Control of Tumourigenesis by the Scribble/Dlg/Lgl Polarity Module

PO Humbert1, NA Grzeschik2, AM Brumby2, R Galea1, I Elsum1 and HE Richardson2

1Cell Cycle and Cancer Genetics Laboratory, Research Division, Peter MacCallum Cancer Center, Melbourne, Victoria, Australia and 2Cell Cycle and Development Laboratory, Research Division, Peter MacCallum Cancer Center, Melbourne, Victoria, Australia

The neoplastic tumour suppressors, Scribble, Dlg and Lgl, is composed of the transmembrane protein Crumbs, originally discovered in the vinegar fly Drosophila Pals (Stardust in Drosophila) and Patj. In this review, we melanogaster, are currently being actively studied for focus on the Scribble polarity module and its role in their potential role in mammalian tumourigenesis. In tumourigenesis. Drosophila, these tumour suppressors function in a common genetic pathway to regulate apicobasal cell polarity and also play important roles in the control of cell proliferation, survival, differentiation and in cell The Scribble/Dlg/Lgl polarity module migration/invasion. The precise mechanism by which Scribble, Dlg and Lgl function is not clear; however, they Scribble, Dlg and Lgl were originally identified in the have been implicated in the regulation of signalling genetically tractable model organism Drosophila,in pathways, vesicle trafficking and in the Myosin II–actin which homozygous mutants in any of the genes results cytoskeleton. We review the evidence for the involvement in the loss of apicobasal cell polarity and neoplastic of Scribble, Dlg, and Lgl in cancer, and how the various tissue overgrowth (Gateff and Schneiderman, 1974; functions ascribed to these tumour suppressors in Droso- Mechler et al., 1985; Woods and Bryant, 1989; Bilder phila and mammalian systems may impact on the process and Perrimon, 2000). Due to the similarity in their of tumourigenesis. mutant phenotypes and the genetic interactions ob- Oncogene (2008) 27, 6888–6907; doi:10.1038/onc.2008.341 served between them, it has been surmised that Scribble, Dlg and Lgl function in a common pathway to regulate Keywords: Scribble; Dlg; Lgl; cell polarity; tumour the establishment and maintenance of apicobasal suppressors polarity in epithelial cells (Bilder et al., 2000). Todate, a single mammalian homologue of Scribble (hScrib, Scrb1) has been identified, whereas four Dlgs, Dlg1 (hDlg, SAP97), Dlg2 (Chapsyn-110, PSD-93), Dlg3 (NE-Dlg, SAP102), Dlg4 (PSD-95, SAP90) and two Introduction Lgls, Lgl1 and Lgl2 (Hugl1 and 2) have been described (for recent reviews see Humbert et al., 2006; Dow and Cancer is a complex, multistep process that involves a Humbert, 2007; Assemat et al., 2008; Yamanaka and series of morphological and physiological changes. The Ohno, 2008). Dlg5, Dlg6 and Dlg7 are erroneously development of epithelial tumours involves loss of named and are not members of the Dlg subfamily of polarized cell architecture, a process fundamental to membrane-associated guanylate kinases (MAGUKs) an epithelial-to-mesenchymal transition (EMT), which (Nechiporuk et al., 2007; te Velthuis et al., 2007, and is a common event in more aggressive tumours and is our unpublished data) and hence will not be referred to correlated with increased invasion and metastasis further in this review. (reviewed by Dow and Humbert, 2007; Wodarz and The structures of Scribble, Dlg and Lgl are rich in Nathke, 2007). Three polarity modules are important in protein–protein interaction domains, and their function the establishment and maintenance of apicobasal cell is highly dependent on their proper localization. polarity in epithelial cells, namely the Scribble, Par and Scribble is an LAP (leucine-rich repeats and PDZ) Crumbs polarity modules (for recent review see Assemat protein that contains 16 leucine-rich repeats (LRR) and et al., 2008). The Scribble polarity module is composed four PSD-95, ZO-1 and Discs large (PDZ) domains of three proteins, Scribble (Scrib), Discs Large (Dlg) and (reviewed by Bryant and Huwe, 2000; Santoni et al., Lethal giant larvae (Lgl). The Par complex is composed 2002). In Drosophila epithelial cells, Scribble is localized of Par3 (Bazooka in Drosophila), Par6 and atypical tothe cortex,basal tothe adherens junction,at the protein kinase C (aPKC), whereas the Crumbs complex septate (basolateral) junctions (Bilder and Perrimon, 2000). In mammalian epithelial cells, Scribble localization overlaps with the adherens junctions and also Correspondence: Dr PO Humbert, Research Division, Peter MacCallum Cancer Centre, Locked Bag 1, A’Beckett Street, Melbourne, Victoria extends basally (Dow et al., 2003; Navarro et al., 2005) 8006, Australia. (see Figure 1). The LRR region and PDZ regions of E-mail: [email protected] Drosophila Scribble are important for its localization Role of the Scribble polarity module in tumourigenesis PO Humbert et al 6889 Drosophila Vertebrate

Par3 apical Subapical Pals Tight Region Crb Par6 aPKC Junction Patj P Adherens Adherens Junction P P Junction P Lgl

