–mesenchymal transition in cancer metastasis: Mechanisms, markers and Epithelial

Transcription

Biochimica et Biophysica Acta 1796 (2009) 75–90
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a c a n
Review
Epithelial–mesenchymal transition in cancer metastasis: Mechanisms, markers and
strategies to overcome drug resistance in the clinic
Angeliki Voulgari, Alexander Pintzas ⁎
Laboratory of Signal Mediated Gene Expression, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation,
48 Vasileos Constantinou Avenue, Athens 11635, Greece
a r t i c l e
i n f o
Article history:
Received 8 October 2008
Received in revised form 5 March 2009
Accepted 7 March 2009
Available online 21 March 2009
Keywords:
Epithelial–mesenchymal transition
Cancer
Clinic
Marker
Metastasis
Drug resistance
a b s t r a c t
Epithelial–mesenchymal transition (EMT) is a key step during embryogenesis. Accumulating evidence
suggests a critical role in cancer progression, through which tissue epithelial cancers invade and
metastasise. Cell characteristics are highly affected during EMT, resulting in altered cell–cell and cell–
matrix interactions, cell motility and invasiveness. Nevertheless, the demonstration of this process in
human cancer has been proven difficult and controversial. Besides the fact that the acquisition of
mesenchymal characteristics is not a prerequisite for cell migration/invasion, it is a transient event that
concerns only few cells in a tumour mass. The induction of EMT depends on the tumour type and its
genetic alterations as well as on its interaction with the extracellular matrix. In parallel, trials for EMT
identification in clinical samples lack of a widely accepted methodology, nomenclature and reliable
markers. This review summarizes the main EMT characteristics and proposes methodologies for better
analysis in vitro. It also highlights recent studies identifying cells with EMT characteristics in human cancer
and proposes certain markers to identify them in tumour samples. Finally, it cites the recent literature
concerning the mechanisms of drug resistance related to EMT in the context of anti-tumour therapies and
proposes related new targets for therapy.
© 2009 Elsevier B.V. All rights reserved.
Contents
1.
2.
3.
Epithelial–mesenchymal transition at a glance . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.
Epithelial to mesenchymal transition in development and homeostasis . . . . . . . . . . . .
1.2.
Tumour progression, EMT and metastasis . . . . . . . . . . . . . . . . . . . . . . . . . .
Induction and mechanisms of EMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Epithelial versus mesenchymal characteristics, in vitro and in vivo . . . . . . . . . . . . . .
2.1.1.
Cell function and morphology . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2.
Cell–cell and cell–matrix interactions in adhesion and migration . . . . . . . . . . .
2.1.3.
Tumour-initiating cell characteristics . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
The E-cadherin-mediated cell–adhesion system in EMT . . . . . . . . . . . . . . . . . . .
2.2.1.
E-cadherin function and regulators . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.2.
The beta-catenin pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Cellular signals inducing EMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.1.
Extrinsic stimuli: the effect of the microenvironment . . . . . . . . . . . . . . . .
2.3.2.
Intrinsic stimuli: mutations in signal transduction molecules . . . . . . . . . . . .
Can EMT detection result in better patient treatment? . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Difficulties in detecting EMT in the clinic . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.
Is EMT associated with cancer progression and metastasis in human disease? . . . . .
3.1.2.
The issue of the spatial heterogeneity and the alternative mechanisms of cell invasion
3.1.3.
The issue of the temporal heterogeneity . . . . . . . . . . . . . . . . . . . . . .
3.1.4.
EMT markers use in the clinic and identification of new markers. . . . . . . . . . .
3.1.5.
In vivo identification of EMT during carcinogenesis . . . . . . . . . . . . . . . . .
⁎ Corresponding author. Tel.: +30 210 7273753; fax: +30 210 7273755.
E-mail address: [email protected] (A. Pintzas).
0304-419X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbcan.2009.03.002
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A. Voulgari, A. Pintzas / Biochimica et Biophysica Acta 1796 (2009) 75–90
3.2.
Drug treatment and resistance related to EMT .
3.2.1.
Drug resistance . . . . . . . . . . .
3.2.2.
Future treatments targeting EMT . . .
4.
Final remarks. . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . .
References. . . . . . . . . . . . . . . . . . . . . . . .
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1. Epithelial–mesenchymal transition at a glance
1.1. Epithelial to mesenchymal transition in development and
homeostasis
The creation of an epithelium which consists of sheets of
continuous and polarized cells along the apical–basal axis, is a basic
point for the formation of a multicellular organism. Such structures
contain closely associated cells that ensure the mechanical integrity of
a tissue and establish a permeability barrier absolutely necessary to
separate different tissues and create an embryo. Nevertheless, during
morphogenic events that accompany early stages of metazoan
development, cells from the early embryonic epithelium are internalised to give rise to the mesodermal tissue. At this stage cells must
be able to detach from the junctions that connect them to the
neighbouring ones, change their shape and polarity, delaminate and
migrate. The event responsible for such profound modifications is
called Epithelial–mesenchymal transition (EMT). Morphogenic movements underlying gastrulating and formation of organs like neural
crest, heart, muscular system, craniofacial structures and peripheral
nervous system, all rely on EMT. A good example can be found in the
development of Drosophila where EMT is present during the
internalisation of future mesodermal cells and their migration along
the side of the ectoderm, to reach the correct position within the
embryo [1,2]. In adults, EMT and stimulation of new fibroblasts can be
accelerated during wound healing or tissue inflammation. However,
these repair responses can disturb the structure of the epithelium by
creating more connective tissue that ultimately invades the stroma.
During these processes and as long as stimuli persist, new fibroblasts
formed by EMT appear and retain a permanent mesenchymal state
resulting in fibrosis [3].
1.2. Tumour progression, EMT and metastasis
The concept of the multistep carcinogenesis in favour of the
tumour progression being a stepwise accumulation of genetic
alterations has been observed in several tumour types. Indeed several
types of pre-malignant lesions are induced by genetic alterations
which offer a growth advantage to the cells and allow their
monoclonal or polyclonal expansion. Further accumulation of genetic
alterations in protooncogenes, tumour suppressor genes and DNA
repair genes will push the pre-malignant cells to malignancy,
initiating thereby a primary tumour formation. Colorectal cancer has
been a model of tumour progression for several years, since the
introduction of the ‘adenoma–carcinoma sequence’ by Fearon and
Vogelstein [4]. This notion describes the progression of the tumour
from an early neoplastic lesion (aberrant crypt foci) to a benign
tumour (adenoma) and finally to a malignant tumour (adenocarcinoma). This is in parallel with the ordered stepwise accumulation of
genetic alterations in genes like the adenomatous polyposis coli
(APC), B-RAF, RAS and p53 [5,6]. However, the hallmark of cancer
malignancy is the metastatic dissemination of the primary tumours
which are originally incapable of invading the surrounding tissue. In
the course of the disease the tumour mass becomes heterogeneous,
since the primary tumour cells independently further accumulate
genetic alterations and interact with their particular local microenvironment [7]. As a result, in localized areas of the carcinoma, a small
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number of cells may bypass fundamental rules of the normal
behaviour, detach from neighbouring ones, migrate and locally invade
the surrounding tissue. Further accession to blood circulation or the
lymphatic vessels leads to their dissemination in the body and finally
to the re-establishment at distant sites. The first and determinant step
of this process is the local invasion through the epithelial basement
membrane, as it requires modifications in cell–cell and cell–matrix
interactions, remodelling of the extracellular matrix, modifications of
the cytoskeleton and enhancement of cell motility. However, many
cancers (like colorectal cancer) are well differentiated, in theory
incapable of fulfilling these activities. Therefore, the hypothesis is that
specific events induce a loss of epithelial and a gain of dedifferentiated
mesenchymal-like phenotype (EMT) in a limited number of cells.
Interestingly, the mechanisms of EMT in development, fibrosis or
cancer appear to be related with common key players and regulators
[8,9] suggesting that similarly to embryonic mesenchymal cells, EMTrelated cancer mesenchymal cells are motile and possibly associated
with the tumour invasive front. Nevertheless, EMT is a rare event in
vivo and some clinical pathologists are sceptical of this idea. This
review provides an overview of the main characteristics of EMT that
will help in its identification, resembles proofs about its existence in
human cancer (more particularly colorectal cancer) and finally
proposes some directives about its characterisation in the clinic and
its potential use in better prognosis and treatment.
