Oncogene (2010) 29, 3173–3184 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc REVIEW TWISTing an embryonic transcription factor into an oncoprotein

S Ansieau1, A-P Morel4, G Hinkal1, J Bastid1,2 and A Puisieux1,2,3,4

1Inserm, U590, Lyon, France; 2Universite´ de Lyon, Lyon I, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France; 3Universite´ de Lyon, Lyon I, France and 4Centre Le´on Be´rard, Lyon, France

Over the past decade, the reactivation of TWIST TWIST proteins are pleiotropic gene regulators embryonic transcription factors has been described as a frequent event and a marker of poor prognosis in an TWIST proteins belong to the large family of basic impressive array of human cancers. Growing evidence now Helix–Loop–Helix (bHLH) transcription factors origin- supports the premise that these cancers hijack TWIST’s ally found to modulate the expression of various embryonic functions, granting oncogenic and metastatic target genes through canonical E-box responsive ele- properties. In this review, we report on the history and ments (Lee et al., 1997; Yin et al., 1997). Well conserved recent breakthroughs in understanding TWIST protein during evolution, two TWIST genes exist in vertebrates, functions and the emerging role of the associated epithelial– TWIST1 and TWIST2 (formerly Dermo-1). The mesenchymal transition (EMT) in tumorigenesis. We then TWIST proteins display a high degree of sequence broaden the discussion to address the general contribution similarity in their C-terminal half, including the bHLH of reactivating embryonic programs in cancerogenesis. domain and a ‘TWIST-box’ protein interaction surface Oncogene (2010) 29, 3173–3184; doi:10.1038/onc.2010.92; and are more divergent in their N terminus (Wolf et al., published online 12 April 2010 1991; Li et al., 1995; Bialek et al., 2004). Among the bHLH proteins, TWIST transcription factors are unique Keywords: TWIST; embryogenesis; EMT; failsafe in their ability to form functional homo- and hetero- program escape; cell dissemination dimers. These two types of complexes were found to display distinct, even antagonistic, properties as exem- plified by the mesoderm specification and the subse- Introduction quent allocation of mesodermal cells to the somatic muscle fate by Twi-Twi (Twi is the Drosophila TWIST Originally shown to be central to embryonic develop- ortholog) homodimers whereas heterodimers between ment, aberrant functions of the TWIST transcription Twi and the E protein homolog Daughterless (Da) factor family have grown and spread into many facets of repress genes required for somatic myogenesis (Casta- tumor development and progression. The initial link non et al., 2001). The balance between TWIST1 homo- between these proteins and cancer identified TWIST1 as and heterodimers (TWIST1-TCF3/E2A (TE) or a pro-metastatic protein in breast carcinomas. Since TWIST1-Hand2 protein complexes) was also shown to then, the roles of this family (TWIST1 and TWIST2) in direct appropriate limb development as well as cranial various types of solid human cancers have expanded to suture patterning and fusion in mice (Firulli et al., 2005; include the development of primary tumor growth, cell Connerney et al., 2006, 2008), suggesting that this dissemination and chemoresistance. Recent progress in TWIST-regulatory mechanism is conserved across understanding TWIST physiological and oncogenic species. These different dimers further differ in stability functions highlights a number of parallels suggesting as the association of TWIST with TCF3/E2A proteins that reactivation of TWIST-associated embryonic pro- protect TWIST from the ubiquitin-dependent protea- grams de facto contribute to tumor progression. The aim some degradation pathway (Hayashi et al., 2007). of this review is to evaluate the status of the field by Multiple factors, including the relative amounts of the highlighting the most recent breakthroughs in under- binding partners, their phosphorylation status as well as standing TWIST functions and identifying future the expression of Id HLH proteins (that preferentially avenues of investigation. We will attempt to substantiate interact with E2A, titrating it and thereby favoring the parallel between the embryonic and oncogenic TWIST homodimers) determine the relative amounts of properties of TWIST proteins and, in particular, address both types of dimers and thereby TWIST activity (for a the role of the associated epithelial–mesenchymal recent review, see Firulli and Conway, 2008). transition in tumor progression and cell dissemination. TWIST proteins behave either as transcriptional repressors, recruiting histone deacetylases or inhibiting Correspondence: Dr S Ansieau, Inserm, U590, Centre Le´on Be´rard, 28 acetyl-transferases, or as transcriptional activators rue Laennec, Lyon, 69008, France. (Hamamori et al., 1999; Gong and Li 2002; Pan et al., E-mail: [email protected] or Professor A Puisieux, Inserm U590, 2009). These functions are independent of the dimer Centre Le´on Be´rard, 28 rue Laennec, 69008 Lyon, France. E-mail: [email protected] type and appear to be tissue-dependent in Drosophila. Received 30 June 2009; revised 9 November 2009; accepted 22 February This switchable behavior is linked to a particular 2010; published online 12 April 2010 conserved domain of Da, which inactivates both Twi TWIST embryonic and oncogenic properties S Ansieau et al 3174 and Da transactivation domains through an intra- pathway (Maestro et al., 1999; Sosic et al., 2003; molecular mechanism (Wong et al., 2008). As the dom- Puisieux et al., 2006; Doreau et al., 2009). Cell death ain is conserved across species, we anticipate that it might also result from inappropriate premature differ- similarly dictates TWIST tissue-specific effects in higher entiation as evidence