EMT in breast cancer stem cell generation

Cancer Letters xxx (2012) xxx–xxx

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Mini-review

EMT in breast cancer stem cell generation Stéphane Ansieau ⇑ INSERM UMR 1052, Lyon, France CNRS UMR 5286, Lyon, France Université de Lyon, UMR S-1052, Lyon, France Centre Léon Bérard, Lyon, France LabEx DEVweCAN, Lyon F-69008, France

a r t i c l e

i n f o

Article history: Available online xxxx Keywords: Cancer stem cells Epithelial-to-mesenchymal transition Cell plasticity Embryonic EMT-inducers Breast

a b s t r a c t The concept of cancer stem cells (CSCs) has been proposed to explain the ability of single disseminated cancer cells to reconstitute tumours with heterogeneity similar to that of the primary tumour they arise from. Although this concept is now commonly accepted, the origin of these CSCs remains a source of debate. First proposed to arise through stem/progenitor cell transformation, CSCs might also or alternatively arise from differentiated cancer cells through epithelial to mesenchymal transition (EMT), an embryonic transdifferentiation process. Using breast carcinomas as a study model, I propose revisiting the role of EMT in generating CSCs and the debate on potential underlying mechanisms and biological significance. Ó 2012 Published by Elsevier Ireland Ltd.

1. Introduction tumour progression models Tumour development is believed to result from a succession of epigenetic/genetic alterations and selection steps leading to the emergence of cells accumulating survival and proliferation advantages. Selection is proposed to take place continuously in the primary tumour [1], giving rise to heterogeneity that simply reflects the coexistence of cell populations evolving independently and displaying distinct oncogenic potentials. While this assumption might be sufficient to interpret the cell diversity observed in tumours, it fails to explain how individual disseminated cancer cells escaping from primary tumours yield secondary tumours with similar diversity. An alternative model (although not exclusive of the first) assumes that primary and secondary tumours arise from cancer cells displaying both self-renewal and differentiation capabilities. By analogy with totipotent stem cells that can differentiate into different lineages, these tumour cells are generally called cancer stem cells or CSCs. Originally, CSCs were identified in chronic myeloid leukaemia (CMLs) as arising through primitive precursor transformation driven by the BCR–ABL fusion protein, product of the recombined Philadelphia chromosome [2]. From solid tumours, CSCs have been isolated by exploiting expected similarities with normal stem cells, including phenotypic features (antigenic phenotype), the ability to export drugs, and the capacity to grow as sin-

⇑ Address: Centre de Recherche en Cancérologie de Lyon, UMR INSERM 1052, CNRS 5186, Centre Léon Bérard, 28 rue Laennec, F-69008, France. Tel.: +33 478785160. E-mail address: [email protected]

gle-cell-originated spheres when cultured under low-adherence conditions. Although current debate focuses on many issues, such as the validity of individual functional assays used to evaluate CSC features and the surprising fact that micro-environmental changes drastically influence the proportion of CSCs in tumour lineages [3], one of the main questions remains their origin. Although tumours obviously arise from cells displaying a self-renewal capacity, the question remains whether the cell of origin always displays such features intrinsically or whether this potential is acquired during transformation, through a combination of a defined cell context with appropriate genetic events and/or microenvironmental conditions. Breast cancers constitute a model of choice for addressing this question, their heterogeneity having long been viewed as arising through transformation of cells of different origins. The present report describes current progress towards elucidating this biological question and discusses the role that the epithelial to mesenchymal transition (EMT) and the associated gene reprogramming might play in generating this diversity. 2. EMT as a cell dedifferentiation process The EMT is a latent embryonic process converting polarized and adjacent epithelial cells to mesenchymal cells. Reversible by definition, this process transiently confers cell motility, allowing the evolution, fusion, or generation of secondary epithelia, essential to both morphogenesis and organogenesis [4,5]. The ability of these embryonic cells to evolve from one state to another, under the direction of extracellular signals, suggests that they display intrinsic cell plasticity. EMT is driven by a restricted number of

