EMT as the ultimate survival mechanism of cancer cells

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Seminars in Cancer Biology 22 (2012) 194–207 Contents lists available at SciVerse ScienceDirect Seminars in Cancer Biology j ourna l ho me p ag e: www.elsevier.com/locate/semcancer Review EMT as the ultimate survival mechanism of cancer cells Neha Tiwari a , Alexander Gheldof b,c , Marianthi Tatari b,c , Gerhard Christofori a,∗ a Institute of Biochemistry and Genetics, Department of Biomedicine, University of Basel, Basel, Switzerland b Department for Molecular Biomedical Research, VIB, 9052 Ghent, Belgium c Department of Biomedical Molecular Biology, Ghent University, 9052 Ghent, Belgium a r t i c l e i n f o Keywords: Cancer EMT Metastasis Signaling Tumorigenesis a b s t r a c t Epithelial cancers make up the vast majority of cancer types and, during the transition from benign ade- noma to malignant carcinoma and metastasis, epithelial tumor cells acquire a de-differentiated, migratory and invasive behavior. This process of epithelial–mesenchymal transition (EMT) goes along with dramatic changes in cellular morphology, the loss and remodeling of cell–cell and cell–matrix adhesions, and the gain of migratory and invasive capabilities. EMT itself is a multistage process, involving a high degree of cellular plasticity and a large number of distinct genetic and epigenetic alterations, as fully differentiated epithelial cells convert into poorly differentiated, migratory and invasive mesenchymal cells. In the past years, a plethora of genes have been identified that are critical for EMT and metastasis formation. Notably, the EMT process not only induces increased cancer cell motility and invasiveness but also allows cancer cells to avoid apoptosis, anoikis, oncogene addiction, cellular, senescence and general immune defense. Notably, EMT seems to play a critical role in the generation and maintenance of cancer stem cells, highly consistent with the notion that metastatic cells carry the ability to initiate new tumors. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction Epithelial-to-mesenchymal transition (EMT) is a cellular process during which epithelial cells lose their polarized organization and cell–cell junctions, undergo changes in cell shape and in cytoskele- tal organization and acquire mesenchymal characteristics, such as fibroblast-like cell morphology and increased cell migration and invasion. EMT has been first recognized as a distinct cell differen- tiation process in the late 70s and, over the years, it has received increasing attention, since it not only occurs in embryonic develop- ment but also contributes to various pathological conditions [1]. On the basis of its functions, EMT can be classified into three different subtypes [2]. Type 1 EMT is associated with embryonic implanta- tion and gastrulation and neural crest cell motility. Type 2 EMT is involved in wound healing, tissue-regeneration, inflammation and fibrosis. Finally, Type 3 EMT represents the conversion of differen- tiated epithelial cancer cells into migratory mesenchymal cancer cells which may lead to cancer invasion, systemic cancer cell dis- semination, and metastasis [2]. EMT involves the loss of epithelial markers, such as the tight junction proteins claudins and occludins, the adherens junc- tion proteins E-cadherin, and -catenin, and cytokeratins. ∗ Corresponding author at: Institute of Biochemistry and Genetics, Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland. Tel.: +41 61 267 35 64; fax: +41 61 267 35 66. E-mail address: [email protected] (G. Christofori). Concomitantly, a number of mesenchymal markers are increased in their expression, including N-cadherin, vimentin, fibronectin, matrix metalloproteinase, integrins V and 1 , and smooth muscle actin [3–5] (Fig. 1). Recent gene expression profiling experiments in various experimental cellular systems of EMT suggest that hun- dreds if not thousands of genes significantly change in their gene expression during EMT [2,6]. It should be noted that in most exper- imental systems a “full” EMT, i.e. a complete change in marker expression, requires up to ten days or longer. On the other hand, our knowledge about the process of EMT during malignant tumor progression and metastasis in patients is still limited. EMT may be transient, as observed in colorectal cancer, where cells at the invasive front lose E-cadherin expression and exhibit nuclear -catenin localization, while the cells of liver metastasis have re- differentiated into epithelial cells and morphologically resemble primary tumor cells [7]. Hence, the actual occurrence of EMT in cancer patients is still debated [8], and better markers to monitor cancer cells during metastatic dissemination are urgently needed to understand the order of events in patients. 2. Loss of E-cadherin During EMT, cells lose their epithelial polarity and dissolve their adherent and tight junctions, favoring a high plasticity in cell–cell adhesion and communication with the extracellular matrix through focal adhesions. Most prominently, E-cadherin exerts a role as repressor of tumor progression in maintaining 1044-579X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcancer.2012.02.013

Transcript of EMT as the ultimate survival mechanism of cancer cells

  • Seminars in Cancer Biology 22 (2012) 194 207

    Contents lists available at SciVerse ScienceDirect

    Seminars in Cancer Biology

    j ourna l ho me p ag e: www.elsev ier .com/

    Review

    EMT as nce

    Neha Tiw rhaa Institute of Bi rlandb Department fc Department o

    a r t i c l

    Keywords:CancerEMTMetastasisSignalingTumorigenesis

    ajoriastasepithoss anlitiesof disferenentireasegeneole intatic

    1. Introduction

    Epithelial-to-mesenchymal transition (EMT) is a cellular processduring whiccellcell juntal organizabroblast-linvasion. EMtiation procincreasing ament but althe basis ofsubtypes [2tion and gainvolved inbrosis. Fintiated epithcells whichsemination

    EMT invjunction prtion protei

    CorresponBiomedicine, UTel.: +41 61 26

    E-mail add

    Concomitantly, a number of mesenchymal markers are increasedin their expression, including N-cadherin, vimentin, bronectin,matrix metalloproteinase, integrins V and 1, and smooth muscle

    1044-579X/$ doi:10.1016/j.h epithelial cells lose their polarized organization andctions, undergo changes in cell shape and in cytoskele-tion and acquire mesenchymal characteristics, such asike cell morphology and increased cell migration andT has been rst recognized as a distinct cell differen-

    ess in the late 70s and, over the years, it has receivedttention, since it not only occurs in embryonic develop-so contributes to various pathological conditions [1]. On

    its functions, EMT can be classied into three different]. Type 1 EMT is associated with embryonic implanta-strulation and neural crest cell motility. Type 2 EMT is

    wound healing, tissue-regeneration, inammation andally, Type 3 EMT represents the conversion of differen-elial cancer cells into migratory mesenchymal cancer

    may lead to cancer invasion, systemic cancer cell dis-, and metastasis [2].olves the loss of epithelial markers, such as the tightoteins claudins and occludins, the adherens junc-ns E-cadherin, and -catenin, and cytokeratins.

    ding author at: Institute of Biochemistry and Genetics, Department ofniversity of Basel, Mattenstrasse 28, 4058 Basel, Switzerland.7 35 64; fax: +41 61 267 35 66.ress: [email protected] (G. Christofori).

    actin [35] (Fig. 1). Recent gene expression proling experimentsin various experimental cellular systems of EMT suggest that hun-dreds if not thousands of genes signicantly change in their geneexpression during EMT [2,6]. It should be noted that in most exper-imental systems a full EMT, i.e. a complete change in markerexpression, requires up to ten days or longer. On the other hand,our knowledge about the process of EMT during malignant tumorprogression and metastasis in patients is still limited. EMT maybe transient, as observed in colorectal cancer, where cells atthe invasive front lose E-cadherin expression and exhibit nuclear-catenin localization, while the cells of liver metastasis have re-differentiated into epithelial cells and morphologically resembleprimary tumor cells [7]. Hence, the actual occurrence of EMT incancer patients is still debated [8], and better markers to monitorcancer cells during metastatic dissemination are urgently neededto understand the order of events in patients.