strongly suggests a role of TWIST organisms. in maintaining myoblast progenitor cells during devel- In addition, TWIST proteins directly interact with opment (Hebrok et al., 1994). In vertebrates, TWIST1 is several other transcription factors, including MyoD, initially expressed throughout the somatic mesoderm MEF2, RUNX1, RUNX2, PGC1-a, p53 and NF-kB, and is subsequently silenced in the forming myotome and inhibit their activities (Spicer et al., 1996; Hama- (Hopwood et al., 1989; Wolf et al., 1991). Similarly, in mori et al., 1999; Sosic et al., 2003; Bialek et al., 2004; Drosophila, Twi is expressed in the somatic mesoderm at Sharabi et al., 2008; Shiota et al., 2008; Pan et al., 2009). high levels before differentiation and is required for As discussed in the following section, inactivation of differentiation of the somatic muscles (Baylies and Bate these transcription factors provides TWIST proteins 1996). Its expression is maintained in clusters of with the ability to inhibit an array of differentiation undifferentiated proliferating cells that will give rise to pathways while endowing survival properties. adult myoblasts and ultimately muscles throughout the larval and early pupal stages (Bate et al., 1991). TWIST1 inhibitory properties rely on their ability to interact and inactivate myogenic factors such as MyoD and MEF2 Physiological functions of Twist proteins (Spicer et al., 1996; Hamamori et al., 1997). The TWIST1 protein is also likely to be important for Embryonic development and tissue specification maintaining the immature phenotype of chondrocytes. Twi was originally identified in Drosophila as a zygotic By comparing chick caudal and cephalic sternal developmental gene crucial for the establishment of the chondrocytes that display distinct capabilities to term- ventral furrow, a prerequisite for the development of all inally differentiate, TWIST1 was found to inhibit mesoderm-derived internal organs (Thisse et al., 1987). chondrocyte differentiation by turning down RUNX2 In mice, Twist1 haploinsufficiency produces viable expression (Dong et al., 2007). Accordingly, in mice, offspring but leads to abnormal craniofacial structures Twist1 mRNA was found to be expressed in immature and polydactyly in the hind limb, a phenotype chrondrocytes whereas the transcript is undetectable in reminiscent to the dominantly inherited Saethre–Chot- more mature ones (Hinoi et al., 2006). Similarly, zen syndrome in humans where TWIST1 is mutated TWIST1 was found to inhibit osteoblastic differentia- (Bourgeois et al., 1998). Twist 1À/À mice, in contrast to tion. In mice, Twist1 is downregulated during endo- flies, undergo normal gastrulation but die at E10.5-11 chondral and intramembranous fetal bone marrow displaying severe defects in closure of the cephalic neural development (Alborzi et al., 1996). During calvaria tube, deficient cranial mesoderm, malformed branchial and suture development, Twist1 is expressed at sides of arches and facial primordium, and retarded limb bud immature bone cells and to less extent in more mature development. TWIST1 therefore acts after mesoderm ones (Rice et al., 2000; Connerney et al., 2006). specification in vertebrates, but remains essential in In addition, in mice and humans (Saethre–Chotzen mesoderm differentiation including muscle, cartilage syndrome), haploinsufficiency of TWIST1 has been and osteogenic cell lineages (Hebrok et al., 1994; Spicer shown to cause craniosynostosis, a premature closure et al., 1996; Lee et al., 1999). Mesoderm formation in of the cranial sutures and limb development abnormal- vertebrates is actually controlled by SNAI1 and SNAI2 ities (Howard et al., 1997; el Ghouzzy et al., 1997; transcription factors (Nieto et al., 1994; Nieto 2002). Bourgeois et al., 1998; Gripp et al., 2000). This All three proteins are functionally related, sharing the phenotype has been linked with premature osteoblastic ability to promote a cellular transdifferentiation process differentiation, mediated by an alteration of the balance known as the epithelial–mesenchymal transition (EMT). of TWIST homo- and TWIST-E2A (TE) heterodimer Originally depicted as a mechanism for mesoderm types and inhibition of RUNX2 by TWIST proteins formation in avian embryos, EMT has since been (Bialek et al., 2004; Connerney et al., 2006). TWIST1 is associated with multiple developmental processes in- also expressed in lateral plate mesoderm, which gen- cluding neural crest formation, somitogenesis, sclero- erates endothelial precursor cells (Hopwood et al., tome formation and cardiac valve development from 1989). Finally, using an elegant approach combining endocardium and consists of a loss of cell to cell contact, knock-down by morpholinos and complementation loss of cell polarity and the acquisition of motility (for assays, both TWIST and SNAI2 transcription factors recent reviews see (Baum et al., 2008; Nakaya and Sheng were found to function downstream of c-MYC and to be 2008)). The mechanisms by which TWIST proteins essential for normal blood vessel formation in Xenopus promote EMT are discussed in the next section. laevis (Rodrigues et al., 2008). Twist1À/À mice also present a significant increase in Although its expression is barely detectable at E9.5, apoptotic cells, especially in the developing sclerotome Twist2 is expressed at E10.5 in the limbs buds and body (Chen and Behringer 1995). This cell death could reflect wall, a pattern progressively restricted to the craniofa- TWIST1 anti-apoptotic properties as TWIST proteins cial mesenchyme, chondrogenic precursors and dermis also behave as key regulators of the p53 oncosuppressive in the skin. Accordingly, Twist2 might have a role in the protein and as major effectors of the NF-kB survival differentiation of distinct subsets of vertebral precursor

