Polarity Proteins in Migration and Invasion

S Etienne-Manneville

Cell polarity and migration group, Institut Pasteur and CNRS URA 2582, Paris cedex 15, France

Cancer is the result of the deregulation of cell prolifera- et al., 1997). In epidermal multilayered epithelium, cells tion and cell migration. In advanced tumors, cells invade that undergo differentiation migrate perpendicularly to the surrounding tissue and eventually form metastases. the basal layer of the epithelium toward the surface of This is particularly evident in carcinomas in which the skin (Kosterand Roop, 2007). In both cases, the epithelial cells have undergone epithelial–mesenchymal directionality of migration is essential for the main- transition. Increased cell migration often correlates with tenance of a functional epithelium. Moreover, intact a weakening of intercellular interactions. Junctions cell–cell contacts maintain the epithelial structure and between neighboring epithelial cells are required to promote coordinated cell migration. Although many of establish and maintain baso-apical polarity, suggesting the molecular mechanisms underlying cell migration are that not only loss of cell–cell adhesion but also common to both normaland tumorcells, tumorcell alteration of cell polarity is involved during invasion. migration is noncoordinated and randomly oriented. Accordingly, perturbation of cell polarity is an important Tumor cells migrate as groups with no clear organiza- hallmark of advanced invasive tumors. Cell polarity is tion oras isolated single cells (Friedl, 2004; Sahai, 2005). also essential for cell migration. Indeed, a front-rear Tumor cell migration leads to alteration of the polarity axis has first to be generated to allow a cell to epithelium structure, disruption of the basal lamina migrate. Because cells migrate during invasion, cell and finally invasion of the tumorinto the underlying polarity is not completely lost. Instead, polarity is tissue. This transition from a collective to an individual modified. From a nonmigrating baso-apically polarized migration recapitulates the developmental process epithelial phenotype, cells acquire a polarized migrating known as epithelial–mesenchymal transition (EMT) mesenchymal phenotype. The aim of this review is to and serves as a good indicator of tumor progression highlight the molecular relationship between the control of (Friedl and Wolf, 2003; Guarino, 2007). During EMT, cell polarity and the regulation of cell motility during polarized epithelial cells that are tightly connected to oncogenesis. one anotherby intercellularjunctions undergotwo Oncogene (2008) 27, 6970–6980; doi:10.1038/onc.2008.347 major functional changes. First, they dissociate from neighboring cells and second, they acquire the ability to Keywords: Par proteins; epithelial mesenchymal transi- migrate away from the original tissue. The molecular tion (EMT); Rho GTPases; Wnt; Scribble mechanisms by which highly polarized and tightly joined epithelial cells transform into randomly migrating cells are likely to have major functions during tumor development.

Introduction

The formation of distant tumorigenic foci and metas- Cell polarity is essential in cell migration tasis are the main cause of morbidity and mortality in patients with cancer. Controling tumor cell migration is Cell migration is a fundamentally polarized process. Cell a central issue in cancer treatment. Nevertheless, the polarity during cell migration encompasses several cascade of events leading to cell invasion during tumor aspects (Figure 1). It is important to distinguish random development is still poorly understood. In a normal cell migration, in which cells migrate in all directions in polarized epithelium, cell migration occurs concomi- a noncoordinated manner, from directed (or oriented) tantly to cell differentiation and is required for normal cell migration, in which cells respond to polarizing cues tissue renewal. In the gut epithelium, for instance, cells to migrate in a given direction. In both cases, cell migrate from the base of the epithelial crypt toward the polarity is required to generate a front-rear axis (Ridley top of villi until they are shed into the gut lumen (Potten et al., 2003). The small G-proteins, Cdc42, Rac and Rho, seem to be at the heart of the initial signals leading to the Correspondence: Dr S Etienne-Manneville, Cell polarity and migration group, Institut Pasteur and CNRS URA 2582, 25 rue du Dr polarization of migrating cells (Charest and Firtel, Roux, 74724 Paris cedex 15, France. 2007). They participate in cell migration by regulating E-mail: [email protected] cytoskeletal reorganization, a prerequisite for cell Polarity proteins S Etienne-Manneville 6971

