Polarity Regulators and the Control of Epithelial Architecture, Cell Migration, and Tumorigenesis

Polarity Regulators and the Control of Epithelial Architecture, Cell Migration, and Tumorigenesis Lukas E. Dow*,{ and Patrick O. Humbert*,{,{ *Cell Cycle and Cancer Genetics Laboratory, Peter MacCallum Cancer Center, Melbourne, Australia Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Australia { Department of Pathology, University of Melbourne, Parkville, Australia {

A large body of work on Drosophila melanogaster has identified and characterized a number of key polarity regulators, many of which are required for the regulation of multiple other processes including proliferation, migration, invasion, and tumorigenesis. Humans possess either single or multiple homologues of each of the Drosophila polarity proteins, and in most cases, these are highly conserved between species, implying an important and conserved function for each of the polarity complexes. Recent studies in cultured mammalian epithelial cells have shed some light on the requirement for the polarity complexes in the regulation of epithelial cell function, including an unexpected link to the regulation of directed cell migration. However, many questions still remain regarding the molecular mechanisms of polarity regulation and whether disruption of polarity protein function is an important step in the development of human cancers. Here we will review what is currently understood about the regulation of cell polarity, migration, and invasion and the level of functional conservation between Drosophila and mammalian tissues. Particular reference will be made as to how the Scribble and Par polarity complexes may be involved in the regulation of apical–basal polarity, migration, and tumorigenesis. KEY WORDS: Scribble, Par, Polarity, Migration, Apical–basal polarity, Epithelium, Tumorigenesis. ß 2007 Elsevier Inc.

International Review of Cytology, Vol. 262 Copyright 2007, Elsevier Inc. All rights reserved.

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0074-7696/07 $35.00 DOI: 10.1016/S0074-7696(07)62006-3

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I. Introduction The generation of a functional three‐dimensional organ requires the cells within a tissue to undergo numerous changes and acquire diVerent functional characteristics at specific developmental stages. In the case of epithelium, cells are required to proliferate, migrate, and form a stable functional barrier that can interact with vastly diVerent microenvironments on each side of the tissue. During the development of an epithelial cancer, the regulation of each of these processes may be disrupted as cells lose normal tissue architecture (including apical–basal cell polarity), lose normal proliferation control, and may become invasive and motile. Thus, understanding the cellular machinery that controls cell polarity, proliferation, and motility is vital to our understanding of normal tissue homeostasis as well as the development and progression of malignant disease. Over the past decade, work in both Drosophila melanogaster and in mammalian model systems has coalesced to provide us with a clearer understanding of the mechanisms of polarity regulation and how the protein networks responsible for polarity establishment may impact on cell migration and tumorigenesis.

II. Epithelial Cell Polarity A. Regulation of Epithelial Cell Polarity Cell polarity describes the asymmetrical distribution of cellular constituents including proteins, carbohydrates, and lipids to particular regions within a cell or group of cells. This asymmetry allows the formation of structurally and functionally distinct domains that cells must have to interact eVectively with variable extracellular environments. Cell polarity takes on many forms but can be broadly classified into three types (illustrated schematically in Fig. 1): (1) apical–basal polarity (as found in epithelial monolayers), (2) anterior–posterior polarity (e.g., migration, asymmetric cell division), and (3) planar (or tissue) cell polarity (e.g., Drosophila wing, mammalian cochlea). The polarization that occurs during T cell immunological synapse formation and axon specification in neurons is a form of anterior–posterior polarity. In these situations, cells form a defined axis of asymmetry but are generally not conformed to specific tissue structure, as is the case for the apical–basal polarity of epithelial cells. The generation of cell polarity in all cases requires active remodeling of the microtubule and actin cell cytoskeletons and is highly dependent upon polarized vesicle traYcking to diVerent

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A

Apical−basal polarity

B

Anterior-posterior polarity T cell

Immunological synapse C

Planar cell polarity

Asymmetric cell division

Migration polarity

Axon specification

FIG. 1 DiVerent forms of cell polarity. Schematic representation of diVerent types of polarity formed in multicellular organisms. (A) Apical–basal polarity of monolayered epithelial cells. (B) Anterior–posterior polarity (examples shown from left to right are asymmetric cell division, T cell synapse formation, migration polarity, and neuronal axon specification). (C) Planar cell polarity or tissue polarity (the example shown represents the arrangement of hair cells in the developing mouse cochlea). Blue‐green color changes indicate segregation of cellular constituents to distinct regions in diVerent cell types; the specific proteins and their localization in each situation vary depending on cell type.

cellular domains. Genetic analysis in Drosophila has demonstrated that establishment of polarity is mediated by at least three interacting protein complexes: (1) the Par/aPKC complex, consisting of Bazooka (Baz), Par6, and atypical protein kinase C (aPKC), (2) the Crumbs complex, consisting of Crumbs (Crb), Stardust (Sdt), and dPATJ, and (3) the Scribble complex, consisting of Scribble (Scrib), Discs large (Dlg), and Lethal giant larvae (Lgl) (see Table I). Most of the work on the function of these complexes has focused on the regulation of apical–basal polarity in epithelial cells; however, it is now being shown that many of the proteins are required to coordinately control polarity in various cell types. It should be noted that in all cases additional cell‐type–specific factors are required for the proper generation of polarity; however, it seems that the three complexes mentioned previously form the conserved core of polarity regulators. While they may be considered as a single polarity control unit, for simplicity each complex and in particular its role in apical–basal polarity will be discussed separately here.

TABLE I Genes Required for Apical–Basal Polarity Role in apical–basal polarity Drosophila

Mammals

Described function

Drosophila

Mammals

References

Par6

Par6 a, b, g

ScaVold protein

þ

þ

Petronczki and Knoblich, 2001; Qiu et al., 2000

Bazooka

Par3

ScaVold protein

þ

þ

Joberty et al., 2000; Muller and Wieschaus, 1996

þ

Atypical PKC

PKC z, i/l

Serine threonine kinase

þ

Cdc42

Cdc42

Rho family GTPase protein

þ

Crumbs

Crumbs3

Transmembrane

þ

þ

Roh et al., 2003; Tepass et al., 1990

Stardust

PALS1

ScaVold protein

þ

þ

Straight et al., 2004; Tepass and Knust, 1993

þ

dPatj

PATJ

ScaVold protein

þ

Scribble

Scribble

ScaVold protein

þ

Discs large

Dlg 1–4

ScaVold protein

þ

Lethal giant larvae

Lgl1, Lgl2

Vesicle traYcking

þ

Avalanche

Vesicle traYcking

þ

vsp25

Vesicle traYcking

þ þ

Rac1

Rac1

Rho family GTPase

Myospheroid

b1 integrin

ECM receptor

Rolls et al., 2003; Suzuki et al., 2001 Hutterer et al., 2004; Kroschewski et al., 1999

Bhat et al., 1999; Shin et al., 2005 Bilder et al., 2000 Woods and Bryant, 1989

þ

Mechler et al., 1985; Musch et al., 2002 Lu and Bilder, 2005 Vaccari and Bilder, 2005

þ

Hakeda‐Suzuki et al., 2002; O’Brien et al., 2001

þ

Ojakian and Schwimmer, 1994

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1. Apical–Basal Polarity in Drosophila a. Bazooka, Par6, and aPKC Epithelial apical–basal polarity is established in the early stages of Drosophila embryogenesis when 5000–6000 syncytial nuclei are segregated into individual cells by invaginations of the embryonic plasma membrane. One of the first events following cellularization is the recruitment of Baz, Par6, and aPKC to the lateral membrane where they form a tripartite complex. Baz and Par6 both contain PDZ domains (PSD95/Dlg/ZO‐1), which are often arrayed in sequence (Baz has three consecutive PDZ domains) and serve to mediate protein–protein interactions (Bilder, 2001). Both Baz and Par6 also possess aPKC binding regions, and Par6 contains a CRIB motif that can associate with GTP‐bound Cdc42 (Hutterer et al., 2004). All three proteins are mutually dependent for proper localization to the ‘‘subapical region’’ (SAR) and for proper formation of the zonula adherens. Mutation or loss of Baz, Par6, or aPKC results in defects in polarity establishment that manifest as a loss of the apical domain, implying a role for these proteins in establishing apical identity. Hutterer et al. (2004) have shown that recruitment of the Par/aPKC complex to the subapical region is dependent on Par6 binding to active, GTP‐bound (but not GDP‐ bound) Cdc42. Consistent with this, a mutant of Par6 that cannot bind Cdc42 (but still binds Baz and aPKC) does not localize properly and does not rescue the mislocalization of Baz and aPKC in par6 mutant embryos. Moreover, expression of a dominant negative Cdc42 (N17) prevents apical accumulation of Par6 and inhibits zonula adherens formation (Hutterer et al., 2004). Interestingly, expression of a constitutively active form of Cdc42 (V12) also inhibits zonula adherens formation, presumably because of ectopic activity following release from localized interaction with regulatory proteins, although this has not been formally proven. Regardless, this point emphasizes the importance of localized signaling in the establishment of polarized protein localization, a theme that has become central to the understanding of cell polarity regulation. b. Scribble, Dlg, and Lgl Little is currently known about the molecular function of Scribble, Dlg, and Lgl in polarity regulation. All are mutually dependent for their correct localization within Drosophila epithelial cells (Scribble and Dlg at the septate junction and Lgl at the cytoplasmic and cortical junctions), and like the Par and Crb complexes (see following), loss of these proteins results in loss of apical–basal cell polarity and a disruption to the zonula adherens. However, in contrast to the Par and Crb complexes, the loss of polarity is manifested as a spreading of the apical domain (Bilder and Perrimon, 2000). Scribble is a large multidomain LAP (leucine‐rich repeats and PDZ domain) protein having 16 leucine‐rich repeats (LRRs) at the N‐terminus and four PDZ domains in the C‐terminal region; Dlg is a