0304-3835/$ - see front matter Ó 2012 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.canlet.2012.05.014

Please cite this article in press as: S. Ansieau, EMT in breast cancer stem cell generation, Cancer Lett. (2012), http://dx.doi.org/10.1016/j.canlet.2012.05.014

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transcription factors that regulate the expression of numerous proteins involved in cell polarity, cell-to-cell contact, cytoskeleton structure, and extracellular matrix degradation. These transcription factors mainly include members of the three protein families ZEB, SNAIL, and TWIST [4,5]. These embryonic proteins are absent from differentiated adult cells but are frequently reactivated in cancers, including carcinomas. As a reminiscence of their embryonic functions, they promote, through EMT induction, cancer cell dissemination at the invasive fronts of tumours [5,6]. Consistently with this observation, they generally constitute poor prognosis factors associated with a high risk of metastasis [4]. Depicted as prometastatic factors, these embryonic proteins do display multiple deleterious properties. They promote escape from failsafe programs (senescence and apoptosis) by down-regulating the RB- and p53-dependant oncosuppressive pathways and provide cells with a survival advantage under numerous stress conditions [7–14]. As such, they likely promote neoplastic transformation. Numerous EMT inducers additionally display intrinsic anti-apoptotic properties, affecting the NF-jB, AKT, and MAP kinase survival pathways and expression of pro- and anti-apoptotic BH3-only proteins [4,15]. Surprisingly, EMT induction has also been found to endow cells with stem-like properties. Transduction of SNAIL1 or TWIST1 into HMLE cells (human mammary epithelial cells (HMECs) sequentially transfected with hTert and SV40 T/t antigens) [16]) has been shown to confer to these cells a mammary stem cell (MaSC)-associated CD44highCD24low antigenic phenotype, the ability to form mammospheres when cultured under low-adherence conditions [17]. When combined with the H-RasG12V mitogenic protein (HMLER cell line), SNAIL1 or TWIST1 expression additionally provides cells with a tumourigenic potential, when xenografted at limiting dilutions [17]. In line with this observation, transduction of the oncoprotein H-RASG12V into the HMLE epithelial cell line leads to emergence of a mesenchymal population with stem-cell-like properties and tumourigenic potential [18]. TWIST1 expression in either MCF10A or MCF7 is similarly associated with a CD44highCD24low antigenic phenotype, increased drug efflux, ALDH1 dehydrogenase activity, increased self-renewal properties, and a higher tumourigenic potential [19]. Collectively, these observations support the view that EMT behaves as a dedifferentiation process converting differentiated epithelial cells into stem-like cells. In support of this view, Mani’s team has recently shown that HMLE cells that have become committed to EMT following TWIST1 or SNAIL1 expression share several cell surface markers with MaSCs and have reacquired pluripotentiality, differentiating into adipocytes, chondrocytes, or osteoblasts when cultured in appropriate media [20]. EMT inducers seem to promote cell dedifferentiation in different ways. TWIST1 has been shown to act downstream from BMI-1 and to cooperate with the Polycomb protein in silencing target genes [21]. The ZEB1 transcription factor has been found to modulate the two stemness genes KLF4 and SOX2 indirectly, through downregulation of miRNAs [22]. More recently, SNAIL2 and SOX9 have been found to cooperate in orchestrating the mammary stem cell state [23]. Various mechanisms seem therefore to link EMT and stemness. What are the consequences? Reversible by definition, EMT has been proposed to modulate dynamically the balance between CSCs and differentiated cells according to micro-environmental variations [24]. This should obviously have profound impacts on the design of future therapeutic approaches. As CSCs were previously believed to represent the major risk of recurrence, they were viewed as the unique targets to be eradicated. Cancer cell death in response to treatment is likely, as a collateral effect, to create a selective pressure forcing differentiated cells to find an escape route. Commitment to the EMT may constitute an appropriate exit, fuelling the CSC population and thus creating an effect opposite to that expected. In line with this hypothesis, selection of cells resistant to chemo- or radio-

