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: apint@eie.gr (A. Pintzas). 0304-419X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbcan.2009.03.002 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 76 76 76 76 76 76 79 79 79 79 80 80 80 80 80 80 81 82 83 84 76 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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 84 85 85 86 86 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 A. Voulgari, A. Pintzas / Biochimica et Biophysica Acta 1796 (2009) 75–90 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 A. Voulgari, A. Pintzas / Biochimica et Biophysica Acta 1796 (2009) 75–90 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 A. Voulgari, A. Pintzas / Biochimica et Biophysica Acta 1796 (2009) 75–90 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 A. Voulgari, A. Pintzas / Biochimica et Biophysica Acta 1796 (2009) 75–90 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 A. Voulgari, A. Pintzas / Biochimica et Biophysica Acta 1796 (2009) 75–90 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 A. Voulgari, A. Pintzas / Biochimica et Biophysica Acta 1796 (2009) 75–90 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- A. Voulgari, A. Pintzas / Biochimica et Biophysica Acta 1796 (2009) 75–90 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 A. Voulgari, A. Pintzas / Biochimica et Biophysica Acta 1796 (2009) 75–90 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 A. Voulgari, A. Pintzas / Biochimica et Biophysica Acta 1796 (2009) 75–90 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 A. Voulgari, A. Pintzas / Biochimica et Biophysica Acta 1796 (2009) 75–90 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. 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