    2. Loss of E-cadherin

    During EMT, cells lose their epithelial polarity and dissolvetheir adherent and tight junctions, favoring a high plasticityin cellcell adhesion and communication with the extracellularmatrix through focal adhesions. Most prominently, E-cadherinexerts a role as repressor of tumor progression in maintaining

    see front matter 2012 Elsevier Ltd. All rights reserved.semcancer.2012.02.013 the ultimate survival mechanism of ca

    aria, Alexander Gheldofb,c, Marianthi Tatarib,c, Geochemistry and Genetics, Department of Biomedicine, University of Basel, Basel, Switzeor Molecular Biomedical Research, VIB, 9052 Ghent, Belgiumf Biomedical Molecular Biology, Ghent University, 9052 Ghent, Belgium

    e i n f o a b s t r a c t

    Epithelial cancers make up the vast mnoma to malignant carcinoma and metand invasive behavior. This process of changes in cellular morphology, the lgain of migratory and invasive capabicellular plasticity and a large number epithelial cells convert into poorly difyears, a plethora of genes have been idthe EMT process not only induces inccells to avoid apoptosis, anoikis, oncoNotably, EMT seems to play a critical rconsistent with the notion that metaslocate /semcancer

    r cells

    rd Christofori a,

    ty of cancer types and, during the transition from benign ade-is, epithelial tumor cells acquire a de-differentiated, migratoryelialmesenchymal transition (EMT) goes along with dramaticd remodeling of cellcell and cellmatrix adhesions, and the. EMT itself is a multistage process, involving a high degree oftinct genetic and epigenetic alterations, as fully differentiatedtiated, migratory and invasive mesenchymal cells. In the pasted that are critical for EMT and metastasis formation. Notably,d cancer cell motility and invasiveness but also allows cancer

    addiction, cellular, senescence and general immune defense. the generation and maintenance of cancer stem cells, highlycells carry the ability to initiate new tumors.

    2012 Elsevier Ltd. All rights reserved.

  • N. Tiwari et al. / Seminars in Cancer Biology 22 (2012) 194 207 195

    Fig. 1. Schema enchythe cytoskelet nt orgthe acquisition

    intact cellmetastatic the E-cadhactivation omigration acadherin exmouse modprogressionfunction caCadherins alevels by meor transcriphuman carcproductionmutation, ation or glycregulation and 2 and Tulated at thhypermethrst genes wfrequencies

    3. EMT and

    During by a large nal transduprotein degtumor-assocytokines, dermal gro(PDGF; [20]sis factor most promof which artional repre

    Zeb1 E-caiptio.eve

    ncomincluWarogenT pro

    of thsion tic representation of epithelial to mesenchymal transition. During epithelial to meson so that they can migrate, invade the neighboring tissue and metastasize to dista

    of mesenchymal markers.

    cell contacts and preventing cell mobility, invasion anddissemination [3,4,9]. In fact, expression or function oferin gene is lost in most carcinomas, and the forcedf E-cadherin is usually sufcient to reduce tumor cellnd invasion [10]. On the other hand, ablation of E-pression in epithelial cells in culture or in transgenicels of cancer in vivo induces EMT and malignant tumor

    and metastasis, indicating that the loss of E-cadherinn be one trigger of EMT and tumor metastasis [1114].re generally regulated at both the mRNA and proteinans of changes in subcellular distribution, translationaltional events and by protein degradation. In various

    and 2,sion oftranscrensues

    Howthe coways, Hippo/morphthe EMvationexpresinomas, functional loss of E-cadherin results from the of a defective protein, which may be a result of genebnormal post-translational modication (phosphoryla-osylation) or increased proteolysis [5,9,15]. Besides itsby the transcriptional repressors Snail-1 and 2, Zeb-1wist [16], E-cadherin gene expression can also be reg-e transcriptional level by silencing through promoterylation [5,9]. In fact, E-cadherin has been one of theith promoter hypermethylation identied at very high

    in human cancer specimen [17].

    growth factors

    malignant tumor progression, EMT can be inducedvariety of stimuli, ranging from growth factor sig-ction pathways to the loss of E-cadherin function byradation or gene mutation. In many carcinomas, theciated stroma produces a variety of growth factors andincluding hepatocyte growth factor (HGF; [18]), epi-wth factor (EGF; [19]), platelet-derived growth factor), broblast growth factor 2 (FGF2; [21]), tumor necro-(TNF; [22]), insulin-like growth factor (IGF; [23]), and,inently, transforming growth factor (TGF; [24]), alle able to induce the expression of various transcrip-ssors of E-cadherin gene expression, including Snail-1

    expression sion of mat(ECM) protthrough motutive activEMT in a vainducer, FGMAPK signa

    In additin inducingactive formPI3K signaliE-cadherin PI3K signaliRho family a major necFurthermorgenic Ras hsurvival du

    EGF is aEGF receptcatenin, leadisruption cytosis of Evia the Wnmal transition (EMT), cells lose their cellcell contacts and re-arrangeans. This metastatic potential is acquired by the loss of epithelial and

    and 2, and Twist [2531]. As a result of the repres-dherin expression and also of the less well-understoodnal activities of these transcriptional repressors, EMT

    r, the actual implementation of EMT is dependent onitant activity of a variety of signal-transduction path-ding MAPK, PI3K, Wnt/-catenin, NFB, Notch, andts signaling for cell proliferation and survival and theic process of EMT [32]. For example, HGF stimulatescess by activating MAPK signaling resulting in the acti-e transcription factor Egr-1 and subsequently in theof Snail-1 and the downregulation of E-cadherin gene

    [25]. Moreover, HGF signaling also leads to the expres-rix metalloproteinases (MMPs) and extracellular matrixeins, which alters cell-ECM and cellcell interactionsdulation of integrin and cadherin functions [33]. Consti-ation of the MAPK Erk1/2 is also required for a completeriety of tumor metastasis models [34,35]. Another EMTF determines the fate of epithelial cells by stimulatingling and also TGF signaling [36,37].ion to MAPK signaling, PI3K signaling plays a key role

    and maintaining EMT. Cells expressing a constitutively of PKB/Akt, the most important downstream effector ofng, induce expression of Snail-1, which in turn repressesgene transcription and induces EMT [38]. Moreover,ng can also be activated by integrins and members of theof small GTPases that control cytoskeleton re-modeling,essity during the morphogenic process of EMT [39,40].e, autocrine PDGFR signaling in the presence of onco-yperactivates PI3K signaling which is required for cellring EMT (Jechlinger et al. [26]).

    potent stimulator of EMT in several cell types, and theor (EGFR) has been shown to directly interact with -ding to tyrosine phosphorylation of -catenin and theof E-cadherin-mediated cell junctions [41,42]. Endo--cadherin results in the release of -catenin, whicht/-catenin signaling pathway promotes Snail-1 gene

  • 196 N. Tiwari et al. / Seminars in Cancer Biology 22 (2012) 194 207

    transcription and consequently E-cadherin repression [43]. On theother hand, E-cadherin located in intact adherens junction com-plexes directly represses the activity of EGFR by inhibiting itstransphosphorylation of Tyr845 [44]. Notably, the transcription fac-tor Dlx2, upstimulates tprovides EGEMT [45]. SII) can induof E-cadhertribute -cathe translocfrom the cy

    Notch siactivated Ndirectly binexpression,dase (Lox) which in tualso able tomammary requires TGlated by NFvimentin ansion is inducmetastatic pinhibitor prpression ofactivators Ycellular tranand EMT [4signaling pEMT (Fig. 2)

    4. EMT and

    Growth of many traSnail-1 andimportant such as E-c[16,51] (Figor MAPK-dmotifs withplex contaigene transclysyl oxidagen synthasubsequentdation [16].