therapy is often associated with EMT [25,26]. Eradication of both CSCs and differentiated cells may thus be necessary to successfully clear tumours. Is EMT essential for cell dedifferentiation or does in fact the associated gene reprogramming only contribute to it? While EMT has been associated with a gain in stem-like properties, induction of the mesenchymal to epithelial transition (MET) by combined inhibition of the TGFb and MEK pathways has conversely been shown to improve the efficiency of induced pluripotent stem cell generation from fibroblasts [27]. This observation suggests that both EMT and MET (depending on the differentiation state of the cell of origin) might be important in enabling cells to exit a differentiation program and adopt an ‘‘intermediate state’’ from which cell dedifferentiation and transformation might be achieved. Consistently with a possible role in tumour initiation, EMT inducers such as TWIST1 and SNAIL1 are frequently detected at the in situ ductal carcinoma (DCIS) stage, long before metastatic dissemination begins [28,29]. Furthermore, as discussed in the next section, combined expression of TWIST1 and RAS in luminal-committed mammary epithelial cells is sufficient to promote breast carcinogenesis [29].

3. EMT and breast cancer typology Gene expression profiling of breast carcinomas has led to identifying five independent intrinsic molecular subtypes: normal-like, HER-2-enriched, luminal (A and B), and basal A/basal-like [30]. An additional subtype, termed basal B/claudin-low, has recently been highlighted [31,32] and found to display EMT- and CSC-associated gene signatures [26,33]. The prevailing concept in the field was that these different subtypes reflect the type of cell from which they originate. Optimization of dissociation techniques, the spectrum of markers for reliably sorting epithelial cell populations out of mouse and human mammary tissues, and use of the in vivo mammary reconstitution assay have led to successful isolation and characterization of differentiated luminal cells, committed luminal progenitors, bipotent progenitors, and mammary stem cells (or, as next discussed, a population enriched in mammary stem cells, MaSC). Transcriptome analysis has highlighted conserved gene expression signatures and pathways between mouse and human, suggesting that despite anatomic differences, an outstanding parallel exists between the epithelial cell hierarchies of these two species [34]. Comparisons of these gene expression profiles with those of human primary breast tumours has unveiled significant similarities between the luminal signature and luminal tumours and between the luminal progenitor signature and basal A/basal-like tumours, strengthening the hypothesis that the different subtypes of breast tumours arise through transformation of cells placed at different branch points of the breast differentiation tree. In support of this hypothesis, BRCA1 depletion in transgenic mice has been found to give rise to different tumour types according to the Cre-recombinase-expressing mouse strain employed. In a p53+/ context, BRCA1 depletion in MaSC/basal cells (driven by a K14-Cre) leads to the development of adenomyoepitheliomas, whereas the same depletion in luminal progenitors (driven by a Bgl–Cre) results in basal-like tumours with metaplastic features and a luminal ER genomic profile, a phenotype coinciding with that of human BRCA1 tumours [35]. These data support the view that BRCA1 tumours arise from luminal progenitors and that the rarely observed adenomyoepitheliomas are an example of breast tumours arising through transformation of mammary stem cells. Although these transgenic models are powerful tools, interpreting these in vivo experiments is often difficult due to the lack of precise information on the Cre expression pattern. It is impossible, for example, to rule out that the Bgl promoter might display residual

Please cite this article in press as: S. Ansieau, EMT in breast cancer stem cell generation, Cancer Lett. (2012), http://dx.doi.org/10.1016/j.canlet.2012.05.014

