Transcriptional Regulation of Cell Polarity in EMT and Cancer

G Moreno-Bueno, F Portillo and A Cano

Departamento de Bioquı´mica, Universidad Autońoma de Madrid, Instituto de Investigaciones Biome´dicas ‘Alberto Sols’, Consejo Superior de Investigaciones Cientı´ficas-Universidad Autońoma de Madrid, Madrid, Spain

The epithelial-to-mesenchymal transition (EMT) is a can lead primary tumours to metastasize (Thiery, 2003; crucial process in tumour progression providing tumour Gupta and Massague´ , 2006).EMT is typically chara- cells with the ability to escape from the primary tumour, cterized by the loss of cell–cell adhesion and apical– to migrate to distant regions and to invade tissues. EMT basal cell polarity, as well as the increased motility of requires a loss of cell–cell adhesion and apical–basal cells (Thiery, 2003).Cell adhesion and polarity in polarity,as well as the acquisition of a fibroblastoid motile epithelia depends on the formation of adherens junc- phenotype. Several transcription factors have emerged in tions in which E-cadherin is a key determinant, recent years that induce EMT,with important implica- providing the physical structure for both cell–cell tions for tumour progression. However,their effects on attachment and the recruitment of signalling complexes cell polarity remain unclear. Here,we have re-examined (reviewed by Knust and Bossinger, 2002; Perez-Moreno the data available related to the effect of EMT related et al., 2003). One of the earliest steps in EMT is the loss transcription factors on epithelial cell plasticity,focusing of E-cadherin function, and in fact it is generally on their impact on cell polarity. Transcriptional and accepted that EMT-inducing factors initiate epithelial post-transcriptional regulatory mechanisms mediated by reorganization by impairing the expression or function several inducers of EMT,in particular the ZEB and of E-cadherin (Peinado et al., 2004; Jeanes et al., 2008). Snail factors,downregulate the expression and/or func- The characterization of E-cadherin regulation during tional organization of core polarity complexes. We also EMT has provided important insights into the mole- summarize data on the expression of cell polarity cular mechanisms involved in the loss of cell–cell genes in human tumours and analyse genetic interactions adhesion and in the acquisition of migratory properties that highlight the existence of complex regulatory net- during carcinoma progression (Peinado et al., 2007). To works converging on the regulation of cell polarity by date, many different extracellular cues have been shown EMT inducers in human breast carcinomas. These to trigger epithelial dedifferentiation and EMT, such as recent observations provide new insights into the relation- those involving transforming growth factor-b (TGF-b), ship between alterations in cell polarity components Notch, fibroblast growth factor and Wnt signalling and EMT in cancer,opening new avenues for their pathways (Barrallo-Gimeno and Nieto, 2005; De Craene potential use as therapeutic targets to prevent tumour et al., 2005a; Huber et al., 2005; Thiery and Sleeman, progression. 2006).Most of the signals inducing EMT exert their Oncogene (2008) 27, 6958–6969; doi:10.1038/onc.2008.346 action through the modulation of transcription factors that repress epithelial genes, such as those encoding Keywords: cell polarity; EMT; Snail; ZEB; tumour E-cadherin and cytokeratins, and that activate markers; interacting networks transcription programmes that specify fibroblast-like motility and an invasive phenotype (Thiery and Sleeman, 2006; Peinado et al., 2007). Several transcription factors have been said to drive EMT, including members of the Snail and basic Helix Loop Helix (bHLH) families, and two double zinc finger Introduction and homeodomain (ZEB) factors (reviewed by Peinado et al., 2007). Extensive analysis of such transcription Metastasis is the most important cause of morbidity and factors has clearly demonstrated their implication in the mortality in human cancers.The epithelial-to-mesench- loss of cell–cell adhesion and in gaining motility, ymal transition (EMT) is an essential process during although an analysis of their impact on cell polarity is embryogenesis (Barrallo-Gimeno and Nieto, 2005) and still pending.Here, we have re-examined the existing its pathological activation during tumour development data on the effect and relationship of the EMT transcription factors on epithelial cell plasticity, focusing our attention on their impact on cell polarity Correspondence: Professor A Cano, Instituto de Investigaciones Biome´ dicas ‘Alberto Sols’ CSIC-UAM, Arturo Duperier 4, Madrid mechanisms.We also summarize the data available 28029, Spain. on the expression of cell polarity genes and their E-mail: [email protected] transcriptional regulators in human tumours, which Polarity regulation in EMT G Moreno-Bueno et al 6959 highlight the existence of complex regulatory networks The assembly and localization of the apical junctional that converge on the regulation of cell polarity in complex require a set of conserved polarity-generating tumours.Apart from their potential as new tumour protein complexes.To date, three core interacting markers, the existence of complex networks involving protein complexes have been identified in mammals EMT regulators that control cell polarity is becoming that participate in apical–basal cell polarity and that evident. influence the assembly and localization of the junctional complexes: (1) the PAR (Par6/Par3/atypical protein kinase C (aPKC)) complex; (2) the CRB (Crb/Pals/Patj) Polarity-generating protein complexes at a glance complex; and (3) the SCRIB (Scrib/Dlg/Lgl) complex (reviewed by Dow and Humbert, 2007; Assemat et al., Although our present understanding of the protein 2008; Aranda et al., 2008; Humbert et al., 2008).The complexes involved in the generation of polarity has PAR complex was initially identified in Caenorabditis been dealt with in more specialized reviews (Dow and elegans mutants (for partitioned defective; Kemphues Humbert, 2007; Lee and Vasioukhin, 2008), to under- et al., 1988), and in mammals, it is composed of two stand the significance of the alteration in the expression scaffold proteins (PAR6 and PAR3) and an aPKC.To of cell polarity genes during tumour development, we date, two PAR3 (PARD3, PARD3B), three PAR6 will briefly summarize the epithelial cell polarity (PARD6A, PARD6B, PARD6G) and two aPKC systems.Epithelial cells maintain two types of cell (PRKCI, PRKCZ) genes have been identified in polarity, planar and apical–basal polarity (Dow and mammalian genomes.The PAR complex is localized Humbert, 2007).Planar polarity is the polarization of to the apical junction domain, and significant evidence cells across the two dimensions of the epithelial sheet indicates that it has an important function in the and it will not be considered here (reviewed by Zallen, assembly of tight junctions (reviewed by Goldstein and 2007).The apical–basal polarity of epithelial cells in an Macara, 2007; Ebnet, 2008).The CRB and SCRIB epithelium is evident from the presence of two specia- complexes were initially identified in Drosophila mela- lized plasma membrane domains, the apical surface nogaster when screening mutations responsible for facing the lumen and a basolateral surface that contacts epithelial defects (reviewed by Assemat et al., 2008). the adjacent cells and the underlying connective tissue. Mammalian CRB complex localizes to the apical Apical–basal polarity may also be present in multilayer membrane and are made up of the transmembrane epithelial tissues, but in this case, the apical cell surface protein Crumbs (Crb) and the cytoplasmic scaffolding is in contact with the upper epithelial layer.The proteins PALS1 (the homologue of Drosophila Stardust, asymmetrical distribution of lipids and proteins between Sdt, a MAGUK protein) and PATJ (Pals-associated both apical and basal domains reflects their different tight junction protein, the homologue of Dpatj).At functions, and it is the result of both polarized present, there are three CRB, two Pals and one PATJ trafficking and the presence of a physical frontier gene known to exist in humans (Assemat et al., 2008). established by the apical junctional complex comprised The composition and function of the CRB complex in of tight junctions and adherens junctions.Tight junc- the mammalian retina have been recently reviewed tions provide a tight seal between neighbouring cells, (Gosens et al., 2008), where mutations in the human which is essential for the epithelium to function as a CRB1 gene cause autosomal recessive retinitis pigmen- barrier, whereas adherens junctions maintain the adhe- tosa and autosomal Leber congenital amaurosis (Ri- sion between neighbouring cells.The protein composi- chard et al., 2006). The mammalian SCRIB complex is tion and barrier properties of this apical junctional localized in the basolateral domain of epithelial cells and complex have recently been reviewed (Perez-Moreno it is comprised of three proteins, Scribble (Scrib), Disc et al., 2003; Perez-Moreno and Fuchs, 2006; Niessen, large (Dlg) and Lethal giant larvae (Lgl) (reviewed by 2007; Hartsock and Nelson, 2008).As such, tight and Vasioukhin, 2006).Four DLG (DLG1-4) and two LGL adherens junctions are composed of transmembrane (LGL1, LGL2) have now been identified in the proteins that adhere to similar proteins in the adjacent mammalian genomes.In addition to these three ‘core’ cell.The transmembrane region of the tight junctions is cell polarity complexes, further components and protein composed mainly of claudins, tetraspan proteins with kinases are increasingly being implicated in the organi- two extracellular loops.In mammals, the claudin gene zation of cell polarity (Assemat et al., 2008). family contains at least 24 members (Anderson et al., There is now convincing evidence that the establish- 2004) and the cytoplasmic domain of these proteins ment of cell polarity is the result of mutually antag- interacts with occludin and several ZO-proteins (ZO1-3) onistic interactions between the PAR, CRB and SCRIB to form the plaque that associates with the cytoskeleton complexes, where the Par6/Par3/aPKC complex has a (Tsukita et al., 1997). Adherens junctions are mediated pivotal function and Rac1/Cdc42 GTPases activation by Ca2 þ -dependent homophilic interactions of cadher- triggers the process (reviewed by Suzuki and Ohno, ins, single-span membrane proteins with more than 100 2006; Etienne-Manneville, 2008; Figure 1).Essentially, members in the vertebrate gene family grouped into six Cdc42 that is activated is recruited by Par6 to the Par subfamilies (Nollet et al., 2000). Cadherins interact with complex where it causes the activation of aPKC.This cytoplasmic catenins that link the cadherin/catenin kinase, in turn, phosphorylates Par3, thereby allowing complex to the actin cytoskeleton (Perez-Moreno and the formation of an active Par complex at the apical Fuchs, 2006). domain and promoting the assembly of the junctional

