- Email: [email protected]
Failsafe program escape and EMT: A deleterious partnership
Seminars in Cancer Biology 21 (2011) 392–396
Contents lists available at SciVerse ScienceDirect
Seminars in Cancer Biology journal homepage: www.elsevier.com/locate/semcancer
Review
Failsafe program escape and EMT: A deleterious partnership Stéphane Ansieau a,b,c,d,∗, Stéphanie Courtois-Cox a,b,c,d, Anne-Pierre Morel a,b,c,d,e, Alain Puisieux a,b,c,d,e,f a
Inserm UMR-S1052, Centre de Recherche en Cancérologie, Lyon F-69008, France CNRS UMR5286, Centre de Recherche en Cancérologie, Lyon F-69008, France UNIV UMR1052, Centre de Recherche en Cancérologie, Lyon F-69008, France d Université de Lyon, F-69000 Lyon, France e Centre Léon Bérard, Lyon, F-69008 Lyon, France f Institut Universitaire de France, 75000 Paris, France b c
a r t i c l e
i n f o
Keywords: EMT Failsafe program escape Tumor initiation
a b s t r a c t The epithelial to mesenchymal transition (EMT) is a latent embryonic process which can be aberrantly reactivated during tumor progression. It is generally viewed as one of the main forces driving metastatic dissemination, by providing cells with invasive and motility capabilities. The aberrant reactivation of embryonic EMT inducers has now been additionally linked to escape from senescence and apoptosis, which suggests a role in tumor initiation. This oncogenic potential relies on the ability of EMT inducers to neutralize both the RB and p53 oncosuppressive pathways. RB and p53 have recently been described as key factors in the maintenance of epithelial morphology, which suggests an unexpected and intimate crosstalk between EMT and the corresponding safety programs. In this review, we attempt to understand how these two cell processes are interlinked and might facilitate cell transformation and tumor initiation. © 2011 Elsevier Ltd. All rights reserved.
Homeostasis in organisms is orchestrated at different levels. It involves subtle control of cell proliferation and survival in tissues, allowing both cyclical and injury-related cell renewal. Cancer cells can be viewed as selfish and renegade, proliferating independently of their neighboring cells and ignoring all alert signals. Their runaway proliferation is the consequence of escape from failsafe programs, particularly through neutralization of programs forcing cells either to undergo permanent growth arrest (senescence) or to commit suicide (apoptosis). Induction of these safety programs, originally observed upon infection of primary cells with mitogenic proteins, has now been unquestionably highlighted in numerous premalignant lesions. It is viewed as a natural barrier erected to preclude the emergence of hyperproliferative cells [1–6]. Neutralization of these programs is thus a determinant step in promoting neoplastic transformation. Failsafe program escape is achieved by thwarting oncosuppressive pathways, the main ones being under the control of the RB and p53 proteins. While genomic alterations contribute largely to loss of function of these proteins, indirect inactivation mechanisms also frequently intervene. Emerging evidence demonstrates that the aberrant reactivation of embryonic
programs governing cell migration and commitment during development, as frequently observed in human cancers, constitutes a recurrent avenue of escape from failsafe programs. One of these embryonic programs involves transdifferentiation of polarized, connected epithelial cells into individual, motile mesenchymal cells, a cell mechanism known as the epithelial to mesenchymal transition (EMT). Embryonic EMT inducers include the bHLH TWIST proteins, the zinc-finger SNAIL family, and the ZEB zinc-fingerhomeobox proteins; which display pleiotropic effects affecting cell shape, migration, survival, and commitment [7]. The multiple functionalities of EMT-inducing transcription factors rely on their ability to drive genetic programs governing various cellular aspects, including intercellular junctions, cytoskeleton organization, cell survival, and plasticity. Recent observations suggest that the aberrant reactivation of EMT inducers promotes failsafe program escape and confers stem-like properties and motility to cells. We discuss herein whether and how these processes might be interlinked.
∗ Corresponding author at: Inserm UMR S1052, CNRS UMR 5286, Centre de Recherche en Cancérologie de Lyon (CRCL). E-mail address: [email protected] (S. Ansieau).
EMT inducers play an important role during morphogenesis and organogenesis by regulating cell migration [8]. A large body of evidence also supports the view that EMT inducers additionally
1044-579X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcancer.2011.09.014
1. Embryonic EMT inducers at the crossroad between cell migration, commitment, and plasticity during development
S. Ansieau et al. / Seminars in Cancer Biology 21 (2011) 392–396
protect cells from anoikis during migration to new territories. The pro-survival properties of EMT inducers are exemplified by those of the members of the SNAIL family of proteins, which all display antiapoptotic properties, protecting cells as diverse as neural crest cells, hematopoietic precursors, and neurons from death [9–12]. Additionally, EMT inducers affect cell commitment, interfering with key regulators of diverse differentiation programs. For example, TWIST proteins are known to inhibit myogenic, osteogenic, and myelomonocytic differentiation by neutralizing the MyoD, MEF2, and RUNX proteins [13]. Collectively, these observations highlight the preponderant role of embryonic EMT inducers in cell migration, survival, proliferation, and differentiation. As these factors are pleiotropic, their aberrant reactivation observed under various pathological conditions is expected to deeply alter cell properties and to have a profound impact on cell behavior and fate. 2. Established roles of EMT in tumor progression Although this is a somewhat simplistic view, EMT inducers are considered undetectable in healthy adult differentiated cells. Their expression is aberrantly induced under pathological conditions such as fibrosis and during neoplastic transformation, where they generally constitute poor prognosis markers. Studies based on the pioneering observation that TWIST1 provides cancer cells with metastatic potential [14] have aimed at evaluating whether all embryonic EMT inducers behave as prometastatic factors. This is likely the case. Their ectopic expression was first found to confer motility and invasive properties to cells in in vitro experiments. Furthermore, detection of EMT inducer expression in human primary tumors was found to be statistically associated with a high incidence of metastatic dissemination [7]. Despite the difficulty of distinguishing epithelial cells committed into an EMT program from stromal cells, the prometastatic role of EMT has now been experimentally demonstrated in both in vitro and in vivo models [15]. Consistently with these findings, EMT inducers have been observed at the invasive fronts of human primary tumors [16]. Efforts to unravel the role of EMT in cancer progression have led to further investigating the consequences of the complete genetic reprogramming that characterizes the transdifferentiation process. In an experimental system, commitment of genetically modified human mammary epithelial cells to EMT has recently been associated with the acquisition of stem-like properties, including the ability to self-renew, an essential requisite for colonizing distant sites and forming secondary tumors [17–19]. Commitment to EMT additionally provides cells with a transforming and tumorigenic potential. This observation has led to the provocative hypothesis that cancer stem cells may arise from differentiated cells through EMT in a reversible and dynamic process [20]. As discussed elsewhere, EMT might be an adaptive process, allowing cancer cells to evade hostile environments generated by hypoxia, mechanical constraints, or nutrient deprivation. Furthermore, EMT might favor escape from radiotherapy, chemotherapy, or hormone therapy, which would link EMT to relapse [21]. Induction of failsafe programs leading to senescence or apoptosis can to some extent also be viewed as causing a stress that cells attempt spontaneously to evade. In the light of the properties of EMT, the possibility that EMT inducers and/or associated plasticity might help cells to escape from senescence or apoptosis is thus an appealing hypothesis. 3. EMT inducers are negative regulators of oncosuppressive pathways As previously mentioned, pioneering experiments have indicated that the embryonic transcription factor TWIST1 promotes cancer cell dissemination through EMT induction [14]. Further
393
studies have demonstrated that such prometastatic properties are actually shared by numerous embryonic EMT inducers [7]. In parallel has emerged a wealth of evidence that these embryonic transcription factors also display anti-apoptotic properties. The TWIST1 protein was successively shown both to counteract c-MYC-induced apoptosis in rat fibroblasts [22] and to act as a survival factor promoting human neuroblastoma development by overriding N-MYC-induced apoptosis [23]. TWIST proteins are now described as pleiotropic regulators of the p53 pathway, preventing the transcriptional activation of ARF in response to mitogenic insults, alleviating p53 activation by phosphorylation, inhibiting the DNA binding of p53 through direct interaction, and titrating its co-activators, thereby preventing activation of downstream target genes [13]. The ability to interfere with p53 now appears to be shared by a large number of EMT inducers, which are able either to interact directly with the oncosuppressive protein (as shown for SNAI1) [24], to alter the upstream ATM pathway (as demonstrated for SIP1) [25], or to hinder the induction of p53 target genes (as demonstrated for SNAI2) [26]. A property shared by many EMT inducers is the ability to alleviate the induction, in response to oncogenic activation, of cyclin kinase inhibitors such as CIP1, CDKN2A, and CDKN2B. This property accounts for the ability of proteins such as TWIST and ZEB1 to override oncogene-induced senescence and to cooperate with oncoproteins such as RAS and the EGF receptor in promoting cell transformation [27,28]. Reduced cyclin kinase inhibitor induction also enables ZEB1 to override replicative senescence, as judged by observations made on Zeb1-knockout mice [29]. Interestingly, TWIST1 and SNAI1 have recently been shown also to alleviate K-RASG12V -induced replicative senescence in the pancreas and lung [24,30]. This may explain the observed addiction (or amnesia as recently discussed [31]) of cancer cells to EMT inducers [32].