Figure 1 Polarity in migrating cells. In response to a chemotactic gradient, both front-rear polarization (green arrow) and direction sensing (red arrow) must occur to transform a nonpolarized and randomly oriented cell (on the left) into a fully polarized migrating cell (on the right). Front-rear polarization is required for migration per se, but is not sufficient forchemotaxis (lowerdrawing).In this case, cells undergo random migration. Cell orientation following direction sensing is crucial to promote directed (or oriented) migration, but is not sufficient to drive cell migration (upper drawing). Orientation of the nucleus-centrosome axis is a good indicator of cell orientation during directed migration of most cell types including fibroblasts, astrocytes or epithelial cells. Actin-driven protrusions are shown in red, microtubules appear in dark green and centrosome in light green. Straight red lines represent stress fibers and red patches represent focal complexes and focal adhesions. N stands for nucleus. motility (Etienne-Manneville and Hall, 2002; Charest along the chemotactic gradient (Allen et al., 1998). and Firtel, 2007). Importantly, the balance of activities Direction sensing is also strongly perturbed, when of the three proteins must be tightly controlled between Cdc42 is depleted from migrating astrocytes (Etienne- the front and the rear of the cell. A simplistic view is that Manneville and Hall, 2001). In contrast with the Cdc42 and Rac are gradually activated toward the front generation of front-rear polarity, which is essentially edge where they control actin and microtubule rearran- actin dependent, cell orientation is largely dependent on gements to promote protrusive activity, whereas Rho is microtubules (Etienne-Manneville, 2004a; Siegrist and rather active at the rear of the cells where, by inducing Doe, 2007). In a wound-healing assay, centrosome and actomyosin contractility, it controls rear-end retraction Golgi reorientation as well as microtubule network and allows forward movement. The role of Cdc42 is polarization do not occur in the absence of Cdc42 actually dual—it not only participates in the protrusive (Osmani et al., 2006) orfollowing perturbationof activity but it is also directly involved in cell orientation microtubule dynamics (Etienne-Manneville and Hall, during directed migration in response to exogenous 2001). Interestingly, expression of a constitutive active polarity cues, such as chemotactic signals or cell Cdc42 or overexpression of an active Cdc42 GEF wounding (Etienne-Manneville, 2004b). Directed migra- perturbs centrosome orientation and microtubule net- tion implies the precise orientation of the front-rear axis work reorganization. This strongly suggests that not following direction sensing. Direction sensing appears to only the activation of a polarity signaling pathway but be a major and evolutionary conserved function of also its spatially restricted localization are required for Cdc42. In chemotactic macrophages, dominant negative correct cell orientation. Cdc42 leads to random migration. In this case, cells can Whereas inhibiting front-rear polarity totally blocks still form a front-rear axis, but are unable to orient it migration, perturbing direction sensing only leads to

Oncogene Polarity proteins S Etienne-Manneville 6972 random migration, during which the direction of the in apicobasal polarity. A growing body of evidence front-rear axis does not need to be determined nor even involves polarity proteins in cell migration. Polarity sustained. Although some tumorcells follow chemotac- proteins are therefore likely to be essential players tic signals, invasion of the surrounding tissue does not, during oncogenesis. in principle, require directed cell migration. This is a Epithelial cell polarity is characterized by the presence major difference with embryogenesis or adult tissue of adherens and tight junctions, which control the renewal where coordinated and directed cell migration is segregation of the plasma membrane in biochemically tightly regulated. In baso-apical polarity, generating a and functionally distinct apical and basolateral do- polarity axis is directly related to its correct orientation mains. A strict separation between these two domains is within the epithelium. This implies that signaling path- essential for an epithelium to function as a barrier. ways are common to both polarization and orientation. Three groups of proteins act together to generate and In contrast, during cell migration, front-rear polariza- maintain baso-apical polarity: (1) the Par complex, that tion and cell orientation are controlled by independent includes Parproteins and atypical proteinkinase C polarity pathways. Growing evidence shows that polar- (aPKC); (2) the Scrib complex, formed of Scrib, Dlg and ity complexes involved in baso-apical polarity have Lgl and (3) the Crb complex which, in addition to Crb, important functions in regulating polarity during cell involves PALS1 and PATJ (Assemat et al., 2008; migration. Figure 2). These polarity proteins have all been ﬁrst identiﬁed using model organisms, such as Caenorhabdi- tis elegans or Drosophila melanogaster ((Kemphues Polarity proteins are common regulators of epithelial cell et al., 1988; Tepass et al., 1990; Knust et al., 1993; polarity and polarized cell migration Bilder et al, 2000) and fora review see (Dow and Humbert, 2007; Goldstein and Macara, 2007; Wang and Because epithelial cells are responsible for a majority of Margolis, 2007), also reviewed in Aranda et al., 2008 cancers, and because, in contrast with leukocyte-derived and Humbert et al., 2008). It was laterrealized that most tumors, the ability of tumor cells to migrate is acquired polarity proteins are highly conserved among multi- during oncogenesis, we will focus on proteins involved cellular organisms from worms to humans (Stern, 2006)