Oncogene Polarity regulation in EMT G Moreno-Bueno et al 6960 Apical activation of a complex genetic programme that

CRB together with the loss of the epithelial character and epithelial polarity implies the acquisition of mesenchy- PALS PATJ CDC42 mal properties and motility.However, there are several TJ/AJCell-cell TJ/AJ interaction important features of EMT and tumour progression that must be considered.The first aspect to bear in mind PAR6 is the fact that, as in embryonic development, EMT is SCRIBB probably a transient event during tumour progression PAR3 aPKC DLG LGL and the converse mesenchymal-to-epithelial transition can also occur during the course of metastasis (Thiery, 2003; Peinado et al., 2004; Peinado et al., 2007). Second, as EMT can be driven by many different signalling factors, most probably in a paracrine fashion (Huber et al., 2005; Thiery and Sleeman, 2006), only some tumour cells might be responsive to EMT-inducing cues acting over short distances in the tumour microenviron- ment (Scheel et al., 2007). Moreover, EMT can probably be considered as the most extreme manifestation of epithelial cell plasticity (Gru¨ nert et al., 2003; Huber Basal et al., 2005; Scheel et al., 2007), and the appearance of Figure 1 Schematic view of polarity-generating mechanism.Cell the full EMT phenotype might be difficult to observe polarity complexes PAR (Par3-Par6-aPKC) and CRB (Crumbs- within tumours.Indeed, the relevance of EMT in human Pals1-Patj) localize to the apical membrane and promote apical tumours is still a matter of considerable debate (Tarin membrane identity.This process is triggered by Cdc42 activation, et al., 2005; Thompson et al., 2005; Christiansen and which is mediated by cell-cell interaction.SCRIB complex is localized at the basolateral domain counteracting the function of Rajasekaran, 2006; Scheel et al., 2007; Talmadge, 2007), the PAR complex and promotes basal membrane identity.SCRIB and some of these considerations await clarification and complex is reciprocally inactivated by aPKC-dependent phospho- the development of appropriate in vivo model systems, rylation of Lgl protein. as well as extensive studies on human tumours. As most of the knowledge on EMT has so far been structure.Localization of active Par complexes to the obtained from cell culture studies, we will first review the apical domain is stabilised by the CRB complex whose existing data on the transcriptional regulation of distribution is reciprocally dependent on the PAR polarity genes in cell culture systems, before examining complex.In this scenario, the basolaterally located Scrib the data available from human tumours. complex functions as an antagonist of the apical localization of the active PAR complex.Lgl proteins of the Scrib complex compete with Par3 for binding to EMTand the loss of cell polarity: strictly coordinated the Par complex, thereby sequestering the active Par events complex away from the apical junction domain (Fig- Concomitant to the EMT process is the disappearance ure 1).Conversely, Lgl phosphorylation by aPKC of the apical–basal polarity of epithelial cells.However, inactivates the SCRIB complex (reviewed by Assemat the mechanisms underlying the downregulation of cell et al., 2008; Lee and Vasioukhin, 2008). polarity determinants and the potential coordination of this with the loss of E-cadherin and other epithelial markers is still poorly understood.Most of the present knowledge regarding the regulation of EMT has come Regulation of cell polarity genes during EMT from studies of the mechanisms underlying E-cadherin loss, which has led to the identification of several EMT: a dynamic process underlying tumour progression transcriptional repressors including members of the Metastasis of epithelial tumours (carcinomas) is a Snail superfamily, SNAI1 (previously known as Snail) complex process in which the tumour cells undergo a and SNAI2 (previously known as Slug), of the bHLH sequential series of events that initiate their exit from the family (E47/TCF3 and Twist), two ZEB factors, ZEB1 primary tissue and lead to the formation of a secondary (also known as dEF1/TCF8) and ZEB2 (also known as tumour focus in a distant tissue (Gupta and Massague´ , SIP1/ZFHX1B; reviewed by Peinado et al., 2007) and 2006).EMT is presently recognized as a key event in FOXC2 (Mani et al., 2007). At present, all these tumour progression, as EMT can be exploited by E-cadherin repressors are recognized as key inducers tumour epithelial cells to acquire the ability to dissociate of EMT.They not only induce EMT when over- from their neighbours and migrate (Thiery, 2003).Thus, expressed in epithelial cells, but indeed they drive EMT is the first step of the metastatic cascade followed genetic EMT programmes that regulate epithelial and by carcinomas, although EMT can also occur at other mesenchymal genes, as well as genes involved in stages of the metastatic process, such as at intra- or extracellular matrix remodelling, cytoskeletal reorgani- extravasation (Gupta and Massague´ , 2006).As indi- zation, cell movements or even cell survival (Barrallo- cated above, the full accomplishment of EMT requires Gimeno and Nieto, 2005; De Craene et al., 2005b;