    Snail-2 different coCTBP [575specic EMcarcinoma [interacting with Smadto suppressrepressed bthese miRNsion is sufZeb-2 activiCbx4 comp[65]). Both Zsion [6668

    Several helix-loop-helix (HLH) proteins have been implicated inEMT: E12 and E47 (alternative splicing products of the E2A gene),Twist-1 and 2, and Id1 to 4 [69]. They have all been shown to repressE-cadherin by directly binding to its promoter. On the other hand,

    pressey ac

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    y reg critiarize-regulated in its expression during TGF-induced EMT,he expression of the EGFR ligand betacellulin and thusFR-dependent survival signaling in cells undergoingimilarly, insulin-like growth factor I and II (IGF-I andce EMT by promoting internalization and degradationin in vesicles located around the nucleus. IGFs also redis-tenin from the cell membrane to the nucleus and induceation of the -catenin partner transcription factor Tcf-3toplasm to the nucleus [23].gnaling synergizes with hypoxia to induce EMT. Uponotch signaling the Notch intracellular domain (ICD)ds to the Snail-1 promoter and activates Snail-1 gene

    while HIF-1 binds to and activates the lysyl oxi-gene promoter. Lox further stabilizes Snail-1 proteinrn induces EMT [46]. NFB transcriptional activity is

    induce EMT, as shown in Ras-transformed murineepithelial (EpRas) cells. Here, the induction of EMTF signaling and oncogenic Ras and is directly regu-B at the promoters of key EMT marker genes, such asd tenascin-C [47]. Furthermore, Snail-1 gene expres-ed by NFb activity, and blocking NFB prevents EMT inrostate cancer cells by the activation of RKIP (Raf kinaseotein), a metastasis suppressor [48]. Finally, overex-

    the Hippo/Warts signaling pathway transcriptionalap1 or Taz prevents contact inhibition and acceleratessformation as well as anchorage-independent growth9,50]. In summary, a variety of classical growth factorathways play a key role in initiating and maintaining.

    transcription factors

    factor signaling in most cases induces the expressionnscription factors which are essential for EMT, such as

    2, Zeb-1 and 2, and Twist, potent repressors of genesfor maintaining epithelial polarity and organization,adherin, claudins, occludins and ZO-family members. 2). Snail-1 expression can be induced in a Smad-3ependent manner [25,52,53]. Snail-1 binds to E-boxin the E-cadherin gene promoter and recruits a com-ning HDAC1, HDAC2 and mSin3A, and thus repressesription [5456]. Snail-1 protein is stabilized by Lox andse-like 2 and 3 (LoxL2/3) and destabilized by glyco-se-kinase 3 (GSK3)-mediated phosphorylation and

    nuclear export, ubiquitylation and proteasomal degra-

    works in a similar way to Snail-1, but it recruits ambination of co-repressors, such as HDAC1/3 and9]. Some of the Snail target genes regulate tissue-T processes, such as HNF-1 in kidney brosis and60,61]. Similarly, Zeb-1 and Zeb-2 (also known as Smad-protein-1 and 2; Sip-1 and 2) form repressive complexes

    proteins and bind to the E-cadherin gene promoter it. The Zeb factors have recently been shown to bey members of the miR-200 family. The expression ofAs is diminished during EMT, and their forced expres-cient to block TGF-induced EMT [6264]. In addition,ty is impaired by post-translational sumoylation by theonent of the polycomb repressive complex 1 (PRC1;eb proteins promote EMT, cell migration and cell inva-].

    the exand thto E2ATGF-sor ATpromoin E-caor Id3 ZO-1 p[71,72the exand 5familyformat

    A ncated factor expresSnail-1obox tand itsexpresinducefactorsdiffereFinallyTGF-represbetacecell prtors arOngoinlated brelatioproces

    5. EM

    miRmolecucodingpost-trmRNAin humNAs haroles inlead toteristic[81,85inuenrespeccytostahave bgrowththese tprocesAgo pr

    In mdirectlto play(summion of Id proteins is decreased in response to TGF,t as antagonists for E-cadherin repression by bindingteins [70]. The expression of Id1 is suppressed duringed EMT by incorporation of the transcriptional repres-

    an ATF3/Smad3/Smad4 complex that binds to the Id11,72]. Loss of Id1 expression correlates with a decreaserin expression. Conversely, ectopic expression of Id2ks TGF-induced repression of E-cadherin expression,in delocalization and smooth muscle actin expression7 also represses desmoplakin expression and inducesion of the mesenchymal proteins N-cadherin, SPARCgrin [73]. Twist-1 and 2, further members of the HLHanscription factors, are major regulators of mesodermand ectopic expression of both leads to EMT [7476].er of additional transcription factors have been impli-gulating the various stages of EMT. The transcriptionga2, a direct target of TGF/Smad-3 signaling, isin mesenchymal cells and induces the expression of

    2, Twist, and represses Id2 expression [77]. The home-ription factor Goosecoid is induced by TGF-signaling,opic expression is sufcient to induce EMT [78]. Theof the forkhead transcription factor FoxC2 is also

    TGF-signaling and by the EMT-inducing transcriptionil, Twist and Goosecoid, and it promotes mesenchymalion and plays a critical role in cancer cell metastasis [79].

    transcription factor Dlx2 is also up-regulated duringed EMT and promotes cell survival during EMT by directof TGF receptor II expression and by the activation of

    gene expression thereby stimulating EGFR-mediatedation and survival [45]. Many more transcription fac-spected to play critical roles in EMT and metastasis.search aims to identify the direct target genes regu-se transcription factors and to delineate their epistatics in the process of EMT and during the metastatic

    microRNAs

    are highly conserved, small 1924 nucleotide RNAwhich are processed from much longer primary non-scripts and are able to control gene expression at theriptional level by specically interacting with targetis estimated that miRNAs regulate 30% of all proteins80]. Besides their roles in physiological processes, miR-en shown to be aberrantly expressed and to play causalriety of pathologies including cancer [8184]. This has

    identication of miRNA signatures that are charac-certain cancers and allow their further classicationRNAs can act as oncogenes or tumor-suppressors ande tumorigenesis process when down or up-regulated,. They can also affect the sensitivity of tumor cells tor radiotherapy [86,87]. For many miRNAs, target genesdentied which are relevant in tumor initiation, tumoror angiogenesis and metastasis. Furthermore, some of

    genes are themselves major components of the miRNAmachinery, including Drosha-DGCR8, Dicer-TRBP2, ands [88].stasis, a large number of miRNAs has been found toulate the expression of even more target genes knowncal roles in the various stages of the metastatic processd in [89]). The complex regulation of EMT by miRNAs

  • N. Tiwari et al. / Seminars in Cancer Biology 22 (2012) 194 207 197

    Fig. 2. Molecuto the inductioof EMT-relatedfactors (for de

    is best exem200a, miR-family reduand 2 and, miR-200 geregulators oand other edirectly affeand thus EMrecent studtasis of mamadditional fous stages oJagged2 canthe transcrithe expressmote EMT. suppress ealar mechanisms regulating EMT. Many signaling molecules, such as TGF, Wnt, HGF, FGFn of EMT. These pathways are largely interconnected to each other to accomplish their fu

    transcription factors. Many miRNAs inuence the EMT process by targeting the expresstails see text).

    plied by the function of the miR-200 family (miR-200b, miR-200c, miR-141 and miR-429). The miR-200ces the expression of the transcription factors Zeb-1conversely, Zeb-1 and 2 repress the expression of thene cluster [6264,9093]. Since Zeb-1 and 2 are criticalf EMT by their transcriptional repression of E-cadherinpithelial marker genes, members of the miR-200 familyct the epithelial vs. mesenchymal status of tumor cellsT, cell migration invasion and metastasis. However, a

    y has reported a miR-200-mediated increase in metas-mary carcinoma cell lines [94], a nding that suggest

    unctions for miR-200 family members during the vari-f the metastatic process. Furthermore, the Notch ligand

    enhance metastasis by repressing the expression ofption factor GATA3. GATA3 has been shown to repression of members of the miR-200 family and thus to pro-GATA3 and miR-200 act in a negative feedback loop toch other and to maintain tissue homeostasis [95].