4. Failsafe program escape and EMT A growing body of evidence supports the view that alteration of RB- and p53-dependent oncosuppressive pathways actually promotes EMT. For instance, RB depletion through RNA interference in MCF10A mammary epithelial cells results in EMT induction, as revealed by morphological changes, an associated shift from epithelial to mesenchymal markers, and transcriptional activation of EMT inducers such as SNAI2 and ZEB1 [33]. In line with a role as a gatekeeper of epithelial morphology, a correlation between RB and E-Cadherin expression has been reported in breast ductal carcinomas [33]. In MEF cells, interestingly, the concomitant knockout of all three genes RB, RBL1, and RBL2 enables cells to form colonospheres when cultured at high density. These colonospheres include cancer stem cells as well as differentiated cells representative of all three embryonic lineages. Formation of these colonospheres is preceded by induction of Zeb1, which proves essential to generating cancer stem cells and to protecting them from anoikis [34]. In addition to substantiating the hypothesis that downregulation of EMT inducer activity might contribute to RB-protein-mediated oncosuppression [35], this observation also strongly suggests that EMT induction might facilitate cell transformation and tumor initiation. The p53 protein is also suggested to behave as a key regulator of epithelial morphology. Mechanistically, ectopic expression of p53 in HMECs or MCF12A cells has been found to induce expression of miRNA200c, previously shown to silence ZEB1 as well as stem cell factors in pancreatic and colorectal cancers [36]. Conversely, p53 depletion in MCF12A cells or its functional inactivation through ectopic expression of a transdominant negative version of p53 promotes EMT and confers stem-like properties to the cells [37]. On the basis of these observations, the authors conclude that loss of p53
394
S. Ansieau et al. / Seminars in Cancer Biology 21 (2011) 392–396
Fig. 1. Schematic representation of the interconnection between EMT and failsafe programs. Reactivation of EMT-promoting embryonic transcription factors, induced in response to various stresses including hypoxia, mechanical constraints, and inflammatory responses, turns down the RB and p53 pathways. Through a positive feedback mechanism, EMT inducer expression is further enhanced, thus promoting EMT. Maintaining RB and p53 down-modulated is essential to generating a permissive environment for genetic and epigenetic reprogramming, providing cells with plasticity and adaptive properties. Unfortunately, these conditions constitute a favorable environment for tumor initiation.
function promotes EMT. Yet many studies suggest that inhibition of the oncosuppressive protein p53 is actually not sufficient to induce complete transdifferentiation. Depletion of p53 in human mammary epithelial cells or its inactivation by the SV40 large T antigen is not associated with any significant morphological changes, mitogenic activation being an additional requisite [38]. Furthermore, the fact that EMT induction in prostatic epithelial cells, associated with transcriptional activation of TWIST1, appears to be specifically triggered by the p53R175H variant and not in response to p53 depletion suggests that this induction results from a gain of function provided by the mutation [39]. Consistently with this view, formation of a wild-type p53-MDM2-SNAI2 complex favors SNAI2 degradation in non-small cell lung cancer (NSCLC) cells, whereas mutant forms of p53 stabilize SNAI2, thereby promoting EMT. Furthermore, a combined “p53 mutation/lack of MDM2/SNAI2 expression” phenotype has been confirmed as a poor prognosis signature in NSCLC, associated with a high proportion of metastasis [40]. How can we combine and interpret jointly these data? If epithelial and mesenchymal cells represent, respectively, the starting and end points of EMT, obviously cells can be partially committed to transdifferentiation, changing certain properties (such as expression of EMT inducers) while retaining their epithelial morphology. The observed concomitant expression of epithelial and mesenchymal markers in carcinomas demonstrates that this is frequently the case. It is likely that alleviation of failsafe program induction in response to mitogenic activation, resulting from a loss of p53 or from its indirect inactivation, is sufficient to promote a partial EMT. The ability of p53 inactivation alone to induce the EMT in MCF12A cells might thus be due to the presence of oncogenic mutations in these cells. Alternatively, as a basal cell line, MCF12A might already be largely committed to the EMT (these cells display an active TGF pathway and express Vimentin) [41]. If so, p53 depletion might trigger completion of the transdifferentiation process. Although EMT regulation through p53 remains a matter of debate, the protein p21CIP1 , a transcriptional target of p53, acts unquestionably as an EMT inhibitor. Ectopic expression of this cyclin kinase inhibitor prevents human mammary epithelial cell commitment to an EMT. Consistently with this, knocking out CDNK2A has been found to significantly increase the incidence of mammary and salivary tumors in MMTV-RAS transgenic mice, the lack of p21CIP1 being associated with EMT and stemness pathway induction [42,43]. Conversely and noticeably p21CIP1 , which has
little impact on c-MYC-induced EMT, reduces the incidence of tumor development in MMTV-MYC transgenic mice [42,43]. On the basis of these observations one might speculate that EMTpermissive conditions facilitate tumor initiation. In line with this hypothesis, artificially substituting N-Cadherin for E-Cadherin during intestine development causes severe dysplasia and the appearance of clusters of cells with neoplastic features [44]. The intimate crosstalk between EMT and failsafe program escape has recently been further confirmed by the finding that BMI1 is a direct target of TWIST1 and that the Polycomb protein is recruited by the embryonic transcription factor to both INK4A and CDH1 promoter sequences. BMI1 and TWIST thus appear to cooperate in regulating EMT, senescence, and the appearance of stem-like properties in HNSCC cells. Accordingly, their coexpression has been associated with the worst outcome in HNSCC patients [45]. 5. EMT and failsafe program escape: the chicken or the egg causality dilemma The intimate crosstalk between failsafe program escape and EMT induction raises the question of the order in which these events take place during tumor progression. Loss of cell polarity and tight junctions might induce EMT inducer expression, as demonstrated by experimental E-Cadherin depletion in SV40T/tantigen-expressing HMECs [46]. Accordingly, CDH1 knockout has been shown to increase breast cancer incidence in WAP-p53−/− mice and to favor cancer cell dissemination [47]. Cell depolarization, potentially initiated in response to a genetic alteration, might thus be a determining event in reactivation of EMT inducer expression and in initiating cell commitment to EMT. Alternatively or additionally, embryonic inducer reactivation might be selected to allow escape from oncogene-induced senescence. As mentioned above, this has been clearly established for the TWIST1 and ZEB1 proteins, shown to abolish senescence induced by RAS and EGFR respectively and to cooperate with these mitogenic oncoproteins in cell transformation [27,28,48]. Consistently with this, it has been demonstrated that Twist1 reactivation is initiated as early as the hyperplasia stage in the MMTV-ErbB2/Neu mammary tumor progression model [49]. Furthermore, the TWIST1 protein is detectable in numerous tumors but absent from corresponding premalignant lesions [27,50–52]. The addiction of RAS-mutation-harboring cancer cells to EMT inducers also supports this hypothesis [30,53].