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MAGUK family member protein (membrane‐associated with guanylate kinase domain) with 3 PDZ, an Src homology 3 domain, and a guanylate kinase‐like domain; and Lgl is a WD‐40 repeat protein (Bilder, 2004; Humbert et al., 2003). Loss of both the maternal and zygotic contribution of Scribble causes a mislocalization of apically polarized proteins and a misdistribution of adherens junctions in the embryonic epithelium that result in embryonic lethality. However, if the maternal contribution of Scribble is unaVected, embryos develop into larvae but fail to pupate, and their tissues continue to grow, eventually forming giant larvae (Bilder et al., 2000). The epithelial and neural tissues (imaginal discs) of these mutants become overgrown and can contain up to five times as many cells as wild‐type discs. Scribble, Dlg, and Lgl have identical epithelial phenotypes and show strong genetic interaction with one another (Bilder et al., 2000), which has led to the hypothesis that they function in a common pathway to regulate specification of the basal domain. Of Scribble, Dlg, and Lgl, each has also been shown to control the polarity of neuroblasts in the Drosophila embryo, implying that they may be general regulators of cell polarity in Drosophila. In Drosophila neuromuscular synapses, Scribble indirectly associates with Dlg through mutual binding of a protein termed GUK‐holder (GUKH) (Mathew et al., 2002). This interaction has not been formally shown in Drosophila epithelia, but the colocalization of the Scribble and Dlg proteins at the septate junction suggests it is likely. Recently, Kallay et al. (2006) have demonstrated that in mammalian epithelial cells Scribble physically interacts with Lgl2, but this interaction has not yet been observed in Drosophila. Structure–function analysis from two groups has shown that both the LRR and PDZ domains of Scribble are required for the appropriate localization of Scribble in epithelia and neuroblasts; however, expression of the LRR domain alone seems to be capable of partially rescuing the loss of polarity in scribble mutants (Albertson et al., 2004; Zeitler et al., 2004). While the LRRs appear to be the primary determinant for cortical localization of Scribble, the function of the PDZ domains is likely to be in binding proteins such as GUKH and stabilizing the localization of Scribble at the septate junctions. In agreement with this is the observation that the second PDZ domain of Scribble, which binds GUKH, is required for eYcient targeting of Scribble to lateral membranes in epithelia (Albertson et al., 2004). c. Crumbs, Stardust, and dPATJ Crumbs (Crb) is a transmembrane protein with 30 epithelial growth factor (EGF)‐like repeats and four laminin A G‐domain‐like repeats in its extracellular domain. It localizes exclusively to the apical domain of polarized epithelial cells and is required for normal photoreceptor diVerentiation in the Drosophila eye (Knust et al., 1993). Stardust (Sdt) is a PDZ domain‐containing protein that binds to Crb on the intracellular membrane (Bachmann et al., 2001), and similarly dPATJ is a

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multi‐PDZ protein that interacts directly with Crb in the subapical domain (Bhat et al., 1999) (dPATJ was previously but erroneously referred to as Discs lost, Dlt). Crb, Sdt, and dPATJ are all required for proper polarity formation in Drosophila epithelium, and loss of these proteins, either through mutation or RNA interference, results in loss of apical identity and a failure to properly form the zonula adherens (Bhat et al., 1999; Muller and Wieschaus, 1996; Tepass, 1996). However, the molecular mechanisms underlying the role of the Crb complex in polarity establishment and maintenance have not yet been fully elucidated. Overexpression of Crb mimics the eVect of loss of Scribble, and this phenotype can be partially suppressed by halving the gene dosage of scribble, indicating that these complexes functionally interact to regulate polarity (Bilder et al., 2003). Moreover, genetic epistasis tests in the embryo have indicated that Crb and Sdt act downstream of Baz/Par6/aPKC to promote formation of the subapical region and the apical domain (Bilder et al., 2003). d. Biochemical Regulation of Crumbs and Scribble Complexes by aPKC Phosphorylation of members of the Crumbs and Scribble complexes by aPKC provides an important functional link to allow coordination of the activity of the various polarity complexes. i. aPKC directly regulates Crb Following proper localization of the Par/ aPKC complex, polarity is regulated through a balance of opposing signals on each of the other two polarity complexes. Most of this regulatory activity is mediated through the kinase activity of aPKC, as expression of a kinase dead dominant negative aPKC is suYcient to disrupt polarity (Sotillos et al., 2004). In Drosophila epithelium, aPKC has been shown to physically interact and phosphorylate both the transmembrane protein Crb and Lgl, thus functionally bridging the three defined polarity complexes. Crb phosphorylation by aPKC is required and is instructive for Crb apical membrane localization (Sotillos et al., 2004) because mutation of the conserved aPKC phosphorylation residues in Crb (T6A, T9A, S11A, S13A) results in mislocalization of the protein into the cytoplasm. Moreover, expression of this construct does not phenocopy the eVect of overexpressing wild‐type Crb (i.e., an expansion of the apical membrane domain) (Sotillos et al., 2004). These data imply that aPKC‐dependent phosphorylation positively regulates Crb (and Sdt) activity in the apical domain. ii. aPKC and Lgl Phosphorylation of Lgl by aPKC, originally described in neuroblasts, is critical for restricting the activity of Lgl to the basal cortex of the cell (Betschinger et al., 2003). Lgl ‘‘activity’’ is not well understood; however, phosphorylation of Lgl by aPKC in the apical domain of both neuroblasts and epithelium is known to prevent it from associating with the cell membrane and presumably inhibits its role in establishing basal identity (in concert with Scribble and Dlg). Interestingly, expression of a

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nonphosphorylatable form of Lgl (Lgl‐3A) does not disrupt polarity in epithelial cells as it does in neuroblasts. This may be because the presence of wild‐type Lgl is suYcient to mask the eVect of this mutant or that interaction with aPKC and phosphorylation of Lgl are critical for its role in regulating epithelial polarity. Consistent with the latter, expression of Lgl‐ 3A fails to rescue the polarity defects of Lgl mutant epithelia (Hutterer et al., 2004). e. Genetic Hierarchy Controlling Polarity Since disruption to Scribble, Dlg, or Lgl results in spreading of the apical domain, the function of these proteins is proposed to be in the repression of the activity of the Par/aPKC and Crb/Sdt complexes, which establish apical identity (Bilder et al., 2003). Likewise, Par/ aPKC and Crb/Sdt are thought to antagonize the activity of Scribble, Dlg, and Lgl because Lgl function is negatively regulated in the apical domain by aPKC‐ dependent phosphorylation (discussed previously), such that cortical localization of Lgl is restricted to the basal domain. Similarly, overexpression of Crumbs mimics the scribble mutant phenotype, and Crb mutants can be partially rescued by reducing the level of Scribble protein (Bilder et al., 2003). These data form the basis for the model that has been well described elsewhere (Bilder, 2004; Humbert et al., 2006). The model proposes that both the Par and Crb complexes act cooperatively to repress the ‘‘basal activity’’ of the Scribble group in the apical domain and the Scribble group restricts the ‘‘apical activity’’ of Par and Crb complexes in the basal domain. The mechanism by which this restriction of ‘‘apical activity’’ is applied remains an open question; however, it is possible that proper formation of the zonula adherens and septate junction (which is dependent on Scribble/Dlg/Lgl) physically retains the protein complexes in their appropriate domains. Having said this, it is unlikely that junction integrity is solely responsible for restriction of apical complexes since mutation of specific structural components of the zonula adherens does lead to the disruption or mislocalization of polarity determinants (Tepass et al., 1996; Uemura et al., 1996). 2. Apical–Basal Polarity in Mammals Throughout evolution and the development of highly complex multicellular organisms (i.e., mammals), the requirement for cell asymmetry has remained essential. Given this, it is not surprising that many of the protein networks that have evolved to regulate cell polarity in Drosophila are conserved through all eukaryotic species. In fact, of the proteins that comprise the Par, Crumbs, and Scribble complexes involved in regulating apical–basal polarity in Drosophila (discussed previously), each has highly conserved mammalian homologues (Table I). Over the past 10 years, the identification and analysis of either single or multiple mammalian homologues of the

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Drosophila polarity proteins have served to validate a conserved functional role for these proteins in regulating epithelial cell polarity. Moreover, the ease of biochemical manipulation in cultured mammalian cells has allowed us to gain new insight into the physical and functional interactions of the polarity proteins and the coordinated development of apical–basal polarity in epithelium that was not possible in Drosophila. a. Crumbs3, PALS1, and PATJ Drosophila Crb has three mammalian homologues, of which only Crumbs3 (CRB3) is expressed widely in mammalian epithelial tissues (Makarova et al., 2003). CRB3 lacks the large extracellular domain of Drosophila Crb but retains a conserved intracellular domain, perhaps suggesting that some of the functions of the single Drosophila Crb protein have diverged during evolution. Importantly, like Crb, CRB3 does function to regulate the establishment of polarity in Madin–Darby canine kidney (MDCK) epithelial cells because overexpression of CRB3 delays the formation of tight junctions following calcium switch (Lemmers et al., 2004; Roh et al., 2003). Calcium switch is a technique used primarily with MDCK cells to examine the early events of epithelial junction formation. It involves the removal of Ca2þ ions from the culture media for 24 h to disrupt cell–cell junctions; Ca2þ is then replaced, and epithelial junctions are examined over time. While in this system, CRB3 expression causes a delay in junction formation; cells that are not Ca2þ, depleted do form normal tight junctions, indicating that CRB3 is likely involved in the formation but not maintenance of tight junctions. In contrast, CRB3 expressing cells cultured as three‐dimensional (3D) cysts in a collagen matrix show complete loss of apical–basal polarity similar to what is observed in Drosophila. As in Drosophila epithelium, in mammals CRB3 exists in a stable molecular complex with the mammalian homologues of Sdt (protein associated with lin seven 1, PALS1) and dPATJ (PALS1 associated tight junction protein, PATJ). The CRB3‐dependent disruption to polarity described in MDCK cells is dependent on the ability of CRB3 to bind PALS1 through a highly conserved C‐terminal tail, indicating the functional importance of this biochemical interaction (Roh et al., 2003). The requirement for both PALS1 and PATJ in the establishment of apical–basal polarity is further demonstrated by the observations that depletion of either protein using RNAi causes a dose‐dependent delay in tight junction formation following Ca2þ switch and loss of polarity in 3D cysts (Shin et al., 2005; Straight et al., 2004). Thus, not only are the physical interactions among Crb, Sdt, and dPATJ. conserved in mammalian epithelial cells, but also the proteins also function similarly to regulate the formation of apical (tight) junctions and cell polarity. b. Par3, Par6, and aPKC Mammalian genomes contain one Baz homologue (also known as Par3 or aPKC specific interacting protein, ASIP), three Par6 homologues (Par6a, b, and g), and two aPKC homologues (aPKCi/l

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and aPKCz) (Table I). Unless specified, these family members will be referred to as Par6 and aPKC, respectively, because although the localization of these proteins can vary (Gao and Macara, 2004) in many cases the functions of the family members are redundant. This redundancy has made loss‐of‐function analysis diYcult, but much has been learned about the activity of these proteins from the study of dominant negative and deletion mutant proteins. The initial characterization of mammalian Par3 provided compelling evidence for conservation of function of the Par protein network. In MDCK cells and rat intestinal epithelia, Par3 was shown to localize to tight junctions where it formed a complex with aPKC (Izumi et al., 1998), and in Ca2þ switch experiments, it was shown to positively regulate the formation of tight junctions in an aPKC‐binding‐dependent manner (Hirose et al., 2002; Nagai‐ Tamai et al., 2002). To complete the identification of the Par tripartite complex identified in Drosophila, Par6 was also shown to bind directly to Par3 and the regulatory domain of aPKC (Joberty et al., 2000; Lin et al., 2000). While the presence of this protein complex at mammalian apical junctions is undisputed, the precise functional role of each of the individual proteins within it remains a controversial issue. What is clear, however, is that as in Drosophila polarity regulation, the crucial signal output of this module seems to be the kinase activity of aPKC. This point is emphasized by the recent observation that expression of an endogenous inhibitor of aPKC, PKCzII, which is 98% identical but lacks the kinase domain of PKCz, negatively regulates the formation of diVerentiated epithelial cell–cell junctions (Parkinson et al., 2004). Likewise, exogenous expression of dominant negative versions of aPKC causes a marked delay in the formation of tight junctions measured by a decrease in transepithelial resistance and increased paracellular diVusion (Suzuki et al., 2001). As in Drosophila, Par3 and Par6 have no catalytic protein domains but may act as scaVolding molecules at cell junctions. In fact, evidence now indicates that Par6 likely regulates the spatiotemporal activity of aPKC through direct interaction with GTP‐bound Cdc42. Par6 is capable of suppressing aPKC kinase activity, and this suppression is dependent on the presence of the CRIB/PDZ motifs within Par6 that are C‐terminal to the actual aPKC binding domain, suggesting other regulatory factors are involved. Yamanaka et al. (2001) demonstrated that Cdc42‐GTP (but not Cdc42‐ GDP) binding to the CRIB/PDZ domain of Par6 partially relieves the suppression of aPKC kinase activity. Thus in this model, Par6 not only acts to restrict the activity of aPKC to regions of localized activated Cdc42, but also provides a means to transduce the Cdc42 signal to aPKC kinase activity. Gao et al. (2002) have proposed an alternate hypothesis whereby Cdc42 and Par6 act to negatively regulate tight junction formation; however, this model does not account for many of the known events that occur during junction formation. For example, the activation of Cdc42 and its subsequent