Oncogene Polarity regulation in EMT G Moreno-Bueno et al 6961 Vandewalle et al., 2005; Bermejo-Rodrı´ guez et al., 2006; (Aigner et al., 2007; Spaderna et al., 2008). A causal Moreno-Bueno et al., 2006; Peinado et al., 2007; Escriva` relationship between ZEB1-induced EMT and the loss et al., 2008). These different EMT regulators share of cell polarity is also supported from these recent a similar basic molecular mechanism of repression, studies, as maintenance of cell polarity depends on binding to conserved E-box sequences (mainly of LGL2 in ZEB1-silenced colorectal carcinoma cells the CAGGTG type) in the proximal promoter of (Spaderna et al., 2008). The biological significance of E-cadherin and other epithelial genes (Peinado et al., the downregulation of LGL2 by ZEB1 is further 2004, 2007).The mechanisms underlying the upregula- emphasized by the observation that ZEB1 silencing tion of mesenchymal motility or survival genes are still suppresses the metastatic ability of colorectal carcinoma poorly understood, but they probably involve indirect cells (Spaderna et al., 2008). Notably, ZEB1 was activation pathways (Vega et al., 2004; Jorda` et al., detected at invasive regions in breast and colorectal 2005; Grotegut et al., 2006; Beltran et al., 2008). tumours and at the tumour–stroma interface, regions Apart from E-cadherin, other epithelial genes initially that display a strong reduction of LGL2 protein and no downregulated by Snail and ZEB factors are compo- signs of polarity (Aigner et al., 2007; Spaderna et al., nents of the tight junctions, including occludin 2008).These recent observations are in line with and several members of the claudin family (Ikenouchi previous studies indicating that EMT occurs at invasive et al., 2003; Ohkubo and Ozawa, 2004; De Craene et al., regions of colorectal tumours (Brabletz et al., 2005) 2005b; Vandewalle et al., 2005; Martı´ nez-Estrada et al., where ZEB1 may have an important function (Spaderna 2006).In fact, direct binding of SNAI1/SNAI2 to et al., 2006). conserved E-box elements in the corresponding promo- Snail factors have also recently been proposed to ters has been reported for occludin, claudin-1 and downregulate polarity genes.Indeed, downregulation of claudin-7 (Ikenouchi et al., 2003; Martı´ nez-Estrada CRB3 and LGL2 expression by SNAI1/SNAI2 was et al., 2006). Interestingly, downregulation of claudin-4, observed in breast and colorectal carcinoma cells, the junctional adhesion molecule-1 (JAM-1/JAM-A)and although less potently than that of ZEB1 (Aigner Dlg3 has been detected in gene profiling studies of et al., 2007; Spaderna et al., 2008). Interestingly, SNAI1 carcinoma cells, as well as in Madin-Darby canine kidney seems to preferentially act through distal E-box (MDCK) cells undergoing EMT after expression of sequences of the CRB3 promoter in breast carcinoma SNAI1, SNAI2, E47 or other EMT regulators (De cells, in contrast to the action of ZEB1 on proximal Craene et al., 2005b; Moreno-Bueno et al., 2006; Peinado E-box elements of the CRB3 promoter in this cell system et al., 2008). Because of the close relationship between (Aigner et al., 2007), suggesting differential binding tight junctions and the organization of epithelial cell specificities for both repressors.However, direct binding polarity, these observations suggest that components of of SNAI1 to the endogenous CRB3 promoter was not the core cell polarity complexes could also be direct analysed in that study.SNAI1 also downregulated the targets of EMT transcriptional regulators. CRB complex but not the PAR complex in MDCK cells (Whiteman et al., 2008), in which SNAI1 strongly Cell polarity regulators as targets of EMTtranscriptional represses the CRB3 promoter but less so the PALS and inducers PATJ promoters.Indeed, direct binding of SNAI1 to Until recently, very little information existed about the proximal E-boxes of the CRB3 promoter was demon- regulation of core polarity components by factors that strated in MDCK-SNAI1 cells (Whiteman et al., 2008), induce EMT.Several recent reports have now provided in contrast to previous suggestions from breast carcino- evidence that members of the CRB and the SCRIB mas (Aigner et al., 2007). As mentioned above, other complexes are direct targets of EMT inducers in core polarity genes like DLG3 are thought to be different cell systems.In a search for ZEB1 targets, downregulated by several EMT inducers, including CRB3, PATJ and HUGl2/LGL2 (the human homolog SNAI1/SNAI2 and bHLH factors (E47/TCF3, E2-2/ of lethal giant larvae 2), as well as several tight junctions TCF4), both in MDCK and mouse carcinoma cells components (JAM-1; occludin, claudin-7), were seen to (Moreno-Bueno et al., 2006; Peinado et al., 2008; be upregulated in undifferentiated breast carcinoma Sobrado V, Moreno-Bueno G et al., unpublished), cells after ZEB1 silencing (Aigner et al., 2007). Promoter although no functional promoter studies are yet avail- analysis and chromatin immunoprecipitation assays able.Taken together, these observations suggest that indicated that CRB3 and PATJ genes are direct targets different EMT transcriptional inducers act coopera- of ZEB1, which bind at specific proximal E-box tively to repress factors involved in determining polarity, sequences leading to their repression (Aigner et al., which could reinforce the suppression of apical–basal 2007).Similarly, ZEB1 silencing upregulates the expres- polarity that is required for cells to undergo EMT. sion of several polarity genes in colorectal carcinoma Alternatively, it is likely that the different EMT inducers cells, including CRB3 and LGL2, the LGL2 promoter could operate by silencing cell polarity genes specifically being a direct target of ZEB1 (Aigner et al., 2007). in different tumour types.Studies on the relationship Interestingly, silencing of ZEB1 in breast and colorectal between the expression of EMT transcriptional repres- carcinoma cells leads to partial reversion of the sors and polarity genes in large series of tumours (see epithelial phenotype and, significantly, to the relocaliza- below) are clearly required to clarify these issues. tion of CRB3 and/or LGL2 to the membrane, which is Regardless of the relationship between the different associated with a reorganization of apical–basal polarity transcriptional inducers of EMT, these recent reports