S. Ansieau et al. / Seminars in Cancer Biology 21 (2011) 392–396
Downregulation of RB and p53 by EMT inducers during transdifferentiation might then be essential to establishing a permissive epigenetic state and to alleviating cell senescence, as demonstrated during iPS generation [54–57]. Cells having undergone mitogenic activation and engaged in an EMT program might thus be particularly prone to evolve to a malignant stage. In keeping with this view, induction of ZEB1, by turning down miR200c expression, can modulate the sensitivity of tumor cells to Cd95-mediated apoptosis [58] and activate the Notch pathway, thus favoring tumor progression [59]. 6. Conclusions Although this remains to be fully demonstrated, EMT inducers might play a biphasic role during tumor progression. Failsafe program escape, when associated with embryonic inducer reactivation, might initiate cell commitment to an EMT program. The resulting intermediate state would provide cells with the potential to transit from an epithelial to a mesenchymal morphology and vice versa in a dynamic process governed by microenvironmental conditions. This plasticity is likely to be determinant in escape from multiple stresses but also in adaptation to novel environments encountered when cells evade from the primary tumor to colonize secondary sites. Maintaining the RB and p53 oncosuppressive pathways at a basic threshold might facilitate the profound epigenetic and genetic changes that drive the transdifferentiation process. As a side effect, a subset of cells might evolve to a malignant stage, this leading to tumor initiation (Fig. 1). In other words, the observed interplay between failsafe programs and EMT may involve distinct mechanisms. The dual properties of numerous embryonic transcription factors might explain the concomitant escape from failsafe programs and EMT induction, while downregulation of oncosuppressive pathways might constitute an intrinsic process essential to EMT induction and cell survival during cell reprogramming. Conflict of interest statement The authors declare no conflict of interest related to this work. Funding Our research is funded by the Ligue Nationale contre Le Cancer, the Association pour la Recherche sur le Cancer, the Institut National du Cancer, and the Project National Cancer Association. Acknowledgments We are grateful to Dr. David Cox and Dr. Katleen Broman for critical reading of the manuscript. SCC is a recipient of fellowships from the Fondation de France (Project #2007-005222). References [1] Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 2005;434:864–70. [2] Braig M, Lee S, Loddenkemper C, Rudolph C, Peters AH, Schlegelberger B, et al. Oncogene-induced senescence as an initial barrier in lymphoma development. Nature 2005;436:660–5. [3] Collado M, Gil J, Efeyan A, Guerra C, Schuhmacher AJ, Barradas M, et al. Tumour biology: senescence in premalignant tumours. Nature 2005;436:642. [4] Chen Z, Trotman LC, Shaffer D, Lin H-K, Dotan ZA, Niki M, et al. Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature 2005;436:725–30. [5] Gorgoulis VG, Vassiliou LV, Karakaidos P, Zacharatos P, Kotsinas A, Liloglou T, et al. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 2005;434:907–13.
395
[6] Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N, et al. Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 2006;444:633–7. [7] Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 2007;7:415–28. [8] Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial–mesenchymal transitions in development and disease. Cell 2009;139:871–90. [9] Tribulo C, Aybar MJ, Sanchez SS, Mayor R. A balance between the anti-apoptotic activity of Slug and the apoptotic activity of msx1 is required for the proper development of the neural crest. Dev Biol 2004;275:325–42. [10] Vega S, Morales AV, Ocana OH, Valdes F, Fabregat I, Nieto MA. Snail blocks the cell cycle and confers resistance to cell death. Genes Dev 2004;8:1131–43. [11] Wu WS, Heinrichs S, Xu D, Garrison SP, Zambetti GP, Adams JM, et al. Slug antagonizes p53-mediated apoptosis of hematopoietic progenitors by repressing puma. Cell 2005;123:641–53. [12] Rodriguez-Aznar E, Nieto MA. Repression of Puma by Scratch2 is required for neuronal survival during embryonic development. Cell Death Differ 2011;18:1196–207. [13] Ansieau S, Morel A-P, Hinkal G, Bastid J, Puisieux A. TWISTing an embryonic transcription factor into an oncoprotein. Oncogene 2010;29:3173–84. [14] Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 2004;117:927–39. [15] Trimboli AJ, Fukino K, de Bruin A, Wei G, Shen L, Tanner SM, et al. Direct evidence for epithelial–mesenchymal transitions in breast cancer. Cancer Res 2008;68:937–45. [16] Brabletz T, Jung A, Hermann K, Gunther K, Hohenberger W, Kirchner T. Nuclear overexpression of the oncoprotein beta-catenin in colorectal cancer is localized predominantly at the invasion front. Pathol Res Pract 1998;194:701–4. [17] Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al. The epithelial–mesenchymal transition generates cells with properties of stem cells. Cell 2008;133:704–15. [18] Morel A-P, Lievre M, Thomas C, Hinkal G, Ansieau S, Puisieux A. Generation of breast cancer stem cells through epithelial–mesenchymal transition. PLoS One 2008;3:e2888. [19] Vesuna F, Lisok A, Kimble B, Raman V. Twist modulates breast cancer stem cells by transcriptional regulation of CD24 expression. Neoplasia 2009;11:1318–28. [20] Gupta PB, Chaffer CL, Weinberg RA. Cancer stem cells: mirage or reality? Nat Med 2009;15:1010–2. [21] Ansieau S, Morel A-P, Puisieux A. From where do cancer-initiating cells originate? Cancer Stem Cells Theories and Practice (InTech) 2011;34–46. [22] Maestro R, Dei Tos AP, Hamamori Y, Krasnokutsky S, Sartorelli V, Kedes L, et al. Twist is a potential oncogene that inhibits apoptosis. Genes Dev 1999;13:2207–17. [23] Valsesia-Wittmann S, Magdeleine M, Dupasquier S, Garin E, Jallas A-C, Combaret V, et al. Oncogenic cooperation between H-Twist and N-Myc overrides failsafe programs in cancer cells. Cancer Cell 2004;6:625–30. [24] Lee SH, Lee SJ, Jung YS, Xu Y, Kang HS, Ha NC, et al. Blocking of p53-Snail binding, promoted by oncogenic K-Ras, recovers p53 expression and function. Neoplasia 2009;11:22–31. [25] Sayan AE, Griffiths TR, Pal R, Browne GJ, Ruddick A, Yagci T, et al. SIP1 protein protects cells from DNA damage-induced apoptosis and has independent prognostic value in bladder cancer. Proc Natl Acad Sci USA 2009;106:14884–9. [26] Perez-Caro