Oncogene TWIST embryonic and oncogenic properties S Ansieau et al 3175 cells during sclerotome maturation and in skin develop- cytokines and chemokines (such as IL-12, IFNg,IL-1, ment suggesting functional divergences and redundancies TNFa, IL-6, MCP-1, MIP-1a) likely through the inactiva- between the two TWIST proteins (Li et al., 1995). tion of both C/EBPa and NF-kB but also by promoting the production of the anti-inflammatory IL-10 cytokine (Sharabi et al., 2008). Finally, TWIST proteins alleviate Twist proteins in adult organisms TNFa induction in response to T-cell receptor (TCR) TWIST proteins might similarly determine cell fate activation and thereby contribute to the negative cross- of adult stem cells. In Drosophila pupals, Twi was found regulation of TNFa by anti-inflammatory pathways (Sharif to be expressed within the adult muscle precursor cells, et al., 2006). TWIST proteins were also found to function which invade the larval oblique muscles and was downstream of TCR in T-helper 1 (Th1) memory determined as essential for their splitting into dorsal lymphocytes and to inhibit these potent inducers of longitudinal muscles found in the thorax of the adult fly inflammation. Recurrent stimulation of Th1 T-lympho- (Cripps and Olson, 1998). Ectopic expression of cytes by the cognate antigen leads to the gradual activation TWIST1 or TWIST2 in human bone marrow-derived of Twist1 through coordinated action of NFAT, NF-kB mesenchymal stromal/stem cells (MSC) was found to and activated STAT4 transcription factors. TWIST1 maintain an immature stromal phenotype, to inhibit expression does not interfere with the homing of the Th1 their osteo-chondrogenic potential and conversely to cell population to the inflamed tissue nor lymph node increase their adipocyte potential, strengthening their draining, instead its expression significantly lowers the role in cell fate determination (Isenmann et al., 2009). histological score of inflammation and tissue destruction TWIST2 was additionally found to be expressed in (Niesner et al., 2008). Overall, these observations depict granulocyte–macrophage progenitors, preventing their TWIST proteins as key-negative mediators of the inflam- proliferation and differentiation into macrophages, matory response. neutrophils and basophils, through the inactivation of both RUNX1 and C/EBPa transcription factors (Shar- abi et al., 2008). On the basis of human multiple tissue Northern blot Overexpression of TWIST oncoproteins in human cancers analysis, TWIST1 expression was originally defined as Both TWIST genes were found to be overexpressed in a being restricted to mesoderm-derived tissues including large set of human and murine tumors including a placenta, heart and skeletal muscles (Wang et al., 1997), variety of carcinomas as well as sarcomas, melanomas, presumably in precursor cells. Recently, the protein was gliomas and neuroblastomas (actual knowledge on found to be additionally expressed in brown fat and to TWIST1 expression in human cancers is recapitulated be essential for its function in adaptative thermogenesis, in Table 1) (Puisieux et al., 2006; Ansieau et al., 2008a). a process that consists of metabolizing fat as heat. TWIST TWIST1 behaves as a negative regulator of the PGC1-a In light of the high frequency of expression in cancers, the following sections will focus on the transcription factor, the key regulator of the entire mechanisms leading to TWIST gene reactivation and program of thermogenesis, by scaffolding together PGC1-a with a class II histone deacetylase to silent its consequences on tumor progression. its target genes. Twist1 depletion is sufficient to provide a remarkable obesity-resistant phenotype, elevated Mechanisms of TWIST gene reactivation in cancers oxygen consumption and mitochondrial biogenesis Although the mechanisms leading to the aberrant linking for the first time TWIST1 to cell metabolism reactivation of TWIST appear to be various and (Pan et al., 2009). complex (Figure 1), they invariably result from the Although Twist1 knockout is lethal at early develop- deregulation of signaling pathways that normally dictate ment stages, Twist2À/À mice die shortly after birth (Chen the expression of the genes during the embryonic and Behringer, 1995; Sosic et al., 2003) from a severe development. Interestingly, stress conditions seem to wasting of body tissue and anemia (cachexia), because control both the physiological and aberrant expression of the overexpression of pro-inflammatory cytokines of TWIST. Concomitant Hif1a and Twist expression (Sosic et al., 2003). This pro-inflammatory cytokine during mouse development have been shown to have a production contributes to pleiotropic effects including role in the development of specific neural populations depletion of lipid and glycogen stores and postnatal (Lee et al., 2009). Hypoxic conditions are similarly atrophy of multiple tissues due to extensive apoptosis. defined as potent inducers of TWIST1 expression in Dissection of the associated molecular mechanisms cancer cells thereby promoting cell dissemination to less firstly pointed out that both Twist genes are direct hostile environment, presumably through its role in targets of NF-kB and contribute to regulate its ability promoting an EMT (Yang et al., 2008; Gort et al., through a negative feedback mechanism. TWIST 2008b). Mechanical strains via integrin-mediated activa- proteins physically interact with NF-kB and specifically tion of the b-catenin pathway were also found to be prevent its ability to activate the TNFa, IL-1b and IL-6 essential in maintaining Twi expression during meso- pro-inflammatory cytokine-encoding genes (Sosic et al., derm invagination in the Drosophila embryo (Farge 2003). Recently, in mature myeloid cells, TWIST2 was 2003; Brouzes et al., 2004; Desprat et al., 2008). Whether confirmed to downregulate the inflammatory responses such torsion-stresses, known to promote tumorigenesis by preventing the production of pro-inflammatory (for a review, see (Butcher et al., 2009)), also lead to