Figure 2 Polarity complexes in polarized epithelial cells (a) and in polarized migrating cells (b). The localization of the three polarity complexes Par (in red), Scrib (in blue) and Crb (in green) are shown. Whereas in epithelial cells, each complex displays a different localization, the three complexes are all localized at the leading edge of migrating cells. The major interactions between polarity complexes (depicted in the lower boxes) strongly differ between epithelial cells (on the left) and migrating cells (on the right). Although the same polarity proteins control baso-apical polarity and oriented cell migration, their regulation, localization and interactions are different in the two situations. The localization of each complex is shown by a line of the respective color (Crb complex in green, Par in red and Scrib in blue). Positive interactions ( þ ) between complexes are indicated by an arrow, negative interactions (À)are shown by bars.

Oncogene Polarity proteins S Etienne-Manneville 6973 and control various aspects of cell polarity, including promote RhoA degradation (Figure 3b). Par6 could not only baso-apical polarity of epithelial cells but also thus participate in establishing front-rear polarity by asymmetric cell division, neuronal differentiation and inhibiting cell contraction at the sites of Cdc42- or Rac- cell migration. Despite the central and conserved role of induced actin polymerization and by favoring protrusive these three polarity complexes, the precise functional activity at the front edge (Wang et al., 2003). Whereas interactions between proteins participating in each Par6 and aPKC seem to act in cell orientation through complex and between the three complexes themselves Cdc42, Par3 has been connected to Rac through the still remain unclear. Studies in model systems indicate Rac-exchange factor Tiam1. Par3 controls tight junction that polarity proteins are at the center of various assembly through Tiam1 (Chen and Macara, 2005; signaling pathways that differdepending on the context Mertens et al., 2006). Recently, however, an additional of cell polarity. One common theme, however, is that the function of Par3 in controlling directionality of cell localization of polarity complexes is essential to their migration during keratinocyte chemotaxis has been function. In polarized epithelial cells, for instance, the described (Pegtel et al., 2007). The Par3–Tiam1 complex Par proteins Par3, Par6 and aPKC are localized at the promotes persistent and directed migration by stabiliz- apical–lateral boundary, whereas the Scrib complex is ing the microtubule network possibly through the associated with the lateral cortex (Izumi et al., 1998) regulation or localization of Par6 and aPKC. (Figure 2a). Basolateral Scrib suppresses apical mem- Anotherinterestingand intricateconnection is the brane extension by excluding the Par complex from the relationship between Par proteins and Wnt signaling. lateral side (Bilder et al., 2003; Tanentzapf and Tepass, Among the many substrates of aPKC, GSK3b has been 2003). Conversely, the Par proteins recruit Crb that shown to be essential for orientation of migrating cells, antagonizes Scrib complex activity at the apical surface suggesting an essential cross talk between Par proteins (Hurd et al., 2003; Lemmers et al., 2004). and Wnt signaling. The intracellular signaling pathway activated by Wnts was originally identified as a b-catenin-dependent pathway (Logan and Nusse, The Par complex 2004). The binding of Wnt to its receptor stabilizes The first polarity complex shown to be involved in the b-catenin through the recruitment of the b-catenin polarity of migrating cells was the Par complex. Cross destruction complex, which includes Axin, Dvl (dishev- talk between Par proteins and small G-proteins of the eled), GSK3b and APC (adenomatous polyposis coli). Rho family seems to be essential formultiple aspects of Stabilized b-catenin enters the nucleus and stimulates cell migration. First, Par6 and aPKC were biochemically the transcription of target genes. In migrating astro- shown to be downstream targets of Cdc42 (Joberty cytes, Cdc42-dependent aPKC activation leads to et al., 2000; Lin et al., 2000). It was then demonstrated phosphorylation and inactivation of GSK3b, which is that Par6 and aPKC downstream of Cdc42 regulate cell in turn responsible for the clustering of APC at polarity in wound-induced directed migration of astro- microtubule plus-ends of the leading edge (Etienne- cytes and fibroblasts (Etienne-Manneville and Hall, Manneville and Hall, 2003). APC and GSK3b regulate 2001; Gomes et al., 2005). Following wounding, Cdc42 microtubules in multiple ways (Barth et al., 2008). is recruited at the wound edge of astrocytes and Inhibition of GSK3b by aPKC and recruitment of APC activated. Cdc42 binds to Par6 and thereby promotes to microtubules at the leading edge of migrating cells aPKC activation. The localized activation of aPKC is control centrosome reorientation and correctly oriented crucial for microtubule polarized organization and cell migration (Etienne-Manneville and Hall, 2003). In orientation. Inhibition of aPKC does not perturb fibroblasts, Wnt5a may control this signaling pathway establishment of the front-rear axis, but affects its independently of Cdc42 (Schlessinger et al., 2007). Par correct orientation and induces random cell migration. proteins and the Wnt pathway are also connected in The contribution of Par6–aPKC as well as Par3 in axonal polarity. Dvl, which acts as a scaffold protein directed cell migration is also illustrated by their downstream of Wnt receptors, has been shown to bind function in Drosophila border cell migration (Pinheiro and activate aPKC to promote polarization of develop- and Montell, 2004). During Drosophila oogenesis, a ing neurons (Zhang et al., 2007). Par3 has also been group of polarized epithelial cells looses their baso- involved in cell polarization during axon extension apical polarity to undergo directed migration. During through APC and GSK3b (Nishimura et al., 2004; Shi the initiation of migration, Bazooka (Drosophila Par3) et al., 2004). APC and GSK3b present in the axon tip and Par6, initially localized at the apical surface of the recruit Par3, which reaches the growing end of axons by cells, concentrate predominantly on the leading edge of moving along microtubules through the plus-end- the cell cluster (Pinheiro and Montell, 2004). In border directed kinesin KIF3A. cell migration as well as in wound-induced migration of In addition to the Par3–Par6–aPKC complex, LKB1 astrocytes, overexpression of Par6 or activated (STK11), the closest ortholog of C. elegans PAR-4, has aPKC delays the migration probably by perturbing received intense attention because of its function in directionality. Peutz–Jeghers syndrome (Hemminki, 1999). LKB1 acts In addition to its connection to Cdc42, Par6 also has a as a tumor suppressor, and alteration of its activity leads function in the regulation of RhoA. Once localized at to the development of polyps along the gastrointestinal the cell leading edge, Par6 recruits Smurf1 (Smad tract and a high risk of epithelial-derived cancer (for a ubiquitination regulatory factor-1), which can locally review, see (Katajisto et al., 2007) and Hezel and