2. Induction and mechanisms of EMT
2.1. Epithelial versus mesenchymal characteristics, in vitro and in vivo
2.1.1. Cell function and morphology
EMT is first identified as a change in cell morphology (Fig. 1).
Epithelial cells display a highly baso-apical polarization essential for
their biological function which includes endocytosis, exocytosis and
vesicle transport. The epithelial cell basolateral surfaces are closely
associated with neighbouring cells, since they display keratin
filaments and regularly spaced membrane-associated specialised
junctions. A classical epithelium plays an important role as
protective barrier, since the movement of individual cell is inhibited
allowing the formation of a space where structure and rigidity are
preserved. In some cases like in colorectal epithelium, the apical
surface of the cell faces a lumen and has a role in secretion or
absorption.
A main characteristic of mesenchymal cells is the loss of their basoapical polarization and the acquisition of front–rear polarization,
necessary for cell migration. In parallel, a distinct organization of the
actin cytoskeleton enhances communication with the extracellular
matrix. In particular at microscopical level, mesenchymal cells appear
to possess points of attachment to the substrate, whereas staining
with phalloidin reveals particular actin-based cytoplasmic structures
in relation with intracellular actin fibres. In contrast to epithelial,
mesenchymal cells become flat and spindle-shaped, are loosely
associated and the source of growth factor production in collaboration
with the surrounding stroma.
2.1.2. Cell–cell and cell–matrix interactions in adhesion and migration
EMT program accomplishment follows a well coordinated process
that includes several steps and will be detailed in this section. It
77
Fig. 1. Epithelial–mesenchymal transition characteristics: Images show the particular cell morphology of Harvey-Ras induced EMT in the intermediate adenoma Caco-2 colorectal cell
line. Immunofluorescence analysis of Vimentin and E-cadherin are adapted from [243]. The table summarises the cells properties that are modified during EMT (on each side of the
table) and indicates some in vitro methods to identify them (in the center of the table).
involves loss of intercellular cohesion, disruption of extracellular
matrix, modifications of the cytoskeleton, increased motility and
invasion. The first step to invasion relies on looser cell–cell contacts at
the tumour leading edge.
2.1.2.1. Intercellular interactions and cell dissociation. The reduction of
intercellular cohesion in mesenchymal cells is mainly the result of
alterations in the sites of mechanical attachment, called the
intercellular junctions composed of desmosomes, adherens junctions
78
and tight junctions which play a central role in regulating the
activity of the entire junctional complex [10]. Epithelial cells use
the transmembrane glycoprotein of type I cadherin superfamilly Ecadherin (encoded by CDH1) as the main molecule in the adherent
junctions. The extracellular domain of E-cadherin consists of five
repeats implicated in the formation of E-cadherin dimers, which
interact with dimers on the membrane of a neighbouring cell
[10,11]. The intracellular domain of E-cadherin is linked to a
protein complex containing beta- alpha- and p120-catenins, which
interact with intracellular actin filaments network. This creates a
communication path between cell contact, regulation of cytoskeleton and cell shape, necessary for keeping the epithelial cells
immobile and physically linked [10,12–14]. Both E-cadherin and
catenins have been found inactivated in human cancer [15]. In
addition to this role, E-cadherin is an important signalling molecule
in EMT in part via its interaction with β-catenin and will be later
discussed. During EMT, the activity of the adherens junctions is
highly modified mainly due to the replacement of E-cadherin by Ncadherin, a process called the ‘cadherin switching’ [16–18]. Indeed,
in contrast to epithelial cells, mesenchymal cells express various
cadherins including N-cadherin, R-cadherin and cadherin-11.
Aberrant expression of N-cadherin seems to have a dominant
effect in cell–cell interaction since even in the presence of Ecadherin it enhances the motility of tumour cells [19] by a
destabilisation of cell–adhesion complexes. Nevertheless, in contrast to E-cadherin down-regulation, up-regulation of N-cadherin is
not always associated with EMT suggesting that its role could be
associated to a subset of tumours. Indeed, other molecules like
Cadherin-11 which is normally expressed in the brain, but
upregulated during tumour progression [20] could play a role in
this process. Moreover, proteins localised in tight junctions like
claudins, connexins, occludins and zonula occludens have also been
found to be involved in EMT [21–24].
As EMT-induced modifications in intracellular interactions are the
key step in cell motility and invasion, it is important to quantify the
intracellular adhesion force. Several in vitro experiments have been
developed towards this direction, like the dispase-based dissociation
assay [25] where cells are treated with dispase before the application
of a mechanical stress, the calculation of cell aggregation rates [26,27]
or measuring the intracellular adhesion force by dual pipette assay
[28].
2.1.2.2. Cell–matrix interactions, cell motility and invasion. In a living
organism, looser cell–cell contacts are necessary but not sufficient to
activate cell motility. Indeed, to reach a particular location cells
must also create a strong relation with the extracellular matrix
(ECM), migrate through it and proceed to its proteolysis. The first
step to migration includes a front-rear polarization affecting surface
receptors, vesicule trafficking, Golgi apparatus localization and
microtubules organization, controlled in part by the Rho family
small guanosine triphosphate (GTP)-binding proteins(Rho-GTRases).
These highly regulated proteins are also implicated in the actin
polymerisation leading to the formation of actin-based cell protrusions in the leading edge of the cell. The two major structures are
called lamellipodia (large structure; actin-filament meshwork) and
philopodia (spike-like structure; radially oriented actin filaments)
but other structures like invadopodia or podosomes exist [29]. These
protrusions are implicated in the attachment to the ECM and are
necessary to the migratory mechanism since they serve as traction
sites. Behind the leading edge, filamentous actin forms contractile
stress fibbers responsible for the contraction of the cell body and
retraction of the trailing edge. Attachment to the extracellular
matrix is mainly performed via transmembrane receptors which
allow the communication between the ECM and the internal actin
cytoskeleton as well as the cell contraction and movement [30]. At
the rear, the disassembly or removal of such adhesion points allows
efficient cell movement, whereas the front continues to elongate.
Contrary to epithelial cells, the mesenchymal cell–matrix adhesion
system is principally based on members of the heterodimeric,
transmembrane integrin family, composed of 24 members, all
having specific functions. Integrin expression and role in migration
is cell-type- and differentiation-stage-specific, as well as dependent
on the ECM constitution [31]. Interestingly, several integrins have
been associated to EMT [32–34], binding to ECM produces an
integrin molecule clustering on the membrane, which enhances the
interaction of their cytoplasmic tail with cellular factors. This results
in the formation of large multi-protein platforms that link the
extracellular matrix to the actin cytoskeleton and appear as a point
of attachment of the cell to the external surface, called focal
adhesions. The transient adhesion complexes linked to the actin
network of the lamellipodia and philopodia important to cell
migration are called focal complexes. Except from mechanically
linking the cell to the ECM, the adhesion complexes allow the
generation of tension and shape changes by orchestrating the
regulation of the ECM binding, the intracellular signal transduction
cascades (‘outside-in signalling’) and the creation of communication
points between the ECM and the actin cytoskeleton [35,36]. The
cell–matrix adhesion complexes are highly altered in response to
signalling or to the environment, as a way to trigger the appropriate
modifications in the cell properties. It is important to notice that the
amoeboid migration is an alternative mode of single-cell migration,
based on high deformability and weak, non-integrin based interactions with the ECM. During amoeboid migration, the cell shape is
modified in order to slide through the matrix rather than degrading
it. A transition between epithelial and amoeboid migration exists in
response to environmental factors or genetic alterations [37,38]. In
addition to actin microfilaments, intermediate filaments (IFs) and
microtubules are major components of the cytoskeleton and play an
important role in mesenchymal migration. IFs are encoded by a
large family of genes and interact with desmosomes, hemidesmosomes, focal adhesions and the ECM to form a complex network
between the cell surface and the nucleus [39,40]. This mediates the
transmission of exterior signals and allows the cell to activate a
mechanism to resist mechanical stress and/or deformation. IFs are
related to the cell physiological function, show high molecular
diversities and are expressed in tissue-specific programs. For
instance, keratins (type I and type II IFs) define epithelial tissues
whereas vimentin (type III, IF) defines mesenchymal origin [41,42].