    E-cadhe9, which rthe up-reggenes requcontributindependent the dissoluttion factor TmiR-10b, wfactor HoxDRhoC gene

    Finally, directly taras Snail anways that pof miR-34arespectivel2 to suppre, IGF, and EGF, by eliciting appropriate signaling pathways contributenction (shown by dashed arrows), ultimately activating transcriptionion of these ligands, receptors, signaling pathways and transcription

    rin gene expression is also directly repressed by miR-esults in the activation of -catenin signaling andulated expression of mesenchymal and angiogenicired for metastatic outgrowth [96]. Another miRNAg to EMT is miR-155. Its expression is induced by Smad-TGF-signaling leading to reduced RhoA expression andion of cell tight junctions [97]. Moreover, the transcrip-wist-1, besides inducing EMT, induces the expression ofhich in turn restrains the expression of the transcription10 known to repress transcription of the pro-metastatic[98].several additional miRNAs have been identied thatget key transcription factors involved in EMT, suchd Zeb, while others modulate various signaling path-romote EMT. For example, p53 induces the expression

    and miR-192, which then repress Snail-1 and Zeb-2,y, to block EMT [99,100]. miR-138 can also repress Zeb-ss EMT [101]. In addition, Twist-1 induces expression of

  • 198 N. Tiwari et al. / Seminars in Cancer Biology 22 (2012) 194 207

    the polycomb repressor complex 1 component B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) to facilitate EMT, whileBmi is repressed by miR-194 [102,103]. miR-27 directly targetsexpression of the tumor suppressor APC, an important repres-sor of the WConversely,expression tetrapolin (regions of presses T-ceactivating GFinally, by scription famiR-335 ex[108]. Togein EMT and

    6. EMT and

    Aberratidiseases, into the prod[109]. Sinceprocess, chthe potentiaFibroblast gbe associatesplicing of tspecicity olial or the the epithelicells, and thin epitheliacan thus leaprostate anbeen subseqing EMT anknown as to the enablfamily and overexpreshMena + 11an epitheliatively splice1 found in msignaling merate varioexpressed imesenchymalternative

    Two rece(epithelial sposition in ESRP1 and the mRNA sgenes, incluthe splicingcancer subt[117]. Yet, Rrevealed thtion of sevRBFOX2, CEFirst functiotors are reqidentity and

    7. Epigenetics and EMT

    Irreversible genetic changes are not the only mechanisms thatcause aberrant gene expression during malignant tumor progres-

    pigentoneuring

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    typeadhnt signaling pathway, and thus promotes EMT [104]. miR-34c counteracts EMT by directly repressing theof Notch-4 [105]. Furthermore, miR-29a suppresses tris-TTP), a protein that binds to AU-rich 3-untranslatedmRNAs for degradation. Moreover, miR-21/31 sup-ll lymphoma invasion and the expression of Tiam-1, anEF for Rac GTPases known to regulate EMT [106,107].inhibiting the expression of the pro-metastatic tran-ctor Sox4 and the ECM molecule tenascin-C, loss ofpression is a hallmark of EMT and tumor metastasisther, these data underscore the importance of miRNAs

    malignant tumor progression (Fig. 2).

    alternative splicing

    ons in RNA splicing have been associated with severalcluding cancer, where altered RNA splicing can leaduction of protein isoforms with oncogenic properties

    EMT is considered an early event in the metastaticanges in splicing patterns associated with EMT havel to become useful prognostic and diagnostic markers.rowth factor receptor 2 (FGFR2) was the rst gene tod with alternative splicing and EMT [110]. Alternativehe third Ig-like domain determines the ligand-bindingf the receptor and generates the alternative IIIb epithe-IIIc mesenchymal isoforms of FGFR2. The ligands foral IIIb isoform are usually expressed in mesenchymale ligands for the mesenchymal-restricted IIIc isoforml cells. A switch from one receptor isoform to anotherd to autocrine signaling, as observed in rat models ofd bladder cancer [110,111]. Several other genes haveuently identied regulated by alternative splicing dur-d malignant tumor progression, including ENAH1 (alsoMena) and CTNND1 (p120 catenin). ENAH1 belongsed/vasodilator-stimulated phosphoprotein (Ena/VASP)regulates actin organization. Human Mena (hMena) issed in human breast tumors, and a splice variant termeda has been identied in a breast cancer cell line withl phenotype [112]. Similarly, p120 catenin is alterna-d into isoform 3 expressed in epithelial cells or isoformesenchymal cells [113]. The hyaluronan receptor and

    olecule CD44 also undergoes alternative splicing to gen-us CD44s and CD44v isoforms. The CD44v isoform isn epithelial cells, while the CD44s isoform is found inal cells and metastatic cancer cells, and the switch insplicing is required for EMT to occur [114].ntly described RNA binding proteins, ESRP1 and ESRP2plicing regulatory protein 1 and 2), have taken a centralcontrolling alternative splicing during EMT [115,116].ESRP2 maintain epithelial homeostasis by promotingplicing of epithelial-specic isoforms of EMT-associatedding Mena, CD44, FGFR2 and CTNND1 [114116]. Also

    factor RBFOX2 has recently been reported to regulateype-specic splicing in a panel of breast cancer cell linesNA sequencing of mRNAs at the various stages of EMTat EMT-associated splicing is regulated by a combina-eral splicing factors, including members of the ESRP,LF, MBNL and hnRNP classes of splicing factors [118].nal experiments clearly indicate that these splicing fac-uired for the maintenance of epithelial or mesenchymal

    thus also for EMT.

    sion. Eand hisroles d

    7.1. DN

    Unsimportexampin primcadherdemetFollowcadherhas bethe memodelexpreshypermcoordithat thmoter epigensuch astainingrecentpromoZeb-1 in DNAtumor

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    Theand poin EMT[1221Aebp2PcG prEMT. Bing thecellula[126]. Bmi-1 Bmi-1 to the and, Bmi-1 are cop16INKacts wrepresactivat

    Amtransfetri-meis highprecurgenic Ezh2 ecancerthe E-cetic modications, such as reversible DNA methylation methylation and acetylation, also appear to play critical

    carcinogenesis.

    ethylation

    uled hypermethylation of promoter CpG islands is anechanism of repressing tumor suppressor genes. For

    -cadherin promoter methylation has been observedprostate cancers and breast cancer lesions where E-lencing can be de-repressed by treatment with theing agent 5-aza-2-deoxycytidine (5-aza) [119,120].hese seminal studies, DNA hypermethylation of the E-romoter concomitant with its diminished expressionbserved in basically all epithelial cancers. To explainisms underlying E-cadherin promoter methylation, twoe been proposed. According to the rst model, Snailis correlated with the silencing of E-cadherin and theylation of its promoter, suggesting a role for Snail in then of both processes [121]. The second model proposesencing of E-cadherin does not necessarily require pro-rmethylation, suggesting the involvement of additionalmodications required for E-cadherin gene repression,one deacetylation. Several co-repressor complexes con-tone deacetylases and DNA methyltransferases haveen described that can be recruited to the E-cadheriny transcriptional repressors, including Snail-1 and 2 or2 [30,56]. The investigation of genome-wide changesthylation during EMT and their relevance to malignantression is certainly warranted.

    modications

    tituents of both polycomb repressive complex 1 (PRC1)mb repressive complex 2 (PRC2) have been implicatedC1 comprises Bmi-1, Ring1, Hph1/2/3, and Hpc1/2/3and PRC2 consists of Ezh2, Eed, Suz12, RbAp46/48 and125]. The PRC1 protein Bmi-1 was the rst reported

    to be associated with carcinogenesis and later on with can inhibit c-Myc-induced apoptosis by directly bind-a-Arf locus and repressing it, thus allowing unrestrictedliferation in response to aberrant mitogenic signalingntly, it has been demonstrated that overexpression of

    to EMT, and its loss prevents EMT-driven cell invasion.ains expression of the tumor suppressor PTEN leadingation of the PI3K/Akt pathway, the stabilization of Snail, to down-regulation of E-cadherin [127]. In addition,cessary for Twist-1-induced EMT, and both of themtively required for the repression of E-cadherin andxpression [103]. Another PRC1 component, Ring1, inter-mi-1 and blocks the expression of the transcriptionalngrailed and increases expression of the transcriptional-Jun and c-Fos, both known to play a role in EMT [128].the components of the PRC2 complex, the methyl-

    Ezh2 has been extensively studied in cancer. Ezh2tes histone 3 at its lysine residue 27 (H3K27me3) andxpressed together with Bmi-1 in prostate metastaticells. Depletion of Bmi1 or Ezh2 diminishes the tumori-etastatic potential of these cancer cells [129]. High

    ssion has been found in many advanced and metastatics, including prostate and breast cancer [130]. Again,erin gene has been identied among the many genes