Oncogene TWIST embryonic and oncogenic properties S Ansieau et al 3176 Table 1 TWIST1 status in cancers Cancer type Poor High or inva- LN or distant Note Expression Reference prognosis sive grade/ metastasis stage

Ameloblastoma Yes Protein Feng et al., 2009 Bladder Yes Yes Benign-malignant mRNA and protein Zhang et al., 2007 conversion Yes Yes Smoker status Protein Fondrevelle et al., 2009 Breast mRNA and protein Watanabe et al., 2004 Yes Yes*/** mRNA Yang et al., 2004 Yes Chromosomal Protein Mironchik et al., 2005 instability Angiogenesis Yes No Yes mRNA and protein Martin et al.,2005 Yes miR10b) — Ma et al., 2007 Yes Protein Cheng et al., 2007 Yes Protein Cheng et al., 2008 mRNA and protein Vesuna et al., 2008 mRNA Ansieau et al., 2008a, b Cervical Yes No Yes*** Protein Shibata et al., 2008 Choroid plexus mRNA Hasselblatt et al., 2009 Colon mRNA Ansieau et al., 2008a, b Yes mRNA Valdes-Mora et al., 2009 Endometrial Yes Yes Yes Protein Kyo et al., 2006 Gastric mRNA Rosivatz et al., 2002 Yes Protein Yan-Qi et al., 2007 Glioma Yes mRNA and protein Elias et al., 2005 Head and neck Yes Yes Induced by hypoxia Protein Yang et al., 2008 Yes Yes Protein Ou et al., 2008 mRNA Kojc et al., 2009 Hepatocellular Yes Protein Lee et al., 2006 carcinoma Benign-malignant mRNA and protein Li et al., 2006 conversion Yes Yes Angiogenesis mRNA and protein Niu et al., 2007 mRNA and protein Xu and Chen, 2007 Yes* mRNA Zhu et al., 2008 mRNA and protein Matsuo et al., 2009 Yes Yes* Recurrence mRNA and protein Yang et al., 2009 Kidney mRNA Ansieau et al., 2008a, b Melanoma Yes Yes mRNA and protein Hoek et al., 2004 Benign-malignant conver- mRNA and protein Ansieau et al., 2008a, b sion Nasopharyngeal Yes Yes mRNA and protein Song et al., 2006 Yes Protein Horikawa et al.,2007 Neuroblastoma mRNA and protein Valsesia-Wittmann et al., 2004 Oesophageal squa- mRNA Ansieau et al., 2008a, b mous cell carcinoma Yes mRNA and protein Yuen et al., 2007 Yes Yes Protein Xie et al.,2009 Ovarian Yes Yes Protein Kajiyama et al., 2006 Yes No Recurrence Protein Hosono et al., 2007 Yes Benign-malignant Protein Yoshida et al., 2009 conversion Pancreas induced by hypoxia mRNA and protein Hotz et al.,2007 Benign-malignant mRNA Ohuchida et al., 2007 conversion mRNA and protein Satoh et al., 2008 Parathyroid Change in protein Protein Fendrich et al., 2009 localization during benign-malignant conversion Pheochromocytoma Yes Benign-malignant mRNA and protein Waldmann et al.,2009 conversion Prostate Yes Yes Benign-malignant Protein Kwok et al., 2005 conversion, drug resistance Yes Yes Benign-malignant Protein Yuen et al., 2007 conversion Sarcoma Protein Maestro et al., 1999 No mRNA Entz-Werle´et al.,2005 Yes mRNA Man et al., 2005 Protein Cates et al., 2008