Oncogene Polarity regulation in EMT G Moreno-Bueno et al 6962 highlight that important polarity genes of the CRB and several microRNAs (Gregory et al., 2008; Korpal et al., SCRIB complexes are transcriptional targets of different 2008; Park et al., 2008) or other non-coding RNAs EMT inducers.Thus, it appears that there is a (Beltran et al., 2008). MicroRNAs are short non-coding coordinated programme of gene regulation underlying RNAs (20–22 nt long) that control gene expression at EMT. the post-transcriptional level by pairing their seed Apart from the direct action of EMT factors on core sequences (2–8 nt at the 50-end) to complementary polarity genes, there are additional possibilities for the sequences located in the 30-UTR region of target transcriptional regulation of cell polarity in association mRNAs (He and Hannon, 2004).This coupling leads with EMT.The distribution of phosphatidylinositol to the degradation of the target mRNA or inhibition of (4,5) bisphosphate at the apical surface is one of the its translation (Filipowicz et al., 2008). Significantly, requirements for the correct apical–basal polarity of evidence for the active participation of specific micro- epithelial cells (Martin-Belmonte et al., 2007). Remark- RNAs in EMT regulation has now been provided and, ably, the PTEN lipid phosphatase that converts in particular, the modulation of ZEB1/ZEB2 mRNA phosphatidylinositol (3,4,5) trisphosphate into phospha- levels by five members of the miR-200 family and by the tidylinositol (4,5) bisphosphate was recently proposed to miR-205 RNA has been reported recently. ZEB1/ZEB2 have an important function in the apical localization of mRNAs, initially described as targets of two members of phosphatidylinositol (4,5) bisphosphate lipids (Martin- the miR-200 family (Christoffersen et al., 2007; Hurteau Belmonte et al., 2007), in addition to its well-known et al., 2007), contain several binding sites for five miR- action counteracting the prosurvival effect of phospha- 200 members and miR-2005 at their 30-UTR regions, tidyl inositol-3OH kinase.Therefore, alterations in the which drive mRNA inactivation and prevent the expression or localization of PTEN could also affect induction of EMT (Gregory et al., 2008; Korpal et al., apical membrane organization.It was recently shown 2008; Park et al., 2008). In accordance with their that PTEN is an additional target of SNAI1 and that it proposed function as negative regulators of EMT, the is strongly repressed when SNAI1 binds directly to the expression of miR-200 and miR-205 RNAs is down- proximal E-boxes of the PTEN promoter (Escriva` et al., regulated in cells that have undergone a full EMT under 2008).Thus, SNAI1-mediated repression of PTEN different stimuli (Gregory et al., 2008; Korpal et al., provides an indirect mechanism to affect both the loss 2008; Park et al., 2008). Similarly, re-expression of of cell polarity and the survival properties of SNAI1- individual miR-200 RNAs in mesenchymal cells induces expressing cells (Vega et al., 2004). This example of partial reversion to an epithelial phenotype and reduces indirect regulation of epithelial cell polarity by SNAI1 the migratory capacity of invasive mouse breast makes it plausible that future studies will identify further carcinoma cells (Gregory et al., 2008; Korpal et al., indirect mechanisms of transcriptional regulation by 2008).Significantly, expression of the miR-200 family is SNAI1 and other EMT inducers that might affect cell lost in invasive breast cancer cells and in metaplastic polarity. breast carcinomas, in conjunction with E-cadherin downregulation (Gregory et al., 2008). Moreover, there Regulating the EMTregulators: transcriptional and is a tight correlation between the expression of the miR- post-transcriptional mechanisms 200 RNAs and E-cadherin in a panel of 60 human Perhaps one of the crucial questions in the EMT field carcinoma cells of the National Cancer Institute relates to the regulation of the different EMT inducers. (NCI60), indicating that the miR-200 family may be a Mechanisms underlying the modulation of EMT strong marker and determinant of the epithelial inducers are just beginning to be revealed.A plethora phenotype in cancer cells (Park et al., 2008). Collec- of signalling pathways are known to control Snai1/Snai2 tively, these results emphasize the participation of the expression both during development and in tumour cells miR-200 family in the regulation of EMT though the (Thiery, 2003; Barrallo-Gimeno and Nieto, 2005). tight control of ZEB1/ZEB2 factors level. Importantly, although many of the signalling pathways Interestingly, regulatory feedback loops between (TGF-b, fibroblast growth factor, Wnt, Notch) impinge miR-RNAs and EMT factors are also starting to on the transcriptional regulation of the Snai1/Snai2 emerge.The expression of two members of the miR- genes (De Craene et al., 2005a; Huber et al., 2005; 200 family is downregulated at the transcriptional level Thiery and Sleeman, 2006), post-translational mechan- by ZEB1 and Snai1 factors in carcinoma cells (Burk isms affecting protein stability and/or nuclear transport et al., 2008), providing a positive regulatory loop to of Snail factors are also emerging as crucial regulators of maintain the expression of ZEB1.On the other hand, their activity (reviewed by Peinado et al., 2007). In other miR-RNAs can act as pro-invasive and pro- contrast to the Snail factors, much less is known about metastatic agents, as exemplified by the miR-10b in the regulation of bHLH and ZEB factors, apart from breast cancer (Ma et al., 2007). Indeed, miR-10b the involvement of some of the aforementioned signal- transcription is upregulated by Twist, another key ling pathways (Peinado et al., 2007). Very recently, inducer of EMT, and its expression inhibits the significant insights have been obtained into the modula- translation of homeobox factor (HOXD10) mRNA, tion of ZEB factors expression, which implicated non- resulting in increased expression of the pro-metastatic coding RNAs in new post-transcriptional mechanisms. gene RHOC (Ma et al., 2007). Although no studies have ZEB1/ZEB2 expression is downregulated in epithelial yet examined the effect of Twist on cell polarity genes, it cells through selective targeting of their mRNAs by is likely that they may also be downregulated during the

