous proteins in cancer progression.

EzrinThe expression of ezrin in over 5000 human cancers, includingbreast, lung and prostate cancers compared with normal tissues,was analysed in a large-scale study that used microarray

immunohistochemistry (Bruce et al., 2007). Although ezrin isexpressed in most normal and cancer tissues, its expression issignificantly higher in cancers of mesenchymal origin (sarcomas)

and in prostate cancer when compared with breast cancer, andshows no change in lung cancer tissue (Bruce et al., 2007). In ezrin,mutation of Y477, a tyrosin residue not conserved in radixin ormoesin, reduced cell migration in the highly metastatic mouse

mammary carcinoma cell line AC2M2 in 3D cultures (which iscommonly used to recapitulate tumourigenesis) (Debnath andBrugge, 2005) as well as local tumour invasion in mice (Mak et al.,

2012). Because Src is responsible for the phosphorylation of ezrinat Y477 (Elliott et al., 2004; Srivastava et al., 2005), it has beensuggested that the ezrin–Src pathway is a potential prognostic

marker for invasive breast cancer in humans (Mak et al., 2012).Because sarcomas have a mesenchymal phenotype, it would beinteresting to determine whether Src phosphorylation of ezrin at

Y477 has a role in the activity of these cancers.The breast cancer cell lines MCF10A (semi-normal) and MDA-

MB-231 (metastatic) have been used in a 3D Matrigel model, inwhich silencing of ezrin by using short hairpin RNA (shRNA)

leads to a decreased invasive potential (Konstantinovsky et al.,2012). Abnormal ezrin distribution has been correlated with poorprognosis of breast cancer patients, where ezrin is found to

relocalise from the apical regions observed in non-tumourigeniccell lines to motile projections in invasive cell lines and thecytoplasm of breast tumours (Sarrio et al., 2006). Phosphorylation

of ezrin Y145 also led to cell spreading in the mouse mammarycarcinoma cell line SP1 (Elliott et al., 2004) and in pig kidneyepithelial (LLC-PK1) cells (Srivastava et al., 2005).

Interestingly, and not observed in the large-scale screen

mentioned above (Bruce et al., 2007), ezrin has also beenimplicated in lung cancer (Li et al., 2012). Here, ezrin expressionwas elevated in the highly metastatic human lung cancer cell lines

LTE, BE1 H446 and H460 when compared with the lowmetastasis human lung adenocarcinoma cell lines SPC andA549. Furthermore, knockdown of ezrin by using small

interfering RNA (siRNA) resulted in significant reduction inproliferation, migration and invasion of these cells in vitro (Liet al., 2012). As in breast cancer cells, localisation of ezrin in lung

cancer cells also appears to be altered, i.e. from the apicalmembrane in normal bronchial epithelium to the cytoplasm inlung cancerous cells. This suggests that the relocalisation of ezrinis a significant factor in altering the activation of signalling

pathways and of actin reorganisation, which both are fundamentalfor cancer progression. In the same study, reduced b-catenin andincreased E-cadherin expression was observed with no change in

the respective mRNA levels. Developmental biology studies havehighlighted a role for ERM proteins in junction remodelling(Dard et al., 2001). For instance, mutations in ezrin within the

conserved threonine residue prevent the formation of E-cadherin-

mediated cel–cell contacts during blastocyst formation in mice(Dard et al., 2001). It has been suggested that the small pool of

ezrin that is present at cell–cell contacts is sufficient to recruit Feskinase – a non-RTK that has also been implicated in cancerprogression (Condorelli et al., 2011) – and to modulate junctionformation by directly interacting with the SH2 domain of Fes

kinase (Naba et al., 2008). This interaction is important for thelocalisation and activation of Fes, which consequently results inthe disassembly of cell–cell contacts. Moreover, ERM proteins

are crucial for Rac-dependent assembly of focal adhesions andadherens junctions (Mackay et al., 1997; Pujuguet et al., 2003).During the formation of cell–cell contacts, fluctuation in the

expression levels and downregulation of Rac and Rho GTPasesare known to occur (Yamada and Nelson, 2007). Ezrin has beenproposed to regulate E-cadherin-dependent adherens junction

assembly through Rac1 activation and the trafficking ofE-cadherin to the plasma membrane (Pujuguet et al., 2003).Therefore, it is possible that overexpression of ezrin prevents therequired fluctuations in the activity of Rac1, as well as the

localisation of E-cadherin at the plasma membrane, consequentlydisrupting cell–cell contacts that are commonly seen in tumourdevelopment (Arpin et al., 2011). As deregulation of E-cadherin

function is believed to promote tumour progression (Canel et al.,2013), it is possible that ezrin has a role in the post-transcriptionalregulation of E-cadherin during cancer progression, although

further investigations are required to better define the molecularmechanisms underlying this role.

Many immunohistochemical studies on high-grade prostate

intraepithelial neoplasia (HGPIN) – the proposed precursor toprostate cancer – and advanced cancerous prostate tissues havealso shown overexpression of ezrin (Pang et al., 2004; Valdmanet al., 2005). However, expression of ezrin is in fact much higher

in HGPIN tissues compared with advanced prostate cancerspecimens (Pang et al., 2004). Importantly, this change inexpression does not appear to be the result of genomic alterations

(Amler et al., 2000). Depletion of ezrin, or overexpression of adominant-negative (T567A) or non-phosphorylatable (Y353F)mutant significantly reduces invasion (Chuan et al., 2006). Thus,

the level of ezrin phosphorylation may be important in thetumourigenesis of precursor lesions – such as that of HGPIN –and, consequently, may trigger carcinogenic tissue to becomeinvasive before ezrin is potentially deregulated in more advanced

stages of the disease (Pang et al., 2004; Valdman et al., 2005).The observed increase of ezrin expression in prostate cancer

has been suggested to be a result of increased expression of the

oncogene Myc (Chuan et al., 2010). In the presence of androgenssuch as testosterone it appears that Myc can bind to the canonicalE-box in the proximal promoter region of ezrin and induce its

transcription. However, ezrin itself can regulate the level of Mycthrough the PI3K/Akt pathway, effectively creating a positive-feedback loop. This regulatory loop has been shown to be

essential for cell proliferation and invasion in both androgen-dependent and -independent metastatic prostate cell lines (Chuanet al., 2010). Under normal physiological conditions, thephosphorylation of ezrin Y353 is important for survival of

epithelial cells through activation of the PI3K/Akt pathway, and3D cell cultures of the epithelial cell line LLC-PK1 showed thatezrin binds to p85, the regulatory subunit of PI3K, in order to

mediate the PI3K/Akt pathway (Gautreau et al., 1999). Therefore,overexpression of ezrin as a result of Myc overexpression could,in turn, maintain the survival of cancer cells through activation of

the PI3K/Akt pathway (Fig. 3).