*, tumor cell lines; **, mouse models; ***, tendency but not statistically significant, LN: lymph nodes.

Oncogene TWIST embryonic and oncogenic properties S Ansieau et al 3177

Figure 1 Schematic representation of the regulation of TWIST expression including the various signals leading to the activation of TWIST transcription, modulating mRNA translation as well as post-translational regulation mechanisms. Induction of TWIST expression in response to stress conditions or to the activation of signaling pathways and transcription factors as well as the regulation of TWIST activity by post-translation modifications are largely discussed in the review. Because of size restriction, the regulation of HMGA2, and potentially of its target gene TWIST1, by the let-7 miRNA has not been discussed. Please refer to (Thuault et al., 2006; Lee and Dutta, 2007; Mayr et al., 2007; Park et al., 2007). T: TWIST protein, E: TCF3/E2A proteins, X: additional TWIST interacting transcription factors such as MyoD, PGC-1a, NF-kB or p53.

TWIST induction during tumor progression remains to cap-dependent translation initiation of growth promoting be determined. Nevertheless, initial arguments seem to mRNAs and enhances the cap-independent translation support this assumption, as compression of colon initiation of EMT-inducing mRNAs including TWIST, sections of APC168N/ þ mice (loss of one APC allele) SNAIL, ZEB1 and HIF1a, thereby promoting growth- is sufficient to induce the nuclear translocation of arrest and cell dissemination (Evdokimova et al., 2009). b-catenin and the resulting transcriptional activa- The methylation of TWIST1 was also depicted as taking tion of both c-MYC and TWIST1 genes (Whitehead part of an epigenetic program essential for EMT et al., 2008). induction (Dumont et al., 2008). Nevertheless, while Post-translational modifications undoubtedly have an TWIST1 promoter was indeed found to be hyper- additional important role in regulating TWIST activity methylated in basal like and/or invasive breast cancers during development. As already mentioned, during (Fackler et al., 2003; Dumont et al.,2008),themethyla- development, TWIST homo- and heterodimers display tion status of the TWIST1 promoter sequences and its distinct, even antagonist functions, impacting TWIST expression are not correlated (Gort et al., 2008a). stability as well as the nature of the genetic program induced (Demontis et al., 2006; Hayashi et al., 2007; Firulli and Conway 2008). Although not yet shown, the Oncogenic and pro-metastatic potentials of TWIST mechanisms regulating TWIST dimerization are likely proteins to also impinge on TWIST oncogenic and/or prometa- The prometastatic potential of TWIST1 was originally static properties. shown by comparison of isogenic murine cell lines. Additional mechanisms susceptible to modulate Successively, Twist1 expression and metastatic potential TWIST expression have been reported. The DNA/ were found to correlate (Yang et al., 2004). In addition, RNA binding protein YB1 was found to suppress Twist1 depletion was shown to prevent lung metastasis

