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)
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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 ([email protected])
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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).
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(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)
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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.
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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).
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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
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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.
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