Oncogene Polarity proteins S Etienne-Manneville 6974

Figure 3 Simplified diagram showing the putative mechanisms involved in the modifications of cell polarity during oncogenesis. Epithelial–mesenchymal transition (EMT) is a crucial event during the transformation of polarized epithelial cells into polarized invading cells. EMT is induced by several oncogenic signals, which lead to the relocalization of polarity proteins. Four of these oncogenic signals are shown here. (a) Alteration of cell–cell junctions results in the relocalization of polarity proteins. In particular, the Par3-Tiam1-Rac pathway leads to the formation of protrusive sites, whereas Par6-aPKC and Cdc42 are likely to promote cell orientation. (b) TGFb signaling induces Par6 recruitment and phosphorylation independently of the Smad pathway (not shown here). Phosphorylated Par6 promotes, through aPKC and Smurf1, the degradation of Rho and the formation of cell protrusion. In addition, Par6 may also recruit Cdc42 and/or Rac to further promote cell protrusion and possibly cell orientation. (c) Activation of tyrosine kinase receptors by various ligands, such as epidermal growth factor, fibroblast growth factor or hepatocyte growth factor, mediates downstream signaling involving the production of PIP3. Polarity proteins, such as Par3, are recruited to sites of PIP3 production, and togetherwith PIP 3-activated exchange factors, recruit and activate the small GTPases Cdc42 and Rac. In these three cases (a, b and c), the exact localization of the polarity complexes determines the orientation of the cell. As the loss of cell–cell contacts (a) as well as nonrestricted growth factor signaling (b and c) induces random positioning of the activated pathways, migration is likely to be random and uncontrolled. In all three cases, relocalization of Par complexes to various membrane sites also affects cell–cell junctions and baso- apical polarity. (d) APC mutations can affect Wnt signaling and its connection to the Par complex. Loss of the carboxy-terminal domain of APC, by preventing the APC–Dlg1 interaction, has a dramatic effect on cell orientation and leads to random cell migration.