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association with Par6 following cell–cell contact do not fit with a role in negative regulation of junction formation. Loss of function analysis will prove invaluable in accurately defining the roles of the Par and aPKC proteins in mediating junction formation and apical–basal cell polarity. i. Association of the CRB3 and Par protein complexes Regulation of aPKC kinase activity and localization of the Par complex are not restricted to the described interactions of these three proteins. Both CRB3 and PALS1 are capable of binding Par6, and mutation of the CRB3 PDZ‐binding domain or expression of a dominant negative PALS1 can cause mislocalization of the Par complex (Hurd et al., 2003; Lemmers et al., 2004). As mentioned, overexpression of Par6 can cause defects in tight junction formation; however, overexpression of a mutant Par6 that cannot bind to either PALS1 or CRB3 does not aVect junction formation; implying that the association of Par6 with PALS1 and/or CRB3 is important for polarity regulation. Interestingly, the association of PALS1 with Par6 is enhanced by the presence of GTP‐Cdc42 (Hurd et al., 2003). Applied to the model described previously, the interaction of PALS1 (and Par3) with the Par6 PDZ domain could serve to stabilize the activity of aPKC by inhibiting Par6‐mediated repression of kinase activity, although this has not been formally proven. c. Scribble, Dlg, and Lgl i. Lgl regulates apical–basal polarity The high level of functional conservation between vertebrate and invertebrate species in the regulation of apical junction formation (discussed previously) leads to the hypothesis that the Scribble group acts to regulate basal identity in mammalian epithelia. The best evidence for a conserved role for these proteins in polarity regulation is the observation that both Lgl1 and Lgl2 bind Par6 and are substrates for aPKC phosphorylation, mirroring the situation in Drosophila (Plant et al., 2003; Yamanaka et al., 2003). The interaction among Lgl, Par6, and aPKC precludes the association of Par3 with this complex and inhibits the formation of the apical domain by binding to and suppressing the kinase domain of aPKC (Yamanaka et al., 2006). aPKC may then reciprocally suppress Lgl activity in the apical domain via phosphorylation of central conserved serine residues, restricting its cortical localization to the basal region of the cell. Consistent with this, the colocalization of Lgl with aPKC is observed only in the early stages of junction formation, and the dissolution of this complex coincides with increased aPKC phosphorylation of Lgl (Yamanaka et al., 2003). Interestingly, substitution of three aPKC phosphorylation sites on Lgl to either alanine (to inhibit phosphorylation) or glutamic acid (to mimic phosphorylation) is suYcient to disrupt tight junction formation following Ca2þ switch. It is not clear whether these mutants act as dominant negative proteins, sequestering other polarity regulators, or represent overactive forms of Lgl, but the data imply that the coordinated regulation of Lgl

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phosphorylation is critical for the establishment of normal epithelial polarity. ii. Scribble and Dlg: Adherens junction formation and apical–basal polarity The biogenesis of cell–cell contacts mediated by E‐cadherin is considered a basic requirement for the formation of cohesive epithelial tissues. Indeed, loss of E‐cadherin is frequently observed in invasive and disorganized epithelial cancers. However, recent evidence suggests that in both Drosophila and cultured epithelial cells, polarity is not absolutely dependent on E‐cadherin engagement (Baas et al., 2004; Harris and Peifer, 2005). In Drosophila, Scribble and Dlg are both required for the proper formation of the septate junction and zonula adherens (Bilder and Perrimon, 2000; Woods et al., 1996). Similarly, in Caenorhabditis elegans, let‐413 (a scribble ortholog) and dlg‐1 mutants show pronounced defects in adherens junction formation (Bossinger et al., 2004; Firestein and Rongo, 2001). However, there is also evidence to implicate E‐cadherin in the recruitment of Dlg to the basolateral membrane (Harris and Peifer, 2004). So are Scribble and Dlg involved in the regulation of adherens junction formation or vice versa? Numerous studies have shown that both Scribble and Dlg1 are localized to the basolateral membrane of epithelial cells below the tight junction marker ZO‐1 and that this polarized localization of Scribble and Dlg1 is correlated with the establishment of adherens junctions (Dow et al., 2003; Laprise et al., 2004; Navarro et al., 2005). Laprise et al. (2004) have shown that Dlg is required for proper adherens junction assembly in human epithelial cells; however, another group claims that E‐cadherin–mediated cell–cell contact is essential for the recruitment of Scribble to the basolateral membrane in polarized epithelia (Navarro et al., 2005). The later conclusions are based on the observation that perturbation of E‐cadherin interactions or loss of E‐cadherin results in mislocalization of Scribble from cell–cell junctions. At least in some cells types, such as T cells, localization of Scribble to the cell cortex is not dependent on cell–cell contact (Ludford‐Menting et al., 2005). Recently, Qin et al. (2005) showed that depletion of Scribble in MDCK cells disrupts E‐cadherin–mediated cell–cell contacts and suggested that Scribble can regulate cell adhesion by mediating the association of E‐cadherin with catenin molecules. However, it has not been shown that Scribble or Dlg physically interacts with any components of the adherens junctions (i.e., E‐cadherin or b‐catenin); thus, it is unclear how these proteins directly aVect junction formation and/or maintenance. Despite the described role for Scribble in coordinating adherens junction integrity, we and others have recently shown that Scribble is not essential for the generation of apical–basal polarity in either human or canine epithelial cells in 3D culture in Matrigel basement membrane (Dow et al., 2006; Qin et al., 2005). However, Yamanaka et al. (2006) have now shown that depletion of Lgl2 in MDCK cells causes defects in polarized cyst formation in collagen I environments. While these results suggest a diVerence in

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mammalian polarity regulation by Scribble and Lgl, they may in fact be explained by the experimental conditions used in each instance. Previous publications examining polarity regulation in in vitro 3D cultures have identified specific diVerences in polarity phenotypes in cells cultured in Matrigel compared to those grown in a collagen I matrix. Forced expression of mammalian Crb3 was shown to cause severe disruption in polarity in cysts cultured in collagen type I gels but did not aVect the polarity of cysts grown in Matrigel (Lemmers et al., 2004; Roh et al., 2003). Similar observations have been made using Src‐transformed MDCK cells (Rahikkala et al., 2001). These findings are significant because in Drosophila overexpression of Crb mimics the Scribble loss of function phenotype. Should the same principles of polarity apply in mammalian cells, the use of Matrigel in these cultures could be masking underlying defects in polarity regulation. Given this caveat, the final verdict on the in vivo requirement for mammalian Scribble in epithelial apical–basal polarity regulation will rest with the generation and analysis of conditional knockout mice. d. Other Regulators of Apical–Basal Polarity The three interacting protein complexes described in detail thus far do not function autonomously in the regulation of apical–basal polarity. A comprehensive discussion of all of the contributing factors is not practical for the purpose of this review. However, the complex role of extracellular signals in the establishment of cell polarity warrants further discussion. During the establishment of multicellular organisms, epithelial cells interact not only with neighboring cells, but also with surrounding extracellular matrix (ECM). The precise ECM composition varies between diVerent tissue types but commonly involves high concentrations of collagen (types I–IV) and laminin polymers, among other protein and carbohydrate molecules. Cell interaction with ECM along the basal surface functions as a structural scaVold for the epithelial monolayer but is also crucial for defining the axis of apical–basal polarity. In Drosophila, it seems that formation of polarity does not require laminin (Brown, 2000); however, in vivo studies in mice have demonstrated a crucial role for normal laminin assembly and cell–ECM interaction in generating functional, polarized tissues (Klinowska et al., 1999; Schuger et al., 1998). Using MDCK cells cultured in 3D collagen gels, O’Brien et al. (2001) and Yu et al. (2005) have shown that proper laminin assembly is required for normal cyst formation and is mediated by activation of Rac1. In this model, the axis of polarity is regulated by the binding of b1 integrin to collagen I, b1‐dependent activation of Rac1, and the polarized secretion and assembly of a laminin network to define the basal region of the cell. While the actual interactions involved in this process are undoubtedly more complex, this pathway provides a good example of the requirement for reciprocal signaling between cells and the ECM to generate epithelial polarity. In addition, a number of guanine nucleotide exchange factors

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(GEFs), which serve to activate GTPase molecules, are required for polarity regulation. In particular, it was recently shown that both Par3 and Par6 interact with the GEFs Tiam1 and ECT2 and that this interaction is required for epithelial polarity establishment (Chen and Macara, 2005; Liu et al., 2004, 2006; Mertens et al., 2005). Thus, our current understanding of apical–basal polarity, while not complete, suggests a model whereby reciprocal signaling between epithelial cells and the ECM mediates polarity and junction formation through the cooperative and antagonistic interactions of three highly conserved protein complexes.