M, Bermejo-Rodriguez C, Gonzalez-Herrero I, Sanchez-Beato M, Piris MA, Sanchez-Garcia I. Transcriptomal profiling of the cellular response to DNA damage mediated by Slug (Snai2). Br J Cancer 2008;98:480–8. [27] Ansieau S, Bastid J, Doreau A, Morel A-P, Bouchet BP, Thomas C, et al. Induction of EMT by twist proteins as a collateral effect of tumor-promoting inactivation of premature senescence. Cancer Cell 2008;14:79–89. [28] Ohashi S, Natsuizaka M, Wong GS, Michaylira CZ, Grugan KD, Stairs DB, et al. Epidermal growth factor receptor and mutant p53 expand an esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors. Cancer Res 2010;70:4174–84. [29] Liu Y, El-Naggar S, Darling DS, Higashi Y, Dean DC. Zeb1 links epithelial–mesenchymal transition and cellular senescence. Development 2008;135:579–88. [30] Lee KE, Bar-Sagi D. Oncogenic KRas suppresses inflammation-associated senescence of pancreatic ductal cells. Cancer Cell 2010;18:448–58. [31] Felsher DW. Oncogene addiction versus oncogene amnesia: perhaps more than just a bad habit? Cancer Res 2008;68:3081–6. [32] Wang Y, Ngo VN, Marani M, Yang Y, Wright G, Staudt LM, et al. Critical role for transcriptional repressor Snail2 in transformation by oncogenic RAS in colorectal carcinoma cells. Oncogene 2010;29:4658–70. [33] Arima Y, Inoue Y, Shibata T, Hayashi H, Nagano O, Saya H, et al. Rb depletion results in deregulation of E-cadherin and induction of cellular phenotypic changes that are characteristic of the epithelial-to-mesenchymal transition. Cancer Res 2008;68:5104–12. [34] Liu Y, Clem B, Zuba-Surma EK, El-Naggar S, Telang S, Jenson AB, et al. Mouse fibroblasts lacking RB1 function form spheres and undergo reprogramming to a cancer stem cell phenotype. Cell Stem Cell 2009;4:336–47. [35] Liu Y, Dean DC. Tumor initiation via loss of cell contact inhibition versus Ras mutation: do all roads lead to EMT? Cell Cycle 2010;9:897–900. [36] Wellner U, Schubert J, Burk UC, Schmalhofer O, Zhu F, Sonntag A, et al. The EMTactivator ZEB1 promotes tumorigenicity by repressing stemness-inhibiting microRNAs. Nat Cell Biol 2009;11:1487–95.
396
S. Ansieau et al. / Seminars in Cancer Biology 21 (2011) 392–396
[37] Chang CJ, Chao CH, Xia W, Yang JY, Xiong Y, Li CW, et al. p53 regulates epithelial–mesenchymal transition and stem cell properties through modulating miRNAs. Nat Cell Biol 2011;13:317–23. [38] Elenbaas B, Spirio L, Koerner F, Fleming MD, Zimonjic DB, Donaher JL, et al. Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells. Genes Dev 2001;15:50–65. [39] Kogan-Sakin I, Tabach Y, Buganim Y, Molchadsky A, Solomon H, Madar S, et al. Mutant p53(R175H) upregulates Twist1 expression and promotes epithelial–mesenchymal transition in immortalized prostate cells. Cell Death Differ 2011;18:271–81. [40] Wang SP, Wang WL, Chang YL, Wu CT, Chao YC, Kao SH, et al. p53 controls cancer cell invasion by inducing the MDM2-mediated degradation of Slug. Nat Cell Biol 2009;11:694–704. [41] Blick T, Hugo H, Widodo E, Waltham M, Pinto C, Mani SA, et al. Epithelial mesenchymal transition traits in human breast cancer cell lines parallel the CD44(hi/)CD24 (lo/-) stem cell phenotype in human breast cancer. J Mammary Gland Biol Neoplasia 2010;15:235–52. [42] Bearss DJ, Lee RJ, Troyer DA, Pestell RG, Windle JJ. Differential effects of p21(WAF1/CIP1) deficiency on MMTV-ras and MMTV-myc mammary tumor properties. Cancer Res 2002;62:2077–84. [43] Liu M, Casimiro MC, Wang C, Shirley LA, Jiao X, Katiyar S, et al. p21CIP1 attenuates Ras- and c-Myc-dependent breast tumor epithelial mesenchymal transition and cancer stem cell-like gene expression in vivo. Proc Natl Acad Sci USA 2009;106:19035–9. [44] Libusova L, Stemmler MP, Hierholzer A, Schwarz H, Kemler R. N-cadherin can structurally substitute for E-cadherin during intestinal development but leads to polyp formation. Development 2010;137:2297–305. [45] Yang MH, Hsu DS, Wang HW, Wang HJ, Lan HY, Yang WH, et al. Bmi1 is essential in Twist1-induced epithelial–mesenchymal transition. Nat Cell Biol 2010;12:982–92. [46] Onder TT, Gupta PB, Mani SA, Yang J, Lander ES, Weinberg RA. Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res 2008;68:3645–54. [47] Derksen PW, Braumuller TM, van der Burg E, Hornsveld M, Mesman E, Wesseling J, et al. Mammary-specific inactivation of E-cadherin and p53 impairs
[48]
[49] [50]
[51]
[52]
[53]
[54]
[55]
[56] [57]
[58] [59]
functional gland development and leads to pleomorphic invasive lobular carcinoma in mice. Dis Model Mech 2011;4:347–58. Kwok WK, Ling MT, Yuen HF, Wong YC, Wang X. Role of p14ARF in TWIST-mediated senescence in prostate epithelial cells. Carcinogenesis 2007;28:2467–75. Husemann Y, Geigl JB, Schubert F, Musiani P, Meyer M, Burghart E, et al. Systemic spread is an early step in breast cancer. Cancer Cell 2008;13:58–68. Kwok WK, Ling MT, Lee TW, Lau TC, Zhou C, Zhang X, et al. Up-regulation of TWIST in prostate cancer and its implication as a therapeutic target. Cancer Res 2005;65:5153–62. Ohuchida K, Mizumoto K, Ohhashi S, Yamaguchi H, Konomi H, Nagai E, et al. Twist, a novel oncogene, is upregulated in pancreatic cancer: clinical implication of Twist expression in pancreatic juice. Int J Cancer 2007;120:1634–40. Zhang Z, Xie D, Li X, Wong YC, Xin D, Guan XY, et al. Significance of TWIST expression and its association with E-cadherin in bladder cancer. Hum Pathol 2007;38:598–606. Singh A, Greninger P, Rhodes D, Koopman L, Violette S, Bardeesy N, et al. A gene expression signature associated with “K-Ras addiction” reveals regulators of EMT and tumor cell survival. Cancer Cell 2009;15:489–500. Hong H, Takahashi K, Ichisaka T, Aoi T, Kanagawa O, Nakagawa M, et al. Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 2009;460:1132–5. Kawamura T, Suzuki J, Wang YV, Menendez S, Morera LB, Raya A, et al. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 2009;460:1140–4. Li H, Collado M, Villasante A, Strati K, Ortega S, Canamero M, et al. The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature 2009;460:1136–9. Utikal J, Polo JM, Stadtfeld M, Maherali N, Kulalert W, Walsh RM, et al. Immortalization eliminates a roadblock during cellular reprogramming into iPS cells. Nature 2009;460:1145–8. Schickel R, Park SM, Murmann AE, Peter ME. miR-200c regulates induction of apoptosis through CD95 by targeting FAP-1. Mol Cell 2010;38:908–15. Brabletz S, Bajdak K, Meidhof S, Burk U, Niedermann G, Firat E, et al. The ZEB1/miR-200 feedback loop controls Notch signalling in cancer cells. EMBO J 2011;30:770–82.