In a subset of cancers, particularly melanoma and breast carcinoma,
vimentin and keratin can both be expressed and represent a
dedifferentiated or interconverted state. Another major protein in
the accomplishment of an epithelial phenotype is the fibroblastspecific protein (Fsp-1 or S100A4), which belongs to the calmodulin-S100-troponin C superfamily of calcium-binding proteins. Apart
from a role in calcium signal transduction, members of this family
have been implicated in microtubule dynamics and cytoskeletalmembrane interactions, cell growth and differentiation. Fsp-1/
S100A4 has been extensively linked to the mesenchymal phenotype
and is considered as a marker for EMT [43,44].
In vitro, the force of cell–matrix interaction can be studied by
‘cell adhesion assays’ performed on cellular supports that mimic
the extracellular matrix like fibronectin, collagen, laminin or
fibrinogen. On the other hand, capacity to migrate is assessed by
‘cell migration assays’ performed in Boyden chambers in response
to a chemotactic gradient [45]. Similarly, in ‘wound healing assay’,
cells are inspected over time as they fill a damaged area in the cell
monolayer.
Nevertheless, a culture dish is only a two-dimensional environment whereas in vivo migration of mesenchymal cells implicates a
three-dimensional extracellular matrix composed of collagens, proteins and proteoglycans which causes several constrains and implicate
the degradation of the barrier. Therefore, function of proteases in
tumour cell spreading seems to constitute a common pathway of all
invasive malignancies, whereas protease activities have been commonly found altered in cancer. To degrade the ECM, the cell sets up
specific mechanisms that concentrate protease activity in the
pericellular environment, mainly by membrane-anchored proteases
called the transmembrane matrix metalloproteinases (MMPs) and the
endogenous proteolytic urokinase-type plasminogen activator (uPA)
system. The most studied mechanism in ECM degradation during
invasion is the activation of MMPs that can be directly linked to the
plasma membrane or localized to invadopodia by specific interactions
with integrins or other cell surface receptors [46]. MMPs also promote
tumourigenesis through limited proteolysis by the activation of
growth factors and the inactivation of protease inhibitors. Interestingly, MMPs have been found upregulated in EMT cells [47] but also
capable of inducing EMT [48].
In vitro, a thin fluorescence-labelled substrate coating can be
utilized to detect and image local proteolytic activity in the
microenvironment of cells [49]. In vitro, cells are allowed to invade
an extracellular matrice in response to a chemotactic agent. Several
matrices exist in order to mimic the stiffness and composition of a
particular tissue, including collagen, fibronectin, laminin or a tumour
basement membrane extract secreted by Engelbreth–Holm–Swarm
(EHS) mouse sarcoma, a tumour rich in ECM proteins (Matrigel TM)
[50]. Recently, the development of tissue-like 3D environment has
been described by the production of cell-derived 3D matrices and in
vitro-produced tissue substitutes, grown by different primary cells like
fibroblasts [51,52]. The circular invasion assay is a modification of this
assay that combines a wound in a cell monolayer and the presence of
matrix barrier component, in order to better mimic the in vivo
conditions [53].
2.1.2.3. Resistance to anoikis. A big majority of normal cells are
adherence-dependent since they grow and divide only if attached to
a solid inert support, in contrast to transformed cells. Indeed,
identification of anchorage independent growth with a ‘soft agar
colony formation assay’ which measures proliferation in a semisolid
culture medium, is a characteristic of malignancy and/or EMT in
vitro [54,55]. Disruption of the interactions between normal
epithelial cells and extracellular matrix or inappropriate anchorage
can induce a programmed cell death called anoikis [56]. Interestingly, resistance to anoikis has been shown to promote metastasis,
since tumour cells can enter and disseminate into the bloodstream.
In agreement with a higher metastatic potential, expression of EMT
cells display resistance to anoikis, whereas loss of the epithelial
adhesion molecule E-cadherin promotes metastasis in part by
inducing anoikis resistance [57].
2.1.3. Tumour-initiating cell characteristics
Cells with stem cell characteristics have been identified in several
solid tumours, like breast [58], colon [59–61] or pancreas [62] and
have been named ‘cancer stem cells’. This term first referred to a
cancer cell able to produce tumours when injected into SCID mice,
having self-renewal properties and giving rise to other cell types in the
tumour were xenografted into immunodeficient mice. This small
population of undifferentiated cells with stem cell characteristics can
perform asymmetrical division to replicate themselves, but also to
create a committed progenitor cell. Interestingly, it has been recently
shown that immortalized human mammary epithelial cells that have
undergone EMT display characteristics of normal embryonic epithelial
or cancer stem cells, mainly characterized by the CD44high/CD24low
antigenic phenotype [63]. This suggests that EMT could result in the
formation of cancer stem cells and further induce the formation of an
undifferentiated cancer, thereby explaining how cells that leave a
primary tumour gain self-renewal capacity. In colorectal cancer,
CD133 positive cells have been shown to have cancer stem cell
characteristics [60].
79
2.2. The E-cadherin-mediated cell–adhesion system in EMT
2.2.1. E-cadherin function and regulators
As detailed earlier, the invasion-suppressor E-cadherin is a very
important molecule in cancer progression and EMT induction [15,64].
Indeed, E-cadherin perturbation in mammalian cell systems is
sufficient to trigger EMT [57,65,66], whereas in transgenic mouse
models of pancreatic beta-cell carcinogenesis loss of E-cadherin is
necessary for tumour progression to invasive forms [67,68]. In parallel,
transgenic mice expressing N-cadherin, which replaces E-cadherin
during EMT, display enhanced metastasis of breast tumours [69]. In a
variety of human cancers, E-cadherin loss is linked to poor prognosis,
tumour progression and metastasis [12,70–72], underlying that its
regulation is a key step in tumour spreading. Among the high number
of factors and mechanisms specifically implicated in E-cadherin
regulation repressors of gene expression have strongly been associated with EMT and tumour progression [73–75]. The first repressors
to be identified were the zinc finger proteins SNAI1 (SNAIL) [74,76]
and SNAI2 (SLUG) [77] and the Smad-interacting proteins ZEB1
(deltaEF1 or ZFHX1A) [78] and ZEB2 (SIP1/ZFHX1B) [79], all capable
of binding the E-pal element on E-cadherin promoter. Other potent
repressors include the basic helix–loop–helix transcription factors
E12/E47 (TCF3) [80] and Twist [81]. These proteins actively repress
transcription by recruiting co-repressors [82,83] and known repressor
complexes [84,85] but also by influencing the activity of other Ecadherin repressors [86]. The activity of E-cadherin repressors is
regulated by central cellular pathways, known to be involved in EMT
like TGFβ [79], β-catenin [87] and Wnt signalling pathways [75,88].
Recently, loss of the Rb protein has been shown to induce EMT in part
by a decrease in E-cadherin levels in breast cancer cells, adding a novel
tumour suppressor function of Rb, related to EMT [89]. Further
knowledge of E-cadherin transcriptional regulators and their mechanism of action would be of great interest for the identification of new
therapeutic targets. As an example, we have recently identified the
TBP associated factor TAF12 as being a repressor of E-cadherin
regulated by the MEK/ERK pathway, showing the implication of new
complexes in this type of regulation [90]. In parallel, hypermethylation
of E-cadherin promoter leading to silencing has emerged as another
important mechanism for the downregulation of the protein during
EMT and in many carcinomas [91–93]. In addition, E-cadherin has
been found to be regulated at the protein level by mechanisms related
to stability [94], but also by a recently discovered mechanism
implicating small non-coding RNA molecules called micro-RNAs
(miRs). Repression of the miR-200 family enhances the mRNA levels
of the E-cadherin repressors ZEB1 and ZEB2 in human colorectal
HCT116 cells, in an in vitro model of EMT in canine kidney epithelial
cells and in a murine mammary epithelial cells model of TGFβinduced EMT [95–97]. On the other hand, the integrity of the cell
adhesion system mediated by E-cadherin can be regulated by posttranslational mechanisms like tyrosine phosphorylation of catenins
[98]. In response to particular signalling pathways like v-Src
oncogenic transformation or EGF treatment, catenins are phosphorylated and subsequently released from the complex containing Ecadherin, leading to decreased cell–cell adhesion [99]. Destabilisation
of E-cadherin and catenin interactions can further lead to a decreased
cellular adhesion by influencing E-cadherin stability [94].