B. Polarity Regulators and Cell Migration Regulated cell motility is essential throughout embryonic development and is crucial for maintaining tissue homeostasis in the adult. In the developing vertebrate embryo, cell migration is evident in many forms, including the coordinated epithelial sheet migration seen during gastrulation and the single cell migration of somites away from the primitive streak. Cell migration is also essential in adult life and occurs following wounding of a tissue, such as those that commonly occur in skin. In this setting, migration of epithelial and underlying mesenchymal cells is required for rapid closure of exposed surfaces to restore protective function. In addition, the recruitment of immune cells to sites of wound infection or infections derived from external pathogens is also dependent on directed migration. Disruption of these processes can be manifested as minor problems such as delayed wound healing and scarring or can lead to serious issues such as a failure to properly fight infections. Moreover, inappropriate activation of the cell migration machinery is thought to be one of the major factors influencing the development of invasive and metastatic malignant cancers in humans. In recent years, it has been shown that the process of directed cell migration is absolutely dependent on the ability of cells to properly polarize the molecular networks that instigate cell migration. This polarization in response to a directional cue is now thought to be dependent on the function of many proteins that also regulate the establishment of apical–basal polarity (discussed previously). In this section, we describe the currently accepted molecular models for directed cell migration in both Drosophila and mammalian settings and discuss how the Scribble, Par, and Crb polarity complexes may impact this essential cell function. 1. Cell Migration in Drosophila a. Dorsal Closure: A Model to Study Cell Migration In Vivo Migration during development has been well studied in Drosophila by examining the process of dorsal closure. Dorsal closure involves bringing together two

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opposing lateral epithelial sheets over an extraembryonic amnioserosa membrane to form a seamless dorsal midline. To date, many genes have been implicated in the regulation of dorsal closure (summarized in Table II). These genes fall into four major categories: cytokine/MAPK signaling, cytoskeleton/ECM receptors, Rho GTPase signaling, and polarity regulators. The first characterized genes required for dorsal closure were hemipterious (hem) and basket (bsk) (Glise et al., 1995; Riesgo‐Escovar et al., 1996; Sluss et al., 1996), both members of the conserved JNK signaling pathway that functions in many varied cellular processes. The identification of JNK signaling as a core component of the dorsal closure machinery established a central pathway around which a complex model of cell migration was built. The following section will describe the currently accepted model for dorsal closure in Drosophila (depicted schematically in Fig. 2A) and the role of polarity proteins in this process. i. JNK regulates dorsal closure At the onset of dorsal closure, JNK activity is detectable in two types of cells: the leading edge epithelial cells and the amnioserosa. JNK activity in the amnioserosa is transient and through an unknown mechanism causes contraction of this tissue (Reed et al., 2001). The mechanical stress caused by amnioserosa contraction is thought to be the initiating event in epithelial cell movement. Indeed, misexpression of actin‐associated proteins that disrupt contraction of the amnioserosa can cause defects in dorsal closure without directly aVecting epithelial cells (Harden et al., 2002). Following initiation of dorsal closure, JNK activity is lost in the amnioserosa but persists in the leading edge epithelial cells, where it is required for the phosphorylation of c‐Jun and thus induction of Jun transcriptional activity. Jun activation is spatially restricted to cells at the leading edge (LE) of the migrating epithelial sheet and is required for the expression of many genes that act to positively regulate closure of the dorsal opening (Glise and Noselli, 1997; Jasper et al., 2001; Zeitlinger et al., 1997). In addition to its role in activating Jun, JNK is also likely to play a more direct role in promoting cell migration and dorsal closure through the phosphorylation and modulation of F‐actin‐associated proteins such as paxillin (Otto et al., 2000). ii. Upstream of JNK The molecular events that initiate movement of LE cells are not well defined but probably involve mechanical stress‐induced activation of a and b integrin membrane receptors scab and myospheroid, both of which are required for dorsal closure (Brown, 1994; Stark et al., 1997). These events are thought to bring about activation of the small GTPase Rac1, similar to the integrin‐mediated activation of Rac1 that promotes apical–basal polarization in mammalian epithelial cells (described previously). Activated, GTP‐bound Rac1 in turn activates JNK via a cascade of signaling kinases including p21‐activated kinase (dPak), misshapen, slipper, and hem, and also acts as a potent inducer of actin polymerization

TABLE II Genes Required for Cell Migration Role in cell migration Mammalian homologue

Drosophila

Described function

Drosophila Cell culture Mouse

References

Cytokine and MAPK signaling

268

Hemipterous

MKK7

MAPKK

þ

Basket

JNK

MAPK

þ

dMEKK1

MEKK1

MAPKK

Jun

Jun

Transcription factor

þ

Misshapen

MAPKKKK

þ

Puckered

Nck‐interacting kinase VH‐1

MAPK phosphatase

þ

Notch

Notch

Cytokine receptor

þ

þ

Decapentaplegic BMP4 (TGF‐b)

Secreted cytokine

þ

þ

EGF

EGF

Secreted cytokine

þ

þ

Blay and Brown, 1985; Mine et al., 2005

TGF‐a

Secreted cytokine

þ

þ

Luetteke et al., 1993; Morelli et al., 1992

þ

Glise et al., 1995 þ

þ

Huang et al., 2003; Riesgo‐Escovar et al., 1996; Sluss et al., 1996; Weston et al., 2004

þ

þ

Xia et al., 2000; Yujiri et al., 2000; Zhang et al., 2003

þ

Hou et al., 1997; Kockel et al., 1997; Li et al., 2003; Riesgo‐Escovar and Hafen, 1997; Zenz et al., 2003 Su et al., 1998 Martin‐Blanco et al., 1998

EGFR

EGFR

Cytokine receptor

þ

ERK

ERK1/2

MAPK

þ

Punt

BMP receptor type II Cytokine receptor

þ

Thick veins

BMP receptor type I Cytokine receptor

þ

Noseda et al., 2004; Zecchini et al., 1999 Padgett et al., 1987; Rothhammer et al., 2005

Chen et al., 1994; Zenz et al., 2003 Matsubayashi et al., 2004

þ

Letsou et al., 1995; Ruberte et al., 1995 AVolter et al., 1994

Integrin and cytoskeleton Myospheroid

b integrin subunit

ECM receptor

þ

þ

þ (b2)

Brown, 1994; Carroll et al., 1995; Guo et al., 1990

Scab

a integrin subunit

ECM receptor

þ

Zipper

Nonmuscle myosin II Motor protein

þ

APC

APC

þ

þ (a2, a5) Carroll et al., 1995; Stark et al., 1997; Yamada et al., 1990 Young et al., 1993

þ

MT binding protein

Etienne‐Manneville and Hall, 2003

Rho GTPase signaling Ras

Ras

Rho family GTPase

Shaggy

GSK3b

Serine threonine kinase

RhoA

Rho1

Rho family GTPase

þ þ

þ

Harden et al., 1999; Kundra et al., 1995

þ

Etienne‐Manneville and Hall, 2003

þ

Harden et al., 1999; Santos et al., 1997

þ

þ

269

ROCK?

ROCK1

Rho kinase

Rap1

Rap1

Rho family GTPase

Myoblast city

DOCK‐180

Rac binding protein

þ

þ

Erickson et al., 1997; Grimsley et al., 2004

DPak

p21‐activated kinase 1 bPix

Rac eVector

þ

þ

Harden et al., 1996; Master et al., 2001

RacGEF

þ

Cau and Hall, 2005

dPix

þ

Fukata et al., 1999; Shimizu et al., 2005 Boettner et al., 2003

GIT1

ARF GAP

þ

Manabe et al., 2002b

Raca

Rac1

Rho family GTPase

þ

þ

Anand‐Apte et al., 1997; Harden et al., 1995

Cdc42a

Cdc42

Rho family GTPase

þ

þ

Etienne‐Manneville and Hall, 2001; Harden et al., 1999

Polarity ZO‐1

ZO‐1

Par6

Par6

þ Protein scaVold

þ

Takahashi et al., 1998 þ

Etienne‐Manneville and Hall, 2003; Pinheiro and Montell, 2004

Atypical PKC

PKCz

Serine threonine kinase

þ

Etienne‐Manneville and Hall, 2001

Lgl

Lgl (1,2)

Vesicle traYcking

þ

þ

Manfruelli et al., 1996; Plant et al., 2003

Dlg

Dlg1

Protein scaVold

þ

þ

Etienne‐Manneville et al., 2005; Perrimon, 1988

Scribble

Scribble

Protein scaVold

þ

þ

a

Rac and Cdc42 are also essential polarity regulators in Drosophila and mammals.

þ

Bilder et al., 2000; Dow et al., 2006; Osmani et al., 2006

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FIG. 2 Directed migration in Drosophila. Dorsal closure. (Ai) Late in Drosophila embryogenesis, opposing lateral epithelial sheets (composed of hundreds of cells) move toward each other over the underlying amnioserosa and ‘‘zipper’’ together at the dorsal midline of the embryo. (Aii) In cells at the leading edge of the epithelial sheet, integrin‐mediated activation of the JNK pathway promotes phosphorylation of the Jun transcription factor and transcription of genes (e.g., decapentaplegic, dpp) that promote cell migration. Expression and secretion of Dpp through an Lgl‐dependent mechanism further promote movement of the epithelial sheet through the receptor‐mediated activation of Cdc42. Rac1 and Cdc42 (and JNK) also directly promote actin polymerization and cell extension. Scribble and Dlg are both required for normal dorsal closure, although the molecular role of these proteins remains undefined. (B) Border cell migration. During Drosophila oogenesis, a small cluster of six or seven border cells (derived from posterior follicle cells) migrates through surrounding nurse cells toward the oocyte in the anterior portion of the egg chamber. A number of polarity regulators have been shown to be essential for this process (listed below the figure).

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(Woolner et al., 2005). Interestingly, although Rac1 is a positive regulator of cell migration in this context, the expression of both active and dominant negative Rac1 in LE cells results in failure of dorsal closure. Woolner et al. (2005) suggest that active Rac1 may block cell migration by premature activation of undefined contact inhibition machinery. iii. Dpp potentiates JNK signals The major downstream target of JNK, the transcription factor c‐Jun, forms heterodimers with Fos, and Jun/Fos complexes promote transcription of many AP‐1‐responsive genes required for cell migration, including decapentaplegic (dpp). Dpp is homologous to mammalian bone morphogenic protein‐4 (BMP4), a member of the mammalian transforming growth factor b (TGF‐b) superfamily. It is a secreted cytokine that binds to its cognate transmembrane receptors punt and thick veins on both LE cells and the more lateral epithelium and activates intracellular signaling events (Glise and Noselli, 1997; Reed et al., 2001). These signaling events are not well defined but are believed to be mediated through activation of the small GTPase Cdc42, which in turn activates dPak. Through this mechanism, it seems that JNK activation promotes a feedforward loop to sustain a continual migration response. Not surprisingly, mutations that inhibit either the production or response to Dpp, such as loss of punt and thick veins, cdc42, or dpp itself, negatively aVect dorsal closure in the embryo (AVolter et al., 1994; Harden et al., 1999; Letsou et al., 1995; Ruberte et al., 1995). iv. Impact of polarity regulators on dorsal closure Loss of the neoplastic tumor suppressor and cell polarity regulator Lgl disrupts dorsal closure and is implicated in the regulation of Dpp signaling (Arquier et al., 2001). Loss of Lgl from various Drosophila tissues, including the LE cells of the embryo, results in decreased expression of many Dpp‐responsive genes. However, Lgl is not required for the downstream response to Dpp because loss of Lgl can be rescued by constitutive activation of the thick veins receptor or the presence of surrounding Dpp‐expressing, wild‐type tissue (Arquier et al., 2001). Rather, Lgl is required for the proper secretion of the Dpp signal (Fig. 2), consistent with a role for Lgl in regulating vesicle traYcking (discussed later). Importantly, in this study, disruption of Dpp secretion in lgl mutants was not due to a general disruption of cell polarity since defects in Dpp secretion could be detected in cells that had established normal apical– basal polarity due to the maternal contribution of Lgl and maintained this polarity as the maternal protein was depleted. Lgl is not the only polarity regulator to have a role in dorsal closure. Suggestive data implicating Dlg in the regulation of dorsal closure were described almost 20 years ago; however, definitive evidence of a direct role for both scribble and dlg has only recently emerged. The reason for this is that the interpretation of dorsal closure in maternal/zygotic scribble and dlg mutants is complicated because they have severely disrupted cellular architecture and body plan. Using only zygotic mutations, Bilder and colleagues (2000)