Oncogene Polarity regulation in EMT G Moreno-Bueno et al 6963 EMT processes mediated by Twist.There is also exposure to TGF-b, TGF-bRII is recruited to the apical evidence that Snai1 and ZEB2 can be upregulated junction complex where it phosphorylates Par6.Phos- by a distinct post-transcriptional mechanism (Beltran phorylated Par6 recruits Smurf1 (an E3 ubiquitin ligase) et al., 2008). In this case, SNAI1 mediates the induction to the junctional complex that in turn targets the small of a natural antisense transcript of ZEB2 that GTPase RhoA for degradation, provoking EMT prevents splicing of a 50-UTR intron containing (Ozdamar et al., 2005). It is notable that the mechanisms an internal ribosomal entry site, thereby favouring behind Par6 phosphorylation and Smurf1 recruitment, ZEB2 mRNA translation (Beltran et al., 2008). Intrigu- and that are required for TGF-b induced EMT, are ingly, ZEB1 and/or ZEB2 expression can be frequently independent of the Smad pathway (Ozdamar et al., found downstream of Snail factors in different model 2005).This further supports the participation of systems of EMT (Guaita et al., 2002; Moreno-Bueno different pathways downstream of TGF-b in the et al., 2006; Sobrado V, Moreno-Bueno G et al., regulation of cell polarity and EMT. unpublished), supporting the existence of a close Besides TGF-b, activation of receptor tyrosine relationship between Snail and ZEB factors in the kinases also modulates the epithelial phenotype in regulation of EMT. development and tumour cells (Huber et al., 2005; These observations, together with the potent repres- Thiery and Sleeman, 2006), suggesting that they may sion of cell polarity genes by Snail and ZEB outlined also participate in the regulation of cell polarity above, support an active function of microRNAs or complexes.Indeed, activation of ErbB2 was shown to other non-coding RNAs in the regulation of apical– disrupt apical–basal polarity of mammary epithelial basal polarity.It will therefore be important to analyse cells and of epithelial acini in three-dimensional cultures the relationship between the expression of non-coding (Muthuswamy et al., 2001). ErbB2 negatively regulates RNAs, EMT inducers and core polarity genes in cell polarity in epithelial cells by associating with Par6- carcinoma cell lines and tumours. aPKC components, thereby dissociating Par3 and generating an inactive PAR complex (Aranda et al., 2006).Interestingly, the action of ErbB2 on cell polarity Post-transcriptional regulation of the PAR complex disruption seems to be independent of cell proliferation during EMT control (Aranda et al., 2006). These studies indicate that The studies described above indicate that members of post-transcriptional regulatory mechanisms of the PAR the CRB and SCRIB complexes can be transcriptionally complex are indeed important modulators of EMT and regulated by known EMT inducers, such as the ZEB and cell polarity. Snail factors.Although still preliminary, the evidence Collectively, the transcriptional and post-transcrip- available suggests that components of the PAR polarity tional regulatory mechanisms outlined above provide complex are not direct targets of EMT transcriptional further evidence of the strict control of polarity regulators, but rather they can be modulated in response components during EMT.Importantly, some of the to different oncogenes, tumour suppressors or EMT regulatory pathways described so far are activated and regulatory signals (reviewed by Lee and Vasioukhin, implicated in human tumours.For example, ErbB2 has 2008).TGF- b is one of the more robust cues inducing been associated with gynaecological tumours as well as EMT in different model systems (reviewed by Zavadil other human cancer types (Hirohashi and Kanai, 2003), and Bottinger, 2005), leading to the upregulation of highlighting the biological relevance of the regulation of several EMT inducers, including SNAI1/SNAI2, ZEB1, cell polarity in cancer. Twist and the Id factors through Smad-dependent and Smad-independent pathways (Peinado et al., 2003; Kondo et al., 2004; Thuault et al., 2006; Moustakas EMT, cell polarity and cancer stem cells and Heldin, 2007).Recent insights into the regulation of The proteins that regulate cell polarity also have an cell polarity by TGF-b during EMT have converged on important function in asymmetric cell division (Wodarz the PAR complex.Par3 protein levels are diminished and Nathke, 2007), a critical event that guarantees the upon exposure to TGF-b in rat intestinal epithelial cells, two essential properties of stem cells: self-renewal and concomitant to E-cadherin suppression and the induc- differentiation.Studies in Drosophila neuroblasts (stem tion of mesenchymal markers, such as a-smooth muscle cell-like progenitors of the nervous system) have shown actin (Wang et al., 2008). The decrease in Par3 mediated that the asymmetric localization of cell-fate determi- by TGF-b treatment results in a redistribution of the nants is controlled by the Par and Scrib complexes, Par6–aPKC complex from the membrane to the mutations in their components provoking unrestricted cytoplasm and the subsequent disorganization of the tumour growth (reviewed by Lee and Vasioukhin, 2008). Par complex, leading to a loss of polarity.By contrast, Recently, the existence of cancer stem cells has taken overexpression of Par3 strongly blocked the EMT- centre stage in cancer biology, as they are increasingly mediated effects of TGF-b treatment, including the considered as the origin of cancer formation, acquiring downregulation of E-cadherin and a-smooth muscle the alternative denomination of cancer initiating cells. actin expression (Wang et al., 2008). Par6 is also affected At present, the origin of cancer stem cells remains by TGF-b, although through a distinct mechanism.In unclear, and the debate revolves around whether they normal mammary cells, TGF-bRI interacts with Par6 in derive from normal stem cells or if they arise from a complex located at the tight junctions.However, after progenitor or more differentiated cells that acquire