Bardeesy, this issue). Like the other Par proteins, LKB1 2008). Moreover, the function of LKB1 in neuronal has a crucial function in epithelial cell polarization (Baas differentiation and migration, and centrosome position- et al., 2004) and has been linked both to Rho GTPases ing during these processes (Barnes et al., 2007; Shelly signaling and to the Wnt pathway. LKB1 is a serine– et al., 2007) is probably, at least partially, conserved. In threonine kinase, which phosphorylates other protein cortical neurons, overactivation of LKB1 leads to the kinases including AMPK, SAD A/B, the Par1-related formation of several axons suggesting that LKB1 proteins MARCKs and GSK3b. A high LKB1 activity regulates cell orientation rather than the ability to can drive the formation of an apical surface in isolated protrude. Localization of the active form of LKB1 to epithelial cells even in the absence of intercellular the extending axon is crucial. Although the mechanisms contacts. Inhibition of LKB1 as well as the over- controlling LKB1 are not clear, its activator Strad and expression of a constitutively active protein alters LKB1 phosphorylation have a function. Several down- polarized astrocyte migration and perturbs centrosome stream targets of LKB1 seem to contribute to LKB1 reorientation (Forcet et al., 2005). The regulation and functions in cell polarity (Alessi et al., 2006). SADs and downstream effectors of LKB1 during cell migration MARKs can phosphorylate microtubule-associated have not been clearly identiﬁed yet. LKB1 has been proteins, regulate microtubule stability and promote shown to interact with active Cdc42 to maintain Cdc42 microtubule network orientation. AMPK contributes to at the leading edge of migrating cells (Zhang et al., cell polarity by directly phosphorylating MRLC (myo-

Oncogene Polarity proteins S Etienne-Manneville 6975 sin-regulatory light chain), which induces acto-myosin Hall, 2005). In these in vitro wound-healing models, contractility. Similarly to aPKC, LKB1 can also inhibit Scrib seems to be required for persistent directed GSK3b by phosphorylation, which is responsible for b- migration by localizing and maintaining the protrusive catenin phosphorylation in Wnt signaling (Ossipova activity at the cell leading edge. As a consequence, loss et al., 2003). Finally, LKB1 is linked to the Wnt of Scrib is likely to lead to an invasive phenotype. pathway through its substrate Par1. Par1 can phosphor- Consistently, mislocalization of Scrib also perturbs cell ylate Dvl and modulate noncanonical Wnt signaling, orientation and overexpression of the wild-type protein whereas a distinct isoform of Par1 regulates canonical or expression of a mutant that cannot localize correctly Wnt signaling through a separate but unknown mechan- at the plasma membrane leads to the generation of ism (Sun et al., 2001; Ossipova et al., 2005). multiple protrusive sites (Osmani et al., 2006). Like Scrib, Dlg1 participates in astrocyte directed migration (Etienne-Manneville et al., 2005). Following The Scrib complex wounding, Dlg1 is targeted to the front edge plasma Recent evidence implicates the Scrib complex in cell membrane where it has an important function in the migration in most animal and cellular models. Scrib, regulation of the microtubule network. Although the Dlg and Lgl are all involved in Drosophila dorsal mechanisms by which Dlg1 associates with the plasma closure. Loss of Scrib together with one allele of Dlg membrane are not elucidated, its recruitment depends strongly perturbs dorsal closure. Similarly, loss of Lgl on Scrib, Cdc42, Par6 and aPKC. Loss of Dlg1 does not inhibits dorsal closure (Manfruelli et al., 1996; Arquier affect protrusion formation and only perturbs cell et al., 2001). During Drosophila oogenesis, absence of orientation. Dlg1 controls microtubule interaction with Dlg induces invasion of follicularepithelial cells (Goode the plasma membrane at the leading edge of migrating and Perrimon, 1997). Interestingly, in this