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have shown that loss of either Scribble or Dlg alone is not suYcient to disrupt cell migration, but halving the gene dosage of dlg on a scribble mutant background results in a failure of these embryos to undergo closure. This genetic interaction between scribble and dlg not only implies a functional role for each in regulating dorsal closure, but also that the proteins likely function to cooperatively coordinate cell migration. It is currently not clear whether the normal dorsal closure apparent in zygotic scribble and dlg mutants is a consequence of a persisting maternal contribution of the proteins or indicates that neither gene individually is essential for this process. We know that at least in some settings, Scribble can regulate cell migration due to the recent identification of the zebrafish scribble homologue as the gene mutated in the landlocked (llk) mutant (Wada et al., 2005). This (zygotic) mutation causes a failure of nVII motor neuron migration (albeit in a non–cell‐autonomous manner), and loss of maternal Scribble expression causes defects in convergent extension during embryogenesis. Scribble and Dlg also positively regulate migration in mammalian cells (discussed later). Together, these data suggest a conserved role for Scribble and Dlg in the regulation of cell motility. What remains to be shown is the mechanism by which these neoplastic tumor suppressor proteins regulate cell migration. One possibility is that Scribble and Dlg operate together with Lgl (as they do in the establishment of apical–basal polarity) to coordinate the secretion of Dpp, although currently there is no direct evidence for this. Another possibility is that Scribble modulates signaling through the JNK pathway. In larval eye imaginal epithelial tissue, loss of scribble in clonal patches of wild‐type tissue results in upregulation of JNK signaling, triggering apoptosis and elimination of scribble mutant cells (Brumby and Richardson, 2003). This observation suggests that Scribble may somehow serve to suppress JNK signaling. While it is normally inactivation of JNK signaling that is implicated in dorsal closure defects, we know that constitutive activation of Rac1 blocks cell movement, demonstrating that any significant deregulation in the balance of JNK activity can result in cell migration defects. b. Border Cell Migration Another type of cell movement that occurs during Drosophila development is border cell migration. Border cell migration is distinct from dorsal closure in that it involves the coordinated movement of a small cluster of cells rather than an epithelial sheet. Border cells are derived from follicle (epithelial) cells, and late in embryogenesis a cluster of six or seven cells migrates from the anterior region of the egg chamber, between nurse cells, toward the oocyte, where migration stops (Montell, 2003) (Fig. 2B). This type of movement involves complex interactions between diVerent cell types and migration of cells through a 3D tissue. In this way, border cell migration more closely resembles tumor cell invasion and depends on a complement of proteins and signals diVerent from those required during dorsal closure, such as

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activation of the JAK/STAT signaling pathway (Montell, 2003). However, like dorsal closure, border cell migration requires directional cell movement and is controlled by a number of apical–basal polarity regulators including members of the Par and Scribble complexes. Baz, Par6, and aPKC are all asymmetrically distributed in migrating border cells, and RNAi studies have shown that both Baz and Par6 are essential for normal cluster migration (Murphy and Montell, 1996; Pinheiro and Montell, 2004). Although Baz and Par6 are asymmetrically localized during border cell migration, they do not appear to be concentrated at the leading edge, in contrast to migrating mammalian cells (discussed following), suggesting diVerent molecular mechanisms may be employed during border cell movement (Pinheiro and Montell, 2004). Both Dlg and Lgl have also been shown to be important in regulating migration during Drosophila oogenesis, although the dlg and lgl mutant phenotypes in this case are distinct from that of the rac, baz, and par6 mutants. Specifically, loss of dlg or lgl causes ectopic invasion of follicle cells, similar to the movement of border cells, although these cells never acquire a border cell fate (Pinheiro and Montell, 2004). Because dlg and lgl mutations disrupt normal oogenesis, it is not clear whether Dlg and Lgl function to regulate conventional border cell migration, but it is clear that the expression of these genes in this tissue can aVect cell migration. 2. Cell Migration and Polarity Regulators in Mammalian Cells Drosophila genetic analysis has been invaluable in characterizing a large number of genes involved in the regulation of cell migration, but an understanding of the molecular mechanics of this process has largely come from the investigation of mammalian cell migration in vitro. Similar to what would occur in the whole animal, many primary cell types and immortalized cell lines will migrate in vitro in response to extracellular cues. Such cues include interaction with specific ECM components, chemokine gradients, wounding, and in some cases a combination of the three. Wounding‐induced migration in vitro has been widely used to examine the cascade of intracellular events that follow sensing of a directional migration cue. Wounding‐induced migration is commonly assessed in vitro by scratching the surface of a monolayer of cells (epithelial, fibroblasts, or astrocytes) with a pipette tip (or similar) and measuring the directional movement of cells into the wounded area. Directed migration can be summarized by the following four steps: (1) directional sensing, (2) establishment of cell polarity, (3) protrusion, and (4) adhesion and forward movement (Ridley et al., 2003, and reviewed following). Interestingly, in this multistep model of cell migration, the mammalian Scribble and Par complexes appear to be required not only for the establishment of migrational polarity, but also for the regulation of other aspects of directed migration.

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a. Directional Sensing and the Establishment of Cell Polarity Directional sensing refers to the process whereby cells can detect and respond to a directional migration stimulus. Examples of this include the recognition of a free cell edge in cells at the edge of a wound or the detection of a directional chemokine gradient. Directional sensing and cell movement are best studied in single cells such as the amoeba Dictyostelium discoideum and in human and murine neutrophils. The factors controlling these events have been reviewed elsewhere (Manahan et al., 2004; Niggli, 2003; Van Haastert and Devreotes, 2004). This discussion of cell migration will largely focus on data obtained from the use of in vitro scratch wounding assays and how these studies have defined an essential role(s) for polarity regulators during directed cell migration. The migration of epithelial cells following wounding is dependent on receptor activation from both soluble and ECM‐associated ligands (Table II). Factors such as EGF and TGF‐b1 have been implicated in epithelial migration because the levels of both are locally elevated during wound healing (Faber‐Elman et al., 1996; Marikovsky et al., 1993). Consistent with this, activation of epidermal growth factor receptor (EGFR) and some TGF‐b/ BMP receptors is essential for normal cell migration in vitro and in vivo (Andl et al., 2004; Chen et al., 1994; Du et al., 2004). The activation of cytokine receptors is coincident with the engagement of ECM molecules through integrin receptors, and this dual activation serves to establish the directional cue. In epithelial cells, the EGFR and the MET receptors physically associate with multiple integrin subunits, and stimulation of these receptors can induce phosphorylation of the cytoplasmic domain of integrins (Falcioni et al., 1997; Trusolino et al., 2001). In the case of human keratinocytes, normal migration is dependent on both the association of EGFR and a6b4 integrin and the binding of each to its respective substrate (Russell et al., 2003). In keratinocytes and primary rat astrocytes, EGFR‐dependent integrin phosphorylation leads to downstream activation of Cdc42 and engagement of the cell polarity machinery (Etienne‐Manneville and Hall, 2001; Russell et al., 2003). b. A Model for Establishment of Polarity in Directed Migration Using primary rat astrocytes as a model, Etienne‐Manneville and colleagues defined a pathway downstream of integrin‐mediated activation of Cdc42 required for the cell polarization that precedes directional migration (depicted in Fig. 3A). In this model, localized GTP‐bound Cdc42 binds to a Par6/ aPKCz complex and promotes signaling through increased aPKCz kinase activity (Etienne‐Manneville and Hall, 2001). While the precise molecular nature of this interaction has not been described in migrating astrocytes, it presumably occurs in a manner identical to that described during the establishment of apical–basal polarity in epithelial cells. Active aPKCz physically associates with and phosphorylates glycogen synthase kinase 3b (GSK3b) on

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FIG. 3 Model for polarity regulation and migration in mammalian cells. (A) Growth factor and integrin‐dependent activation of Cdc42 at the leading edge leads to a Par6‐dependent increase in PKCz kinase activity. PKCz phosphorylates GSK3b at the inhibitory serine residue (Ser9), and GSK3b dissociates from APC, which then binds to the positive ends of growing microtubules. Dlg is recruited to the leading edge in a Par6/PKCz‐dependent manner and binds APC at the positive ends of microtubules, anchoring them at the leading edge. The microtubule organizing center (MTOC) and Golgi apparatus are repositioned (polarized) in the plane of migration. Polarization of the MTOC/Golgi is dependent on Cdc42, Par6, PKCz, GSK3b, APC, Dlg1, and Scribble activity. bPix is polarized to the leading edge of the cell through a Cdc42‐Pak‐dependent mechanism and in concert with Rac1 is required for localized actin polymerization and forward protrusion. Scribble colocalizes to the leading edge with bPix and regulates the recruitment of Rac1 and Cdc42 to this site. Scribble is also required to establish polarity following a migration cue (through an unidentified mechanism). (B) Ozdarma et al. (2005) showed that Cdc42‐ and TGF‐bRI‐dependent activation of the Par6/aPKC complex leads to recruitment of Smurf1 to the leading edge. Smurf1 activity at this site promotes membrane protrusion and migration by inducing degradation of the small GTPase RhoA, which is thought to antagonize Rac1 (and Cdc42) activity. (C) A study by Nishiya et al. (2005) showed that activity of GIT1 bound to paxillin at nonphosphorylated a4 integrin at cell–cell contacts promotes Arf6‐mediated Rac1 GTP hydrolysis, thereby restricting active Rac1 to the leading edge (where a4 integrin is phosphorylated).

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serine 9, blocking GSK3b catalytic activity and allowing the association of a GSK3b substrate, adenomatous polyposis coli (APC), with the positive end of growing microtubules (Etienne‐Manneville and Hall, 2003). APC at the plus ends then interacts with Dlg1 at the cell membrane, and microtubules become ‘‘anchored’’ at the leading edge (Etienne‐Manneville et al., 2005; Iizuka‐Kogo et al., 2005). Disruption to any of the processes described here, using dominant negative proteins, siRNA, or pharmacological inhibitors, leads to a failure to polarize the microtubule‐organizing center (MTOC) and Golgi apparatus in the plane of migration between the nucleus and leading edge of the cell. Similar mechanisms have been described in migrating primary rat fibroblasts and in 3T3 fibroblasts subjected to sheer flow, where Cdc42 regulates the repositioning of the nucleus and MTOC relative to the direction of flow in a Par6/aPKCz‐ dependent manner (Cau and Hall, 2005; Lee et al., 2005). Gomes et al. (2005) propose a model whereby Cdc42 acts to induce MTOC reorientation by initiating actin polymerization and retrograde flow through its eVector myotonic dystrophy kinase‐related Cdc42 binding kinase (MRCK) and controls the position of the MTOC at the cell centroid through activation of Par6/aPKCz. Given the role of Par6/aPKCz in regulating the capture of microtubules at the membrane, maintenance of MTOC positioning may occur through pulling forces exerted from the leading edge. In addition to the microtubule– APC–Dlg1 interaction described in astrocytes, capture and anchoring of microtubules at the leading edge membrane can occur through the binding of microtubule–APC to the Rac1/Cdc42 eVector IQGAP1 identified in Vero fibroblasts (Watanabe et al., 2004), as well as other docking proteins. Interestingly, in Vero cells, Rac1 activity is required for MTOC reorientation, whereas in astrocytes it is not, suggesting there may be subtle cell type‐ specific diVerences in the machinery that controls MTOC positioning. Nevertheless, the scaVolding of multiprotein complexes with microtubules at the leading edge is thought to provide a means by which microtubule dynamics can positively regulate the activity of Rho GTPases and vice versa, creating a positive feedback loop for maintenance of polarity (Wittmann and Waterman‐Storer, 2001). Recently, we described an essential role for Scribble in promoting cell polarization and the regulation of Rho GTPase recruitment to the leading edge of migrating cells (Dow et al., 2006). Moreover, we showed that Scribble is required for wound healing in vivo in the mouse. Similarly, Scribble is also required for polarity and Rho GTPase recruitment during migration in astrocytes (Osmani et al., 2006). It should also be noted that in some settings, Scribble appears to restrict cell movement rather than facilitate it, as Macara and colleagues showed that depletion of Scribble in MDCK cells increases cell migration (Qin et al., 2005). In each case, however, loss of Scribble disrupts the directionality of cell movement. In all, the data describe a model whereby localized activation of GTPases (primarily Rac1 and Cdc42) and