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activity in bipotent progenitors, explaining the ability of these ‘‘luminal precursor cells’’ to reconstitute a complete mammary gland when implanted in cleared mammary fat pats. BRCA1 depletion in these bipotent progenitors might actually foster differentiation to committed luminal precursors, as suggested by Liu and colleagues [36]. Alternatively, BRCA1 depletion might promote dedifferentiation of luminal-committed cells to bipotent or pluripotent cells. Whatever the exact cell of origin, the available data cast doubt on the notion that the phenotype of a tumour directly reflects the cell of origin. Comparison of the MaSC signature with tumour gene expression profiles has highlighted significant similarity to the basal B/ claudin-low subtype, suggesting that these tumours might arise from MaSC [37,38]. Nonetheless, the sorted out CD49highCD29high CD24+Sca1 epithelial cell population from which the MaSC signature was established comprises less than 5% MaSC and also includes intermediates such as bipotent or committed basal progenitors and differentiated myoepithelial cells exhibiting a common cell surface phenotype and gene expression profile [39]. The similar profiles of claudin-low breast tumours and MaSC might thus reflect altered or incomplete myoepithelial differentiation [40,41]. In line with this, the Charlotte Kuperwasser team isolated luminal and basal subpopulations from human mammary epithelial cells on the basis of differential EpCAM and CD10 expression [42] and found the gene expression profile of the EpCAMl°CD10+ basal cells to match the MaSC signature [33,43]. This supports the hypothesis that this signature might reflect basal/myoepithelial differentiation. Transformation of these cells with various combinations of cellular and/or viral oncoproteins led to the development of claudin-low/metaplastic tumours [42]. This suggests that basal B/claudin-low tumours arise through transformation of basal/myoepithelial progenitor cells. Interestingly, transduction of similar oncoprotein cocktails into the luminal cell subpopulation gave rise to oestrogen-receptor-positive and -negative tumours, supporting the view that basal A/basal-like tumours need not originate from basal progenitor cells but might arise through transformation of luminal progenitor cells [42]. The absence of specific antigens for sorting out mammary stem cells and their rarity in adults obviously make it hard to ascertain their expression signature. To circumvent this problem, Benjamin Spike and co-workers used mammary rudiments of E18.5 murine embryos (eMaSC) as an alternative source of MaSC (fetal MaSC or fMaSc). Their results again raise the question of the significance of the MaSC signature, as the gene expression profiles of fMaSc and adult MaSC diverge significantly. Basal A/basal-like tumours and many Her2+ tumours show enrichment in the fMaSC signature, while claudin-low and metaplastic tumours appear enriched in a signature established from the foetal–stromal-cell-enriched population present in E18.5-embryo-derived mammary rudiments, a subpopulation lacking functional progenitor competency [44]. fMaSC express luminal and myoepithelial markers concomitantly with vimentin, a frequent hallmark of basal A/basal-like breast tumours [44]. These observations led the authors to hypothesize that EMT, commonly observed in aggressive tumours, may result in reversion to an embryonic-like state resembling that of the fMaSC and/or fSTR compartments. In vitro cell culture conditions are also likely to affect gene expression profiles, making it hard to establish correlations between cell-derived signatures and tumours. Profiles of freshly sorted-out cells and of cells cultured even for short periods of time may indeed significantly differ. In support of this view, Gupta and co-workers have recently demonstrated that breast cancer cell lines are composed of different cell subpopulations with different antigenic phenotypes, and that these subpopulations vary in relative proportion until an equilibrium is reached. Although these populations have been defined as stem-like, basal, and

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luminal, their antigenic phenotypes suggest that they might actually be described as epithelial, partially committed to EMT, and mesenchymal [41,45]. In line with this view, several genes expressed in the cell population defined as stem-like (e.g. ZEB1, VIM, and DCN) have been shown previously to contribute to the EMT signature [46]. The observed equilibrium might thus reflect the reversibility of EMT and the influence of micro-environmental changes. Do the basal B/claudin-low tumours displaying a profile similar to the MaSC signature thus reflect altered myoepithelial differentiation or, rather, dedifferentiation? A first piece of evidence to be considered is the demonstration that EMT induction in a Neutransgenic-mouse-derived epithelial cell line leads to a claudinlow gene expression profile with acquisition of several stem-like features, including the ability to form mammospheres, an increased tumourigenic potential, and activation of signalling pathways associated with cancer stem cells (e.g. homeobox gene pathways, the TGFb, Notch, and Hedgehog pathways) [47]. We have additionally demonstrated that commitment of basal A/basal-like cell lines to EMT, either through ectopic expression of EMT inducers or in response to TGFb, confers to cells a claudinlow gene expression profile and stem-like properties. This further supports the hypothesis of EMT-promoted cell dedifferentiation rather than altered myoepithelial differentiation [29]. In support of this notion of plasticity, we have recently demonstrated in transgenic mouse models that combined expression of RAS and of the EMT inducer TWIST1 in luminal-committed cells (differentiated cells and potential luminal progenitors, transgene directed by a WAP-Cre) induces the development of claudin-low breast tumours in vivo [29]. An interpretation that remains to be demonstrated is as follows: TWIST1, by promoting EMT, might promote cell dedifferentiation and increase the pool of cells displaying a self-renewal potential and thus susceptible to be transformed by RAS. Collectively, these observations suggest that mammary cells may be able to shift from one phenotype to another, and that breast tumour typology might actually reflect the degree of cell commitment to EMT. As micro-environmental changes affect EMT [48], the typology might evolve during tumour progression.