Oncogene TWIST embryonic and oncogenic properties S Ansieau et al 3178 without affecting primary tumor formation, suggesting venting both p53- and RB-dependent pathways and that TWIST1 specifically contributes to late cancer cell cooperate with these mitogenic oncoproteins to fully dissemination (Yang et al., 2004). This potential was transform murine cells, providing them with a tumori- suggested to rely on the ability of TWIST1 to induce an genic potential, when xenografted in nude mice (Ansieau EMT and thereby promote motility, reminiscent of its et al., 2008a). Escape from senescence relies on the embryonic biological properties. E-cadherin, encoded ability of TWIST proteins to abrogate p21CIP/WAF1 by the CDH1 gene, is generally defined as a guardian of and p16INK4A transcriptional activation (Ansieau the epithelial phenotype and loss of its expression is et al., 2008a). By inhibiting both senescence and associated with tumor invasiness, metastatic dissemina- apoptotic programs, TWIST proteins are suggested to tion and poor patient prognosis (Schipper et al., 1991; promote malignant conversion and to provide cells with Oka et al., 1993; Umbas et al., 1994). Repression of a growth advantage at the primary site. Accordingly, CDH1 gene by TWIST proteins is either direct (Yang several studies reported the induction of TWIST1 et al., 2004; Vesuna et al., 2008) or mediated by SNAI1, expression during the progression toward malignant according to the cell type (Smit et al., 2009). When stages in several cancer types including ovary, prostate, direct, transcriptional repression might actually require bladder, pancreas and hepatic carcinomas, as well as TWIST stabilization through heterodimerization poten- melanomas and pheochromocytomas (Kwok et al., tially in response to environmental signals, giving a 2005; Li et al., 2006; Ohuchida et al., 2007; Yuen rationale to the detection of TWIST-positive, et al., 2007; Zhang et al., 2007; Ansieau et al., 2008a; E-cadherin-negative cells at the invasive fronts of Waldmann et al., 2009; Yoshida et al., 2009). Originally malignant parathyroid neoplasia (Fendrich et al., described as independent processes, inhibition of failsafe 2009). Interestingly, TWIST was inversely found to be program induction and EMT may turn out to be directly activated following loss of E-cadherin expression (Onder linked. By performing standard cooperation assays in et al., 2008), raising the possibility that TWIST epithelial cells, both TWIST proteins were found to contributes to EMT induction by inducing the expres- abrogate ERBB2 or RASV12-induced senescence and to sion of mesenchymal genes rather than by repressing concomitantly cooperate with these mitogenic oncopro- CDH1. Accordingly, a correlation between VIM, CDH2 teins in promoting an EMT permitting cellular migra- (the vimentin and N-cadherin-encoding genes, respec- tion (Ansieau et al., 2008a). Therefore, reactivation of tively) and TWIST gene expressions is observed in TWIST genes might simultaneously favor malignant breast cancer cell lines (Lombaerts et al., 2006). In conversion through the inhibition of Rb- and p53- addition, TWIST1 was proposed to favor metastasis by dependent pathways and early cancer cell dissemination inducing the miR-10b/HoxD10/RhoC signaling path- through EMT induction. It is noteworthy that the way (Ma et al., 2007). Nevertheless, the correlation knock-down of the ZEB1, another embryonic transcrip- between miR-10b and distant metastasis or poor tion factor known to promote EMT, is also associated prognosis remains a matter of discussion (Gee et al., with senescence program induction (Liu et al., 2008), 2008; Tavazoie et al., 2008). strengthening the link between EMT and senescence Another aspect of the TWIST proteins’ dark side escape (Smit and Peeper, 2008; Ansieau et al., 2008b). relies on their ability to override oncogene-induced Importantly, our laboratory and the Weinberg senescence and apoptosis, the natural barriers activated laboratory recently showed that EMT endows normal to constrain cell growth in premalignant lesions and transformed mammary epithelial cells with stem- (Bartkova et al., 2005; Braig et al., 2005; Chen et al., like properties, including self-renewal capabilities (Mani 2005; Collado et al., 2005; Gorgoulis et al., 2005). et al., 2008; Morel et al., 2008). Interestingly, both Originally, the anti-apoptotic properties of TWIST TWIST1 and TWIST2 genes are overexpressed in proteins were deciphered through a functional screen CD44 þ CD24– cells, an antigenic phenotype allotted to using the escape of c-MYC-induced apoptosis as the breast cancer stem cells. Furthermore, ectopic expres- determinant. The two related transcription factors were sion of TWIST1 or SNAI1 in immortalized differen- found to alleviate ARF induction and the consequent tiated mammary epithelial cells triggers the gain of the p53 stabilization in response to the oncogenic insult CD44 þ CD24– phenotype (Mani et al., 2008). Collec- (Maestro et al., 1999). In agreement with these conclu- tively, these observations strongly suggest that, in the sions, our group identified TWIST1 reactivation as the course of tumor progression, reactivation of EMT- prevalent mechanism of p53 functional inactivation in inducing transcription factors, including TWIST1 and neuroblastomas, preventing N-MYC-induced apoptosis TWIST2, triggers the generation of cells with self- and thus tumor progression (Valsesia-Wittmann et al., renewal capabilities, favoring both the growth of the 2004). Various studies have since shown that TWIST primary tumor and the initiation of secondary tumors proteins actually negatively regulate each step leading to following cell dissemination. Taking into account the p53 target gene activation, including p53 stabilization, role of the microenvironnement in the control of EMT, its activation through post-translational modifications, these observations also suggest that, within a tumor, the its DNA binding and trans-activation potential (Hama- acquisition and the maintenance of stem cell properties mori et al., 1999; Xie et al., 2001; Stasinopoulos et al., by tumor cells might be a dynamic and unstable process 2005; Shiota et al., 2008). (Gupta et al., 2009a). Recently, in addition our group showed that TWIST Additional oncogenic properties have been allotted proteins override oncogene-induced senescence by pre- to TWIST proteins (Figure 2). By turning-down p53,