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their eVectors permits the coordination of microtubule and actin cytoskeletal dynamics to polarize the nucleus and MTOC in the plane of migration. It is important to note that recent publications on Rac1 and Cdc42 knockout cell phenotypes have indicated that some studies using dominant negative constructs may not reflect the inhibition of specific GTPases (Czuchra et al., 2005; Wells et al., 2004). For example, Cdc42 knockout fibroblastoid cells show defects in filopodium formation, cell polarization, and directed migration, but the phenotypes can be exacerbated by expression of dominant negative Cdc42 in these cells (Czuchra et al., 2005). This diVerence is presumably because dominant negative proteins sequester regulatory GEF molecules that often control numerous eVectors. It should be stressed that such studies do not exclude a role for Rac1 and Cdc42 in cell migration, but rather highlight the importance of considering the potential importance of other factors when interpreting results from studies using dominant negative approaches. c. Polarity Regulators and the Control of Localized GTPase Activity Polarization of the MTOC as well as actin polymerization and membrane protrusion are dependent on tightly controlled and spatially restricted activation of many signaling components. This is most clearly demonstrated by the observation that both dominant negative and constitutively active versions of Rac1, Cdc42, Par6, aPKCz, and GSK3b disrupt polarization of the MTOC (Cau and Hall, 2005; Etienne‐Manneville and Hall, 2001, 2003), implying that the presence of signaling is not suYcient to induce polarity. So how is the activity of these signals properly localized at the leading edge? As is the case in the establishment of apical–basal polarity (and probably all polarized cell types), the delivery and recycling of vesicles toward the leading edge of migrating cells are likely to contribute to the polarized localization of receptor and downstream signaling components such as integrins and small GTPases Rac and Cdc42 (Ivaska et al., 2005; Palacios et al., 2001; Schmoranzer et al., 2003), but the question of how these complexes are activated in specific locations has been a diYcult one to answer. Nishiya and colleagues (2005) have recently shown that the phosphorylation of a4 integrin specifically at the leading (free) edge of a scratch wound can directly promote the localized activation of Rac1 by disrupting a multiprotein complex that negatively regulates Rac1 activity (Fig. 3C). Interestingly, a key component of this regulatory complex is the Arf‐GAP, GIT1, which has been shown to regulate vesicle traYcking (Manabe et al., 2002). GIT1 also associates constitutively with Scribble in mammalian epithelial cells, although the functional consequences of this interaction are not clear (Audebert et al., 2004). In addition, Scribble and GIT1 also bind bPix, a GEF for Rac1 (Audebert et al., 2004). bPix also shows some GEF activity toward other GTPases such as Cdc42, and recent data from Etienne‐Manneville et al. support the notion that

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Scribble can regulate migration through this signaling network (Osmani et al., 2006). bPix is necessary and suYcient for recruitment and activation of Rac1 at membrane ruZes, and knockdown of bPix in fibroblasts decreases actin‐based cell protrusions and migration (Cau and Hall, 2005; ten Klooster et al., 2006). The presence of GIT1 and bPix (that have opposing eVects on Rac activity) in a stable complex suggests that other factor(s) must modulate the balance of activity of each protein to spatially control Rac1 signaling. The known role of Scribble in regulating migration and Rac1 targeting (Dow et al., 2006) makes it an attractive candidate for the control of this process. i. Balancing GTPase activity at the leading edge In addition to controlling the localized activation of Rac1 and Cdc42 at the leading edge, polarity regulators also seem to promote migration through the direct inhibition of negative regulators of cell movement. While activation of Rac1 and Cdc42 promotes protrusion, another small GTPase, RhoA, is thought to induce membrane retraction because the activation of RhoA induces actin stress‐ fiber formation and contractile force (Hall, 2005). Rac1 and Cdc42 have been shown to antagonize RhoA and as such must be spatially segregated in migrating cells to allow Rac1/Cdc42 to promote protrusion at the leading edge and RhoA to promote retraction at the rear of the cell (Evers et al., 2000). Recent data from Wrana and colleagues suggest that the Par6 polarity complex may play a central role in regulating the balance of Rac1/Cdc42 and RhoA activity at the leading edge (Bose and Wrana, 2006). They have shown that both TGF‐b‐dependent phosphorylation of Par6 on S345 and activation of PKCz can recruit the E3 ubiquitin ligase Smurf1 to cellular protrusions, where it promotes the ubiquitin‐mediated degradation of RhoA (Ozdamar et al., 2005; Wang et al., 2003) (Fig. 3B). Considered in the context of the ability of the Par and Scribble polarity complexes to regulate Rac1/ Cdc42 localization and activity, it is likely that these networks serve as key mediators of cell migration machinery. d. Protrusion and Movement Cell protrusions are often classified into two distinct types based on physical characteristics. Lamellipodia are broad flat, actin‐rich protrusions that have classically been linked to activation of Rac1, while filopodia are small finger‐like cell projections containing thin bundles of actin filaments that are thought to rely on Cdc42 activity (Hall, 2005). Both types of membrane protrusions can occur simultaneously, and both play a role in cell migration. GTP‐bound Rac1 and Cdc42 interact with many downstream eVectors, particularly the WASP/WAVE family of proteins that activates the Arp2/3 complex. Arp2/3 is a seven protein complex that catalyzes the polymerization of actin monomers to form a branched actin meshwork to promote membrane protrusion (Cory et al., 2003). As actin polymerization drives membrane protrusion, focal adhesions are formed at the protruding edge following interaction of integrins with

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underlying ECM substrate. Focal adhesions provide two very important functions: (1) they allow the attachment of cells to the ECM and thus generate a tractile force that cells use for locomotion and (2) they stabilize many integrin‐ mediated signals such as the stimulation of Src family kinases and the Src‐ dependent activation of the MEK‐ERK pathway (Fincham et al., 2000; Matsubayashi et al., 2004). Blocking ERK phosphorylation using selective MEK1 or Src inhibitors blocks cell migration (Matsubayashi et al., 2004). Integrin/Src‐induced ERK phosphorylation has been proposed to be involved in the formation of focal adhesions at the leading edge (Fincham et al., 2000). One mechanism for this is through the direct phosphorylation of the focal adhesion protein paxillin that coordinates activation of Rac1 and focal adhesion kinase (FAK) (Ishibe et al., 2004). Activated FAK is required not only for the assembly of focal adhesions, but also because the polarity of migrating cells and loss of FAK or Src kinase activity disrupt Golgi polarization in migrating epithelial cells (Tilghman et al., 2005; Timpson et al., 2001). This eVect on cell polarity suggests a connection with the activity of known polarity regulators. Indeed, Scribble, Dlg, and members of the Par complex are all concentrated at the leading edge of migrating cells, juxtaposed with focal adhesions (Humbert et al., 2006), although it is currently unclear whether these polarity regulators directly aVect focal adhesion dynamics to promote cell movement or vice versa, or in fact whether they functionally interact at all. It is clear, however, that both processes are essential for proper cell migration. The regulated turnover of focal adhesion complexes is also required for cell migration, since it allows release of the migrating cell from the underlying substrate to enable net forward movement. This process is governed by many of the same molecular networks that are employed to promote focal adhesion formation, including ERK and FAK. Significant disruption to the coordination of either focal adhesion formation or turnover inhibits appropriate cell migration. 3. In Vivo Models for Mammalian Epithelial Cell Migration a. Eyelid Closure Studying cell migration in vitro has been essential to progressing our understanding of the mechanisms of cell movement, but it has obvious limitations. Specifically, most techniques fail to recapitulate the complex interaction of multiple cell types and tissue‐specific cytokine/ECM compositions. To fully appreciate these factors and their potential role in cell migration, it is necessary to examine cell behavior in vivo in a whole organism. Some studies have used adult wound healing as a model for in vivo cell migration, but this process is often complicated by infection and the initiation of an inflammatory response at wound sites. For this reason and the fact that many mouse mutants do not survive to adulthood, the most extensively used system for analyzing in vivo epithelial sheet migration has become the morphogenesis and closure of the mouse eyelid (Xia and Karin, 2004).

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Similar to Drosophila dorsal closure, migration of the eyelid epithelium occurs late during embryonic development as two opposing epithelial sheets move as a cohesive unit toward one another, fuse at the lateral midline, and ‘‘zip’’ shut. Not surprisingly, many of the proteins identified as regulators of dorsal closure in Drosophila also mediate eyelid closure (Table II). Specifically, several knockout mouse studies have delineated two overlapping pathways downstream of TGF‐b/activin and EGF‐like ligands that are essential for migration of the eyelid epithelium. In this model, the activation of MAPK kinases MEKK1, ERK, and JNK, downstream of receptor stimulation leads to dynamic changes in actin cytoskeleton and c‐jun‐dependent transcription of further genes required for cell migration, including EGF and the EGF receptor (Li et al., 2003; Mine et al., 2005; Zenz et al., 2003; Zhang et al., 2003). By analogy to Drosophila, JNK/Jun signaling would also be expected to promote the expression of TGF‐b/BMP‐like ligands, although this has not yet been shown. Receptor activation also stimulates activity of the small GTPase RhoA and its downstream eVector ROCKI (Shimizu et al., 2005). This arm of the pathway promotes phosphorylation of myosin light chain (MLC) and the subsequent assembly of actomyosin cables to stabilize the leading edge of the migrating eyelid epithelium. The generation of transgenic animals overexpressing a2, a5, and b1 integrin subunits in the suprabasal epidermis has identified a role for these transmembrane receptors in eyelid closure (Carroll et al., 1995). Together, these genetic studies, in combination with some biochemical evidence, have confirmed a physiologically relevant role for a large number of signaling molecules previously identified as regulators of cell migration in vitro. b. Polarity Regulators and Migration in the Mouse Supporting the notion that polarity regulators control cell migration in vivo in mammals is the recent identification of severe eyelid closure defects in two independent Scribble mutant mouse strains: circletail (crc) and rumpelstilztchen (rumz) (Murdoch et al., 2003) (our unpublished observations). The Scribble crc allele is a single base pair insertion after position 3182, resulting in a non‐ sense mutation in the coding sequence and premature termination of the protein product. The rumz allele is a single base pair mutation causing an isoleucine to lysine substitution at amino acid 285 (I285K) (Zarbalis et al., 2004). The specific eVects of the crc mutation remain uncharacterized, but we have recently shown that the rumz mutation results in reduced protein stability and mislocalization of Scribble away from cell–cell junctions in murine keratinocytes (Dow et al., 2006). Interestingly, both Scribble mutations, which are presumably hypomorphic, also result in the most severe form of neural tube defects termed craniorachischisis (Doudney and Stanier, 2005). This defect is distinct from the related conditional lumbosacral spina bifida in that almost the entire brain and spinal cord remain exposed,