model, Dlg cells. Dlg1 interacts with APC, and the PDZ-mediated cooperates with Bazooka to favor this invasive cell interaction between these two tumor suppressors is behavior(Abdelilah-Seyfried et al., 2003). In zebrafish, likely to be involved in the capture of microtubule plus- Scrib is required for convergent extension during ends at the plasma membrane. The Dlg1–APC interac- embryogenesis and for motor neuron migration (Wada tion is particularly interesting as it provides another link et al., 2005). In mice, point mutations in the Scrib gene between a polarity protein and Wnt signaling, APC alterepithelial sheet migration and leads to defects in being an essential regulator of the Wnt pathway. neural tube and abdominal wall closure (Murdoch et al., Lgl mutant cells form metastatic tumors in Drosophila 2003; Zarbalis et al., 2004). In mammalian epithelial (Woodhouse et al., 1998), but the function of Lgl in cells and astrocytes, depletion of Scrib affects cell polarized cell migration has not been studied in detail. migration in wound healing assays. In MDCK cells, Biochemically, Lgl is phosphorylated by aPKC suggest- loss of Scrib leads to increased random cell migration ing that it may also be involved in the control of cell associated with weakened adherens junctions (Qin et al., orientation. Inhibiting Lgl phosphorylation only has 2005). Although the exact role of Scrib in cell–cell limited effect on protrusion formation (Plant et al., junctions has not been deciphered to date, Scrib 2003). However, the same nonphosphorylatable mutant function in cell–cell adhesion appears to be at least of Lgl dramatically inhibits centrosome reorientation in partially responsible for the increased epithelial cell astrocytes (S E-M, unpublished results). In epithelial migration. In mammary epithelial cells and in astro- cells, Par6 and aPKC form a complex with Lgl, which cytes, loss of Scrib rather slows down migration prevents Par3 interaction with aPKC (Yamada et al., (Osmani et al., 2006; Dow et al., 2007). Cell velocity 2003; Yamanaka et al., 2006). Whetherthe same may then vary between cell types depending on the competition between Lgl and Par3 also occurs during strength of cell–cell junctions and on how cell–cell cell migration is still unclear. junctions restrain cell migration. In all cases, however, Scrib depletion induces random migration suggesting that Scrib controls cell orientation rather than cell The Crb complex motility per se (Dow et al., 2007). This idea is confirmed Little is known about the function of the Crb complex in by the finding that, in wound healing assays, in the the regulation of polarity during cell migration. How- absence of Scrib, centrosome and Golgi reorientation ever, K Shin et al. have recently shown that PATJ is toward the wound edge does not occur and the required for directional epithelial migration in an in vitro microtubule network does not polarize. wound-healing assay (Shin et al., 2007). PATJ is How may Scrib control cell orientation? Scrib is recruited to the leading edge of migrating cells and required for Cdc42 activation and localization to the control the localization of Par3 and aPKC possibly leading edge plasma membrane following wounding. through PALS1 and Par6 (Hurd et al., 2003; Wang During cell migration, Scrib interacts with the Cdc42- et al., 2004). Depletion of PATJ perturbs cell orientation and Rac-exchange factor bPIX, which is also recruited and slows down wound closure either by reducing cell to the leading edge and promotes Cdc42 activation and velocity or inhibiting persistence of cell migration. Crb subsequent cell orientation. It appears, however, that does not seem to be involved in this phenomenon; bPIX is not responsible for Rac activation and Rac- however, future studies are likely to point to a function induced protrusive activity but may be involved in of all the Crb complex proteins in the regulation of controling Rac localization at the leading edge (Cau and polarized migration.