2.2.2. The beta-catenin pathway
In addition to its important role in physical association of the cells
E-cadherin has a central role in signalling, mainly via its interaction
with the β-catenin multi-functional armadillo repeat protein which
exists as a cadherin-associated (membrane bound) or -free form
[100]. When β-catenin is released in the cytosol, it is phosphorylated
in a complex containing the APC protein, axin and GSK3beta kinase,
responsible for leading β-catenin to degradation through the
ubiquitin–proteasome system [101]. During transduction of a
80
Winglesss/Wnt-related signal, GSK3beta phosphorylation inhibits
this process [102] and allows β-catenin to accumulate in the
cytoplasm and translocate to the nucleus, where it functions as a
cofactor for members of the Tcf (T-cell factor)/Lef family of
transcription factors [103–105] which further activate the transcription of important genes. Altered expression of these genes, like c-myc
has been implicated in carcinogenesis and EMT [106]. The importance
of the E-cadherin molecule in partial control or amplification of Wntsignals has been demonstrated in different systems and underlines a
second mechanism of action of E-cadherin in EMT [64]. In human
cancer, mutations in proteins implicated in the Wnt signalling and/or
the β-catenin degradation are mainly responsible for the accumulation of β-catenin to the cytoplasm and further translocation to the
nucleus. For instance, alterations leading to an APC protein lacking the
ability to bind to β-catenin are observed in 40–80% of colorectal
cancers and are responsible for familial adenomatous polyposis (FAP).
Importantly, staining for β-catenin provides an indication of the
integrity of the Wnt signalling and β-catenin degradation pathways.
In normal fibroblasts and endothelial cells, β-catenin staining is
limited to cytoplasm and/or cell membranes, whereas it is strictly
membranous in normal epithelial cells [107]. In mesenchymal cells, βcatenin appears to be mainly nuclear [57,108].
2.3. Cellular signals inducing EMT
2.3.1. Extrinsic stimuli: the effect of the microenvironment
The tumour microenvironment composed of extracellular matrix,
cells and soluble factors, plays an important role in EMT induction and
further in metastasis. Indeed, interaction of tumour cells with their
local microenvironment can induce the autocrine and/or the paracrine secretion of growth factors, cytokines and extracellular matrix
proteins further leading to EMT [8,7,109–111]. In agreement, breast
cancer-associated fibroblasts have been implicated in the proliferation
and migration of tumour cells via secretion of chemokines [112].
Similarly, conditioned media cultures from cancer-associated but not
normal fibroblasts induce EMT in breast cancer cells [113]. During the
past years, a great number of growth factors and signalling pathways
have been associated with EMT induction, like the epidermal growth
factor (EGF) via the Janus-activated kinase (JAK) pathway [114], the
fibroblast growth factor (FGF) via the ERK/MAPK [115–117] and the
hepatocyte growth factor (HGF) [118,119]. In agreement, the expression of EMT-key molecules like the members of the SNAIL family (Ecadherin repressors) have been found to be directly controlled by
numerous extracellular signals and pathways [75,120,121]. The most
extensively studied effect, is that of the transforming growth factor
beta (TGFβ) acting via the Smad proteins or the ERK and PI3 K
signalling pathways [111,122,123]. Nevertheless, it is becoming clear
that the interplay between different stimuli and the activation of
different signalling pathways is more likely to be involved in EMT
induction. Wnt, TGF-β, Hedgehog, Notch, and nuclear factor-κB (NFκB) signalling pathways have been found to be critical for EMT
induction [109,111]. In vivo, and in agreement with the idea that the
microenvironment plays a central role in induction and/or maintenance of EMT, a reversion from mesenchymal cancer cells to more
differentiated epithelial cells has been shown in metastatic sites of
human colorectal adenocarcinomas, suggesting that the dedifferentiated mesenchymal phenotype is dynamic and reversible [124]. The
authors of this study postulated that since the reversion of EMT is
possible in the case of well differentiated carcinomas, the tumour
microenvironment rather than permanently acquired gene mutations
would be the driving force of EMT induction. Indeed, tumour cells
with EMT but not the normal mucosa, share common pathways and
signalling molecules with the surrounding stroma, suggesting a strong
cooperation in EMT induction [125]. These cancer related modifications of the stroma could in turn lead to exposure of cancer cells to
EMT-inducing growth factors. In agreement with the central role of
the environment in EMT induction, E-cadherin expression was not
affected in an in vitro culture of epithelial-like subclones of MDCK cells
transformed with H-Ras, but was partially reduced in metastasis
derived from the injection of these cells into mice. Similarly, tumourexcised fibroblastic-like MDCK cells transformed with H-Ras, partially
start to re-express E-cadherin when put into culture [126].
2.3.2. Intrinsic stimuli: mutations in signal transduction molecules
The prominent idea is that even though some factors like TGFβ
could have the possibility to trigger EMT, they would need the
accumulation of particular gene mutations to unlock or maintain the
EMT program. Indeed, gene alterations concerning extracellular
receptors and/or consequent signal transduction are common in
cancer. For example, TGFβ receptor mutations are frequently involved
in human tumours [127] including colorectal tumours [128] but also
induce EMT in human cell lines [129]. On the other hand, it has been
shown in a skin carcinogenesis model in vivo [130], that accumulation
of several mutations affecting the TGFβ receptor or the components of
its transduction pathway was necessary for an EMT induction. Indeed,
loss of Smad4 (a central molecule of the TGFβ pathway) has been
shown to abolish the tumour-suppressive functions of TGFβ but not its
tumour-promoting functions leading to cell transformation [131].
Others suggest that TGFβ treatment alone is sufficient to induce a
transient state resembling EMT in normal epithelial cells [132]
whereas in other cell types, cell mutations finally affecting the
MAPK and PI3 K pathways seem to be necessary to achieve this effect
[133]. Similarly, tumour-suppressive functions of Notch signalling can
be abolished in the presence of particular oncogenic events [134].
Interestingly, an activated Harvey Ras oncogene, which is a potent
activator of the MAPK pathway, has been found to cooperate with
TGFβ1 signalling in hepatocytes and mammary cells to induce
autocrine production levels of TGFβ1 [135] and is involved in the in
vivo maintenance of EMT and cell survival during metastasis [133]. On
the contrary, introduction of a mutated, constantly active Harvey RAS
in different cell systems was sufficient to induce and maintain EMT,
suggesting that the unique genetic background of each cell may be of
great importance in the mechanisms of EMT [55,136]. In agreement to
this idea, only a minority of human cancer cell lines treated with
TGFbeta underwent EMT, underlying that particular set of mutations
might be necessary [137]. In parallel, since EMT induction is the result
of a tumour cell adaptation to its interaction with the extracellular
matrix, as well as a combination between the activation of several
pathways, it is possible that a two dimensional environment could not
offer the right conditions. Accordingly, a subclone of MDCK cells
transformed with H-Ras shows a reduction of E-cadherin only when
interacting with an in vivo environment [126]. In addition and
contrarily to the in vitro situation where particular gene mutations
allow a complete EMT, a partial EMT could be preferred in vivo. Further
importance of the microenvironment is pointed out by the fact that a
mutated Kirsten RAS (Ki-Ras) isoform is unable to induce EMT when
stably expressed into the intermediate colon adenocarcinoma Caco-2
cell line [55], but has been associated with high degree of tumour
budding and invasion in primary colorectal adenocarcinomas [138].