4. An emerging concept: EMT-inducers as bona fide regulators of mammary stem cell state and commitment The observation that SNAIL2 is present in mammary stem cells and that it cooperates with SOX9 in orchestrating the stem cell state is unquestionable evidence that EMT inducers play a role in regulating stemness [23]. Yet a set of previous experiments suggests a more complex relationship between EMT and stemness than initially believed. First, on the basis of their different antigenic phenotypes, epithelial and mesenchymal subpopulations have been isolated from non-transformed human mammary basal/myoepithelial cell lines. The sorted-out EpCam+ CD49f+ epithelial cell contingent displays ALDH1 activity (a stem-like property), a differentiation potential when cultured on collagen, and a gene expression profile similar to that of luminal progenitors. Through spontaneous EMT, these cells give rise to a CD44highCD24low mesenchymal cell contingent lacking ALDH1 activity and differentiation potential but displaying an increased ability to generate mammospheres under low-adherence conditions [41]. Collectively, these data dissociate EMT from stemness and further demonstrate that the CD44highCD24low antigenic phenotype is a hallmark of EMT. Supporting this point of view, we recently have demonstrated that transduction of ZEB1 into HMECs is sufficient to confer to cells a mesenchymal phenotype, motility, and a CD44highCD24low phenotype without enabling them to generate mammospheres under low-adherence culture conditions [29].

Please cite this article in press as: S. Ansieau, EMT in breast cancer stem cell generation, Cancer Lett. (2012), http://dx.doi.org/10.1016/j.canlet.2012.05.014

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Considering that CSCs might, rather, arise through transformation of stem cells, Rangarojan’s team recently reproduced the HMEC transformation assay, using the same strategy as developed by Weinberg’s laboratory (consecutive transduction of the h-TERT and SV40T/t antigens [16]) but modifying the culture conditions so as to increase significantly the precursor stem cell population (culture under low-adherence conditions). Surprisingly, unlike the HMLE cells produced when this strategy is applied to a population containing few precursor stem cells, the resulting cell line displays multiple features of CSCs: tumourigenic potential, self-renewal properties (assessed by 10 consecutive passages in a mammosphere formation assay), differentiation potential when cultured in collagen, and activated Notch, Hedgehog, and Wnt pathways [49]. This obvious improvement in CSC-generating efficiency supports the view that the observed CSCs arose through transformation of normal stem cells or committed precursor cells. Stem/ precursor cells would thus possess intrinsic properties making them prone to transformation. As previously mentioned, inducing EMT through ectopic expression of SNAIL1, TWIST1, or H-RASG12V provides HMLE cells with stem-like properties [17,18]. This suggests that EMT induction confers to differentiated adult cells a capacity to generate CSCs similar to that of stem/progenitor cells. What transformation-favouring properties normal stem/precursor cells possess remains to be determined, but an exciting possibility is that they might intrinsically express embryonic EMT inducers