  • N. Tiwari et al. / Seminars in Cancer Biology 22 (2012) 194 207 199

    repressed by the Ezh2-mediated repressive mark H3K27me3, andforced expression of E-cadherin can attenuate Ezh2-mediated inva-sion in prostate and breast epithelial cells [131,132]. These resultsare similar to ES cell data where Ezh2 is required for ES cell pluripo-tency and Notably, msion by couG-protein-cpathway anOverexpresand the proDZNep [138tion of Adrbmodel of pr

    The traninducer of the ATPase to the E-cathat Snail-1in ES cells aPRC2, leadsSnail-1. Thiand Ezh2 apromoter [1histone lysition of a teof specic ttranscriptio3 and togethRecently, a repressive measured bEMT [142]. Lsd1, and dechemo-resi

    Besides trolled by expression relates witH3K9Ac anmodicatiore-expressimarks. Thesing fate cha

    8. EMT and

    Acquisitties of neop[144]. Althocommon inety in mecphysiologicticipates incell prolifercer, TGF eof carcinoging proliferhave escapsion, migraresponse tonisms, as SmEMT and bcells that hacells to the

    Smad-independent TGF signaling regulating pro-apoptotic andsurvival effects have also been reported [150].

    The most extensive studies on evasion of apoptosis through EMThave been performed on the Snail family of transcriptional repres-

    scribF-miatedr stu

    in my TGng inbula

    to iNone

    EMTy a pealeonser proK/Errestsvolv

    1 cellassocll cyc

    TGFding f cellediatle, inor atts a pAppasion.cle cropeg caresuld Zee den of ve to-1 increas cah Smle, TGptosiy, wo TGre p

    epithents ed in

    tumin ralit

    mbero apotopicthroust-1 irradn cane byime adifferentiation by repressing E-cadherin [133135].iR-101 has been found to maintain E-cadherin expres-nteracting Ezh2 expression [136]. In addition, Adrb2, aoupled receptor (GPCR) of the -adrenergic signalingd a target of Ezh2, has been implicated in EMT [137].sion of Ezh2 can repress Adrb2 at both the transcripttein levels and the recently discovered Ezh2 inhibitor,], can prevent Adrb2 repression by Ezh2. Downregula-2 blocks EMT and prevents tumor growth in a xenograftostate cancer.scriptional repressor of E-cadherin expression andEMT, Zeb-1, can also induce EMT by recruiting Brg1,subunit of the Swi/Snf chromatin-remodeling complex,dherin promoter [139]. Recently, it has been reported-mediated repression of E-cadherin is PRC2-dependentnd that depletion of Suz12, one of the components of

    to partial de-repression of the E-cadherin promoter bys study also shows that Snail-1 interacts with Suz12nd increases the binding of Suz12 at the E-cadherin40]. Snail-1 also recruits the amino oxidase domain ofne-specic demethylase 1 (Lsd1) resulting in a stabiliza-rnary Snail1-Lsd1-CoREST complex and the repressionarget genes, including the E-cadherin gene [141]. At thenal target sites, Lsd1 demethylates lysine 4 on histoneer with Snail-1 supports the repression of target genes.global reduction in heterochromatin, measured by themark H3K9me2, and increased levels of euchromatin,y the active mark H3K4me3, has been reported duringThese changes are largely dependent on the activity ofpletion of Lsd1 prevents TGF--induced migration andstance.E-cadherin, 4 integrin has been shown to be con-polycomb-mediated histone modications [143]. Theof 4 integrin is lost during EMT and this loss cor-h a decrease in the activating histone modicationsd H3K4me3 and an increase in the repressive histonen H3K27me3. Furthermore, reversal of EMT leads to aon of 4 integrin and the restoration of active epigenetice results argue for a dynamic epigenetic regulation dur-nges of epithelial cells.

    apoptosis: TGF rules

    ion of resistance to cell death is one of the basic proper-lastic transformation of cells and a hallmark of cancerugh a gain of resistance to apoptosis through EMT is

    many different cellular systems, the substantial vari-hanisms reects the importance of the cellular andal contexts. TGF is a multifunctional cytokine that par-

    the regulation of different cellular functions, includingation, differentiation, apoptosis, and EMT [145]. In can-xerts two rather opposing roles. In the early stagesenesis, it functions as a tumor suppressor by inhibit-ation and inducing apoptosis. However, in cells thated its pro-apoptotic effects, TGF enhances cell inva-tion and evasion of immune surveillance [146]. EMT in

    TGF is mediated mainly by Smad-dependent mecha-ad3-null mice treated with TGF are protected fromrosis [147,148]. Restoration of Smad3 levels in MDCKve undergone EMT re-establishes the sensitivity of the

    growth inhibitory effects of TGF [149]. Nonetheless,

    sors dethe TG1-medAnothenalingEMT bsignaliimal tuunable[154]. lead toment bhas revcells ction) othe MEalso arnism inp21Cip

    An the cewhichdepenEMT o1/2-mexampsible fit exer[158]. exprescell cyation pcell lunZeb-1 reducewith thablatiosensitiof Zeband in

    TGFvia botexampto apoNotablcells texposubreast2 previnvolv

    Theptosis abnormily meleads tyet ecptosis of Twiing -ovariasistancsame ted above. For example, fetal rat hepatocytes overcomeediated pro-apoptotic effects during TGF- and Snail-

    EMT by a mechanism involving BclXL and Akt [151].dy on fetal rat hepatocytes points to a role for EGFR sig-aintaining survival and proliferation after induction ofF treatment [152]. Likewise, a role for EGF and Akt

    survival and EMT has been shown in human prox-r cells [153]. However, in adult hepatocytes TGF isnduce EMT or to mediate an anti-apoptotic responsetheless, forced expression of Snail-1 in these cells does, and Snail-1 blocks the apoptotic effects of TGF treat-rocess requiring NFB activation [155]. Another studyd that Snail-1 confers resistance to cell death in MDCKquent to withdrawal of survival factors (serum deple--apoptotic stimuli (TNF administration) by activatingk and PI3K/Akt pathways [156]. In this setting, Snail-1

    the cells in the G1/S phase of the cell cycle by a mecha-ing repression of cyclin D2 and increased expression of

    cycle inhibitor.iation of EMT-mediated cell survival with the stage ofle has also been observed in AML-12 hepatocytes, in

    treatment resulted in growth arrest, apoptosis or EMT,on the cell cycle stage [157]. Notably, TGF-induceds in G1/S, but provoked apoptosis of cells in G2/M. Zeb-ed EMT has also been linked to cell cycle regulation. For

    RT112 bladder cells, Zeb-2 but not Zeb-1 is respon-tenuation of the G1/S cell cycle phase transition androtective role against DNA damage-induced apoptosisrently, Zeb-2 arrests cells in G1 by repressing cyclin D1

    Conversely, forced expression of cyclin D1 uncouplesontrol from EMT, generating cells that retain prolifer-rties and are also able to migrate [159]. In non-smallncer cell lines with high Zeb-1 expression, ablation ofts in suppression of cell growth, while in some casesb-1 expression leads to apoptosis [160]. In accordancescription of Zeb-1 as a survival mediator in cancer cells,Zeb-1 renders head and neck squamous carcinoma cells

    the EGFR inhibitor erlotinib [161]. Similarly, silencing mesenchymal pancreatic cancer cell lines reverts EMTes sensitivity to DNA-damaging drugs [162].n activate both Snail-1 and 2 and thus promote EMTad-dependent and -independent pathways [163]. ForF-induced EMT has been shown to provide resistances in three murine mammary epithelial cell lines [164].hile short exposure of mammary epithelial NMuMGF leads to proliferation arrest and apoptosis, longromotes EMT and cell survival [150]. In MCF7 humanelial cancer cells, expression of either Snail-1 or Snail-DNA-damage-induced cell death by repressing genes

    the DNA damage response pathway [165].or suppressor p53 induces cell cycle arrest and apo-esponse to DNA damage and other chromosomalies, a function that seems to be modulated by Snail fam-s and EMT. For example, DNA damage by -irradiationptosis of MDCK cells and RWP1 pancreatic cancer cells,

    expression of Snail-1 protects these cells from apo-gh activation of Akt [166]. Moreover, phosphorylation

    by Akt reduces the efciency of p53 activation follow-iation, and apoptosis is eventually inhibited [167]. Incer cells, both Snail family members mediate chemore-

    antagonizing p53-mediated apoptosis, while at thectivating a stemness program [168]. Snail-2 can protect

  • 200 N. Tiwari et al. / Seminars in Cancer Biology 22 (2012) 194 207

    Fig. 3. Mechancells provides outgrowth of cinhibitors (p16upstream effec

    normal hemapoptosis bthrough thtein and, lik[169171]. sis by represtabilizationing combinthe therapy

    In summplays a critcells arresteferent apopinvolved inof p53 functal systemscues, a functherapy.