Contents lists available at SciVerse ScienceDirect
Seminars in Cancer Biology journal homepage: www.elsevier.com/locate/semcancer
Review
Failsafe program escape and EMT: A deleterious partnership Stéphane Ansieau a,b,c,d,∗, Stéphanie Courtois-Cox a,b,c,d, Anne-Pierre Morel a,b,c,d,e, Alain Puisieux a,b,c,d,e,f a
Inserm UMR-S1052, Centre de Recherche en Cancérologie, Lyon F-69008, France CNRS UMR5286, Centre de Recherche en Cancérologie, Lyon F-69008, France UNIV UMR1052, Centre de Recherche en Cancérologie, Lyon F-69008, France d Université de Lyon, F-69000 Lyon, France e Centre Léon Bérard, Lyon, F-69008 Lyon, France f Institut Universitaire de France, 75000 Paris, France b c
a r t i c l e
i n f o
Keywords: EMT Failsafe program escape Tumor initiation
a b s t r a c t The epithelial to mesenchymal transition (EMT) is a latent embryonic process which can be aberrantly reactivated during tumor progression. It is generally viewed as one of the main forces driving metastatic dissemination, by providing cells with invasive and motility capabilities. The aberrant reactivation of embryonic EMT inducers has now been additionally linked to escape from senescence and apoptosis, which suggests a role in tumor initiation. This oncogenic potential relies on the ability of EMT inducers to neutralize both the RB and p53 oncosuppressive pathways. RB and p53 have recently been described as key factors in the maintenance of epithelial morphology, which suggests an unexpected and intimate crosstalk between EMT and the corresponding safety programs. In this review, we attempt to understand how these two cell processes are interlinked and might facilitate cell transformation and tumor initiation. © 2011 Elsevier Ltd. All rights reserved.
Homeostasis in organisms is orchestrated at different levels. It involves subtle control of cell proliferation and survival in tissues, allowing both cyclical and injury-related cell renewal. Cancer cells can be viewed as selfish and renegade, proliferating independently of their neighboring cells and ignoring all alert signals. Their runaway proliferation is the consequence of escape from failsafe programs, particularly through neutralization of programs forcing cells either to undergo permanent growth arrest (senescence) or to commit suicide (apoptosis). Induction of these safety programs, originally observed upon infection of primary cells with mitogenic proteins, has now been unquestionably highlighted in numerous premalignant lesions. It is viewed as a natural barrier erected to preclude the emergence of hyperproliferative cells [1–6]. Neutralization of these programs is thus a determinant step in promoting neoplastic transformation. Failsafe program escape is achieved by thwarting oncosuppressive pathways, the main ones being under the control of the RB and p53 proteins. While genomic alterations contribute largely to loss of function of these proteins, indirect inactivation mechanisms also frequently intervene. Emerging evidence demonstrates that the aberrant reactivation of embryonic
programs governing cell migration and commitment during development, as frequently observed in human cancers, constitutes a recurrent avenue of escape from failsafe programs. One of these embryonic programs involves transdifferentiation of polarized, connected epithelial cells into individual, motile mesenchymal cells, a cell mechanism known as the epithelial to mesenchymal transition (EMT). Embryonic EMT inducers include the bHLH TWIST proteins, the zinc-finger SNAIL family, and the ZEB zinc-fingerhomeobox proteins; which display pleiotropic effects affecting cell shape, migration, survival, and commitment [7]. The multiple functionalities of EMT-inducing transcription factors rely on their ability to drive genetic programs governing various cellular aspects, including intercellular junctions, cytoskeleton organization, cell survival, and plasticity. Recent observations suggest that the aberrant reactivation of EMT inducers promotes failsafe program escape and confers stem-like properties and motility to cells. We discuss herein whether and how these processes might be interlinked.
∗ Corresponding author at: Inserm UMR S1052, CNRS UMR 5286, Centre de Recherche en Cancérologie de Lyon (CRCL). E-mail address: [email protected] (S. Ansieau).
EMT inducers play an important role during morphogenesis and organogenesis by regulating cell migration [8]. A large body of evidence also supports the view that EMT inducers additionally
1044-579X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcancer.2011.09.014
1. Embryonic EMT inducers at the crossroad between cell migration, commitment, and plasticity during development
S. Ansieau et al. / Seminars in Cancer Biology 21 (2011) 392–396
protect cells from anoikis during migration to new territories. The pro-survival properties of EMT inducers are exemplified by those of the members of the SNAIL family of proteins, which all display antiapoptotic properties, protecting cells as diverse as neural crest cells, hematopoietic precursors, and neurons from death [9–12]. Additionally, EMT inducers affect cell commitment, interfering with key regulators of diverse differentiation programs. For example, TWIST proteins are known to inhibit myogenic, osteogenic, and myelomonocytic differentiation by neutralizing the MyoD, MEF2, and RUNX proteins [13]. Collectively, these observations highlight the preponderant role of embryonic EMT inducers in cell migration, survival, proliferation, and differentiation. As these factors are pleiotropic, their aberrant reactivation observed under various pathological conditions is expected to deeply alter cell properties and to have a profound impact on cell behavior and fate. 2. Established roles of EMT in tumor progression Although this is a somewhat simplistic view, EMT inducers are considered undetectable in healthy adult differentiated cells. Their expression is aberrantly induced under pathological conditions such as fibrosis and during neoplastic transformation, where they generally constitute poor prognosis markers. Studies based on the pioneering observation that TWIST1 provides cancer cells with metastatic potential [14] have aimed at evaluating whether all embryonic EMT inducers behave as prometastatic factors. This is likely the case. Their ectopic expression was first found to confer motility and invasive properties to cells in in vitro experiments. Furthermore, detection of EMT inducer expression in human primary tumors was found to be statistically associated with a high incidence of metastatic dissemination [7]. Despite the difficulty of distinguishing epithelial cells committed into an EMT program from stromal cells, the prometastatic role of EMT has now been experimentally demonstrated in both in vitro and in vivo models [15]. Consistently with these findings, EMT inducers have been observed at the invasive fronts of human primary tumors [16]. Efforts to unravel the role of EMT in cancer progression have led to further investigating the consequences of the complete genetic reprogramming that characterizes the transdifferentiation process. In an experimental system, commitment of genetically modified human mammary epithelial cells to EMT has recently been associated with the acquisition of stem-like properties, including the ability to self-renew, an essential requisite for colonizing distant sites and forming secondary tumors [17–19]. Commitment to EMT additionally provides cells with a transforming and tumorigenic potential. This observation has led to the provocative hypothesis that cancer stem cells may arise from differentiated cells through EMT in a reversible and dynamic process [20]. As discussed elsewhere, EMT might be an adaptive process, allowing cancer cells to evade hostile environments generated by hypoxia, mechanical constraints, or nutrient deprivation. Furthermore, EMT might favor escape from radiotherapy, chemotherapy, or hormone therapy, which would link EMT to relapse [21]. Induction of failsafe programs leading to senescence or apoptosis can to some extent also be viewed as causing a stress that cells attempt spontaneously to evade. In the light of the properties of EMT, the possibility that EMT inducers and/or associated plasticity might help cells to escape from senescence or apoptosis is thus an appealing hypothesis. 3. EMT inducers are negative regulators of oncosuppressive pathways As previously mentioned, pioneering experiments have indicated that the embryonic transcription factor TWIST1 promotes cancer cell dissemination through EMT induction [14]. Further
393
studies have demonstrated that such prometastatic properties are actually shared by numerous embryonic EMT inducers [7]. In parallel has emerged a wealth of evidence that these embryonic transcription factors also display anti-apoptotic properties. The TWIST1 protein was successively shown both to counteract c-MYC-induced apoptosis in rat fibroblasts [22] and to act as a survival factor promoting human neuroblastoma development by overriding N-MYC-induced apoptosis [23]. TWIST proteins are now described as pleiotropic regulators of the p53 pathway, preventing the transcriptional activation of ARF in response to mitogenic insults, alleviating p53 activation by phosphorylation, inhibiting the DNA binding of p53 through direct interaction, and titrating its co-activators, thereby preventing activation of downstream target genes [13]. The ability to interfere with p53 now appears to be shared by a large number of EMT inducers, which are able either to interact directly with the oncosuppressive protein (as shown for SNAI1) [24], to alter the upstream ATM pathway (as demonstrated for SIP1) [25], or to hinder the induction of p53 target genes (as demonstrated for SNAI2) [26]. A property shared by many EMT inducers is the ability to alleviate the induction, in response to oncogenic activation, of cyclin kinase inhibitors such as CIP1, CDKN2A, and CDKN2B. This property accounts for the ability of proteins such as TWIST and ZEB1 to override oncogene-induced senescence and to cooperate with oncoproteins such as RAS and the EGF receptor in promoting cell transformation [27,28]. Reduced cyclin kinase inhibitor induction also enables ZEB1 to override replicative senescence, as judged by observations made on Zeb1-knockout mice [29]. Interestingly, TWIST1 and SNAI1 have recently been shown also to alleviate K-RASG12V -induced replicative senescence in the pancreas and lung [24,30]. This may explain the observed addiction (or amnesia as recently discussed [31]) of cancer cells to EMT inducers [32].