Oncogene Polarity regulation in EMT G Moreno-Bueno et al 6964 specific mutations that endow them with stem cell correlated with more invasive types of diffuse gastric properties, such as self-renewal (Reya et al., 2001; carcinomas (Boussioutas et al., 2003). Other recent data Bjerkvig et al., 2005). Recent data have shed new light have shown decreased expression of DLG and/or on the relationship between EMT and stem cells, SCRIB in colorectal tumours, associated with the lack showing that EMT generates cells with stem cell of epithelial cell polarity and a disorganized tissue properties (Mani et al., 2008). It is noteworthy that architecture (Gardiol et al., 2006), as well as in other both non-transformed and transformed mammary cells tumour types (reviewed by Dow and Humbert, 2007). that undergo EMT following SNAI1 or Twist expres- Decreased LGL1 expression has also been reported in sion acquire stem cell markers and properties, including several tumours, including colorectal carcinomas (Schi- self-renewal and the ability to form mammospheres in manski et al., 2005), endometrial carcinomas (Tsuruga suspension cultures.Moreover, cells similar to stem cells et al., 2007) or melanomas (Kuphal et al., 2006). In isolated from mammary glands or mammary carcino- several cases, the downregulation of LGL1 is associated mas express EMT markers that are associated with the with more advanced stages of the tumour, lymph node increased expression of several key inducers of EMT metastasis and/or poor survival, supporting its use as a (SNAI1/SNAI2, Twist and/or ZEB2) (Mani et al., marker of poor prognosis.In addition to expression 2008).For the first time, these data provide a direct data on core polarity genes, recent studies on LKB1, a link between EMT and the acquisition of stem cell Ser/Thr kinase involved in the regulation of AMPK and properties.Although not formally proven, these data cell polarity (Baas et al., 2004), support its activity as a suggest a link between EMT and the generation of tumour suppressor and its involvement in the control of cancer stem cells that undergo self-renewal, contributing cell polarity in non-small cell lung cancer (Zhang et al., to the generation of secondary tumours at distant sites 2008) and endometrial carcinomas (Contreras et al., (Mani et al., 2008). This proposal is in accordance with 2008). the existence of cancer stem cells with migratory All these observations are evidence that cell polarity properties generated by EMT at invasive regions components participate in human tumours.However, suggested previously (Brabletz et al., 2005) and with there is little information regarding the loss of cell the induction by Snail and Twist of cell survival factors polarity during tumour progression and in relation to and local tumour recurrence (reviewed by Peinado et al., EMT regulators.Indeed, it is presently unclear 2007; Cobaleda et al., 2007). Therefore, it is tempting to whether alterations in the elements involved in main- speculate that the alterations to cell polarity factors taining cell polarity in human tumours are secondary induced in tumour cells due to EMT may underlie the consequences or if they are causally linked to tumour acquisition of a stem-like phenotype.Further studies progression.An inverse correlation has been established into these phenomena in the near future will certainly between ZEB1, an inducer of EMT, and LGL2 clarify these important issues. expression in invasive regions of colorectal and breast carcinomas (Aigner et al., 2007; Spaderna et al., 2008), supporting a causal function for the loss of polarity in tumour progression.However, neither the degree of Cell polarity genes as tumour markers temporal regulation nor the extent of transcriptional programming of polarity components in human tu- Early indications that cell polarity genes are involved in mours has been studied previously.On the other hand, tumorigenesis came from observations in D. melanoga- genetic links between human cancer and DLG, SCRIB ster.Mutations in three neoplastic genes, disc large (dlg), and LGL homologues have not yet been reported.In the scribble (scrib)andlethal giant larvae (lgl), revealed a absence of studies to address these fundamental issues, link between the regulation of cell polarity and cell we have reviewed the analyses of existing gene data sets proliferation.Indeed, because inactivating mutations in for human breast carcinomas to define the correlation these three genes led to neoplastic overgrowth in between the expression of core polarity genes and EMT imaginal discs, they were initially characterized as inducers with the clinical pathological data where tumour suppressors in Drosophila (reviewed by Dow available. and Humbert, 2007; Wodarz and Nathke, 2007; Lee and Vasioukhin, 2008).In mammals, the Dlg, Scrib and Lgl proteins are highly conserved, although different iso- Networking of EMTinducers and cell polarity forms of these proteins have been found.At least four determinants in human tumours DLG (DLG1-4) and two LGL (LGL1-2) genes have been To understand complex biological processes such as the described, whereas only one Scrib homologue was loss of cell polarity and cancer progression, it is identified in humans (Assemat et al., 2008). These genes important to consider differential gene expression in have been considered as tumour suppressors in humans, the context of complex molecular networks.To analyse as they are downregulated in a variety of tumour types. the relationship between EMT inducers and the DLG3 expression is virtually abolished in some oeso- hypothetical function of polarity genes in human phagus tumours (Hanada et al., 2000) and it is strongly tumours, we have analysed gene expression profiles in repressed in gastric carcinoma (Liu et al., 2002).A two different data sets of breast adenocarcinomas (van’t decrease in DLG1 is a hallmark of gastric carcinomas Veer et al., 2002; Ma et al., 2004). We have used these and the additional decrease of DLG4 expression is models because they permit the expression of individual