COMMENTARY Journal of Cell Science (2014) 127, 267–275 doi:10.1242/jcs.133108

271

Jour

nal o

f Cel

l Sci

ence

As mentioned above, high expression of ezrin has beenobserved in breast cancer cell lines, as well as in tissue samples of

breast cancer patients (Bruce et al., 2007; Konstantinovsky et al.,2012; Mak et al., 2012; Sarrio et al., 2006). The recent discoveryof a new role for BRCA1, a well-established tumour suppressorgene in breast and ovarian cancer, in cell motility and invasion

might shed light on the regulation of ezrin in breast cancer (Coeneet al., 2011). Here, depletion of BRCA1 resulted in increased cellspreading and motility of single cells, as well as increased

invasive behaviour of cells in wound healing assays in vitro

(Coene et al., 2011). BRCA1 was also suggested to colocalisewith radixin and moesin, therefore this interaction is not unique to

ezrin (Coene et al., 2011). Could BRCA1 target ezrin in order toregulate its expression? The BRCA1 C-terminus (BRCT) domain,the binding domain of BRCA1, has been shown to interact

directly with ezrin in complex with F-actin at the plasmamembrane, as well as at membrane extensions and focal adhesionsites. In addition, it has been proposed that BRCA1, through itsE3 ubiquitin ligase activity, regulates ezrin expression. Therefore,

loss of BRCA1, as commonly seen in breast cancer cells, couldresult in an accumulation of ezrin that not only leads to itsreported high expression in breast cancer cells but also an

increased activity of specific signalling pathways that involveezrin and regulate cell-cell contacts, cell adhesion and cellmigration (Fig. 3) (Coene et al., 2011).

A so far unknown interaction between ezrin and thedesomosome protein desmoglein 3 (Dsg3) has recently beenidentified; and Dsg3 appears to colocalise with ezrin at the plasma

membrane (Brown et al., 2013). Overexpression of Dsg3 in the oralsquamous cell carcinoma (OSCC) cell lines A431 and SqCC/Y1caused a marked increase in phosphorylation of the conservedezrin residue T567, which resulted in increased migration and

invasion in 3D cell culture assays. It has been suggested that ezrinis phosphorylated by PKC after its colocalisation with Dsg3, aninteraction that might be responsible for the metastatic potential of

OSCC cell lines (Brown et al., 2013). Ezrin has previously beenproposed to act as a downstream signalling molecule in PKC-induced cell migration; here, PKCa phosphorylates specifically the

conserved T567 residue on ezrin (Ng et al., 2001), which results inCD44-dependent directional migration (Legg et al., 2002).Therefore, Dsg3, through its interaction with PKC, mightmediate the activation of ezrin and, consequently, contribute to

an invasive phenotype (Fig. 3).As well as breast, lung and prostate cancers, and oral squamous

cell carcinomas (OSCCs), ezrin overexpression has also been

suggested to influence tumour metastasis in other cancers, such asosteosarcoma, rhabdomyosarcoma and carcinomas of thepancreas (Akisawa et al., 1999; Hunter, 2004; Khanna et al.,

2004; Meng et al., 2010; Shang et al., 2012; Yu et al., 2004).

RadixinCompared with ezrin, only very little is known with regard to therole of radixin in cancer. However, significantly differentexpression patterns of radixin have been recently observedbetween HGPIN and prostate cancer samples (Bartholow et al.,

2011). This is the first report that describes the differences in theexpression profiles of radixin between normal donor prostates,HGPIN, prostate cancer and normal tissue adjacent to

adenocarcinoma. Similarly to ezrin, radixin expression wassignificantly higher in HGPIN compared with prostate cancer,suggesting that radixin has an initial role in the progression of the

tumour but is then deregulated as the tumour progresses. A role of

radixin in pancreatic cancer has also recently been suggested(Chen et al., 2012). Here, knockdown of radixin expression using

shRNA decreased proliferation, survival, adhesion and invasivepotential of human pancreatic carcinoma, epithelial-like cells(PANC-1) in vitro. Similar effects, i.e. the significant inhibitionof tumour growth, was also seen when cells, in which radixin has

been silenced, were implanted into nude mice (Chen et al., 2012).Furthermore, knockdown of radixin in PANC-1 cells resulted inan increase in E-cadherin expression, which is also seen in ezrin-

depleted cells (Chen et al., 2012). This implies that both ezrin andradixin may have an important role in the disruption of cell–cellcontacts through the downregulation of E-cadherin and its

relocalisation during cancer progression (Chen et al., 2012;Condorelli et al., 2011; Li et al., 2012; Pujuguet et al., 2003).

We recently found a new role for radixin in cell–cell adhesion

and cell migration in cells of the commonly used highly metastaticprostate cell line PC3 that may further elucidate the degree ofredundancy between the three ERM proteins. We observed thatdepletion of radixin by using siRNA not only promotes an

increase in the spreading of PC3 cells and in N-cadherin-mediatedcell–cell adhesion, but that their morphology also changed andappeared to become more epithelial-like (Valderrama et al., 2012).