4. Failsafe program escape and EMT A growing body of evidence supports the view that alteration of RB- and p53-dependent oncosuppressive pathways actually promotes EMT. For instance, RB depletion through RNA interference in MCF10A mammary epithelial cells results in EMT induction, as revealed by morphological changes, an associated shift from epithelial to mesenchymal markers, and transcriptional activation of EMT inducers such as SNAI2 and ZEB1 [33]. In line with a role as a gatekeeper of epithelial morphology, a correlation between RB and E-Cadherin expression has been reported in breast ductal carcinomas [33]. In MEF cells, interestingly, the concomitant knockout of all three genes RB, RBL1, and RBL2 enables cells to form colonospheres when cultured at high density. These colonospheres include cancer stem cells as well as differentiated cells representative of all three embryonic lineages. Formation of these colonospheres is preceded by induction of Zeb1, which proves essential to generating cancer stem cells and to protecting them from anoikis [34]. In addition to substantiating the hypothesis that downregulation of EMT inducer activity might contribute to RB-protein-mediated oncosuppression [35], this observation also strongly suggests that EMT induction might facilitate cell transformation and tumor initiation. The p53 protein is also suggested to behave as a key regulator of epithelial morphology. Mechanistically, ectopic expression of p53 in HMECs or MCF12A cells has been found to induce expression of miRNA200c, previously shown to silence ZEB1 as well as stem cell factors in pancreatic and colorectal cancers [36]. Conversely, p53 depletion in MCF12A cells or its functional inactivation through ectopic expression of a transdominant negative version of p53 promotes EMT and confers stem-like properties to the cells [37]. On the basis of these observations, the authors conclude that loss of p53
394
S. Ansieau et al. / Seminars in Cancer Biology 21 (2011) 392–396
Fig. 1. Schematic representation of the interconnection between EMT and failsafe programs. Reactivation of EMT-promoting embryonic transcription factors, induced in response to various stresses including hypoxia, mechanical constraints, and inflammatory responses, turns down the RB and p53 pathways. Through a positive feedback mechanism, EMT inducer expression is further enhanced, thus promoting EMT. Maintaining RB and p53 down-modulated is essential to generating a permissive environment for genetic and epigenetic reprogramming, providing cells with plasticity and adaptive properties. Unfortunately, these conditions constitute a favorable environment for tumor initiation.
function promotes EMT. Yet many studies suggest that inhibition of the oncosuppressive protein p53 is actually not sufficient to induce complete transdifferentiation. Depletion of p53 in human mammary epithelial cells or its inactivation by the SV40 large T antigen is not associated with any significant morphological changes, mitogenic activation being an additional requisite [38]. Furthermore, the fact that EMT induction in prostatic epithelial cells, associated with transcriptional activation of TWIST1, appears to be specifically triggered by the p53R175H variant and not in response to p53 depletion suggests that this induction results from a gain of function provided by the mutation [39]. Consistently with this view, formation of a wild-type p53-MDM2-SNAI2 complex favors SNAI2 degradation in non-small cell lung cancer (NSCLC) cells, whereas mutant forms of p53 stabilize SNAI2, thereby promoting EMT. Furthermore, a combined “p53 mutation/lack of MDM2/SNAI2 expression” phenotype has been confirmed as a poor prognosis signature in NSCLC, associated with a high proportion of metastasis [40]. How can we combine and interpret jointly these data? If epithelial and mesenchymal cells represent, respectively, the starting and end points of EMT, obviously cells can be partially committed to transdifferentiation, changing certain properties (such as expression of EMT inducers) while retaining their epithelial morphology. The observed concomitant expression of epithelial and mesenchymal markers in carcinomas demonstrates that this is frequently the case. It is likely that alleviation of failsafe program induction in response to mitogenic activation, resulting from a loss of p53 or from its indirect inactivation, is sufficient to promote a partial EMT. The ability of p53 inactivation alone to induce the EMT in MCF12A cells might thus be due to the presence of oncogenic mutations in these cells. Alternatively, as a basal cell line, MCF12A might already be largely committed to the EMT (these cells display an active TGF pathway and express Vimentin) [41]. If so, p53 depletion might trigger completion of the transdifferentiation process. Although EMT regulation through p53 remains a matter of debate, the protein p21CIP1 , a transcriptional target of p53, acts unquestionably as an EMT inhibitor. Ectopic expression of this cyclin kinase inhibitor prevents human mammary epithelial cell commitment to an EMT. Consistently with this, knocking out CDNK2A has been found to significantly increase the incidence of mammary and salivary tumors in MMTV-RAS transgenic mice, the lack of p21CIP1 being associated with EMT and stemness pathway induction [42,43]. Conversely and noticeably p21CIP1 , which has
little impact on c-MYC-induced EMT, reduces the incidence of tumor development in MMTV-MYC transgenic mice [42,43]. On the basis of these observations one might speculate that EMTpermissive conditions facilitate tumor initiation. In line with this hypothesis, artificially substituting N-Cadherin for E-Cadherin during intestine development causes severe dysplasia and the appearance of clusters of cells with neoplastic features [44]. The intimate crosstalk between EMT and failsafe program escape has recently been further confirmed by the finding that BMI1 is a direct target of TWIST1 and that the Polycomb protein is recruited by the embryonic transcription factor to both INK4A and CDH1 promoter sequences. BMI1 and TWIST thus appear to cooperate in regulating EMT, senescence, and the appearance of stem-like properties in HNSCC cells. Accordingly, their coexpression has been associated with the worst outcome in HNSCC patients [45]. 5. EMT and failsafe program escape: the chicken or the egg causality dilemma The intimate crosstalk between failsafe program escape and EMT induction raises the question of the order in which these events take place during tumor progression. Loss of cell polarity and tight junctions might induce EMT inducer expression, as demonstrated by experimental E-Cadherin depletion in SV40T/tantigen-expressing HMECs [46]. Accordingly, CDH1 knockout has been shown to increase breast cancer incidence in WAP-p53−/− mice and to favor cancer cell dissemination [47]. Cell depolarization, potentially initiated in response to a genetic alteration, might thus be a determining event in reactivation of EMT inducer expression and in initiating cell commitment to EMT. Alternatively or additionally, embryonic inducer reactivation might be selected to allow escape from oncogene-induced senescence. As mentioned above, this has been clearly established for the TWIST1 and ZEB1 proteins, shown to abolish senescence induced by RAS and EGFR respectively and to cooperate with these mitogenic oncoproteins in cell transformation [27,28,48]. Consistently with this, it has been demonstrated that Twist1 reactivation is initiated as early as the hyperplasia stage in the MMTV-ErbB2/Neu mammary tumor progression model [49]. Furthermore, the TWIST1 protein is detectable in numerous tumors but absent from corresponding premalignant lesions [27,50–52]. The addiction of RAS-mutation-harboring cancer cells to EMT inducers also supports this hypothesis [30,53].