Oncogene Polarity regulation in EMT G Moreno-Bueno et al 6965 Table 1 Statistical signification between EMT inducer expression and region of the DLG3, LLGL2 and PARD6A genes, polarity genes in two data sets from breast carcinomas (Ma et al., 2004, respectively.These data suggest that the expression of up; van’t Veer et al., 2002: bottom) these genes could be modulated by the EMT-related SNAI1 expression TCF8 expression transcription factors, although further studies are (n %) (n %) required to clarify this assumption. DLG3 expression We also investigated the potential network between Negative (n ¼ 32) 15 (60.0) EMT inducers, cell adhesion and polarity genes at the Positive (n ¼ 33) 10 (40.0) level of direct interactions (Figure 2).We included 15 P ¼ 0.043 genes involved in these processes: DLG, LGL and PARD family members and SNAI1, SNAI2, TCF3, DLG4 expression 19 (67.9) Negative (n ¼ 31) 9 (32.1) ZEB1 and ZEB2/TCF8, as well as five genes related to Positive (n ¼ 29) P ¼ 0.019 cell adhesion, such as CDH1, CLND1, CLDN7, OCLN and JAM1.These genes were used to produce a protein– LLGL2 expression protein interaction network for the direct interactions Negative (n ¼ 32) 17 (68.0) Positive (n ¼ 28) 8 (32.0) between their products (Figure 2).This analysis was P ¼ 0.048 carried out using PathArchitect software incorporating information about these genes from the literature or PARD6 from the experimental data included in different Negative (n ¼ 32) 18 (72.0) databases.The protein–protein interaction network Positive (n ¼ 28) 7 (28.0) P ¼ 0.014 obtained is the first such network available to analyse the function of EMT modulators in the context of cell SNAI2 expression (n %) polarity.We found that the network is typical of the DLG3 expression Negative (n ¼ 45) 30 (75.0) complex cell systems, and that whereas the majority of Positive (n ¼ 34) 10 (25.0) nodes have few links, a few nodes (CDH1, TCF8 or P ¼ 0.001 DLG4) have many links, ensuring that the elements are PARD6 fully connected. Negative (n ¼ 43) 29 (72.5) Positive (n ¼ 36) 11 (27.5) Two different regions were identified in this network: P ¼ 0.001 the ‘cell polarity’ region and the ‘adhesion-EMT’ region. The former included interactions at the protein binding The microarray data from breast tumour samples in both series were level between cell polarity and cell adhesion genes, available as background-corrected and normalized log-10 ratios.The highlighting the important function of the CLND1 and expression values of genes related to cell polarity, EMT inducers and OCLN proteins as they are regulated by the LLGL2 or some cell adhesion molecules were extracted.We categorized the expression of the different genes in terms of each variable, considering DLG2 proteins.In addition, this region marks a zone a tumour as positive or negative using the 75th percentile value as the where the apical junction was regulated and other cutoff.The significance of the associations between categorical interactions between DGL1, 2 and 4, LLGL2 and variables was tested in contingency tables with the Yates correction PARD6A proteins were identified at the same level.In or Fisher’s exact test. the ‘adhesion-EMT’ region, TCF8/ZEB1 appears to modulate the adhesion junctions by interacting with CDH1; interestingly, this interaction appears to be either directly or indirectly regulated by the DLG1 gene. genes to be analysed in both non-metastatic and Importantly, DLG1 and DLG4 seem to link both metastatic primary tumours in situ. regions, and they could be considered as important The correlation between EMT repressors and cell members of this network that connect cell polarity and polarity genes in both series was analysed using the the EMT. categorized expression data test.Accordingly, although More interesting results were obtained when the SNAI1 expression was associated with repression of expression profile of the different transcription factors DLG3, LLGL2 and PARD6 (Ma et al., 2004), the were compared with the clinical-pathological para- upregulation of TCF8/ZEB1 and DLG4 downregulation meters of the data set.In the analysis of lymph node was observed (Table 1, up).However, no significant negative (N0) breast adenocarcinoma series (van’t Veer correlation was found between either the SNAI2 or the et al., 2002), TCF8 emerges as the most important factor TCF3 transcription factors and any of the selected influencing cell polarity.Indeed, the level of TCF8 polarity genes.By contrast, in another series (van’t Veer expression was correlated with metastasis (Table 2) and et al., 2002), the only significant correlation detected was almost all tumours studied that developed metastasis between SNAI2 expression and the downregulation of expressed TCF8 (30/30, Pp0.001). In addition, 26 out DLG3 and PARD6 genes (Table 1, bottom).These of 30 breast tumours that expressed TCF8 displayed a results suggest that the regulation of the selected poor tumour differentiation grade (P ¼ 0.004). More- polarity genes by EMT inducers depends on the tumour over, a highly significant correlation was found between context. TCF8 expression and clinical survival (w2: 6.928, 1 df, The correlation between the transcription factors and P ¼ 0.014, Figure 3). These results indicate that TCF8 polarity genes indicated was reinforced by the identifica- expression can be considered as a new marker of poor tion of 9, 11 and 8 canonical E-boxes in the promoter prognosis in N0 breast adenocarcinoma.