Oncogene Polarity proteins S Etienne-Manneville 6976 In summary, polarity complexes essential to baso- polarity proteins and polarity proteins regulate cell–cell apical polarity of epithelial cells share a conserved junction stability. In epithelial cells, upregulation or fundamental regulatory function during cell migration. downregulation of polarity proteins or modification The signaling through polarity complexes is strongly of their localization perturbs tight and adherens junc- coupled with both small G-proteins signaling and the tions and leads to a loss of baso-apical polarity of the Wnt pathway. Although only few of the proteins epithelium (Helfrich et al., 2007). participating in these complexes have been directly involved in front-rear polarization, they are all essential for cell orientation. Upregulation, downregulation and Activation of TGFb signaling mislocalization of these proteins dramatically affect In addition to the downregulation of E-cadherin, which sustained cell orientation, and therefore coordinated may occur as a consequence rather than as a first cause migration. of EMT, oncogenic signal transduction pathways as well as extracellular signals including growth factors, cyto- kines and proteases, secreted by the surrounding cells has an important function in EMT (Kopfstein and Oncogenic signals lead to the relocalization of polarity Christofori, 2006). TGFb is one of the most potent and proteins better-studied inducers of EMT (Levy and Hill, 2006). In polarized epithelial cells, TGFb promotes EMT A recurrent theme in the regulation of cell polarity is the associated with a loss of baso-apical polarity and apical need for a restricted localization of the polarity junctions (Thiery and Huang, 2005). TGFb acts through complexes. How are these molecules affected during serine–threonine kinase receptors that phosphorylate the oncogenic process? Is their expression level and/or Smads to regulate gene transcription. In addition, the theirlocalization modified and how can these events TGFb-receptor recruits and phosphorylates Par6 at the affect cell motililty? On the basis of the findings plasma membrane, independently of the Smad pathway summarized above, signals that regulate the activity (Ozdamar et al., 2005). This regulation of Par6 is likely and the localization of polarity proteins in order to to participate in the disruption of intercellular junctions maintain baso-apical polarity in normal conditions may and also in the local degradation of Rho, which may be lost or modified during tumor progression. Polarity contribute to the generation of a protrusive site proteins may be diverted toward regions of the plasma (Figure 3b). Accordingly, TGFb-induced EMT seems membrane to promote protrusive activity, participate in to be Rho dependent (Bhowmick et al., 2001). establishing front-rear polarization and control cell orientation. Cross talks between oncogenic signals and polarity complexes were recently described ((Dow Alteration of the Wnt pathway and Humbert, 2007; Wodarz and Nathke, 2007; Lee and Alteration of the Wnt signaling pathway is a well Vasioukhin, 2008) and Humbert et al., 2008), recognized characteristic of early stages in epithelial and may provide mechanisms explaining how basal tumor formation. Wnts control directed movements and apical polarity complexes can relocalize to the during gastrulation where cells undergo transient leading edge of migrating cells (Figure 3). epithelial to mesenchymal transitions, allowing the cells to dissociate and migrate. In the Wnt signaling pathway, Loss of cell–cell junctions APC interacts with b-catenin to control its degradation E-cadherin is considered as an invasion suppressor, as by the proteasome and to regulate the levels of cytosolic loss of E-cadherin-mediated junctions promotes transi- b-catenin (Huang and He, 2008). APC mutations that tion from adenoma to carcinoma ((Hirohashi, 1998; Perl cause familial adenomatous polyposis and colonic et al., 1998; Cavallaro and Christofori, 2004), and also cancerhave been extensively studied (fora review,see reviewed in Jeannes et al., 2008). Upon disruption of (Segditsas and Tomlinson, 2006; Kikuchi and Yama- adherens junctions, epithelial polarity is lost, Rho moto, 2008)). Most of these mutations lead to the GTPases, Par proteins and the Scrib complex are truncation of the carboxy terminal part of the molecule. released from cell–cell contacts, and change localization Although they do not induce a total loss of the protein, to promote cell migration (Figure 3a). For instance, loss which has been shown to prevent epithelial migration of tight junctions affects the localization of Par3, which along the intestinal crypt, these truncations perturb the is normally associated with the junctional adhesion interaction between APC and b-catenin and deregulate protein JAM-A (Itoh et al., 2001). It is likely that the canonical Wnt signaling. They also prevent APC relocalization of Tiam1 following Par3 uncoupling from from interacting with the microtubule plus-end asso- junctional sites, has a function in cell invasion as mice ciated protein EB1 and with Dlg1 (Figure 3d). It is very deficient for Tiam1 are resistant to Ras-induced skin likely that these truncations, in addition to their effect tumors (Malliri et al., 2002). Loss of Par6 from cellular on gene expression, strongly perturb microtubule contacts during HGF-induced epithelial cell scattering regulation and thereby cell orientation. This would has also been observed (Johansson et al., 2000). The again favorrandom motility without totally inhibiting close relationship between polarity proteins and epithe- cell migration. Indeed, most mutations do not affect the lial junctions leads to a typical chicken and egg situation armadillo repeats that are required for the Kif3–KAP3 in which cell–cell junctions control the localization of interaction, and are probably needed for Par3, Par6 and