S. Ansieau et al. / Seminars in Cancer Biology 21 (2011) 392–396
Downregulation of RB and p53 by EMT inducers during transdifferentiation might then be essential to establishing a permissive epigenetic state and to alleviating cell senescence, as demonstrated during iPS generation [54–57]. Cells having undergone mitogenic activation and engaged in an EMT program might thus be particularly prone to evolve to a malignant stage. In keeping with this view, induction of ZEB1, by turning down miR200c expression, can modulate the sensitivity of tumor cells to Cd95-mediated apoptosis [58] and activate the Notch pathway, thus favoring tumor progression [59]. 6. Conclusions Although this remains to be fully demonstrated, EMT inducers might play a biphasic role during tumor progression. Failsafe program escape, when associated with embryonic inducer reactivation, might initiate cell commitment to an EMT program. The resulting intermediate state would provide cells with the potential to transit from an epithelial to a mesenchymal morphology and vice versa in a dynamic process governed by microenvironmental conditions. This plasticity is likely to be determinant in escape from multiple stresses but also in adaptation to novel environments encountered when cells evade from the primary tumor to colonize secondary sites. Maintaining the RB and p53 oncosuppressive pathways at a basic threshold might facilitate the profound epigenetic and genetic changes that drive the transdifferentiation process. As a side effect, a subset of cells might evolve to a malignant stage, this leading to tumor initiation (Fig. 1). In other words, the observed interplay between failsafe programs and EMT may involve distinct mechanisms. The dual properties of numerous embryonic transcription factors might explain the concomitant escape from failsafe programs and EMT induction, while downregulation of oncosuppressive pathways might constitute an intrinsic process essential to EMT induction and cell survival during cell reprogramming. Conflict of interest statement The authors declare no conflict of interest related to this work. Funding Our research is funded by the Ligue Nationale contre Le Cancer, the Association pour la Recherche sur le Cancer, the Institut National du Cancer, and the Project National Cancer Association. Acknowledgments We are grateful to Dr. David Cox and Dr. Katleen Broman for critical reading of the manuscript. SCC is a recipient of fellowships from the Fondation de France (Project #2007-005222). References [1] Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 2005;434:864–70. [2] Braig M, Lee S, Loddenkemper C, Rudolph C, Peters AH, Schlegelberger B, et al. Oncogene-induced senescence as an initial barrier in lymphoma development. Nature 2005;436:660–5. [3] Collado M, Gil J, Efeyan A, Guerra C, Schuhmacher AJ, Barradas M, et al. Tumour biology: senescence in premalignant tumours. Nature 2005;436:642. [4] Chen Z, Trotman LC, Shaffer D, Lin H-K, Dotan ZA, Niki M, et al. Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature 2005;436:725–30. [5] Gorgoulis VG, Vassiliou LV, Karakaidos P, Zacharatos P, Kotsinas A, Liloglou T, et al. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 2005;434:907–13.
395
[6] Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N, et al. Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 2006;444:633–7. [7] Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 2007;7:415–28. [8] Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial–mesenchymal transitions in development and disease. Cell 2009;139:871–90. [9] Tribulo C, Aybar MJ, Sanchez SS, Mayor R. A balance between the anti-apoptotic activity of Slug and the apoptotic activity of msx1 is required for the proper development of the neural crest. Dev Biol 2004;275:325–42. [10] Vega S, Morales AV, Ocana OH, Valdes F, Fabregat I, Nieto MA. Snail blocks the cell cycle and confers resistance to cell death. Genes Dev 2004;8:1131–43. [11] Wu WS, Heinrichs S, Xu D, Garrison SP, Zambetti GP, Adams JM, et al. Slug antagonizes p53-mediated apoptosis of hematopoietic progenitors by repressing puma. Cell 2005;123:641–53. [12] Rodriguez-Aznar E, Nieto MA. Repression of Puma by Scratch2 is required for neuronal survival during embryonic development. Cell Death Differ 2011;18:1196–207. [13] Ansieau S, Morel A-P, Hinkal G, Bastid J, Puisieux A. TWISTing an embryonic transcription factor into an oncoprotein. Oncogene 2010;29:3173–84. [14] Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 2004;117:927–39. [15] Trimboli AJ, Fukino K, de Bruin A, Wei G, Shen L, Tanner SM, et al. Direct evidence for epithelial–mesenchymal transitions in breast cancer. Cancer Res 2008;68:937–45. [16] Brabletz T, Jung A, Hermann K, Gunther K, Hohenberger W, Kirchner T. Nuclear overexpression of the oncoprotein beta-catenin in colorectal cancer is localized predominantly at the invasion front. Pathol Res Pract 1998;194:701–4. [17] Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al. The epithelial–mesenchymal transition generates cells with properties of stem cells. Cell 2008;133:704–15. [18] Morel A-P, Lievre M, Thomas C, Hinkal G, Ansieau S, Puisieux A. Generation of breast cancer stem cells through epithelial–mesenchymal transition. PLoS One 2008;3:e2888. [19] Vesuna F, Lisok A, Kimble B, Raman V. Twist modulates breast cancer stem cells by transcriptional regulation of CD24 expression. Neoplasia 2009;11:1318–28. [20] Gupta PB, Chaffer CL, Weinberg RA. Cancer stem cells: mirage or reality? Nat Med 2009;15:1010–2. [21] Ansieau S, Morel A-P, Puisieux A. From where do cancer-initiating cells originate? Cancer Stem Cells Theories and Practice (InTech) 2011;34–46. [22] Maestro R, Dei Tos AP, Hamamori Y, Krasnokutsky S, Sartorelli V, Kedes L, et al. Twist is a potential oncogene that inhibits apoptosis. Genes Dev 1999;13:2207–17. [23] Valsesia-Wittmann S, Magdeleine M, Dupasquier S, Garin E, Jallas A-C, Combaret V, et al. Oncogenic cooperation between H-Twist and N-Myc overrides failsafe programs in cancer cells. Cancer Cell 2004;6:625–30. [24] Lee SH, Lee SJ, Jung YS, Xu Y, Kang HS, Ha NC, et al. Blocking of p53-Snail binding, promoted by oncogenic K-Ras, recovers p53 expression and function. Neoplasia 2009;11:22–31. [25] Sayan AE, Griffiths TR, Pal R, Browne GJ, Ruddick A, Yagci T, et al. SIP1 protein protects cells from DNA damage-induced apoptosis and has independent prognostic value in bladder cancer. Proc Natl Acad Sci USA 2009;106:14884–9. [26] Perez-Caro