Oncogene Polarity regulation in EMT G Moreno-Bueno et al 6966

Figure 2 Epithelial-to-mesenchymal transition (EMT) and cell polarity protein networks at the direct interaction level.To obtain the protein–protein interaction network, we selected cell polarity genes (DLG, LGL and PARD family members), EMT inducers (SNAI1, SNAI2, TCF3, ZEB1/TCF8, ZEB2/SIP1) and cell adhesion-related (CDH1, CLND1, OCLN, JAM1) genes, and we used the PathArchitect software (Stratagene).( a) ‘Cell polarity region’, (b) ‘EMT-region’ (ellipses with a blue outline indicate the proteins included in the analysis, those without an outline represent proteins closely related according to the databases used).Symbol code indicates different interaction levels.

Table 2 Statistical correlation between TCF8 expression and 1,0 clinicopathological features TCF8 expression (n %) 0.8 Metastasis TCF8-negative Negative (n ¼ 44) 0 Positive (n ¼ 35) 30 (85.7) Pp0.001 0.6

Grade WD (n 8) 0 ¼ Probability 0,4 MD (n 20) 4 (20) ¼ TCF8-positive PD (n ¼ 51) 26 (51.0) P ¼ 0.004 0,2 Well (WD), moderately (MD), poorly (PD) differentiated tumours.

0,0 p=0.014

Concluding remarks and future directions 0.00 2,505,00 7,50 10,00 12,50 There has been a significant advance in recent years Follow up (years) regarding our understanding of the mechanisms involved Figure 3 Kaplan–Meier analysis of TCF8/ZEB1 expression in in EMT and cell polarity regulation.There appears to be a lymph node-negative (N0) breast adenocarcinomas (van’t Veer et al., 2002). Positive (white) and negative (black) expression values direct link between EMT inducers and the transcriptional of TCF8 mRNA were obtained from the average expression ratio. downregulation of several cell polarity genes.Indeed, P-values were derived from log-rank tests. direct and indirect mechanisms that regulate cell polarity components at the transcriptional and post-transcriptional level occur after induction of EMT.Moreover, studies to address the many questions that remain functional interacting networks linking inducers of EMT unresolved.In particular, we must define the relationship to cell polarity and cell junction molecules have identified between EMT and cell polarity, and the cancer stem cell potential regulators in human breast carcinomas, propos- phenotype, as well as the establishment of metastasis and ing a pivotal function of ZEB1 factor.Finally, a link tumour recurrence.Nevertheless, these advances indicate between EMT and stem cell properties has also emerged that EMT inducers may be suitable targets for new recently, which will certainly pave the way for future therapeutic interventions to impair tumour progression.

Oncogene Polarity regulation in EMT G Moreno-Bueno et al 6967 Acknowledgements Education and Science (SAF2007-63051, NAN2004-09230- C04 and CONSOLIDER-INGENIO 2010 CSD2007-00017 to We thank members of A Cano’s laboratory for their excellent AC, and SAF2007-63075 to GMB), the EU (MRTN-CT-2004- work and encouraging discussions, and Gonzalo Gomez 005428) to AC and from the Fundacio´ n Mutua Madrilen˜ a (CNIO, Spain) for helping with the protein network analysis. (2006) to GMB.GMB is a junior researcher contracted on the Our work is supported by grants from the Spanish Ministry of Ramon y Cajal program, 2004.

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