Oncogene TWIST embryonic and oncogenic properties S Ansieau et al 3179 Cell dissemination Failsafe program escape ectopically expressing the protein in human breast or Self-renewal properties Growth and survival advantage Chemoresistance Genomic instability murine melanoma cell lines (Mironchik et al., 2005; Hu et al., 2008). Interestingly, TWIST1 was also shown to RB EMT p53 take place downstream of thrombin and to drive thrombin-induced tumor growth, migration and neo- angiogenesis (Hu et al., 2008). Multiple growth factors and receptors including Ang-2, VEGF, KDR, and Gro- TWIST a were defined as positively regulated TWIST target genes (Hu et al., 2008), highlighting a novel mechanism

AKT2 by which thrombosis contributes to a more malignant phenotype (Nierodzik and Karpatkin, 2006). VEGF BCL-2/BAX

Angiogenesis Chemoresistance Follow-up questions and perspectives Figure 2 The oncogenic properties of TWIST proteins stem from their multiple functions. By turning down both RB- and p53- During cancer progression, reactivation of embryonic dependent pathways, TWIST proteins override failsafe programs, EMT-inducing transcription factor-encoding genes, in- trigger chromosomal instability and provide cells with chemoresis- tance. Chemoresistance relies also on the increase of the BCL-2/ cluding TWIST1 and TWIST2, is a frequently observed Bax ratio and, in epithelial cells, is also associated with the event that should provide cells with critical properties induction of an epithelial–mesenchymal transition (EMT). This including self-renewal capabilities, resistance to oncogene- transdifferentiation process additionally provides cells with moti- induced failsafe programs and invasive capabilities. lity and stem-like properties. p53 and RB silencing were found to Although EMT is a transient process, the acquisition promote EMT suggesting a potential crosstalk with failsafe program escape. Conversely, EMT might directly contribute to of a propensity to undergo this transdifferentiation escape from failsafe programs. These assumptions are represented process likely favors both the primary tumor growth by a dashed line with arrows on each side. By inducing VEGF and cell dissemination. Associated with malignant production, TWIST proteins finally promote the neoangiogenesis. conversion, reactivation of EMT-inducing transcription factors might foster early cancer cell dissemination (Ansieau et al., 2008a) (Figure 3). This assumption is TWIST proteins also promote chromosomal instability now supported by various results from the Klein and consequently tumor progression. Accordingly, laboratory. This group first showed that disseminated ectopic expression of TWIST1 in the MCF7 breast cell tumor cells, isolated from the bone marrow of breast line induces chromosomal instability and frequencies of cancer patients, display few chromosomal abnorma- chromosomal amplifications are significantly higher in lities suggesting that they arise from premalignant TWIST-expressing breast carcinomas (Mironchik et al., lesions before the telomeric crisis (Schardt et al., 2005). TWIST proteins additionally provide cells with 2005). In line with this hypothesis, using a transgenic significant higher resistance to genotoxic and cytotoxic mouse model expressing the ERBB2-activated oncogene stresses through their ability to induce AKT2 expression in mammary epithelial cells (Di et al., 1999), they or to differently modulate the ratio between the pro- and recently confirmed that premalignant cells disseminate anti-apoptotic members of the BCL-2 family (see from atypical ductal hyperplasia, an early stage in Table 2). Associated-EMT might also contribute to tumorigenesis, in association with Twist1 reactivation TWIST-induced chemoresistance. Growing evidence (Husemann et al., 2008). indeed supports the assumption that EMT is as a Overall, these data suggest that EMT constitutes a transient state providing cells with a higher stress major force driving tumor growth and progression. threshold and could thereby constitute an escape from Although the role of EMT in cancer has long been a apoptosis. In vitro, oxaliplatin-resistant colorectal can- matter of discussion, particularly because epithelial cells cer cells, tamoxifen-resistant breast cancer cells, gefinti- that have undergone an EMT are phenotypically nib-resistant lung cancer cells as well as gemcitabine- indistinguishable from native fibroblastic cells, direct resistant pancreatic cells have been shown to have evidence of EMT in breast cancer recently arose out of undergone an EMT (Hiscox et al., 2006; Yang et al., mouse mammary tumor progression model studies 2006; Rho et al., 2009). Additional characteristics of (Trimboli et al., 2008). EMT turns into a mechanism ‘resistance’ in cancer stem cells might also be provided endowing a cell with plasticity and stem-like properties through an EMT, such as lower levels of ROS and as well as various advantages including chemoresistance, consequent protection of their genomes (Diehn et al., immuno-editing and immune suppression (Moody et al., 2009). Therefore, it appears that the acquisition of 2005; Mani et al., 2008; Morel et al., 2008; Kudo-Saito mesenchymal phenotypes, if even temporarily, engen- et al., 2009). Directly linked to metastatic dissemination ders tumor cells with a multifaceted capacity to and cancer recurrence, it is also likely to have a role in proliferate, migrate, avoid cell death and permanent facilitating cell transformation. For these various arrest, as well as protection from extracellular signals reasons, signaling pathways promoting EMT and the and chemistries. At last, TWIST1 promotes tumor reverse MET process, have been deeply investigated, formation by favoring neo-angiogenesis, as shown by highlighting a preponderant role of multiple embryonic