    9. EMT and

    Once tuentered themicroenvirolack of cellthe main tuadherens juthe surrounisms leading to EMT contribute to inhibition of senescence, apoptosis and anoikis in neoplthe means for bypassing senescence, apoptosis and anoikis. By mitigating these anti-tumoancerous lesions. For instance, both Snail-1 and 2 have been shown to affect p53 functionaInk4a, p21Cip1), as well as p63 and p73. Also Twist is a negative modulator of p16Ink4a andtors, such as TGF and EGF, are able to elicit similar responses (for details see text).

    atopoietic progenitor cells from DNA damage-inducedy antagonizing the p53-mediated apoptotic pathwaye repression of Puma, a pro-apoptotic BH3-only pro-e Snail-2 itself, a target of p53 transcriptional controlTwist-1 can also protect against p53-mediated apopto-ssing the expression of p19ARF, thus preventing p53

    and function [172]. As a consequence, Snail-2 silenc-ed with pro-apoptotic treatment shows high efcacy in

    of metastatic neuroblastoma [173].ary, during the process of EMT the cell cycle stage

    ical role in the execution of an apoptotic program, asd in the G1 phase of the cell cycle are resistant to dif-totic stimuli (Fig. 3). Among the signaling pathways

    this regulation, the activation of Akt and a repressiontions appear to be a recurrent scheme in experimen-

    in which EMT is driven by various EMT-inducingtional connection that should be exploited for cancer

    anoikis resistance

    mor cells have disengaged from the tumor mass and circulation, they face new challenges by their newnment, namely anoikis, i.e. apoptosis induced by theular adhesion [174]. During EMT, cells detach frommor mass by partial or complete downregulation ofnctions, tight junctions and desmosomes, invade intoding tissue, and eventually enter the blood stream.

    Apparentlyable not onof signaling

    Inductiothe generataddition, mby sequeste[145]. In thinducing EMectopic Trkby a mechadown-regu1 and Snailcells after Texpression recently idethe Zeb famprovides a Twist-1 expsensitivity t

    Wnt sigology and cancer [181ily of Wnt aIt has been1 results inexpression sion. Moreoastic cells. The emergence of EMT-inducing mechanisms in neoplasticral defense mechanisms, the appearance of EMT can contribute to thelity, while Zeb-1 is able to modulate several cyclin-dependent kinase

    p19Arf and thus interfering with cell cycle progression. Additionally,

    , cells that have been separated from the tumor mass arely to actively migrate but also to survive in the absence

    cues from the tumor environment [2].n of EMT results in the suppression of anoikis and inion of invasive and metastatic cancer cells [11,13]. Inutant p53 enhances the metastatic activities of TGFring p63 and thus inhibiting its pro-anoikis functionsis context, TGF functions as a tumor promoter byT and enabling cancer cells to survive [146]. Similarly,

    B expression results in EMT and resistance to anoikisnism involving Twist-1 and Snail-1 and the subsequentlation of E-cadherin expression [175]. Thereby, Twist--1 cooperate to promote Zeb-1 expression in NMuMGGF treatment [176]. Likewise, Snail-2 induces Zeb-2in melanoma cell lines [177]. Notably, TrkB has beenntied as a target of miR200c [93], a major repressor ofily of EMT inducers, and by repressing TrkB, miR200c

    link between EMT and anoikis resistance [178]. Loss ofression has also been reported to restore tumor cello anoikis and to TNF-induced apoptosis [179,180].naling is involved in many aspects of cellular physi-its de-regulation is associated with different types of]. Soluble frizzled-related proteins (SFRPs) are a fam-ntagonists with reduced expression in various cancers.

    recently demonstrated that down-regulation of SFRP- EMT characterized by down-regulation of E-cadherinand up-regulation of vimentin and Zeb-1 and 2 expres-ver, the resistance of these cells to anoikis points to a

  • N. Tiwari et al. / Seminars in Cancer Biology 22 (2012) 194 207 201

    potential link between low SFRP-1 levels in tumors and poor prog-nosis [182]. Finally, activated Wnt/-catenin signaling has beenfound to protect malignant hepatocytes against anoikis and therebyto favor metastatic dissemination [183]. Also, constitutive activa-tion of NFBwith EMT aappears to ptosis and [47,184,185mechanismNFB activi

    Taken totumor cellsfully underappears to ies are requsurvive dur(Fig. 3).

    10. EMT an

    Cellular tects an orgCellular senends has binduced senbeen descrimulation oftheir crucia[188]. Indeepathways. of OIS in paOIS seems thypothesis mation [189

    Thus, forexist that ayears severto mitigateto be neceand of bothhomologs ocellular senincluding thinduced serepress cycsion resultican repressmoter [194

    Experimregulation ysis of Zebzone of theonic perichknockout mciated withinhibitors [Bmi-1 nullcence of n[196].

    Remarkawhich is pothat while msenescencethis dual ne

    that both Zeb-1 and 2 can overcome EGFR-mediated senescence,which subsequently results in an EMT phenotype [198]. However,these functions of Zeb-1 and 2 seem to depend on a simultaneousinactivation of p53 function. The relation of the p53miR200cZeb

    sene H200c. Stype

    thatZeb-

    cellZeb-also y attppor

    arel of tancyt to em t1 canurthe

    canctogensfothat rmorm sressiced

    mber, althences in il-1, wtes i

    SnapresD2. Ipopttion

    progt maenesessio

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    relail-2 e

    cell cress

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    cell, theOn ty p5s p53Sincet cleescen transcriptional activity has been found to be associatednd metastasis in several experimental models. It alsoplay a key role in the induction of resistance to apo-anoikis by activating the PI3K/Akt signaling cascade]. Finally, HGF/Met inhibits anoikis during EMT by a

    involving ERK and Akt signaling yet independent ofty [186].gether, the signaling pathways mediating survival of

    once they detach from their surroundings are notstood. The PI3K/Akt signaling that accompanies EMTplay a role in overcoming anoikis, yet further stud-ired to obtain a comprehensive picture of how cellsing EMT and metastatic dissemination of cancer cells

    d cellular senescence

    senescence has been proposed as a mechanism that pro-anism against the initiation and progression of cancer.escence that correlates with erosion of the telomereeen termed replicative senescence [187]. Oncogene-escence (OIS) is a different form of senescence that hasbed in cancer cells, and goes hand in hand with an accu-

    the p53 and p16Ink4a cell cycle regulators, underscoringl role in the induction of this type of cell cycle arrestd, OIS can be overruled by inactivating the p53 and RbNumerous reports have demonstrated the occurrencetients and various experimental models. Interestingly,o be mainly induced in benign lesions, supporting thethat it provides protection against malignant transfor-].

    a tumor to progress to malignancy, mechanisms mustllow a cancer cell to bypass OIS. Indeed, during recental EMT-inducing transcription factors have been shown

    the effects of OIS. For instance, Zeb-1 has been shownssary for the transcriptional repression of deltaNp63

    isoforms of p73 [190]. Both p63 and p73 are closef p53 and have been implicated in the induction ofescence. p63 can induce several p53 target genes,e cell cycle inhibitor p21Cip1, and is necessary for Ras-nescence [191,192]. Furthermore, both p73 and p63lin-dependent kinase (Cdk1) and cyclin B gene expres-ng in a senescence phenotype [193]. Reciprocally, p63

    Zeb-1 expression by binding directly to the Zeb-1 pro-].ental evidence for an involvement of Zeb-1 in theof senescence has also been obtained from the anal--1-decient mice. Progenitor cells of the ventricular

    embryonic brain, and proliferating cells of embry-ordial tissue as well as embryonic broblasts of Zeb-1ice undergo premature replicative senescence asso-

    up-regulation of the p15Ink4b and p21Cip1 cell cycle195]. These ndings are similar to observations in

    mice, in which Bmi-1 prevents premature senes-eural stem cells by repressing p16Ink4a and p19Arf

    bly, both Zeb-1 and Bmi-1 are targets of miR200c,sitively regulated by p53. These relationships indicateediators of EMT can repress senescence, modulators of

    can also negatively affect EMT [197]. The existence ofgative feedback loop is further supported by the ndings

    axis towheremiR20phenomRNA[199]. tion ofWhile it has therebing sugrowthcontromalign

    Nextors seTwist-[74]. Fbreastarrest Ras-traarrest Furthecue froby repis indu[201].