Radixin depletion has also been shown to lead to an increase inRac1 activity, in a manner that is dependent on Vav, a GEFresponsible for activation of Rac1 (Abe et al., 2000). As

mentioned above, ERM proteins can act as scaffolds for boththe activation and deregulation of Rac1; so, potentially, radixinacts as a scaffold to deregulate Vav expression. In the absence of

radixin, Vav could increase its expresssion and/or activity and,consequently, reduce Rac1 activity, thereby mediating epithelialpolarity (Fig. 3). Importantly, the depletion of ezrin or moesindoes not induce a similar response, suggesting that the regulation

of Vav is specific to radixin (Valderrama et al., 2012). Although,an alteration in the localisation of radixin in normal epitheliumcompared with cancerous tissues remains to be investigated, these

recent results suggest that the role of radixin is important indefining cell polarity and subsequent acquisition of malignancy,independently of ezrin and moesin (Valderrama et al., 2012).

MoesinThe expression of moesin, like that of the other two ERM proteins,has been correlated to cancer progression. Its expression pattern

has been linked to increased tumour size, as well as mode ofinvasion and differentiation in OSCC (Kobayashi et al., 2004). Inaddition, moesin relocalises from the plasma membrane to the

cytoplasm in tumour cells that have a higher incidence of lymphnode metastasis. This suggests that, in OSCC, mislocalisation ofmoesin together with increased expression levels influences the

invasive or metastatic ability of the tumour cells (Kobayashi et al.,2004). Moesin has also been linked to early lung colonisation bymelanoma cells in vivo (Estecha et al., 2009). Here, established

metastatic lung melanoma cell lines that have been depleted ofmoesin by using siRNA were injected into the tails of micetogether with ezrin-depleted cells. Moesin-depleted cells thenarrived at the lungs, but were unable to colonise and disappeared

from the lungs, whereas ezrin-depleted cells remained, suggestingthat moesin is essential for colonisation of tumour cells in thelungs. When the moesin-depleted cells where analysed in a 3D

collagen invasion assay, they exhibited a flattened morphology andshowed significantly less invasive potential compared with thecontrol cells, which quickly acquired an elongated phenotype.

During the initial adhesion of moesin-depleted cells in the 3D

COMMENTARY Journal of Cell Science (2014) 127, 267–275 doi:10.1242/jcs.133108

272

Jour

nal o

f Cel

l Sci

ence

assay, there was an increase in phosphorylated moesin, which wasrepressed when PKC and ROCK were inhibited. Therefore, PKC

and ROCK might contribute to the initial adhesion-dependentactivation of moesin. Moesin is also required to activate RhoAsignalling in response to initial attachment and spreading.Therefore, it is possible that moesin is activated initially by PKC

– independently of Rho – and, subsequently enters a positivemoesin and RhoA feedback loop to promote Rho activation andcell migration (Estecha et al., 2009) (Fig. 3).

Many studies have shown that epithelial–mesenchymaltransition (EMT) of epithelial cells is induced by transformedgrowth factor-beta (TGF-b) (Nawshad et al., 2005; Wendt et al.,

2009). This transition is fundamental in tumour development andmetastasis and allows epithelial cells to develop a mesenchymaland, therefore, more invasive phenotype. Moesin has been

predicted to be a potential EMT marker in breast and pancreaticcancer (Abiatari et al., 2010; Haynes et al., 2011; Wang et al.,2012). Furthermore, expression profiles of both breast and basalbreast carcinomas have shown strong upregulation of moesin

(Charafe-Jauffret et al., 2007; Condeelis et al., 2005; Kobayashiet al., 2004; Wang et al., 2012). Moesin was also shown to beessential for EMT in the human mammary cell line MCF-10A,

whereas ezrin and radixin were not (Haynes et al., 2011); afterstimulation with TGF-b, moesin was observed to relocalise fromcell–cell adhesions to filopodia and large membrane protrusions.

Moreover, depletion of moesin reduces the number of actin stressfibres, and the bundled filaments of the actin cytoskeleton werethinner, shorter and less uniformly aligned along the main cell axis,

causing cells to be less elongated. Haynes et al. suggested thatROCK is responsible for the increase of phosphorylated moesinduring EMT (Haynes et al., 2011). Moesin has been previouslyreported to colocalise with CD44 (Fehon et al., 2010; Legg and

Isacke, 1998; Matsui et al., 1998; Ng et al., 2001). During EMT,CD44 is most abundant at dorsal protrusions to promote cell–substrate adhesion, and this localisation is drastically reduced when

moesin is depleted (Haynes et al., 2011). This suggests that moesinhas a dual role in promoting EMT by affecting the reorganisationof the actin cytoskeleton and by regulating the localisation of

CD44.When cells are stimulated with TGF-b in the absence of

moesin, there is also a significant reduction in the level ofautophosphorylated focal adhesion kinase (FAK) (Haynes et al.,

2011), a non-RTK that localises at focal adhesions (Parsons, 2003).Like ERM proteins, FAK also contains a FERM domain (Ceccarelliet al., 2006), with the characteristic F1, F2 and F3 lobes; and,

through F2, FAK directly binds Met, the transmembrane receptorthat is activated in response to HGF (Chen and Chen, 2006).Inhibition of FAK impairs cell migration (Mitra et al., 2005) and,

therefore, increased expression of moesin upon stimulation ofTGF-b may have a role in the regulation of FAK activation, whichin turn promotes cancer progression (Fig. 3) (Chen and Chen,

2006). This study describes the first evidence of a functional linkbetween moesin and FAK, but it remains unclear how moesinmight mediate the phosphorylation of FAK during EMT.