Oncogene Polarity proteins S Etienne-Manneville 6977 aPKC transport to the lamellipodia. (Nishimura et al., sensing and correct cell orientation and overall stimu- 2004; Shi et al., 2004). lates random migration leading to the uncontrolled cell invasion. Although a clearimage of the mechanisms controlling Growth factor-induced PI3K signaling polarity proteins during cancer development is only The relocalization of polarity proteins could also be beginning to appear, it seems that the molecules and mediated by phosphoinositides. Tumorcells as well as signaling pathways involved in EMT lead to mislocali- cells of tumor microenvironment secrete a variety of zation of polarity proteins from cell–cell contacts and growth factors, which act on tyrosine kinase receptors. affects polarity signaling pathways. Upon stimulation, these receptors dimerize and autho- phosphorylate allowing the binding to SH2 domain containing molecules, such as PI3K, which, in turn, locally generates PIP . A large number of studies on 3 Conclusion isolated migrating cells, such as Dictyostelium cells or leukocytes, indicate that PI3K and the phophoinositide Little is known about the direct contribution of polarity phosphatase, PTEN (Merlot and Firtel, 2003; Kolsch proteins during cancer cell invasion in vivo. Evidence, et al., 2008) have an important function in establishing however, converges to indicate that polarity proteins front-rear polarity. The levels of PIP and PIP are 2 3 may indeed be acting as positive or negative regulators sufficient to specify basolateral and apical membrane in during the development of invasive cancers. A number epithelial MDCK cells (Martin-Belmonte et al., 2007). of studies have shown decreased expression or a The link between PIP signaling and polarity proteins is 3 complete loss of Scrib, Dlg or Lgl in primary tumors not clearly identified, but addition of PIP to the apical 3 forhuman patients (Cavatorta et al., 2004; Nakagawa surface is sufficient to induce transient localization of et al., 2004; Schimanski et al., 2005; Gardiol et al., 2006; basolateral proteins to this membrane and exclude Kuphal et al., 2006). It is not surprising to find that loss apical proteins. Drosphila Par3 (Bazooka) binds directly of or decreased Scrib, Dlg or Lgl correlates with more to PTEN (von Stein et al., 2005). Although this invasive tumors as these molecules are essential for interaction has not been confirmed in mammalian cells, maintaining directed migration (Humbert et al., 2006). PI3K activity is required for Par3 localization either at Interestingly, the E6 oncoprotein from HPV16 and cell–cell contacts orin lamellipodia, and is criticalfor HPV18 papilloma viruses, which are responsible for a Par3 localization at the tip of differentiating axons vast majority of cervical cancers, target Scrib and Dlg (Sander et al., 1998; Shi et al., 2003). Furthermore, fordegradation ((Thomas et al., 2005), also reviewed in numerous exchange factors for Cdc42 and Rac are Thomas et al., 2008). Von-Hippel–Lindau, a tumor recruited and/or activated by phosphoinositides, which suppressor involved in the von-Hippel–Lindau disease, may indirectly be responsible for the recruitment and which leads to the development of hemagioblastoma, activation of the Par6-aPKC complex. PTEN also clear-cell renal carcinoma and pheochromocytomas interacts with LKB1 and controls its localization (Kaelin, 2005), interacts with the Par3–Par6–aPKC (Mehenni et al., 2005). complex and regulates microtubule network organization (Schermer et al., 2006). It also controls ubiquitin- Activation of the Ras pathway mediated degradation of activated aPKC (Okuda et al., Activation of the small G-protein Ras and of the Raf– 2001). MEK–ERK downstream pathway is observed in a large Downregulation of most polarity proteins is likely to proportion of human cancers (Dhillon et al., 2007). In strongly affect apico-basal polarity and promote ran- Drosophila, molecules of the Scrib complex have been dom noncoordinated migration, leading to the disrup- shown to cooperate with oncogenic Ras or Raf to tion of the epithelial structure and to tissue infiltration promote tumor progression and favor metastasis of by cancer cells. Upregulation of polarity proteins and/or activated Ras cells (Pagliarini and Xu, 2003; Tapon, overactivation of polarity pathways generally perturb 2003). In the absence of Scrib, activated Ras affects cell orientation and coordinated migration. Studies on epithelial cell–cell junctions leading to the increased PKCz and PKCi, have shown that these proteins are cell migration. Recently, Le Dow et al.(2008) have overexpressed in colon, ovarian and lung human cancers shown that loss of Scrib or Dlg1 similarly promotes (Eder et al., 2005; Regala et al., 2005; Zhang et al., Ras-dependent invasion of mammary epithelial cells. 2006). PKCz has also been implicated in human cancers. Activated Ras prevents normal epithelial junction PKCz is hyperactivated in head-and-neck carcinoma formation, perturbs the polarity of epithelial cysts in (Cohen et al., 2006) and has a crucial function in the matrigel and is likely, therefore, to alter polarity regulation of tumor-cell invasion and metastasis protein localization. Overexpression of Scrib regulates (Sun et al., 2005; Kuribayashi et al., 2007). But, Ras target gene expression, restores normal cadherin expression level of polarity proteins is not the only expression and epithelial morphology of Ras parameter that can dramatically affect migratory mutant cells. In contrast, knockdown of Scrib behavior. It is highly probable that the modification of promotes cell protrusion, induces cell invasion out of theirlocalization by oncogenic signals is also crucial the cellularacini. It is tempting to speculate that forthe initiation of an invasive phenotype. More in these conditions, loss of Scrib perturbs direction refined analysis of gene expression combined with

Oncogene Polarity proteins S Etienne-Manneville 6978 biochemical studies and precise localization of polarity Acknowledgements proteins is likely to provide novel insights into tumor cell invasion. We apologize for those authors whose work was not cited directly owing to space limitation. This work was supported by grants from the l’Agence Nationale pour la Recherche, Abbreviations la Fondation de France, La Ligue contre le Cancer et l’Association pour la Recherche sur le Cancer, la Fondation APC, adenomatous polyposis coli; aPKC, atypical protein kinase Schlumberger pour l’Enseignement et la Recherche, the C; Dvl, disheveled; EMT, epithelial–mesenchymal transition. EMBO Young Investigator Program.

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