    MeIndeedsenescof K-Raof Snaabrogarole ofoverexcyclin from aprotectumorcontexaffect s2 exprfactorsbeen sInterestionalicells [2

    Theof SnaG0/G1D1 expof theD1 levloop scancerSnail-2[207]. lated binhibit[171]. it is noor senscence has been further conrmed in endothelial cells,2 treatment leads to p53-dependent up-regulation ofubsequently, Zeb-1 is down-regulated and a senescentis established. Over-expression of a version of Zeb-1

    is not responsive to miR200c can inhibit senescence2 has also been reported to be involved in the modula-ular senescence, however with contradictory ndings.2 is implicated in bypassing EGFR-induced senescence,been found to directly inhibit cyclin D1 expression,enuating cell cycle progression [159,198]. This nd-ts the hypothesis that occurrence of full EMT and cell

    mutually exclusive and that only cells that maintainhe cell cycle and undergo EMT can contribute to tumor.the ZEB family, other EMT-inducing transcription fac-o be involved in the regulation of cellular senescence.

    override c-Myc-induced senescence in rat broblasts 2rmore, ablation of Twist-1 or 2 expression in murineer cells or in melanoma cells results in G1 growthther with enhanced cellular senescence. In addition,rmed murine embryonic broblasts undergo growthcan be rescued by overexpressing Twist-1 or 2 [74].e, Twist is responsible for the hypoxia-induced res-enescence in human mesenchymal stem cells (hMSC)ng p21Cip1 expression [200]. Notably, Twist expressionby HIF1 to overcome senescence and to induce EMT

    s of the Snail family can also modulate senescence.ough H-Ras and N-Ras expression immediately induce, this does not seem to be the case for K-Ras. Expressionmouse broblasts results in ATR-mediated stabilizationhich then binds the DNA-binding domain of p53 and

    ts function [202]. While these examples emphasize theil-1 as an inhibitor of the senescent phenotype, Snailsion can also block the cell cycle by directly repressingn addition, cells over-expressing Snail-1 are protectedosis [156], indicating that senescence induction andfrom apoptosis are interconnected during malignantression. These results also suggest that the cellulary determine how EMT-inducing transcription factorscence. For example, the balance between p53 and Bcl-n inuences the effect of EMT-inducing transcriptionenescence or apoptosis; increased levels of Bcl-2 have

    to convert apoptotic cells into a senescent state [203].ly, Bcl-2 overexpression is known to inhibit p53 func-d to lead to partial EMT in squamous cell carcinoma05].tion of Snail-2 to senescence is not as clear. Ablationxpression reduces cyclin D1 expression and inducesycle arrest. Yet in contrast to Zeb-2, repression of cyclinion by Snail-2 is not direct; Snail-2 inhibits expressionbiquitin conjugase UbcH5c, leading to higher cyclin206]. As mentioned earlier, a dual negative feed back

    to exist between p53 and Snail-2. Indeed, in lungs wild type p53 is responsible for the degradation ofreby inhibiting the development of an EMT phenotypehe other hand, in irradiated mice Snail-2 is upregu-3 in hematopoietic progenitor cells and it subsequently-dependent transcription of Puma and thus apoptosis

    p53 is a mediator of both senescence and apoptosis,ar whether Snail-2 acts mainly by inhibiting apoptosisce and whether the cellular context and the specic

  • 202 N. Tiwari et al. / Seminars in Cancer Biology 22 (2012) 194 207

    type of molecular damage may determine the actual outcome(Fig. 3).

    11. EMT an

    If EMT-iovercome oto oncogeninates frompathways aone oncogeingly, lung ctheir growtand Zeb-1 kdependencywhich seemexpressing K-Ras [2102 activity, may involvtion of EMMCF-10A cp65 undergand ablatioably, p65 icells, contrasidered as These obserment permprimary onEMT can mobservationtumors. Forinducible Mafter a latehigh levels [212].

    These nability to overating cancevents.

    12. EMT an

    Accumuinuence imFor examplcells with sin CD4+ T suppressivecells mediaTGF and tIn vivo, Snaapy and shto yet anotdisseminatiused to reinduce epithlial cells, e.induction oenchymal vmatrix protsive agentsto maintainobliterative

    be involved in immune cell motility, as the recruitment of acti-vated macrophages is regulated by Snail-1. The expression ofSnail-1 in oral keratinocytes results in repression of terminal dif-ferentiation and the induction of cyclooxygenase 2 (Cox2) and

    ammocytocytl immmacr

    heacroped tieeme maonclr of une

    e.

    T an

    istaneatmies aformesenost

    indinroteiultid

    P-gltherae anent eme ence t, or ent oadriawithed inltidre) an1. Abhanctical nd cne-d4ARF

    1 alsducionclus EMsearcnce.

    T an

    lethoiferam cet to hey ogened id oncogene addiction

    nducing transcription factors can enable tumor cells toncogene-induced senescence, they may also contributee addiction. The concept of oncogene addiction orig-

    the nding that, although a multitude of regulatoryre deregulated in cancer cells, cells strictly depend onnic event for proliferation and survival [208]. Interest-arcinoma cells that are dependent on K-Ras activity forh can lose this dependency upon TGF-mediated EMT,nockdown in K-Ras-independent cells can restore this

    [209]. A similar effect has been reported for Snail-2,s to be necessary for the survival of colon cancer cellsmutant K-Ras, but not for cells expressing wild-type]. Since Ras-activated pathways are inducers of Snail-an intrinsic mechanism for evasion of Ras addictione the stabilization of Snail-2 protein and the induc-T. Similar observations have been made with NFB;ells forced to express high levels of the NFB subunito EMT as the consequence of Zeb-1/2 upregulation,n of Zeb-1 or 2 reduces cell viability [211]. Remark-s initially not necessary for the viability of MCF-10Ary to the above-mentioned ndings where Ras is con-one of the initiating main drivers of tumor growth.vations suggest that the creation of a cellular environ-issive for EMT may sufce to overcome addiction to thecogenic event. Additional support for the notion thatodulate oncogene addiction comes from the frequent

    of mesenchymal-like transformations in recurrent instance, after regression of mammary tumors in theMTV-Her2/Neu mouse model, new tumors emerge

    ncy period that lack Her2/Neu expression but exhibitof Snail-1 expression and with it an EMT phenotype

    dings support the notion that EMT, in concert with itsercome oncogene-induced senescence, can free prolif-er cells from their dependency on the initial oncogenic

    d immunosuppression

    lating experimental evidence indicates that EMT canmunosuppression, which affects cancer progression.