Recently, Zhu et al. demonstrated a correlation between the

high expression of moesin and high-grade glioblastoma tumourswithout any significant changes in the expression levels of ezrinor radixin (Zhu et al., 2013) and also observed elevated moesin

expression in established glioblastoma cell lines (Zhu et al.,2013). Here, moesin was found to colocalise with CD44 atmembrane extensions, and the authors propose that moesin

competes with the tumour suppressor NF2 for binding to CD44 in

order to activate CD44-induced growth signalling. NF2 has beenpreviously shown to colocalise with CD44 and to form a

molecular switch that results in arrest of cell growth (Morrisonet al., 2001). Overexpression of moesin might, therefore, displacethe tumour suppressor NF2 during glioblastoma progression andpromote growth-signalling pathways. Interestingly, one pathway

that is predominantly activated downstream of moesin–CD44 isthe Wnt/b-catenin pathway (Zhu et al., 2013). The interactionbetween moesin and CD44 was found to induce transcription of

b-catenin, as well as translocation of b-catenin from themembrane to the nucleus. Mechanistically, this can be achievedby PI3K/Akt-mediated phosphorylation (downstream of CD44

activation) of b-catenin at S552, as previously suggested toinduce b-catenin translocation to the nucleus (Fang et al., 2007).The results presented in this recent study, therefore, suggest that

moesin represents a target for glioblastoma therapy (Zhu et al.,2013).

Conclusions and perspectivesERM proteins have different fundamental roles in tumour

development and the ability to subsequently promote theprogression of tumours to more advanced stages. They take partin a number of signalling pathways that depend, for example, on

RhoGTPases, PI3K/Akt, Wnt/b-catenin, CD44 and RTKs – suchas EGFR and HGFR – all of which are crucial for cancerprogression (Fig. 3).

Although there is some overlap in the involvement of ERM

proteins in tumour progression, it is important to emphasise thatmany of the roles discussed in this Commentary highlight theindividuality of each of them. However, further investigations

that focus on how each protein exerts their role in the abovementioned pathways are needed, and more information regardingthe activation mechanisms and interaction and/or regulatory

partners is required to reinforce their potentially specific roles indifferent epithelial cancers, and their possible use as relevantprognostic markers.

AcknowledgementsWe thank Guy Whitley, Francesc Miralles (St George’s University of London, UK),Aleksander Ivetic and Valerie Vivancos (King’s College London, UK) for theircomments and insights during the preparation of this manuscript.

Competing interestsThe authors declare no competing interests.

FundingThis work is supported by a SGUL PhD studentship to J.C.

ReferencesAbe, K., Rossman, K. L., Liu, B., Ritola, K. D., Chiang, D., Campbell, S. L.,Burridge, K. and Der, C. J. (2000). Vav2 is an activator of Cdc42, Rac1, andRhoA. J. Biol. Chem. 275, 10141-10149.

Abiatari, I., Esposito, I., Oliveira, T. D., Felix, K., Xin, H., Penzel, R., Giese, T.,Friess, H. and Kleeff, J. (2010). Moesin-dependent cytoskeleton remodelling isassociated with an anaplastic phenotype of pancreatic cancer. J. Cell. Mol. Med.14, 1166-1179.

Akisawa, N., Nishimori, I., Iwamura, T., Onishi, S. and Hollingsworth, M. A.(1999). High levels of ezrin expressed by human pancreatic adenocarcinomacell lines with high metastatic potential. Biochem. Biophys. Res. Commun. 258,395-400.

Algrain, M., Turunen, O., Vaheri, A., Louvard, D. and Arpin, M. (1993). Ezrincontains cytoskeleton and membrane binding domains accounting for itsproposed role as a membrane-cytoskeletal linker. J. Cell Biol. 120, 129-139.

Amler, L. C., Agus, D. B., LeDuc, C., Sapinoso, M. L., Fox, W. D., Kern, S., Lee,D., Wang, V., Leysens, M., Higgins, B. et al. (2000). Dysregulated expressionof androgen-responsive and nonresponsive genes in the androgen-independentprostate cancer xenograft model CWR22-R1. Cancer Res. 60, 6134-6141.

Arpin, M., Chirivino, D., Naba, A. and Zwaenepoel, I. (2011). Emerging role forERM proteins in cell adhesion and migration. Cell Adh. Migr. 5, 199-206.

COMMENTARY Journal of Cell Science (2014) 127, 267–275 doi:10.1242/jcs.133108

273

Jour

nal o

f Cel

l Sci

ence

Autero, M., Heiska, L., Ronnstrand, L., Vaheri, A., Gahmberg, C. G. andCarpen, O. (2003). Ezrin is a substrate for Lck in Tcells. FEBS Lett. 535, 82-86.

Bartholow, T. L., Chandran, U. R., Becich, M. J. and Parwani, A. V. (2011).Immunohistochemical staining of radixin and moesin in prostatic adenocarcinoma.BMC Clin. Pathol. 11, 1.

Baumgartner, M., Sillman, A. L., Blackwood, E. M., Srivastava, J., Madson, N.,Schilling, J. W., Wright, J. H. and Barber, D. L. (2006). The Nck-interactingkinase phosphorylates ERM proteins for formation of lamellipodium by growthfactors. Proc. Natl. Acad. Sci. USA 103, 13391-13396.

Belkina, N. V., Liu, Y., Hao, J. J., Karasuyama, H. and Shaw, S. (2009). LOK is amajor ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangementthrough ERM phosphorylation. Proc. Natl. Acad. Sci. USA 106, 4707-4712.

Berryman, M., Franck, Z. and Bretscher, A. (1993). Ezrin is concentrated in theapical microvilli of a wide variety of epithelial cells whereas moesin is foundprimarily in endothelial cells. J. Cell Sci. 105, 1025-1043.

Bonaccorsi, L., Carloni, V., Muratori, M., Formigli, L., Zecchi, S., Forti, G. andBaldi, E. (2004). EGF receptor (EGFR) signaling promoting invasion isdisrupted in androgen-sensitive prostate cancer cells by an interaction betweenEGFR and androgen receptor (AR). Int. J. Cancer 112, 78-86.

Bretscher, A. (1983). Purification of an 80,000-dalton protein that is a componentof the isolated microvillus cytoskeleton, and its localization in nonmuscle cells.J. Cell Biol. 97, 425-432.