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owing to a failure to initiate closure of the dorsal midline. Neural tube closure involves coordinated cell and tissue migration, further emphasizing the essential role for Scribble in this process in vivo. A Dlg1 mutant mouse strain produced as a result of a gene trap insertion screen does not cause craniorachischisis; however, homozygotes do show defects in palate fusion, another process that requires cell sheet migration (Caruana and Bernstein, 2001). It is possible that functional redundancy among the four known Dlg family members accounts for the lack of migration phenotypes observed in this mouse, although a definite answer to this question will involve the generation of genetically complex mouse models. The notable omissions to the pathways described previously are the migration and polarity regulators recognized in mammalian cell culture such as Rac1, Cdc42, and aPKCz. A role for these proteins is yet to be described in vivo, although given their essential role in Drosophila, future development of appropriate mouse models will likely indicate a function in regulating cell migration in vivo. In the coming years, the development of conditional mouse alleles in many diVerent polarity regulators will serve to define the role of these proteins in regulating diVerent stages of directed cell migration during both embryonic development and adult homeostasis. However, the evidence presented here suggests that polarity regulators, particularly those of the Scribble and Par complexes, are essential for the control of cell migration in Drosophila, the mouse, and humans.

III. Polarity Regulators and Tumorigenesis In addition to the essential role for polarity regulators in epithelial morphogenesis during eukaryotic development, many lines of evidence have implicated polarity proteins in the control of mammalian tumorigenesis (Bilder, 2004; Humbert et al., 2003). First, in Drosophila, Scribble, Dlg, and Lgl function as tumor suppressors to restrict neoplastic overgrowth of epithelium. Second, Scribble and Dlg mammalian homologues are targeted for degradation by the E6 oncoprotein from high‐risk (but not low‐risk) human papilloma viruses (HPV) HPV16 and HPV18, which are causally linked to greater than 90% of cervical cancers (Burger et al., 1996; Gardiol et al., 1999; Nakagawa and Huibregtse, 2000; zur Hausen, 2000). Significantly, the transforming ability of the E6 protein from these viruses is dependent on direct binding to Scribble, Dlg, and other PDZ proteins because E6 mutants that lack the PDZ‐binding motif, but can still bind and inactivate p53, cannot transform rodent cells or induce epithelial hyperplasia like wild‐type E6 (Kiyono et al., 1997; Nguyen et al., 2003). However, it remains to be shown whether E6‐mediated degradation of Scribble and Dlg specifically is required for the transformation of

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HPV‐infected cells. Finally, many studies have now demonstrated that Scribble and/or Dlg protein are reduced or lost during the development of tumors in vivo in the mouse and in human cancers. In humans, Scribble and Dlg expression is decreased in a variety of tumor types, including those associated with HPV (Massimi et al., 2004; Nakagawa et al., 2004) and some cancers unrelated to HPV infection such as breast and colon cancer (Gardiol et al., 2006; Navarro et al., 2005). Similarly, Lgl1 has been shown to be reduced in a variety of human malignancies including breast, lung, prostate, colon, and melanoma (Grifoni et al., 2004; Kuphal et al., 2006; Schimanski et al., 2005). Moreover, the loss of Scribble, Dlg, and/or Lgl expression in these tumors correlates with more invasive and aggressive cancers. Together this evidence suggests that loss of Scribble, Dlg, or Lgl may be an important step in the development of mammalian cancers, in particular the progression to invasive and malignant disease. However, the way in which loss of these polarity proteins may contribute to oncogenic progression is currently poorly understood.

A. Polarity and Cancer Progression Using 3D basement membrane cultures, the Bissell and Weaver groups have been able to directly examine the requirements of tissue architecture and consequences of polarity disruption for disease progression. Using a series of human mammary epithelial cells established almost 20 years ago (HMT‐ 3522 series) (Briand et al., 1987), Weaver and colleagues (1997) showed that the progression of breast cancer and tumor formation is highly correlated with polarity and tissue structure. This cell series progresses from nonmalignant, polarized S‐1 cells to highly malignant, nonpolar T4–2 cells. The genetic and epigenetic changes that have resulted in this altered phenotype are not known; however, manipulating the interaction of these cells with ECM components using integrin‐blocking antibodies can either suppress or enhance polarity disruption. Blocking b1 integrin interactions not only causes a reversion of the disorganized nonpolar phenotype in culture, but it also suppresses tumor formation in mouse xenografts (Weaver et al., 1997). In contrast, blocking either a6 or b4 integrin subunits disrupts polarization in nonmalignant S‐1 cells, indicating an involvement of the a6b4 integrin heterodimer in regulating normal polarized architecture. In malignant T4–2 cells, a6 and b4 are required, but rather than maintaining cell polarity, they serve to protect cells from sensitivity to apoptosis through the autocrine production of laminin 5 and a6b4‐dependent Rac activation (Zahir et al., 2003). This increased sensitivity to drug/cytokine‐induced apoptosis of T4–2 cells, provoked by disrupted 3D architecture, is not specific to this cell type but is a more general feature of nonpolarized cells growing in 3D environments (Park et al., 2006; Weaver et al., 2002). A similar sensitivity to apoptotic signals has

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been noted in scribble mutant (nonpolarized) cells in mosaic Drosophila epithelial tissue (Brumby and Richardson, 2003). The apoptosis of abnormal cells within normal tissues may represent a default mechanism that restricts inappropriate, perhaps invasive, behavior of cells that have lost normal polarity. It is plausible to imagine that in the example described here, the default ‘‘death pathway’’ of nonpolarized T4–2 cells is bypassed by ectopic a6b4 interaction with polarity regulators such as Rac, essentially fooling the transformed cell into believing it is polarized and thus evading programmed cell death. In such a scenario, loss of polarity or tissue structure cooperates with other survival/transformation signals to promote tumor development and progression to malignant disease. In fact, recent work using Drosophila to examine tumorigenesis has shown that polarity regulators play a significant role in mediating the tumorigenic and invasive properties of transformed epithelial cells, particularly in cooperation with known oncogenes.

B. Drosophila, Cell Invasion, and Cooperative Tumorigenesis 1. Loss of Dlg or Lgl Promotes Cell Invasion As described, loss of scribble, dlg, or lgl in Drosophila gives rise to neoplastic overgrowth of epithelial tissues, which results in lethality of the animal before pupation. To examine the consequence of dlg, and lgl mutations on cell invasion and metastasis, Woodhouse et al. (1998) used a Drosophila transplant model where cells from mutant larval imaginal discs are injected into the abdomen of an adult host. In this model, loss of Dlg or Lgl alone was suYcient to allow tumor formation at multiple secondary sites. This eVect may be due to the increased production of matrix‐modifying proteins such as collagenase IV, which is increased in lgl mutants (Woodhouse et al., 1994). The ability of these mutant cells to become invasive is not unique to transplant models. In the early embryo, just prior to the onset of border cell migration, loss of dlg causes ectopic invasion of follicle cells (Goode and Perrimon, 1997; Goode et al., 2005). While invasive dlg mutant follicle cells do not acquire a border cell fate, the invasion does rely on the expression of Baz to mediate cell–cell interactions as occurs during normal border cell migration (Abdelilah‐ Seyfried et al., 2003). Together these data suggest that loss of dlg or lgl may provide cells with the intrinsic ability for invasion and metastasis, although this eVect may be context dependent (Humbert et al., 2006). 2. Cooperative Tumorigenesis in the Fly The use of Drosophila as a model to study all the facets of tumorigenesis is complicated by the fundamentally diVerent physiology of invertebrates and vertebrate organisms (i.e., open versus closed circulatory system). However,

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one way in which the Drosophila model can be applied to cancer is through examination of cooperative genetic events that lead to tumorigenic cell behavior (Brumby and Richardson, 2005). Using the MARCM system to induce clonal somatic mutations in epithelial tissue, Brumby and Richardson (2003) and Pagliarini and Xu (2003) identified a cooperation between activation of the protooncogene Ras and loss of polarity regulators, including Scribble, Dlg, Lgl, Baz, Sdt, Crb, and Cdc42. In the Drosophila eye, activation of Ras in somatic clones causes hyperproliferation and ectopic photoreceptor diVerentiation, but mutant cells never form overgrown tumor masses. However, in combination with loss of expression of polarity regulators, particularly Scribble, epithelial cells fail to diVerentiate and overgrow existing wild‐type cells within the tissue. Pagliarini and Xu also report that in some cases these cells become invasive, moving locally into the ventral nerve cord and forming secondary tumors at distant sites in the Drosophila larvae. Such cell movement has obvious parallels to the metastasis of human cancers, although the lack of vasculature in flies makes a clear distinction between these two events. Activated alleles of the Notch receptor (NotchACT) and Raf also synergized with loss of Scribble to promote neoplastic overgrowth in this assay, although overexpression of potent growth regulators dMyc or dAkt did not, suggesting that the tumorigenic cooperation with loss of polarity regulators was not a general phenomenon of all oncogenes. Invasion and metastasis was not assessed in RafACT‐scribble or NotchACT‐ scribble mutant larvae. As discussed earlier, loss of scribble in Drosophila embryos and C. elegans disrupts the ordered arrangement of the zonula adherens. Forced overexpression of the Drosophila E‐cadherin homologue (shotgun) was able to block invasion and metastasis of RasACT‐scribble mutant tumors, but loss of shotgun and activation of Ras did not produce invasive tumors, implying that loss of scribble (or polarity) does not lead to invasion simply by compromising junction integrity (Pagliarini and Xu, 2003). In summary, these two significant publications have clearly demonstrated that defects in the machinery that regulates normal tissue structure and polarity can collaborate with known oncogenes (i.e., Ras) to induce neoplastic overgrowth and metastatic disease. These studies have obvious implications for the understanding of human disease, as activating mutations in Ras are present in approximately 30% of all human cancers (Malumbres and Pellicer, 1998). In some cases, such as pancreatic cancer, more than 95% of tumors harbor Ras mutations, and the 5‐year survival rates for patients with these tumors are very low (Hingorani and Tuveson, 2003). The eVect(s) of Ras mutation on cell function is dependent on many factors including cell type, developmental context, and genetic background. A full discussion of Ras‐mediated transformation is beyond the scope of this review but has been presented elsewhere (for example, see Hancock, 2003). Rather, the following section will review the evidence to support the notion

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that oncogenic activation of Ras (and perhaps other protooncogenes) can cooperate with disruption of polarity regulation to promote malignant transformation in a mammalian tissue.