Finally, genetic and epigenetic alterations in several genes have been
implicated in the initiation and completion of EMT, including Ecadherin [91], v-SCR [139] and Rb [89].
3. Can EMT detection result in better patient treatment?
3.1. Difficulties in detecting EMT in the clinic
3.1.1. Is EMT associated with cancer progression and metastasis in human
disease?
Recognition of epithelial to mesenchymal transition is relatively
new in oncology, since morphological changes in tumours were in the
past simply called metaplasia. Despite the strong arguments in favour
of the EMT in vitro, its real existence in human cancer is still
controversial as elegantly described by pathologists [140]. Indeed,
EMT has not been identified in a majority of human tumours [141,142].
Whether these discrepancies come from artefacts related to the in
vitro cell manipulation, the overstated use of mesenchyme specific
markers and the inappropriate usage of the term ‘EMT’ or alternatively, from the lack of clinical evidence due to inefficient tools or
limited awareness, is still unknown. Indeed, a number of issues
arguing against EMT include the fact that it has been seen only in
limited cases of human cancer and could not therefore be necessary
for metastasis. In the same direction, metastasis and poor prognosis in
human cancer are not always associated with expression of the most
common EMT markers. In parallel, the theory based on EMT-related
metastasis should produce metastatic tumours histologically different
from the epithelial primary tumour from which they are derived. To
answer these questions, clinicians have tried during the past years to
identify this phenomenon in humans, but the success is still limited.
As the observation of histological sections is the main tool used by
surgical pathologists, the spatial and temporal heterogeneity of EMT
in human cancer, the lack of good markers and the possibility of partial
EMT resulting in cells expressing some but not all the mesenchymal
markers, can represent important obstacles in the safe EMT
identification. On the other hand, lack of evidence of EMT in clinical
81
samples could be related to a preferential use of alternative
mechanisms of cell migration/invasion, based on the preservation of
epithelial characteristics [37]. These mechanisms are described below.
Spatial and temporal heterogeneity are also summarised in Fig. 2.
3.1.2. The issue of the spatial heterogeneity and the alternative
mechanisms of cell invasion
Even though histopathological observations are a valuable tool in
the clinic, metastatic capacity may be difficult to classify by this means
since cells with metastatic capacity are only a subpopulation of the
primary tumour and located in particular areas of the tumour [7]. In
practice, the architecture of the invasive front of a non diffuse
carcinoma is characterized by individual cells that are detached from
the main tumour mass, are less differentiated, less cohesive and
appear in tight connection with the surrounding stroma [[143] and
Fig. 2a]. Importantly, recent studies have located expression of EMT
markers in the invasive front of a number of tumours, suggesting that
it could be one of the mechanisms allowing tumour spreading [144–
146]. Nevertheless, and since the presence and the role of EMT in
cancer metastasis has been suggested only recently, no nomenclature
has yet been accepted in the clinic which makes the communication in
this field difficult. For instance tumour buds, a term which describes
single cancer cells and/or small cancer clusters, have been identified
Fig. 2. Spatial and temporal heterogeneity of EMT. (a) Spatial heterogeneity: Increased contacts with the tumour stoma induce epithelial–mesenchymal transition (EMT) in a
minority of cells of an epithelial colon carcinoma. This allows the dissemination of single carcinoma cells from the site of the primary tumour and results in local invasion, which is the
first step to metastasis. In practice, single cells or clusters of cells with EMT are found away from the central tumour mass. (b) Temporal heterogeneity: Cells with EMT that have
locally invaded a tissue, intravasate into blood vessels and are transported to distant organs. At the site of metastasis, carcinoma cells extravasate and are allowed to form a metastatic
carcinoma after revertion to an epithelial state, by a process called mesenchymal–epithelial transition (MET).
82
in the invasive front of several tumours and their presence has been
shown to have prognostic significance in rectal and breast cancer
[147,148] and has been associated with metastasis in colorectal cancer
[149,150]. Interestingly enough, switching from an epithelial to a
mesenchymal phenotype has been referred as tumour budding in
vitro [151] whereas it has been parallel to EMT and used as an
independent prognostic factor in the context of colorectal cancer
[152]. In parallel, buds show reduced E-cadherin expression, nuclear
β-catenin activation [153] and cancer stem cells characteristics [154],
suggesting that it may indeed reflect the presence of invasive
mesenchymal-like cells. Nevertheless, reports about the identification
of cells with EMT in human tumours are rare, given the fact that the
majority of human tumours give local and/or distant metastasis.
Moreover, inhibition-based targeting of proteases involved in ECM
degradation, that should in theory inhibit the mesenchymal migration, has only offered weak benefit to patients [155], suggesting that
the overall metastatic capacity remained intact. The existence of
alternative mechanisms to the epithelial individual cell migration
could give a solution to this paradox. Indeed, the amoeboid individual
cell migration which allows cell movement across the ECM without
degrading it, has been observed in 3D cultures and in certain types
cancers [37] as well as in response to protease inhibition [156]. On the
other hand, interesting studies demonstrated that epithelial cells are
also able to migrate without acquiring mesenchymal characteristics,
through a collective cell migration process based on strong cell–cell
interactions. In this type of migration, groups of cells function as large
units which appear as sheets connected to the primary site or as
detached strands and which migrate along rays of altered ECM
[37,157]. The mechanisms of collective migration induce a front–rear
polarization of the entire migration unit, with the appearance of
protrusions necessary for traction and ECM alteration at the leading
edge. At the end of a migration cycle, the conserved strong
intercellular contacts allow dragging of the trailing edge by the
leading edge, through the path created in the ECM. Notably, the use of
collective migration mechanisms implicates strong tissue architecture
as well as a functional differentiation in the unit and may therefore
appear in highly differentiated cancers. Thus, the lack of mesenchymal
cells in the invasive front of human samples could suggest a
preferential use of collective migration mechanisms offering an
advantage in tissue colonisation, via spreading of a large number of
clustered heterogeneous epithelial cells [140]. Notably, different types
of cell movement can be interchangeable depending on parameters
like the type of ECM, the genetic alterations and a potential drug
treatment [37,38,158], pointing out the possibility that they could
alternatively used as a result of an adaptation of tumour cells to
facilitate metastasis. In agreement and as suggested by recent studies
in mice, mesenchymal cells could constitute the driving force for
epithelial cells to fulfil local invasion and distant metastasis [159–161].
Moreover, several examples in the literature agree to diversity in the
mechanisms of cell invasion in human tumours, since metastatic
capacity is not always associated with loss of expression of epithelial
markers like E-cadherin. For instance, infiltrating lobular carcinomas
of the breast show a concomitant loss of E-cadherin expression
independently of the clinical outcome, whereas E-cadherin reduction
has been associated with poor prognosis in node-negative breast
cancer patients [162] and in invasive non-lobular breast carcinomas
[163]. In parallel, other studies failed to associate E-cadherin reduction
with cell polarity, invasion and survival in grade I ductal carcinoma
[164]. Important variability in collection and annotation of the
samples, tumour grading, levels of lymph node involvement and
arbitrarily set standards as well as different adjuvant therapies could
be responsible for discrepancies. Alternatively, these contradictory
results may suggest that an epithelial-like morphology would be
preferred to the mesenchymal one, during the metastatic process of
certain types of tumour cells [164]. In agreement to this, expression of
E-cadherin and α/β catenins were altered in invasive lobular
carcinomas (ILC) but not in invasive ductal carcinomas (IDC) [165],
pointing out the existence of tumour type-specific mechanisms of
invasion. Interestingly enough, a recent study based on the comparison between the expression signature of a set of breast tumours
showed that genes overexpressed in IDCs coded preferentially for
promoters of cell proliferation whereas those overexpressed in ILCs
coded for proteins involved in cell adhesion and the E-cadherin
related pathways [166]. The choice of the one or the other types of
migration may arise from a particular genetic background or an
adaptation to the tumour local microenvironment. As an argument
supporting tissue particularities affecting invasion, P-cadherin but not
E-cadherin alterations have been correlated with differentiation of
breast carcinomas possibly via specialized intercellular junctions
important for cell cohesion [167]. Similar reasons can be evoked to
explain discrepancies in E_cadherin prognostic value in other types of
cancer, like gastric carcinoma [168]. In addition, since E-cadherin
function can be abolished by other alterations whilst retaining a
normal expression/localization [15], testing the functionality of the
cell–adhesion complex is necessary, by assessing for example the
localization of catenins [169,170]. Alternatively, alteration in the
expression molecules involved in cell–matrix rather in cell–cell
interactions like integrins may be important for the identification of
the mechanism by which the cells invade the surrounding stroma
[169]. Finally, recent studies show that partial EMT phenomena are
more likely to occur in human disease than complete EMT, suggesting
that a low or a moderate down-regulation of E-cadherin could be of
great clinical significance, as well as the proportion of cells with
altered expression in a tumour bulk [171].