that determine their fate under the control of micro-environmental signals. Consistently with this hypothesis, stabilization of SNAIL2 has been found to be a determinant of luminal progenitor cell enrichment in BRCA1-deficient mice [50]. Through their ability to down-regulate oncosuppressive pathways, EMT inducers would cooperate with mitogenic oncoproteins to promote stem/progenitor cell transformation. Experimental conditions favouring differentiated cancer cell commitment to EMT would thus generate CSCs by simply recreating the oncogenic cooperation between oncoproteins and EMT inducers that normally contributes to stem/progenitor cell transformation (Fig. 1). How efficiently CSCs are generated from stem/progenitor cells likely depends on the oncoprotein employed in the transformation assay. To promote cell transformation successfully, functional inactivation of p53, as experimentally achieved through expression of the SV40 large T antigen, might be required in addition to endogenous expression of embryonic EMT inducers [49]. This might explain the observed cooperation between loss of E-cadherin and p53 depletion in breast carcinoma development in transgenic mice [51]. It might also explain why p53 depletion or inactivation affects cell morphology differently in different cellular settings [16,52]. The detection of EMT inducers in stem/progenitor cells and their differential efficiency in cooperating with various oncoproteins might thus constitute a first explanation of how combinations of specific cell types and genetic alterations dictate the typology of cancers, an old concept recently updated [53]. 5. A model transposable to other cancer types?

Fig. 1. EMT as a driver of committed precursor cell transformation. Because they display self-renewal properties, stem/progenitor cells are believed to constitute the cells of origin of cancers. The degree of plasticity determined by endogenously expressed embryonic transcription factors (ETFs), genetic alterations, and microenvironmental parameters determines the phenotype of the resulting tumours. Transformation of differentiated cells in vitro can be achieved (in red) by induction of complete genetic reprogramming through EMT or MET (according to the cell type employed) combined with an oncogenic insult. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The link between EMT and the acquisition of stem-like properties has been demonstrated in multiple cell lines of various origins [21,47,54–56] (Table 1), suggesting that the dedifferentiation/ transformation model might theoretically be extrapolated to other, if not all solid tumours. Is this model applicable to cancers such as gastrointestinal tumours, which likely arise through transformation of stem cells? In patients having a mutation in APC and displaying a genetic predisposition to familial adenomatous polyposis (FAP) leading to colorectal cancer, the first observable change affects the proliferative compartment, where a shift from asymmetrical to symmetrical proliferation of stem cells is observed [57–59]. Progression from adenoma to carcinoma is proposed to result from further amplification of the Wnt pathway and consequent activation of EMT, as demonstrated by the presence of nuclear b-catenin staining in cells at the invasive fronts of colon cancers [60]. This amplification is likely due to multiple factors, including loss of feedback mechanisms [61] and micro-environmental changes such as secretion of factors by stromal myofibroblasts [62]. Partial and complete commitment to EMT might thus play different roles during tumour progression. Interestingly, while the ZEB1 EMT inducer is detectable only at the invasive fronts of sporadic colon cancers, in FAP it is detected in epithelial cells of both adenomas and carcinomas [63], supporting the hypothesis that partial commitment to EMT may contribute to tumour

Table 1 Listing of cell lines and experimental conditions for which EMT commitment was found to be associated with a gain in stem-cell like properties. Cell line

Genetic alterations

EMT-inducer

Reference

HMEC-hTERT HMEC-hTERT MCF-10A, MCF-7 MCF-10A, MCF-7 MCF-10A, MMC FaDu, OECM-1 Pten / hepatic tumor derived cell line

SV40 T/t SV40 T/t, H-RASG12V – – – – –

SNAIL1, TWIST1 – TWIST1 TWIST2 TGF-b, TNF-a HIF1a, TWIST1, BMI1 TGFb, SNAIL1

[17] [18] [19] [55] [48] [21] [56]