Oncogene TWIST embryonic and oncogenic properties S Ansieau et al 3180 Table 2 TWIST and chemoresistance Protein Cell type Drug Mechanism EMT Reference

TWIST1 Nasopharingeal carcinoma Vincristin-paclitaxel Reduction of BAX ND Wang et al., 2004 TWIST1 Breast carcinoma Paclitaxel Induction of AKT2 Yes Cheng et al., 2007 TWIST1-2 Fibroblasts, prostate carcinoma Daunorubicin–cisplatin BCL-2 dephosphorylation NR Pham et al., 2007 TWIST1 Lung adenocarcinoma Cisplatin Increase of BCL-2/BAX ratio Yes Zhuo et al., 2008 TWIST1 Ovarian carcinoma Paclitaxel ND Yes Kajiyama et al., 2007 TWIST1 Prostate carcinoma Paclitaxel Reduction of BAX Yes Kwok et al., 2005

Abbreviations: ND, not defined; NR, not relevant.

Figure 3 Reminiscent of their embryonic functions, Twist proteins promote malignant conversion and early cancer cell dissemination through EMT. The mitogenic effect of activated oncoproteins leads to the induction of senescence and apoptosis in premalignant lesions. By downregulating the RB and p53-dependent pathways, TWIST1 and TWIST2, and potentially other functionally related transcription factors promote the progression from benign tumor to malignancy. Importantly, malignant cells co-expressing these embryonic transcription factors and mitogenic oncoproteins are prone to undergo a complete EMT. Consequently, in response to permissive microenvironmental signals, some cells acquire stem-like properties as well as motility and invasive capabilities through this transdifferentiation process.

transcription factors, frequently expressed in cancers, nately limit the chance to completely abrogate their such as ZEB1, ZEB2, the SNAIL zinc-finger protein activity or to interfere with their activation. However, as family, the E47 and TWIST bHLH proteins as well as exemplified by the increased sensitivity of A549 cells to Goosecoid and FOXC2 (for a recent review see (Polyak cisplatin when SNAI1 expression is knocked-down and Weinberg, 2009)). (Zhuo et al., 2008), combined chemotherapies and In light of their emerging role in cancer development, EMT inhibition might be fruitful. As a transdifferentia- targeting EMT-inducers (particularly when their neu- tion process, EMT might additionally be associated with tralization is associated with restoration of failsafe the expression (or re-expression) of a set of particular program functions) may open novel routes for futures antigens, that could potentially constitute future targets therapies. Multiple parameters including the restricted for immune therapies. Interestingly, the Weinberg accessibility of nuclear factors, their interplay and laboratory recently showed that CD44 þ CD24– breast functional redundancies, and the large number of cancer cells, that have undergone an EMT, display pathways downstream in which they partake, unfortu- particular sensitivity to salinomycin, a potassium

Oncogene TWIST embryonic and oncogenic properties S Ansieau et al 3181 ionophore inhibitor, opening a novel route to specifi- Acknowledgements cally target these cells (Gupta et al., 2009b). Hopefully, further knowledge will help in deciphering additional Because of size restriction, unfortunately we could not refer to Achille’s heels of this process. all significant contributions in this field. We therefore apologize to non-cited scientists. Our researches are supported by the Ligue contre le Cancer (Comite´s de l’Ain, de la Droˆme et Rhoˆne-Alpes), l’Association pour la Recherche sur le Conflict of interest Cancer (ARC), l’Institut National du Cancer (INCa), la Fondation de France and la Fondation Albert et Jeanne The authors declare no conflicts of interest Rollet du Coudray.

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