3.1.3. The issue of the temporal heterogeneity
EMT researchers agree that cancer cells probably undergo only
partial EMT more as an intermediate state. Indeed, once cells have
invaded the primary tumour and penetrate the surrounding tissue,
they must be able to colonise the new tissue and form a tumour mass
(Fig. 2b). As increased cell–cell adhesion is needed, cells must return to
an epithelial phenotype with re-expression of E-cadherin, by undergoing a reverse conversion called mesenchymal to epithelial transition
(MET), which is a fundamental embryologic process [75,172]. In
support to the idea that EMT is a reversible state, abolishment of Snail
or Twist expression in mouse is enough to loose the EMT-related cell
properties [63]. The existence of the MET could explain the difficulties
in finding strong proof about the existence of EMT in human cancer.
MET has been demonstrated in a series of bladder cancer cell lines,
where a transit between EMT and MET facilitated the escape from the
primary tissue and the colonization of another tissue respectively
[173]. In agreement, dedifferentiated mesenchyme-like tumour cells
expressing nuclear β-catenin were found at the invasive front of a
colorectal carcinoma but not in the subsequent metastatic tumour of
epithelial morphology [124]. The mechanisms inducing the MET
phenomenon are not yet elucidated, but a possible mechanism would
be redrawing of the EMT inducers, probably resulting from a change of
the surrounding microenvironment. In support to this idea, experiments performed in mice showed that Ras-transformed epithelial-like
MDCK cells expressing E-cadherin, reacted to a host environment by
producing cells clusters with lower E-cadherin. Expression of Ecadherin was gradually recovered when cells were put back in culture,
suggestive of a MET. Interestingly, these cells were creating metastatic
tumours expressing E-cadherin, suggesting than EMT might be
necessary to the first steps of invasion but the epithelial phenotype
could possibly be preferred for the rest of the metastatic process [126].
Nevertheless, studies demonstrating the existence of MET in vivo are
limited. In addition, the idea of the MET existence received criticism
about the fact that a cell with severe modifications induced by EMT
could revert and create a tumour identical to the one from which it
originally derived [140–142]. Moreover, several studies have demonstrated the presence of clustered cells expressing keratins and E-
cadherin, in the blood of patients with metastasis, suggesting that the
bloodstream transport would be facilitated by epithelial characteristics. These cells could derive from collective migration from the
epithelial primary tumour or by a reversion of the mesenchymal
phenotype after local migration. Nevertheless, the use of EMT and nonEMT cells in a mouse system showed that both invasive epithelial and
mesenchymal cells were found in the blood of mice during the
metastatic process suggesting that both types could collaborate [161].
Interestingly, mechanisms of induction of collective cell migration
have been associated with stromal derived growth factors, suggesting
that collective migration could be an alternative to EMT [174]. Whether
the use of the one or the other mechanism is used by the tumour to
metastasise and whether this depends on the cell type, the genetic
background or the environmental conditions, remains to be demonstrated in vivo.
3.1.4. EMT markers use in the clinic and identification of new markers
Being able to identify EMT in tumour samples could be of
tremendous help in better prognosis and treatment, but also towards
the understanding of the metastasis-related mechanism. Nevertheless, the difficulty of objective judgement makes this parameter
difficult to use in routine practice by histopathologists, as the more
‘mesenchymal’ the tumour cells get, the more difficult is to distinguish
them from the mesenchymal cells that surround the tumour (i.e.
83
fibroblasts or myoblasts) in haematoxylin and eosin staining. It is also
important to underline that in order to identify EMT one should give
attention to particular morphological features at the invasive front of
the tumour away from the tumour mass. Notably, the frequency of
budding in colorectal cancer has been reported to correlate with
microsatellite stability and high frequency of APC mutations [175],
suggesting the need for particular genetic alterations and building the
first rules to its identification. Nevertheless, the fact that cells that
undergo EMT within a tumour seem to represent only a small minority
of the entire cellular mass, markers should be preferentially based on
histological studies that allow a spatial separation of the cells than on
other methods based in analysis of a bulk of tumour cells. Another
advantage of histological studies is that they are widely used in the
clinic and samples can be easily processed, given that the antibodies
are specific. By this method, several proteins implicated in the EMT
mechanisms have been found to differentially mark particular sets of
tumour cells, located at the invasive front of colorectal and/or other
cancers and have been correlated with invasion and metastasis. These
proteins and their cellular localization after immunohistochemical
detection are summarized in Table 1 and could potentially be used as
markers for the identification of cells with mesenchymal characteristics on tumour samples. This list includes proteins implicated in cell–
matrix interactions, cell structure and motility like N-cadherin,
Vimentin, Fibronectin, Integrins and FSP-1/S100A4. Secretion of
Table 1
Markers for detecting EMT in clinical samples.
Potential marker
Characteristics
Mesenchymal markers; up-regulated during EMT
FSP1/S100A4
Fibroblast calcium-binding protein
Vimentin
Mesenchymal intermediate filament
MMPs
Matrix metalloproteinases,
zinc-dependent endopeptidase
SNAIL (SNAI1)
Snail homolog 1 (Drosophila);
zinc finger transcriptional repressor
ZEB1
Zinc finger E-box binding homeobox 1
SLUG (SNAI2)
Snail homolog 2 (Drosophila);
zinc finger transcriptional repressor
Twist
Up-regulated in mesenchymal
N-cadherin
Type-1 transmembrane protein
Fibronectin
High-molecular-weight extracellular
matrix glycoprotein
Integrins αvβ6; α5β1
Cell surface receptors
Epithelial markers; down-regulated during EMT
E-cadherin
Type-1 transmembrane glycoprotein
in adherent junctions
cdx-2
Caudal type homeobox transcription factor 2
Desmoplakin
Protein associated with desmosomes
Cytokeratin
Intermediate filament keratins found in
the intracytoplasmic cytoskeleton
ZO-1
Zona occludens 1; found in intercellular
tight junctions
Claudin 1
Others
Laminin-5 (alpha 3,
beta 3, gamma 2)
Beta-catenin
Ki-67
Limitations
Cellular localization
Reference
Occasional staining of the extracellular matrix
activated fibroblasts, endothelial and smooth
muscle cells as well as leucocytes
Nucleus and/or cytoplasm
Mainly cytoplasm
Cytoplasm and/or
extracellular space
Nucleus
[43,186]
[187]
[188,153]
Nucleus
Nucleus
[191]
[192–194]
Nucleus
Membrane
Cytoplasm and/or
extracellular space
Membrane
[192,195]
[192]
[196]
Membrane
[199]
nucleus
membrane
cytoplasm
[200]
[201]
[202,203]
Membrane and cytoplasm;
diffuse cytoplasmic and/or
nuclear in migrating cells
Membrane
[204]
Protein abundance is decreased
during EMT. Mesenchymal
cancer cells will not be stained.
Member of the claudin family found in
tight junctions
[145,189,190]
[197,198]
[204]
Basement membrane glycoprotein.
Modifications in the expression
of the different isoforms in invasive
cancer; gamma 2 usually overexpressed
Subunit of the cadherin protein
complex. Armadillo family of proteins.
Modifications in expression and/or
localization may be tissue specific.
Basal Membrane and/or
cytoplasm accumulation
[179,205,176]
Diffuse staining, difficult to localize
catenin in different compartments.
Risk of confusion mesenchymal and
stromal cells.