Bretscher, A., Edwards, K. and Fehon, R. G. (2002). ERM proteins and merlin:integrators at the cell cortex. Nat. Rev. Mol. Cell Biol. 3, 586-599.

Brown, L., Waseem, A., Cruz, I. N., Szary, J., Gunic, E., Mannan, T., Unadkat,M., Yang, M. and Valderrama, F., O’Toole, E. A. et al. (2013). Desmoglein 3promotes cancer cell migration and invasion by regulating activator protein 1and protein kinase C-dependent-Ezrin activation. Oncogene [Epub ahead ofprint] doi:10.1038/onc.2013.186.

Bruce, B., Khanna, G., Ren, L., Landberg, G., Jirstrom, K., Powell, C.,Borczuk, A., Keller, E. T., Wojno, K. J., Meltzer, P. et al. (2007). Expression ofthe cytoskeleton linker protein ezrin in human cancers. Clin. Exp. Metastasis 24,69-78.

Canel, M., Serrels, A., Frame, M. C. and Brunton, V. G. (2013). E-cadherin-integrin crosstalk in cancer invasion and metastasis. J. Cell Sci. 126, 393-401.

Carreno, S., Kouranti, I., Glusman, E. S., Fuller, M. T., Echard, A. and Payre,F. (2008). Moesin and its activating kinase Slik are required for cortical stabilityand microtubule organization in mitotic cells. J. Cell Biol. 180, 739-746.

Ceccarelli, D. F., Song, H. K., Poy, F., Schaller, M. D. and Eck, M. J. (2006).Crystal structure of the FERM domain of focal adhesion kinase. J. Biol. Chem.281, 252-259.

Charafe-Jauffret, E., Monville, F., Bertucci, F., Esterni, B., Ginestier, C.,Finetti, P., Cervera, N., Geneix, J., Hassanein, M., Rabayrol, L. et al. (2007).Moesin expression is a marker of basal breast carcinomas. Int. J. Cancer 121,1779-1785.

Chen, S. Y. and Chen, H. C. (2006). Direct interaction of focal adhesion kinase(FAK) with Met is required for FAK to promote hepatocyte growth factor-inducedcell invasion. Mol. Cell. Biol. 26, 5155-5167.

Chen, S. D., Song, M. M., Zhong, Z. Q., Li, N., Wang, P. L., Cheng, S., Bai, R. X.and Yuan, H. S. (2012). Knockdown of radixin by RNA interference suppressesthe growth of human pancreatic cancer cells in vitro and in vivo. Asian Pac.J. Cancer Prev. 13, 753-759.

Chuan, Y. C., Pang, S. T., Cedazo-Minguez, A., Norstedt, G., Pousette, A. andFlores-Morales, A. (2006). Androgen induction of prostate cancer cell invasionis mediated by ezrin. J. Biol. Chem. 281, 29938-29948.

Chuan, Y. C., Iglesias-Gato, D., Fernandez-Perez, L., Cedazo-Minguez, A., Pang,S. T., Norstedt, G., Pousette, A. and Flores-Morales, A. (2010). Ezrin mediatesc-Myc actions in prostate cancer cell invasion. Oncogene 29, 1531-1542.

Coene, E. D., Gadelha, C., White, N., Malhas, A., Thomas, B., Shaw, M. andVaux, D. J. (2011). A novel role for BRCA1 in regulating breast cancer cellspreading and motility. J. Cell Biol. 192, 497-512.

Condeelis, J., Singer, R. H. and Segall, J. E. (2005). The great escape: whencancer cells hijack the genes for chemotaxis and motility. Annu. Rev. Cell Dev.Biol. 21, 695-718.

Condorelli, F., Stec-Martyna, E., Zaborowska, J., Felli, L., Gemmi, I., Ponassi,M. and Rosano, C. (2011). Role of the non-receptor tyrosine kinase fes incancer. Curr. Med. Chem. 18, 2913-2920.

Crepaldi, T., Gautreau, A., Comoglio, P. M., Louvard, D. and Arpin, M. (1997).Ezrin is an effector of hepatocyte growth factor-mediated migration andmorphogenesis in epithelial cells. J. Cell Biol. 138, 423-434.

Dard, N., Louvet, S., Santa-Maria, A., Aghion, J., Martin, M., Mangeat, P. andMaro, B. (2001). In vivo functional analysis of ezrin during mouse blastocystformation. Dev. Biol. 233, 161-173.

Debnath, J. and Brugge, J. S. (2005). Modelling glandular epithelial cancers inthree-dimensional cultures. Nat. Rev. Cancer 5, 675-688.

Easty, D. J., Gray, S. G., O’Byrne, K. J., O’Donnell, D. and Bennett, D. C.(2011). Receptor tyrosine kinases and their activation in melanoma. PigmentCell Melanoma Res. 24, 446-461.

Elliott, B. E., Qiao, H., Louvard, D. and Arpin, M. (2004). Co-operative effect ofc-Src and ezrin in deregulation of cell-cell contacts and scattering of mammarycarcinoma cells. J. Cell. Biochem. 92, 16-28.

Estecha, A., Sanchez-Martın, L., Puig-Kroger, A., Bartolome, R. A., Teixido, J.,Samaniego, R. and Sanchez-Mateos, P. (2009). Moesin orchestrates corticalpolarity of melanoma tumour cells to initiate 3D invasion. J. Cell Sci. 122, 3492-3501.

Fang, D., Hawke, D., Zheng, Y., Xia, Y., Meisenhelder, J., Nika, H., Mills, G. B.,Kobayashi, R., Hunter, T. and Lu, Z. (2007). Phosphorylation of beta-cateninby AKT promotes beta-catenin transcriptional activity. J. Biol. Chem. 282,11221-11229.

Fehon, R. G., McClatchey, A. I. and Bretscher, A. (2010). Organizing the cellcortex: the role of ERM proteins. Nat. Rev. Mol. Cell Biol. 11, 276-287.