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progression. EMT commitment has been confirmed as a determinant in colon cancers by the increased number of CK+a-SMA+ cells during the adenoma-carcinoma transition (a special form of EMT termed epithelial-to-myofibroblast transition (EMyT)) [64] and by recent gene expression profiling highlighting an EMT signature as predictive of recurrence, advancing stage, and poor prognosis [65]. Although a role of ZEB1 in promoting tumour initiation remains to be demonstrated, the protein has interestingly been found to regulate members of a set of stemness-inhibiting miRNAs [22] and additionally to facilitate escape from oncogene-induced senescence [14]. It is also proposed, by promoting cell commitment to EMT, to substitute for RAS pathway activation [66,67]. 6. Concluding remarks While EMT has long been viewed as a cell conversion process occurring at initiation of the metastatic cascade, a growing body of evidence now supports the concept that the associated genetic reprogramming might be determinant in promoting cell transformation. When combined with appropriate genetic events, EMT, and likely MET, according to the cell type, might thus bring cells into a state facilitating their dedifferentiation or transformation. Although this remains to be demonstrated, dedifferentiation might contribute directly to cell transformation. Acknowledgements I am grateful to Anne-Pierre Morel and Alain Puisieux for insightful discussions, Agnès Tissier, Arnaud Vigneron and Kathleen Broman for critical reading of the manuscript. Our research is funded by la Ligue Nationale contre le Cancer, l’Institut National sur le Cancer (INCa), la Fondation de France, l’Association pour la Recherche sur le Cancer (ARC), the project National Cancer Association and grants from the LabEX DEVweCAN. Sponsors have no role in the writing of the review and in the decision to submit it for publication. References [1] R. Bernards, R.A. Weinberg, A progression puzzle, Nature 418 (2002) 823. [2] D. Bonnet, J.E. Dick, Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell, Nat. Med. 3 (1997) 730– 737. [3] E. Quintana, M. Shackleton, M.S. Sabel, D.R. Fullen, T.M. Johnson, S.J. Morrison, Efficient tumour formation by single human melanoma cells, Nature 456 (2008) 593–598. [4] H. Peinado, D. Olmeda, A. Cano, Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat. Rev. Cancer 7 (2007) 415–428. [5] J.P. Thiery, H. Acloque, R.Y. Huang, M.A. Nieto, Epithelial-mesenchymal transitions in development and disease, Cell 139 (2009) 871–890. [6] J. Yang, S.A. Mani, J.L. Donaher, S. Ramaswamy, R.A. Itzykson, C. Come, P. Savagner, I. Gitelman, A. Richardson, R.A. Weinberg, Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis, Cell 117 (2004) 927–939. [7] R. Maestro, A.P. Dei Tos, Y. Hamamori, S. Krasnokutsky, V. Sartorelli, L. Kedes, C. Doglioni, D.H. Beach, G.J. Hannon, Twist is a potential oncogene that inhibits apoptosis, Genes Dev. 13 (1999) 2207–2217. [8] S. Valsesia-Wittmann, M. Magdeleine, S. Dupasquier, E. Garin, A.C. Jallas, V. Combaret, A. Krause, P. Leissner, A. Puisieux, Oncogenic cooperation between H-Twist and N-Myc overrides failsafe programs in cancer cells, Cancer Cell 6 (2004) 625–630. [9] W.S. Wu, S. Heinrichs, D. Xu, S.P. Garrison, G.P. Zambetti, J.M. Adams, A.T. Look, Slug antagonizes p53-mediated apoptosis of hematopoietic progenitors by repressing puma, Cell 123 (2005) 641–653. [10] S. Ansieau, J. Bastid, A. Doreau, A.P. Morel, B.P. Bouchet, C. Thomas, F. Fauvet, I. Puisieux, C. Doglioni, S. Piccinin, R. Maestro, T. Voeltzel, A. Selmi, S. ValsesiaWittmann, F.C. Caron de, A. Puisieux, Induction of EMT by twist proteins as a collateral effect of tumor-promoting inactivation of premature senescence, Cancer Cell 14 (2008) 79–89. [11] M. Perez-Caro, C. Bermejo-Rodriguez, I. Gonzalez-Herrero, M. Sanchez-Beato, M.A. Piris, I. Sanchez-Garcia, Transcriptomal profiling of the cellular response to DNA damage mediated by Slug (Snai2), Br. J. Cancer 98 (2008) 480–488.

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