Normal fibroblasts: cytoplasm
and/or membrane. Normal
epithelium: membrane.
Mesenchymal cells after
EMT: mainly nuclear
[124]
Marker of cell proliferation; Epithelial
cells proliferate very fast, mesenchymal not
Difficult to distinguish a resting tumour
cell from the normal surrounding cells
Nucleus
[206,207,124]
Several EMT-targets have been shown in the literature to be associated with budding cells or with the tumour metastatic front by immunohistochemistry on human samples
(references indicate the corresponding studies). These targets could potentially be considered as markers of EMT in clinical samples and possibly used as prognostic of tumour
progression and metastasis.
84
proteolytic enzymes like MMP2, MMP9 and increased activity of
cathepsin B has also been reported in these cells. Notably, a laminin 5
alpha3 subunit downregulation has been determined in colorectal
carcinoma budding cells, leading to laminin5 gamma2 and beta3
subunits cytoplasmic accumulation [176]. On the contrary, accumulation of the gamma 2 subunit has been shown in the invasive front of
many cancer types [177–179], suggesting that the nature of the lamin
5 deregulation in cells with metastatic potential is tissue specific. In
parallel, transcription factors implicated in the down-regulation of the
epithelial E-cadherin and widely used as markers for EMT like SNAIL1,
SNAIL2, Twist, EF1/ZEB1 and SIP1/ZEB2 have been associated with the
cancer invasive front [75,172]. On the contrary, epithelial-specific
proteins related to the absence or the reversion of EMT have been
correlated with the non metastatic, central part of tumours. This
category includes mainly molecules of the cell–cell communication
system like E-cadherin, Desmoplakin, MUC1, Cytokeratins, Occludin,
ZO-1 and Claudins. The above cited markers have shown to be very
valuable tools for identification and characterization of EMT in vitro, in
cell culture or in homogeneous cell populations. Nevertheless,
clinicians are confronted with two major problems related to the in
vivo identification of EMT in a high heterogeneous cell population.
First, it is important to underlie that the transition of an epithelial cell
to a mesenchymal aggressive phenotype is not an ‘all or nothing’
event. Tumour cells can express partial EMT, where the markers and
the overall cell characteristics could be altered at different degrees and
therefore difficult to assess [140]. For instance, we have observed
variable levels of E-cadherin/Vimentin expression in a panel of
established colorectal cancer cell lines in vitro [90] whereas partial
EMT has been detected in 3D cultures suggesting that this phenotype
could be more relevant to the in vivo situation than the complete EMT
[180]. In addition, commonly used markers besides not being able to
differentiate between a cancer-derived mesenchymal cell and a
normal fibroblast, are also not proven to be absolute markers of
mesenchymal characteristics. In parallel, the idea that altered gene
expression in some tumour cells is more the result of a high degree of
disorder due to malignancy than the accomplishment of a particular
EMT-induced gene expression program makes the marker identification even more difficult. Even though the combinatorial use of several
markers could occasionally provide a solution to this problem, the
need for new and better markers comes along with the increasing
importance of EMT identification in the field of cancer treatment. New
insights in the mechanisms of EMT induction could potentially allow
the development of specific markers able to discriminate cells with a
potential to acquire a complete EMT, before this mechanism actually
takes place. For example, the reduction of miR-200 expression
recently identified as a key event for EMT could be followed by in
situ detection from paraffin-embedded sections [181]. On another
hand, the cancer stem cell marker CD44high/CD24low recently
identified in mammary cells with EMT could potentially represent a
valuable marker of metastatic potential [63]. During the past years,
several studies deal with gene expression profile generated from DNA
microarray analysis in order to identify EMT specific gene signatures in
which researchers could rely on, to safely identify EMT. Microarray
analysis have been performed in in vitro models of EMT induced by
TGFβ [182] and EGF [183] treatment, or expression by the Harvey Ras
[55] (Joyce et al., under revision) and Myc oncogenes [184].
Interestingly, these in vitro systems have turned to be good models
of the in vivo situation, since many agreed in an important number of
common genes regulated during EMT. Also, studies based on
recurrence-free survival rates on patients with head and neck
squamous cell carcinoma [185], are in favour of the presence of EMT
signature in human disease. This show promise for the use of
emerging signatures as predictive biomarkers of clinical outcome
and will further allow the development of better markers. Nevertheless, problems arise by the lack of a natural environment in the in
vitro studies and the degree of variability in terms of genetic
background in human samples, which underlies the need for
approaches in the animal modelling front (Table 1).
3.1.5. In vivo identification of EMT during carcinogenesis
It has become clear that different states of EMT exist, depending on
the cell-models and the combination of inducers. These can express
some but not all the EMT markers, result in a stable or transient EMT
phenomenon and implicate a diversity of actors and pathways
[109,208]. Therefore, during the past years a great effort has been
made to prove the existence of EMT in vivo, to link it directly to the
metastatic process and thereby to create appropriate mouse models.
Indeed, as spindle-shaped tumour-associated cells cannot be correctly
marked and traced through the progression of the disease, the real
existence of EMT in cancer remains unclear. The first study to
demonstrate the existence of EMT in cancer dissemination was
performed in engineered mice with mammary carcinomas [209].
The authors followed tumour spread via detection of the FSP1/S100A4
promoter activation and showed that when injected back in mice, cells
with EMT were the ones responsible for metastatic tumours. Another
recently developed system based on marking and independently
following the fate of tumour-epithelial and stromal cells in mouse
mammary cancer progression, demonstrated the induction and
accomplishment of EMT in tumour epithelial cells during highly
metastatic myc-induced carcinogenesis [184]. In parallel, it has been
shown that non-EMT cells were unable to metastasise without the
action of EMT cells when inoculated sub-cutaniously into mice,
suggesting that at least in some cases, EMT or EMT-like phenomena
could be a prerequisite for metastasis [161]. The development of
techniques like the array based gene expression analysis of live
invasive cells from primary tumours in intact animals [210] could
allow the identification of the particular gene expression signature of
invading cells.
3.2. Drug treatment and resistance related to EMT
3.2.1. Drug resistance
Studies dealing with gene expression profiling on in vivo invasive
cells or cells undergoing EMT in vitro, revealed a switch from a
proliferative to an invasive phenotype. In agreement to this observation, the cell proliferation marker Ki-67 has been shown to stain only a
small minority of cells at the invasive front comparing to the
differentiated central part of the tumour, suggesting that non
proliferating tumour cells that have escaped the tumour mass could
have metastatic potential [124]. This implies that treatments that
target cell growth pathways might not be effective in killing these
cells. Indeed, increasing amount of data relate drug resistance of
tyrosine kinase inhibitors to the existence of EMT. For instance,
epithelial but not mesenchymal gene signature has been associated
with sensitivity to the small molecule-EGFR-inhibitor erlotinib
(Tarceva) mediated growth inhibition, after Affymetrix oligo microarrays performed from 42 non-small cell lung carcinoma (NSCLC)
tumour cell lines [211]. Further clinical trials confirmed a clinical
benefit in patients with NSCLC with high expression of E-cadherin and
who received erlotinib, contrarily to the E-cadherin-negative patients
who had a worsened overall situation after erlotinib treatment. These
results were confirmed in xenografts of NSCLC [212], in other types of
tumours like head and neck squamous cell carcinoma and hepatocellular carcinoma as well as for treatement with other EGFR inhibitors
like gefitinib (Iressa) [213] and cetuximab (Erbitux) [39]. In agreement, a causal association between silencing of E-cadherin expression
(and EMT) and resistance to cetuximab has been established in
urothelial carcinoma cell lines [214]. In parallel, gemcitabine-resistant
pancreatic cells with increased invasive capacities, oxaliplatinresistant colorectal cancer cells and post-ionizing radiation related
tumour distant metastasis in patients with advanced lung cancer, have
all been associated with EMT [215–217]. The list of EMT implication in
therapeutic drug resistance was recently increased with Lapatinib
(Tyverb) resistance in breast cancer [218] and paclitaxel resistance in
epithelial ovarian carcinoma [219].