Fievet, B. T., Gautreau, A., Roy, C., Del Maestro, L., Mangeat, P., Louvard,D. and Arpin, M. (2004). Phosphoinositide binding and phosphorylation actsequentially in the activation mechanism of ezrin. J. Cell Biol. 164, 653-659.

Gandy, K. A., Canals, D., Adada, M., Wada, M., Roddy, P., Snider, A. J., Hannun,Y. A. and Obeid, L. M. (2013). Sphingosine 1-phosphate induces filopodiaformation through S1PR2 activation of ERM proteins. Biochem. J. 449, 661-672.

Garbett, D. and Bretscher, A. (2012). PDZ interactions regulate rapid turnover ofthe scaffolding protein EBP50 in microvilli. J. Cell Biol. 198, 195-203.

Gary, R. and Bretscher, A. (1995). Ezrin self-association involves binding of anN-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site. Mol. Biol. Cell 6, 1061-1075.

Gautreau, A., Poullet, P., Louvard, D. and Arpin, M. (1999). Ezrin, a plasmamembrane-microfilament linker, signals cell survival through thephosphatidylinositol 3-kinase/Akt pathway. Proc. Natl. Acad. Sci. USA 96, 7300-7305.

Gautreau, A., Louvard, D. and Arpin, M. (2000). Morphogenic effects of ezrinrequire a phosphorylation-induced transition from oligomers to monomers at theplasma membrane. J. Cell Biol. 150, 193-204.

Harris, B. Z. and Lim, W. A. (2001). Mechanism and role of PDZ domains insignaling complex assembly. J. Cell Sci. 114, 3219-3231.

Haynes, J., Srivastava, J., Madson, N., Wittmann, T. and Barber, D. L. (2011).Dynamic actin remodeling during epithelial-mesenchymal transition depends onincreased moesin expression. Mol. Biol. Cell 22, 4750-4764.

Hebert, M., Potin, S., Sebbagh, M., Bertoglio, J., Breard, J. and Hamelin,J. (2008). Rho-ROCK-dependent ezrin-radixin-moesin phosphorylation regulatesFas-mediated apoptosis in Jurkat cells. J. Immunol. 181, 5963-5973.

Hsu, Y. Y., Shi, G. Y., Kuo, C. H., Liu, S. L., Wu, C. M., Ma, C. Y., Lin, F. Y., Yang,H. Y. and Wu, H. L. (2012). Thrombomodulin is an ezrin-interacting protein thatcontrols epithelial morphology and promotes collective cell migration. FASEB J.26, 3440-3452.

Hunter, K. W. (2004). Ezrin, a key component in tumor metastasis. Trends Mol.Med. 10, 201-204.

Jeon, S., Kim, S., Park, J. B., Suh, P. G., Kim, Y. S., Bae, C. D. and Park,J. (2002). RhoA andRho kinase-dependent phosphorylation of moesin at Thr-558in hippocampal neuronal cells by glutamate. J. Biol. Chem. 277, 16576-16584.

Jung, Y. and McCarty, J. H. (2012). Band 4.1 proteins regulate integrin-dependent cell spreading. Biochem. Biophys. Res. Commun. 426, 578-584.

Khanna, C., Wan, X., Bose, S., Cassaday, R., Olomu, O., Mendoza, A., Yeung,C., Gorlick, R., Hewitt, S. M. and Helman, L. J. (2004). The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis. Nat. Med.10, 182-186.

Kobayashi, H., Sagara, J., Kurita, H., Morifuji, M., Ohishi, M., Kurashina,K. and Taniguchi, S. (2004). Clinical significance of cellular distribution ofmoesin in patients with oral squamous cell carcinoma. Clin. Cancer Res. 10,572-580.

Konstantinovsky, S., Davidson, B. and Reich, R. (2012). Ezrin and BCAR1/p130Cas mediate breast cancer growth as 3-D spheroids. Clin. Exp. Metastasis29, 527-540.

Krieg, J. and Hunter, T. (1992). Identification of the two major epidermal growthfactor-induced tyrosine phosphorylation sites in the microvillar core proteinezrin. J. Biol. Chem. 267, 19258-19265.

Lamb, R. F., Ozanne, B. W., Roy, C., McGarry, L., Stipp, C., Mangeat, P. andJay, D. G. (1997). Essential functions of ezrin in maintenance of cell shapeand lamellipodial extension in normal and transformed fibroblasts. Curr. Biol. 7,682-688.

Lan, M., Kojima, T., Murata, M., Osanai, M., Takano, K., Chiba, H. and Sawada,N. (2006). Phosphorylation of ezrin enhances microvillus length via a p38 MAP-kinase pathway in an immortalized mouse hepatic cell line. Exp. Cell Res. 312,111-120.

Legg, J. W. and Isacke, C. M. (1998). Identification and functional analysis of theezrin-binding site in the hyaluronan receptor, CD44. Curr. Biol. 8, 705-708.

Legg, J. W., Lewis, C. A., Parsons, M., Ng, T. and Isacke, C. M. (2002). A novelPKC-regulated mechanism controls CD44 ezrin association and directional cellmotility. Nat. Cell Biol. 4, 399-407.

Li, Q., Gao, H., Xu, H., Wang, X., Pan, Y., Hao, F., Qiu, X., Stoecker, M., Wang,E. and Wang, E. (2012). Expression of ezrin correlates with malignantphenotype of lung cancer, and in vitro knockdown of ezrin reverses theaggressive biological behavior of lung cancer cells. Tumour Biol. 33, 1493-1504.

Mackay, D. J., Esch, F., Furthmayr, H. and Hall, A. (1997). Rho- and rac-dependent assembly of focal adhesion complexes and actin filaments inpermeabilized fibroblasts: an essential role for ezrin/radixin/moesin proteins.J. Cell Biol. 138, 927-938.

Mak, H., Naba, A., Varma, S., Schick, C., Day, A., SenGupta, S. K., Arpin,M. and Elliott, B. E. (2012). Ezrin phosphorylation on tyrosine 477 regulatesinvasion and metastasis of breast cancer cells. BMC Cancer 12, 82.