    e, co-culture of Snail-expressing B16-F10 melanomapleen cells results in the increased expression of FoxP3regulatory (TReg) cells, thus supporting the immuno-

    function of TReg cells. The Snail-expressing melanomate this effect partly by the enhanced expression ofhrombospondin, known mediators of TReg cells [213].il-expressing melanomas are resistant to immunother-ow increased metastatic dissemination. This pointsher mechanism by which EMT promotes metastaticon. It is noteworthy that immunosuppressive agentsduce host rejection after organ transplantation alsoelial de-differentiation. Treatment of bronchial epithe-g. with cyclosporine or mycophenolate, results in anf partial EMT with loss of E-cadherin, induction of mes-imentin, and excessive production of the extracellulareins bronectin and collagen. These immunosuppres-

    can also induce the production of TGF, which serves this bro-proliferative transformation that leads to

    bronchiolitis [214]. Moreover, EMT also appears to

    pro-inkeratinof moncontrovated wounding mainamand it sof thes

    In cnumbeate immdefens

    13. EM

    Restive trtherapmajor the prcells. MATP bglycopand msion ofchemoinvasiv

    Recovercoresistavationtreatmagent ously enhancare mucristinTwist-and enthe critance aoncogeing p1Twist-thus re

    In cinducether reresista

    14. EM

    A pto prolcer stethoughcells: tone pridentiatory cytokines like Il-6 and Il-8. Snail-1 thus enablese invasion and concomitantly promotes the migrationes [215]. Interestingly, Snail-1 also seems to directlyune cell migration, such as the recruitment of acti-

    ophages during acute inammation and toward earlyling sites. High Snail-1 expression is found in migrat-hages of the mucosal immune system in chronicallyssues in Crohns disease and ulcerative colitis patients,s to play an essential role in the TGF-induced motilitycrophages [216].usion, cells undergoing EMT gain the expression of acytokines and secreted factors that dampen appropri-

    responses, thus allowing EMT cells to survive immune

    d chemoresistance

    ce to chemotherapy is a major obstacle to the effec-ent of most cancer types. Although most anti-cancerlter tumor growth, the effect is not long lasting. One

    of chemotherapy resistance has been associated withce of molecular pumps that transport drugs out ofprevalent transporters are members of the family ofg cassette (ABC) membrane proteins, also called P-ns, such as multiple drug resistance protein 1 (MDR1)rug resistance-related protein 1 (MRP1). Overexpres-ycoproteins in cancer cells not only results in increasedpy resistance but in many cases also correlates withd metastatic behavior.xperimental evidence indicates that EMT aids cells tondogenous safeguard mechanisms as well as to induceo cell death triggered by DNA damage, serum depri-direct apoptotic stimuli such as TNF [156]. Indeed,f epithelial breast cancer cells with the DNA-damagingmycin results in induction of apoptosis simultane-

    an increase in the number of EMT-like cells withvasiveness. These newly formed dedifferentiated cellsug-resistant (for example against pacilitaxel and vin-d express high levels of P-glycoprotein and also oflation of Twist-1 expression blocks drug-induced EMTes adriamycin-induced apoptosis, further underliningrole of Twist-1 and EMT in the modulation of drug resis-ell survival [217]. Twist-1 has been reported to inhibitependent and p53-dependent cell death by inhibit-expression or MDM2 activity [172,218]. Interestingly,o stimulates the direct association of p53 and Mdm2,ng p53 levels and p53-dependent p21Cip1 expression.sion, it is still not clear whether chemotherapy directlyT or selects for cells that have undergone EMT. Fur-h is warranted to delineate the role of EMT in therapy

    d cancer stem cells

    ra of recent studies suggests that the ability of tumorste and propagate relies on stem-like cells, called can-lls (CSCs) or cancer-propagating cells (CPCs). CSCs areshare fundamental characteristics with somatic stemdivide asymmetrically to produce one stem cell anditor cell (self-renewal; [219]). CSC-like cells have beenn several solid tumors, including breast, brain, colon,

  • N. Tiwari et al. / Seminars in Cancer Biology 22 (2012) 194 207 203

    pancreas, prostate, lung, and head and neck tumors [220223].Depending on cancer tissue type they share surface markers withsomatic stem cells of the original tissue, which allows their identi-cation and isolation. Several of the molecular signaling pathwaysassociated wand Notch sopment andCSCs. In faca number oin CSCs.

    In a semlated whetmetastatic growth. SecEMT expresstem cells. F(P-glycoprocells that hachemotheraalso found i

    While soformation osuggest thaan adaptiveCells that hsuspensiontumorigenicompromishave underels of CD24,[229]. It is species. EMcell populaCD24high eptial [22622trans-differferentiateddiscovery tCSCs encounot only wi

    15. Outloo

    EMT, ana critical prby various many casesby cancer chave made ular mechainto the fun

    CurrentlEMT-associing EGFR acknown to reation. Whilegrowth, in tance, and tmetastasis hing the actiin the inducEGFR, IGF1Rpre-clinicalmetastatic such analys

    metastasis that can be evaluated by non-invasive methods duringtherapy. It is hoped that the rapid progress in EMT research andthe various facets of innovative insights into the molecular mecha-nisms underlying EMT and metastasis will open novel avenues for

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    HW,ceptoduce f TWISith normal stem cell homeostasis, including Wnt, Shhignaling, are well-known to be active in cancer devel-, hence, have been implicated in the maintenance of

    t, recent gene expression proling of CSCs has revealedf genes commonly expressed in somatic stem cells and

    inal review, Brabletz and colleagues have specu-her metastatic cells could resemble CSCs [224]. First,cells by denition are able to initiate de novo tumorond, metastatic cells and also cells that have undergones an over-proportional number of genes associated withinally, by the increased expression of ABC transportersteins) and by other mechanisms, metastatic cells andve passed through EMT develop high resistance againstpy and other apoptosis-inducing agents, a hallmarkn somatic stem cells [225].me studies suggest that CSCs may arise from the trans-f their normal counterparts, recent observations rathert they originate from fully differentiated cells through

    trans-differentiation program, such as EMT [226228].ave undergone EMT are able to grow as spheroids in

    culture, another hallmark of CSCs, and they are highlyc when xenografted at limited dilution in immune-ed mice. Transformed human breast epithelial cells thatgone EMT also express high levels of CD44 and low lev-

    a marker combination found in human mammary CSCsof note that these marker combinations vary betweenT in most cases is a reversible process, and the stemtion within EMT cells continuously generates CD44low

    ithelial cells that however lack a tumorigenic poten-8]. With regard to the EMT-associated properties, theentiation process is thus considered to convert dif-

    epithelial cancer cells into CSCs. Of note, the recenthat the cytotoxic drug salinomycin preferentially killsrages the screening for drugs that specically interfereth CSCs but also with EMT and metastasis [230].

    k

    d with it cancer cell migration and invasion, is certainlyocess in the metastatic cascade. EMT can be inducedsignaling pathways and regulatory networks which in

    overlap with developmental processes that are hijackedells and the tumor microenvironment. Yet, while wesubstantial progress in the understanding of the molec-nisms underlying EMT, we still lack sufcient insightsctional contribution of EMT in cancer patients.y, no therapeutic approach specically targets EMT,ated cancer cell migration or invasion. Drugs repress-tivity are already in clinical use to combat cancer typesly on up-regulated EGFR activity for cancer cell prolifer-

    these therapies show initial efcacy in reducing tumormost cases cancers recur by developing evasive resis-he effects of the targeted therapy against EGFR on tumorave not been specically evaluated. Novel drugs target-vities of different receptor tyrosine kinases implicatedtion of EMT, cancer cell invasion and metastasis, such as, PDGFR, c-Met, and FGFR, are in rst clinical trials or in

    testing. However, also here the specic efcacy againstspread remains to be determined. A major problem inis is the lack of appropriate surrogate markers of tumor

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    [19] Loreinohment of appropriate surrogate markers for improvednd prognosis, and, most importantly, for the design ofi-metastasis therapies.

    interest

    clared

    gments

    ogize to all colleagues whose important work we could to space restrictions. Research in the laboratory of theated to this review has been supported by the EU-FP6

    program BRECOSM LSHC-CT-2004-503224, the EU-FP7 program TuMIC 2008-201662, the Swiss Cancer League,a Beider Basel, the FWO, the Stichting tegen Kanker, anderteerde Onderzoeksacties of Ghent University.

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