Apart from a predictive advantage concerning drug sensitivity and
the use of appropriate treatment in selected patients, identification of
EMT opens a new perspective in the correct time window of drug
administration. For example, Erlotinib is currently used in advanced
stage metastatic pancreatic tumours even though cells appear to have
an increased sensitivity only in the epithelial early-stage disease [212].
In parallel, studies dealing with drug combinations using classical
therapies together with substances targeting the EMT-related
mechanisms are now in process. This should help to develop new
strategies to fight against a number of drug resistance cases and the
more aggressive types of tumours.
3.2.2. Future treatments targeting EMT
3.2.2.1. Targeting the EMT related pathways. EMT-related pathways
constitute main targets for novel drug development. For example, the
expression of the integrin-linked kinase (ILK) has been shown to be
responsible for the increased activation of AKT, further leading to EMT
and associated drug resistance in hepatocellular carcinoma. Interestingly, inhibition of ILK activity increases mesenchymal sensitivity of
these cells to EGFR-targeted therapies in xenografts models [39]. In
parallel, Artesunate (an antimalarial agent) has been found to induce
changes resembling to a MET, cell cycle arrest and apoptosis possibly
by affecting the hyperactive Wnt pathway in colorectal cell lines with
characteristics of EMT but not in cells with epithelial phenotype [220].
On the other hand Lupeol, a triterpene found in fruits and vegetables,
has been recently proven to specifically induce a reversion of head and
neck squamous cell carcinoma with NF-kappaB-dependent-EMT and
could be used alone or in combination with other agents in the case of
chemoresistance and radioresistance [221]. The cysteine protease
inhibitor cystatin C (CystC) has been found to interfere with the TGFβ
signalling in normal and cancer cells [222], whereas the smallmolecules cyclopamine and IPI-269609 can limit pancreatic cancer
metastases via inhibition of the Hedgehog signalling, Snail downregulation and up-regulation of E-cadherin in cells with tumourinitiating properties and EMT [223,224]. Interestingly, use of Src kinase
inhibitors such as dasatinib, have been shown to be more effective in
inhibiting growth of cells with EMT in vitro [225]. Interestingly, we
have found that overexpression of oncogenic Harvey-Ras and
subsequent EMT sensitise Caco-2 colorectal cell lines to the tumour
necrosis factor-related apoptosis-inducing ligand (TRAIL) [226], opening an interesting perspective in the use of TRAIL in cells with EMT.
3.2.2.2. Targeting the cancer stem cells characteristics. On the basis of
the observations that EMT cells have cancer stem cell-like characteristics, it appears that the elimination of these cells is essential for the
development of more effective treatments. Indeed, therapies that
eradicate the bulk of tumour cells fail to kill cancer stem cells [227],
mainly due to their quiescence and the expression of drug membrane
transporters [228]. Nevertheless, stem/progenitor cancer cells display
some particular properties that could be exploited for targeted
therapies to invasive and metastatic tumours. For instance, inhibitors
of the main transporters of chemotherapy drugs are tested as
therapeutics as they may overcome drug resistance and eliminate
tumour cells [228]. Interestingly enough, a recent study dealing with
promoter-controlled oncolytic viruses activated only in target cells,
reported the specific in vivo killing of a proportion of CD44CD24−/low
breast cancer cells [229]. In parallel, another therapeutic approach was
proposed in mice transplanted with human acute myelogenous
leukaemia, using an activating monoclonal antibody directed to the
adhesion molecule CD44 [230]. Finally, gene insertion into stem cells
followed by direct specific delivery into the tumour has been reported
in animal models [231].
85
3.2.2.3. Targeting the tumour–stroma interactions. The design of new
curative treatments that target the interactions between tumour and
stroma may also be effective on the EMT process. For example,
targeting the cellular components of the stroma like the tumourassociated macrophages with liposome-encapsulated clodronate, was
found to have an effect on tumour burden and metastasis [232]. In
parallel, inhibiting the effect of the soluble factors secreted by the
stroma could have positive effects in treatment, like for example the
TGFβ receptors inhibitor LY2109761 that reduces metastases in vivo
[233] or the small interfering RNA construct targeting TGFβ1 which
displayed similar effects in mouse lung metastases [234]. Alternatively, neutralizing antibodies of the soluble factors could be very
useful in new treatments like in the case of the tumour-interactive
monocyte chemoattractant protein 1 [235]. On the other hand,
peptide and antibody-based reagents that block molecules involved
in cell–matrix communication, like fibronectin [236], have been
developed to treat cancers. Finally, targeted therapies using anticancer
agents attached to antibodies or peptides have been described, like
the recent interesting fusion of the TRAIL ligand to a peptide
recognised by αVβ3 and αVβ5 integrins and which improved the
antitumour activity of TRAIL in tumour endothelial and integrinpositive cells [237].
3.2.2.4. New technologies in drug development: RNA interference,
microRNA and antagomirs. Small RNA molecules can regulate the
posttranscriptional gene silencing of theoretically any given protein.
This opens the way for therapeutic approaches based on RNA
interference, as a new strategy to target EMT and/or metastasis.
Short hairpin (shRNA) can be specifically synthesised and delivered to
the cell to target a specific mRNA. In the context of EMT, the Ecadherin inhibitor SNAIL has been stably silenced by shRNA leading to
a derepression of E-cadherin and a MET in an in vitro system and after
injection into mice [238]. A decrease in metastatic potential was also
observed during stable inhibition of vimentin expression [239].
Recently, microRNAs (miRNA) have been identified as key regulators
of gene expression. One way to use miRNAs in therapeutics could rely
on the targeting of endogenous mRNAs with artificial synthetic
miRNAs. For instance, down-modulation of the CXCR4 protein linked
to EMT and tumour metastasis has been achieved using a miRNA
expressive plasmid with a pre-microRNA sequence in breast cancer
cell lines [240]. An alternative strategy could be based on the control
of miRNA expression by antisense oligonucleotides modified to singlestranded RNA analogues, complementary to a specific miRNA (antimiRNA antisense oligonucleotide (AMO) or antagomirs). Such constructs have already been used with success in mice [241]. Nevertheless, the success of miRNA therapy depends on effective systems to
deliver interference molecules to the targeted cell or tissue. The main
candidate are the viral-based vectors including retroviruses and
lentiviruses (that stably integrate into the targeted genome) as well as
adenoviruses, adeno-associated viruses (AAV), and herpes simplex
virus-1 (HSV-1) (that stay mainly as episomes) [242].
4. Final remarks
During the past few years, EMT has emerged as one of the hot spots
of clinical research. Its existence in human tumours can form the basis
for explaining characteristics of cancer progression and metastasis, as
well as certain cases of drug resistance and relapses after treatment.
Nevertheless, its existence in vivo has been very controversial and
argued. In reality, results coming from studies performed using
human samples agree on the fact that EMT in vivo may exist, but is a
transient phenomenon that concerns only a minority of cells. An
important issue for EMT identification to be regarded as having a high
prognostic and therapeutic value is to determine the use of specific
markers. These should in theory be able to recognise a cancer EMTderived mesenchymal cell from a normal mesenchymal cell. This is
86
possible in theory, since an epithelial cancer cell that progresses by
initiating EMT must have a unique set of mutations that would
differentiate it from a normal cell. To better understand EMT
mechanisms and develop better markers, EMT has to be proved in
animal models. A number of studies dealing with this problem have
managed to identify and follow EMT, but additional models and better
techniques must be developed. As a help in this direction, several in
vitro models agree on the existence of reliable markers. New
discoveries will elucidate the complex mechanisms of EMT and will
hopefully allow on one hand a better selection of patients and on the
other hand the development of new drugs targeting metastatic
mechanisms and more aggressive cancers.
Acknowledgements
A.V. and A.P. are supported by the European Union grant LSHC-CT2006-037278 to A.P. and by grants of General Secretariat of Research
and Technology of Greece. We would like to thank Prof George
Kontogeorgos and Dr Despoina Mourtzoukou, Department of Pathology, General Hospital of Athens G. Gennimatas for helpful discussions.
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–mesenchymal transition in cancer metastasis: Mechanisms, markers and Epithelial

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