Martin, T. A., Harrison, G., Mansel, R. E. and Jiang, W. G. (2003). The role of theCD44/ezrin complex in cancer metastasis.Crit. Rev. Oncol. Hematol. 46, 165-186.

Matsui, T., Maeda, M., Doi, Y., Yonemura, S., Amano, M., Kaibuchi, K., Tsukita,S. and Tsukita, S. (1998). Rho-kinase phosphorylates COOH-terminal

COMMENTARY Journal of Cell Science (2014) 127, 267–275 doi:10.1242/jcs.133108

274

Jour

nal o

f Cel

l Sci

ence

threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J. Cell Biol. 140, 647-657.

Matsui, T., Yonemura, S., Tsukita, S. and Tsukita, S. (1999). Activation of ERMproteins in vivo by Rho involves phosphatidyl-inositol 4-phosphate 5-kinase andnot ROCK kinases. Curr. Biol. 9, 1259-1262.

Meng, Y., Lu, Z., Yu, S., Zhang, Q., Ma, Y. and Chen, J. (2010). Ezrin promotesinvasion and metastasis of pancreatic cancer cells. J. Transl. Med. 8, 61.

Mitra, S. K., Hanson, D. A. and Schlaepfer, D. D. (2005). Focal adhesion kinase:in command and control of cell motility. Nat. Rev. Mol. Cell Biol. 6, 56-68.

Morrison, H., Sherman, L. S., Legg, J., Banine, F., Isacke, C., Haipek, C. A.,Gutmann, D. H., Ponta, H. and Herrlich, P. (2001). The NF2 tumor suppressorgene product, merlin, mediates contact inhibition of growth through interactionswith CD44. Genes Dev. 15, 968-980.

Naba, A., Reverdy, C., Louvard, D. and Arpin, M. (2008). Spatial recruitment andactivation of the Fes kinase by ezrin promotes HGF-induced cell scattering.EMBO J. 27, 38-50.

Nakamura, F., Amieva, M. R. and Furthmayr, H. (1995). Phosphorylation ofthreonine 558 in the carboxyl-terminal actin-binding domain of moesin bythrombin activation of human platelets. J. Biol. Chem. 270, 31377-31385.

Nakamura, N., Oshiro, N., Fukata, Y., Amano, M., Fukata, M., Kuroda, S.,Matsuura, Y., Leung, T., Lim, L. and Kaibuchi, K. (2000). Phosphorylation ofERM proteins at filopodia induced by Cdc42. Genes Cells 5, 571-581.

Nawshad, A., Lagamba, D., Polad, A. and Hay, E. D. (2005). Transforminggrowth factor-beta signaling during epithelial-mesenchymal transformation:implications for embryogenesis and tumor metastasis. Cells Tissues Organs179, 11-23.

Ng, T., Parsons, M., Hughes, W. E., Monypenny, J., Zicha, D., Gautreau, A.,Arpin, M., Gschmeissner, S., Verveer, P. J., Bastiaens, P. I. et al. (2001).Ezrin is a downstream effector of trafficking PKC-integrin complexes involved inthe control of cell motility. EMBO J. 20, 2723-2741.

Niggli, V. and Rossy, J. (2008). Ezrin/radixin/moesin: versatile controllers ofsignaling molecules and of the cortical cytoskeleton. Int. J. Biochem. Cell Biol.40, 344-349.

Niggli, V., Andreoli, C., Roy, C. and Mangeat, P. (1995). Identification of aphosphatidylinositol-4,5-bisphosphate-binding domain in the N-terminal regionof ezrin. FEBS Lett. 376, 172-176.

Orian-Rousseau, V., Morrison, H., Matzke, A., Kastilan, T., Pace, G., Herrlich,P. and Ponta, H. (2007). Hepatocyte growth factor-induced Ras activationrequires ERM proteins linked to both CD44v6 and F-actin. Mol. Biol. Cell 18, 76-83.

Pang, S. T., Fang, X., Valdman, A., Norstedt, G., Pousette, A., Egevad, L. andEkman, P. (2004). Expression of ezrin in prostatic intraepithelial neoplasia.Urology 63, 609-612.

Parsons, J. T. (2003). Focal adhesion kinase: the first ten years. J. Cell Sci. 116,1409-1416.

Pearson, M. A., Reczek, D., Bretscher, A. and Karplus, P. A. (2000). Structureof the ERM protein moesin reveals the FERM domain fold masked by anextended actin binding tail domain. Cell 101, 259-270.

Pietromonaco, S. F., Simons, P. C., Altman, A. and Elias, L. (1998). Proteinkinase C-theta phosphorylation of moesin in the actin-binding sequence. J. Biol.Chem. 273, 7594-7603.

Pujuguet, P., Del Maestro, L., Gautreau, A., Louvard, D. and Arpin, M. (2003).Ezrin regulates E-cadherin-dependent adherens junction assembly throughRac1 activation. Mol. Biol. Cell 14, 2181-2191.

Reczek, D., Berryman, M. and Bretscher, A. (1997). Identification of EBP50: APDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family. J. Cell Biol. 139, 169-179.

Ridley, A. J. (2001). Rho GTPases and cell migration. J. Cell Sci. 114, 2713-2722.Ridley, A. J., Comoglio, P. M. and Hall, A. (1995). Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cells. Mol.Cell. Biol. 15, 1110-1122.

Sarrio, D., Rodrıguez-Pinilla, S. M., Dotor, A., Calero, F., Hardisson, D. andPalacios, J. (2006). Abnormal ezrin localization is associated with clinicopathologicalfeatures in invasive breast carcinomas. Breast Cancer Res. Treat. 98, 71-79.

Sato, N., Funayama, N., Nagafuchi, A., Yonemura, S., Tsukita, S. and Tsukita,S. (1992). A gene family consisting of ezrin, radixin and moesin. Its specificlocalization at actin filament/plasma membrane association sites. J. Cell Sci.103, 131-143.