C. Cooperation between Polarity Proteins and Ras in Mammalian Tumorigenesis 1. aPKC Is Required for Ras‐Mediated Invasion The best evidence for a key Drosophila polarity regulator being influential in the development of invasive human cancer is that of atypical protein kinase C. Of the two mammalian isoforms of aPKC (i and z), most studies have focused on the role of aPKCi in tumorigenesis. aPKCi is overexpressed in ovarian and lung cancers, and high aPKCi expression is correlated with reduced disease‐free survival (Eder et al., 2005; Regala et al., 2005a,b; Weichert et al., 2003). aPKCi is also overexpressed in human colon cancer and in azoxymethane (AOM)‐induced colonic tumors in mice (Murray et al., 2004). Significantly, both of these tumor types are correlated with activating K‐Ras mutations. Consistent with a role for aPKCi in Ras‐mediated tumorigenesis, constitutive expression of kinase dead aPKCi reduces the incidence of AOM and K‐Ras‐induced colonic tumors in mice, and constitutive overexpression of wild‐type aPKCi causes an increased incidence of AOM‐ induced colonic tumors of a more aggressive subtype. Expression of aPKCi alone in this context does not induce tumor formation; in fact, it acts downstream of Ras with Rac1 and is required for Ras‐mediated eVects on cell invasion but not proliferation. 2. E6 and Ras: Implication for Polarity Regulators In addition to deregulation of aPKCi, there is circumstantial evidence to suggest that loss of other polarity proteins, perhaps Scribble specifically, cooperates with oncogenic mutations to promote tumorigenesis in vivo. As discussed earlier, E6 proteins from high‐risk HPV strains target Scribble, Dlg, and other PDZ proteins for degradation. It was recognized over 10 years ago that activated alleles of Ras can synergize with these E6 proteins (Storey and Banks, 1993) to promote cell transformation. Since this time, numerous studies have shown that HPV infection is correlated with Ras mutation in mouse models and in human tumor samples (Buyru et al., 2006; Dokianakis et al., 1999; Greenhalgh et al., 1994; Schreiber et al., 2004). In addition, two groups have shown cooperation between HPV E6/E7 and activation of upstream cytokine receptors EGFR and Notch (Rangarajan et al., 2001; Woodworth et al., 2000). It is important to note that E6 and E7 proteins

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can also directly inhibit the p53 and Rb pathways that have been shown to restrict various aspects of Ras‐mediated transformation. Thus, directly examining the requirement for loss of Scribble and other E6 targets will be essential to determine the molecular nature of the cooperation observed between these two well‐known human oncogenes. 3. The Interrelationship of Polarity Complexes in Cancer Analysis of human cancers indicates that loss of Scribble/Dlg/Lgl and overexpression of PKCi are often correlated with aggressive and invasive disease (Eder et al., 2005; Gardiol et al., 2006; Murray et al., 2004; Nakagawa et al., 2004; Regala et al., 2005a,b; Weichert et al., 2003). However, it is not known whether these events are correlated, mutually exclusive, or unrelated. Interestingly, many of the polarity and overproliferation defects associated with loss of Lgl in Drosophila can be rescued by inactivating aPKC, suggesting deregulated aPKC activity is at least partially responsible for the Scribble/ Dlg/Lgl mutant phenotype (Humbert et al., 2006; Rolls et al., 2003). Moreover, the invasion of dlg mutant follicle cells in Drosophila embryos is dependent on Par/aPKC complex activity (Abdelilah‐Seyfried et al., 2003), suggesting the two complexes functionally interact to control cell behavior in atypical situations such as cancer. In mammals, many studies now show that aPKCi is required for eYcient Ras transformation and is amplified in many cancers (Murray et al., 2004; Regala et al., 2005b; Weichert et al., 2003); perhaps loss of Scribble, Dlg, or Lgl represents a means other than gene amplification of activating signaling through this pathway. Recently, Muthuswamy and colleagues showed that both Scribble and the Par6–aPKC pathway are important mediators of an ErbB2‐mediated ‘‘tumorigenic’’ transformation in MCF10A cells. Consistent with the concepts described above, in this instance the Scribble and Par polarity complexes seem to act antagonistically to modulate cell behavior, although the molecular details of this genetic interaction are yet to be defined (Aranda et al., 2006). 4. Ras, Rho‐GTPases, and Polarity Regulators In most cases, expression of activated alleles of Ras elicit drastic changes in cell morphology. Classic ‘‘cobblestone’’ epithelia lose regular cell contacts and become elongated and ‘‘spindle’’ shaped. Not surprisingly, these dramatic changes are largely mediated through eVects on the cell cytoskeleton by members of the Rho GTPase family: Rho, Rac, and Cdc42 (Malumbres and Pellicer, 1998). In fact, in many cases, Rac1 and Cdc42 can synergize with Ras eVectors (e.g., Raf1) to promote transformation and are essential for positively regulating cell invasion. However, some studies have reported that sustained Ras signaling results in a reduction in active Rac1 (and Cdc42) and

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an increase in Rho activity and that forced expression of Rac1, or its activator Tiam1, can revert the Rho‐dependent transformed morphology (Hordijk et al., 1997; Sahai et al., 2001; Zondag et al., 2000). This is due largely to the ability of Rac and Tiam1 to promote formation of E‐cadherin‐containing cell–cell junctions, which are frequently lost in Ras‐transformed cells. Interestingly, in Drosophila, loss of Cdc42 cooperates with activated Ras to allow tumorigenic overgrowth of epithelium (Pagliarini and Xu, 2003). This cooperation is thought to be a direct result of cell polarity disruption following loss of Cdc42. Thus, Rho GTPases (Rac1 and Cdc42) can either inhibit or promote cell invasion downstream of Ras signaling, depending on cell context. Some in vitro evidence suggests their precise role is dependent on the ability of these GTPases to control cell behavior and junction formation downstream of known polarity regulators. Recently, Zhang and Macara (2006) showed that Par3 controls dendritic morphogenesis by spatially restricting the subcellular localization of the RacGEF, Tiam1. In the absence of Par3, cells showed ectopic formation of lamellipodia and filopodia, similar to the phenotype of cells expression activated Rac. This Rac–Tiam1 localization‐dependent signaling has also been reported in other cell systems whereby activation of the Tiam1–Rac1 complex in epithelial cell types promotes cell adhesion and polarity, while activation under conditions that inhibit E‐cadherin‐mediated cell contacts or in ‘‘nonpolarized’’ fibroblasts promotes cell motility and invasion (Hordijk et al., 1997; Michiels et al., 1995; Sander et al., 1998). In fact, disruption to the normal localization of regulatory GEF proteins may be a more general feature in transformed cells because misdistribution of ECT2 results in ectopic activation of RhoA and promotes malignant transformation (Saito et al., 2004). It is possible that the Scribble complex, like Par3 (described previously), acts to suppress malignant transformation by controlling the localization and activity of Rac1 and Cdc42 as has been shown in migrating cells (Dow et al., 2006; Osmani et al., 2006).

D. A Common Mechanism for the Regulation of Polarity, Migration, and Tumorigenesis? In this review, we have discussed the potential role for polarity determinants in regulating cell polarity, migration, and tumorigenesis. The critical question that must now be considered is whether these polarity regulators act to control such seemingly diverse processes through a common molecular mechanism. A number of recent observations suggest that polarity proteins, particularly those of the Scribble complex, may regulate cell behavior in all contexts through the control of endocytosis and vesicle traYcking. Two pieces of functional evidence suggest that the Scribble/Dlg/Lgl group (particularly Lgl) may contribute to polarization through the regulation of

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vesicle traYcking. First, mammalian Lgl can rescue the secretion defects of yeast homologues Sro7/Sro77p, and this is dependent on the binding of Lgl to the vesicle docking t‐SNARE protein, Sec9 (Gangar et al., 2005). Second, Musch et al. (2002) have shown that mammalian Lgl binds to Syntaxin 4, a member of the exocytic machinery specific for basolateral targeting. The requirement for Scribble (Dlg and Lgl) in regulating endocytosis and polarized recycling of signaling receptors during cell migration has not yet been directly investigated. However, Lahuna et al. (2005) recently showed that Scribble is essential for the proper recycling of the thyrotropin receptor, and in glutamatergic synapses in Drosophila, mutation of scribble disrupts vesicle recycling dynamics (Roche et al., 2002), indicating that it is important for this process. Dlg is also required for proper synaptic transmission in glutamatergic synapses (Guan et al., 1996), and similarly, mammalian Dlg family members (in particular Dlg4/PSD‐95) bind to many receptors in postsynaptic neurons, and disruption to these interactions can impair synaptic transmission (Funke et al., 2005), suggesting a possible role in vesicle function. In addition, the Rac‐GEF bPix, which interacts constitutively with Scribble, has been shown to bind to the E3 ubiquitin ligase c‐Cbl and modulate the balance between EGFR recycling and degradation (Feng et al., 2006; Wu et al., 2003). In Drosophila border cells, loss of Cbl (and thus decreased receptor tyrosine kinase degradation) causes profound defects in directed migration (Jekely et al., 2005). Interestingly, this same protein complex has been shown to be an essential node during Src‐mediated cell transformation. In NIH/3T3 cells, overexpression of bPix compromises c‐Cbl function and causes EGFR accumulation and excessive mitogenic stimulation. In combination with v‐Src, this leads to increased cell growth in soft agar and tumorigenesis in nude mice (Feng et al., 2006). Finally, the further suggestion that Scribble, Dlg, and Lgl restrict tumorigenesis through regulation of endocytosis and vesicle traYcking comes from the recent identification of two novel neoplastic tumor suppressors in Drosophila, avalanche (avl) and vsp25. Both Avl and Vsp25 are core components of the endocytic traYcking machinery (Lu and Bilder, 2005; Vaccari and Bilder, 2005), and loss of function mutations in these genes causes loss of polarity and neoplastic overgrowth of epithelial tissues, mirroring the eVect of scribble, dlg, or lgl mutation in flies.

IV. Concluding Remarks Polarity regulators of the Scribble and Par complexes function in many diVerent cellular processes including apical–basal cell polarity, planar cell polarity, and cell migration and can have an impact on the development of

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aggressive cancers. Therefore, understanding the mechanisms of polarity regulation represents an important step in our knowledge of normal and aberrant cellular function. We expect that in the coming years, information gained from multiple ‘‘model’’ biological systems will converge to elucidate the mechanism(s) of polarity regulation in diVerent cell types and delineate the precise role of the various polarity regulators discussed here. With this framework in place, we can begin to understand exactly how the disruption of polarity regulation aVects the progression of human cancer and instigate the development of new therapeutic strategies to treat invasive and malignant cancers that have thus far been diYcult to treat eVectively.

Acknowledgments We would like to thank Anthony Brumby and Sarah Russell for critical reading of the manuscript and numerous discussions. P.O.H was supported by a Career Development Award from the Australian NHMRC and L.E.D. by a Postgraduate Cancer Research Scholarship from the Cancer Council Victoria. The studies that formed the foundation of this work were supported by grants from the Association for International Cancer Research and Cancer Council Victoria (P.O.H.).

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