M, Bermejo-Rodriguez C, Gonzalez-Herrero I, Sanchez-Beato M, Piris MA, Sanchez-Garcia I. Transcriptomal profiling of the cellular response to DNA damage mediated by Slug (Snai2). Br J Cancer 2008;98:480–8. [27] Ansieau S, Bastid J, Doreau A, Morel A-P, Bouchet BP, Thomas C, et al. Induction of EMT by twist proteins as a collateral effect of tumor-promoting inactivation of premature senescence. Cancer Cell 2008;14:79–89. [28] Ohashi S, Natsuizaka M, Wong GS, Michaylira CZ, Grugan KD, Stairs DB, et al. Epidermal growth factor receptor and mutant p53 expand an esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors. Cancer Res 2010;70:4174–84. [29] Liu Y, El-Naggar S, Darling DS, Higashi Y, Dean DC. Zeb1 links epithelial–mesenchymal transition and cellular senescence. Development 2008;135:579–88. [30] Lee KE, Bar-Sagi D. Oncogenic KRas suppresses inflammation-associated senescence of pancreatic ductal cells. Cancer Cell 2010;18:448–58. [31] Felsher DW. Oncogene addiction versus oncogene amnesia: perhaps more than just a bad habit? Cancer Res 2008;68:3081–6. [32] Wang Y, Ngo VN, Marani M, Yang Y, Wright G, Staudt LM, et al. Critical role for transcriptional repressor Snail2 in transformation by oncogenic RAS in colorectal carcinoma cells. Oncogene 2010;29:4658–70. [33] Arima Y, Inoue Y, Shibata T, Hayashi H, Nagano O, Saya H, et al. Rb depletion results in deregulation of E-cadherin and induction of cellular phenotypic changes that are characteristic of the epithelial-to-mesenchymal transition. Cancer Res 2008;68:5104–12. [34] Liu Y, Clem B, Zuba-Surma EK, El-Naggar S, Telang S, Jenson AB, et al. Mouse fibroblasts lacking RB1 function form spheres and undergo reprogramming to a cancer stem cell phenotype. Cell Stem Cell 2009;4:336–47. [35] Liu Y, Dean DC. Tumor initiation via loss of cell contact inhibition versus Ras mutation: do all roads lead to EMT? Cell Cycle 2010;9:897–900. [36] Wellner U, Schubert J, Burk UC, Schmalhofer O, Zhu F, Sonntag A, et al. The EMTactivator ZEB1 promotes tumorigenicity by repressing stemness-inhibiting microRNAs. Nat Cell Biol 2009;11:1487–95.
396
S. Ansieau et al. / Seminars in Cancer Biology 21 (2011) 392–396
[37] Chang CJ, Chao CH, Xia W, Yang JY, Xiong Y, Li CW, et al. p53 regulates epithelial–mesenchymal transition and stem cell properties through modulating miRNAs. Nat Cell Biol 2011;13:317–23. [38] Elenbaas B, Spirio L, Koerner F, Fleming MD, Zimonjic DB, Donaher JL, et al. Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells. Genes Dev 2001;15:50–65. [39] Kogan-Sakin I, Tabach Y, Buganim Y, Molchadsky A, Solomon H, Madar S, et al. Mutant p53(R175H) upregulates Twist1 expression and promotes epithelial–mesenchymal transition in immortalized prostate cells. Cell Death Differ 2011;18:271–81. [40] Wang SP, Wang WL, Chang YL, Wu CT, Chao YC, Kao SH, et al. p53 controls cancer cell invasion by inducing the MDM2-mediated degradation of Slug. Nat Cell Biol 2009;11:694–704. [41] Blick T, Hugo H, Widodo E, Waltham M, Pinto C, Mani SA, et al. Epithelial mesenchymal transition traits in human breast cancer cell lines parallel the CD44(hi/)CD24 (lo/-) stem cell phenotype in human breast cancer. J Mammary Gland Biol Neoplasia 2010;15:235–52. [42] Bearss DJ, Lee RJ, Troyer DA, Pestell RG, Windle JJ. Differential effects of p21(WAF1/CIP1) deficiency on MMTV-ras and MMTV-myc mammary tumor properties. Cancer Res 2002;62:2077–84. [43] Liu M, Casimiro MC, Wang C, Shirley LA, Jiao X, Katiyar S, et al. p21CIP1 attenuates Ras- and c-Myc-dependent breast tumor epithelial mesenchymal transition and cancer stem cell-like gene expression in vivo. Proc Natl Acad Sci USA 2009;106:19035–9. [44] Libusova L, Stemmler MP, Hierholzer A, Schwarz H, Kemler R. N-cadherin can structurally substitute for E-cadherin during intestinal development but leads to polyp formation. Development 2010;137:2297–305. [45] Yang MH, Hsu DS, Wang HW, Wang HJ, Lan HY, Yang WH, et al. Bmi1 is essential in Twist1-induced epithelial–mesenchymal transition. Nat Cell Biol 2010;12:982–92. [46] Onder TT, Gupta PB, Mani SA, Yang J, Lander ES, Weinberg RA. Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res 2008;68:3645–54. [47] Derksen PW, Braumuller TM, van der Burg E, Hornsveld M, Mesman E, Wesseling J, et al. Mammary-specific inactivation of E-cadherin and p53 impairs
[48]
[49] [50]
[51]
[52]
[53]
[54]
[55]
[56] [57]
[58] [59]
functional gland development and leads to pleomorphic invasive lobular carcinoma in mice. Dis Model Mech 2011;4:347–58. Kwok WK, Ling MT, Yuen HF, Wong YC, Wang X. Role of p14ARF in TWIST-mediated senescence in prostate epithelial cells. Carcinogenesis 2007;28:2467–75. Husemann Y, Geigl JB, Schubert F, Musiani P, Meyer M, Burghart E, et al. Systemic spread is an early step in breast cancer. Cancer Cell 2008;13:58–68. Kwok WK, Ling MT, Lee TW, Lau TC, Zhou C, Zhang X, et al. Up-regulation of TWIST in prostate cancer and its implication as a therapeutic target. Cancer Res 2005;65:5153–62. Ohuchida K, Mizumoto K, Ohhashi S, Yamaguchi H, Konomi H, Nagai E, et al. Twist, a novel oncogene, is upregulated in pancreatic cancer: clinical implication of Twist expression in pancreatic juice. Int J Cancer 2007;120:1634–40. Zhang Z, Xie D, Li X, Wong YC, Xin D, Guan XY, et al. Significance of TWIST expression and its association with E-cadherin in bladder cancer. Hum Pathol 2007;38:598–606. Singh A, Greninger P, Rhodes D, Koopman L, Violette S, Bardeesy N, et al. A gene expression signature associated with “K-Ras addiction” reveals regulators of EMT and tumor cell survival. Cancer Cell 2009;15:489–500. Hong H, Takahashi K, Ichisaka T, Aoi T, Kanagawa O, Nakagawa M, et al. Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 2009;460:1132–5. Kawamura T, Suzuki J, Wang YV, Menendez S, Morera LB, Raya A, et al. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 2009;460:1140–4. Li H, Collado M, Villasante A, Strati K, Ortega S, Canamero M, et al. The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature 2009;460:1136–9. Utikal J, Polo JM, Stadtfeld M, Maherali N, Kulalert W, Walsh RM, et al. Immortalization eliminates a roadblock during cellular reprogramming into iPS cells. Nature 2009;460:1145–8. Schickel R, Park SM, Murmann AE, Peter ME. miR-200c regulates induction of apoptosis through CD95 by targeting FAP-1. Mol Cell 2010;38:908–15. Brabletz S, Bajdak K, Meidhof S, Burk U, Niedermann G, Firat E, et al. The ZEB1/miR-200 feedback loop controls Notch signalling in cancer cells. EMBO J 2011;30:770–82.