Shang, X., Wang, Y., Zhao, Q., Wu, K., Li, X., Ji, X., He, R. and Zhang,W. (2012). siRNAs target sites selection of ezrin and the influence of RNA

interference on ezrin expression and biological characters of osteosarcomacells. Mol. Cell. Biochem. 364, 363-371.

Shiue, H., Musch, M. W., Wang, Y., Chang, E. B. and Turner, J. R. (2005). Akt2phosphorylates ezrin to trigger NHE3 translocation and activation. J. Biol.Chem. 280, 1688-1695.

Simons, P. C., Pietromonaco, S. F., Reczek, D., Bretscher, A. and Elias,L. (1998). C-terminal threonine phosphorylation activates ERM proteins to linkthe cell’s cortical lipid bilayer to the cytoskeleton. Biochem. Biophys. Res.Commun. 253, 561-565.

Sperka, T., Geissler, K. J., Merkel, U., Scholl, I., Rubio, I., Herrlich, P. andMorrison, H. L. (2011). Activation of Ras requires the ERM-dependent link ofactin to the plasma membrane. PLoS ONE 6, e27511.

Srivastava, J., Elliott, B. E., Louvard, D. and Arpin, M. (2005). Src-dependentezrin phosphorylation in adhesion-mediated signaling. Mol. Biol. Cell 16,1481-1490.

Takahashi, K., Sasaki, T., Mammoto, A., Takaishi, K., Kameyama, T., Tsukita,S. and Takai, Y. (1997). Direct interaction of the Rho GDP dissociation inhibitorwith ezrin/radixin/moesin initiates the activation of the Rho small G protein.J. Biol. Chem. 272, 23371-23375.

Takeuchi, K., Sato, N., Kasahara, H., Funayama, N., Nagafuchi, A., Yonemura,S., Tsukita, S. and Tsukita, S. (1994). Perturbation of cell adhesion andmicrovilli formation by antisense oligonucleotides to ERM family members. J.Cell Biol. 125, 1371-1384.

ten Klooster, J. P., Jansen, M., Yuan, J., Oorschot, V., Begthel, H., DiGiacomo, V., Colland, F., de Koning, J., Maurice, M. M., Hornbeck, P. et al.(2009). Mst4 and Ezrin induce brush borders downstream of the Lkb1/Strad/Mo25 polarization complex. Dev. Cell 16, 551-562.

Turunen, O., Wahlstrom, T. and Vaheri, A. (1994). Ezrin has a COOH-terminalactin-binding site that is conserved in the ezrin protein family. J. Cell Biol. 126,1445-1453.

Valderrama, F., Thevapala, S. and Ridley, A. J. (2012). Radixin regulates cellmigration and cell-cell adhesion through Rac1. J. Cell Sci. 125, 3310-3319.

Valdman, A., Fang, X., Pang, S. T., Nilsson, B., Ekman, P. and Egevad,L. (2005). Ezrin expression in prostate cancer and benign prostatic tissue. Eur.Urol. 48, 852-857.

Viswanatha, R., Ohouo, P. Y., Smolka, M. B. and Bretscher, A. (2012). Localphosphocycling mediated by LOK/SLK restricts ezrin function to the apicalaspect of epithelial cells. J. Cell Biol. 199, 969-984.

Wald, F. A., Oriolo, A. S., Mashukova, A., Fregien, N. L., Langshaw, A. H. andSalas, P. J. (2008). Atypical protein kinase C (iota) activates ezrin in the apicaldomain of intestinal epithelial cells. J Cell Sci. 21, 644-654.

Wang, C. C., Liau, J. Y., Lu, Y. S., Chen, J. W., Yao, Y. T. and Lien, H. C. (2012).Differential expression of moesin in breast cancers and its implication inepithelial-mesenchymal transition. Histopathology 61, 78-87.

Wendt, M. K., Allington, T. M. and Schiemann, W. P. (2009). Mechanismsof the epithelial-mesenchymal transition by TGF-beta. Future Oncol. 5,1145-1168.

Yamada, S. and Nelson, W. J. (2007). Localized zones of Rho and Rac activitiesdrive initiation and expansion of epithelial cell-cell adhesion. J. Cell Biol. 178,517-527.

Yang, H. S., Alexander, K., Santiago, P. and Hinds, P. W. (2003). ERM proteinsand Cdk5 in cellular senescence. Cell Cycle 2, 517-520.

Yokoyama, T., Goto, H., Izawa, I., Mizutani, H. and Inagaki, M. (2005). Aurora-Band Rho-kinase/ROCK, the two cleavage furrow kinases, independently regulatethe progression of cytokinesis: possible existence of a novel cleavage furrowkinase phosphorylates ezrin/radixin/moesin (ERM). Genes Cells 10, 127-137.

Yu, Y., Khan, J., Khanna, C., Helman, L., Meltzer, P. S. and Merlino, G. (2004).Expression profiling identifies the cytoskeletal organizer ezrin and thedevelopmental homeoprotein Six-1 as key metastatic regulators. Nat. Med.10, 175-181.

Zhou, R., Cao, X., Watson, C., Miao, Y., Guo, Z., Forte, J. G. and Yao, X. (2003).Characterization of protein kinase A-mediated phosphorylation of ezrin in gastricparietal cell activation. J. Biol. Chem., 278, 35651-35659.

Zhu, J., Feng, Y., Ke, Z., Yang, Z., Zhou, J., Huang, X. and Wang, L. (2012).Down-regulation of miR-183 promotes migration and invasion of osteosarcomaby targeting Ezrin. Am. J. Pathol. 180, 2440-2451.

Zhu, X., Morales, F. C., Agarwal, N. K., Dogruluk, T., Gagea, M. andGeorgescu, M. M. (2013). Moesin is a glioma progression marker that inducesproliferation and Wnt/b-catenin pathway activation via interaction with CD44.Cancer Res. 73, 1142-1155.

COMMENTARY Journal of Cell Science (2014) 127, 267–275 doi:10.1242/jcs.133108

275