Scrib Basolateral Lgl Basolateral Junction Region Dlg

CyclinE DIAP1

Nucleus basolateral Integrin ECM

Figure 1 The regulation of apicobasal cell polarity, cell proliferation and survival by the Scribble/Dlg/Lgl polarity module. Epithelial cells are polarized along their apicobasal axis, due to the action of three polarity modules; the Crumbs complex (composed of Crumbs (Crb), Pals (Sdt in Drosophila) and Patj), the Par complex (composed of Par3 (Bazooka in Drosophila), Par6 and aPKC) and the Scribble polarity module (composed of Scribble (Scrib), Dlg and Lgl). The Crumbs and Par complex are localized to the subapical region and the Scribble polarity module in localized to the basolateral region (in Drosophila, at Septate or basolateral junctions). In Drosophila neuromuscular junctions, Scribble and Dlg interact via Gukh. Mutually antagonistic interactions exist between these polarity modules, which are required for restricting the activity of each module to speciﬁc cortical domains and the positioning of the adherens junctions (composed of E-cadherin, a-catenin and b-catenin). In Drosophila embryonic epithelia, genetic epistasis studies have shown that the Par complex is required ﬁrst for the establishment of apicobasal cell polarity, and acts to repress the activity of the Scribble polarity module and promote the activity of the Crumbs complex. Furthermore, Lgl inhibits aPKC activity in the basolateral domain, and aPKC phosphorylates and excludes Lgl from the apical cortex. The Scribble polarity module also plays a role in negatively regulating cell proliferation by inhibiting the expression of the key cell cycle regulator, Cyclin E, and in promoting apoptosis by blocking expression of the apoptosis inhibitor, DIAP1 (see text for further details).

and stabilization at the plasma membrane (Albertson Wirtz-Peitz and Knoblich, 2006; Yamanaka and Ohno, et al., 2004; Zeitler et al., 2004). Furthermore, the 2008), which are involved in protein–protein inter- importance of the LRR domain of Scribble in localiza- actions. In contrast to Scribble and Dlg, which are tion is evolutionarily conserved, as highlighted by the constitutively localized to the plasma membrane, the fact that point mutations in the LRR domain of mouse localization of Lgl depends on its phosphorylation and the worm Caenorhabditis elegans Scribble result status; phosphorylated Lgl is unable to localize to the in abnormal protein localization (Legouis et al., 2003; cortex and is inactive (reviewed by Wirtz-Peitz and Zarbalis et al., 2004; Dow et al., 2007), and the Knoblich, 2006; Yamanaka and Ohno, 2008). In structure–function analysis of human Scribble indicates Drosophila and mammalian cells, phosphorylation of that both LRR and PDZ domains are required for Lgl is mediated by aPKC and depends on its binding to correct localization (Navarro et al., 2005; Nagasaka Par6 and aPKC (Betschinger et al., 2003; Plant et al., et al., 2006). 2003; Yamanaka et al., 2003, 2006) (see below). Dlg contains three PDZ domains and is a member of Despite the evidence from Drosophila mutants that the MAGUK family of proteins (Woods et al., 1996). Scribble, Dlg and Lgl act in a common genetic pathway MAGUK proteins contain both Src homology 3 (SH3) (Bilder et al., 2000; Wodarz, 2000), physical interactions and guanylate kinase-like (GUK) domains (reviewed by between members of this polarity module have proved Anderson, 1996). In Drosophila and mammalian cells, harder toidentify. In Drosophila synapses, the Dlg- Dlg colocalizes with Scribble at the basolateral cortex dependent localization of Scribble is mediated through (Bilder and Perrimon, 2000; Dow et al., 2003) (see the linker protein Guk-Holder, which binds to both the Figure 1). The structure–function analysis in Drosophila GUK domain of Dlg and the second PDZ domain of has shown that the second PDZ motif is required for the Scribble (Mathew et al., 2002); however, such an correct localization of Dlg (Hough et al., 1997). interaction has yet to be shown in mammalian cells. Lgl contains at least four WD40 motifs or b-propeller More recently, Kallay et al. (2006) reported that the domains (Mechler et al., 1985; Betschinger et al., 2005; LRR region of mammalian Scribble directly interacts

Oncogene Role of the Scribble polarity module in tumourigenesis PO Humbert et al 6890 with Lgl2; however, again no such interaction has yet when scrib mutant clones also expressed oncogenic been described in Drosophila or confirmed in any other versions of the Ras or Notch signalling proteins that systems. Regardless of the lack of evidence for clear massive tumour overgrowth occurred and, at least in the physical interactions between Scribble, Dlg and Lgl in case of oncogenic Ras, the tumour exhibited all the epithelial cells, the analysis of Drosophila scrib, dlg and hallmarks of cancer that can be modelled in Drosophila lgl mutants has shown that the plasma membrane (Brumby and Richardson, 2003; Pagliarini and Xu, localization of each protein depends on the function of 2003). The difference between the behaviour of the the others (Bilder and Perrimon, 2000). Whether this tumours when the whole tissue is mutant to that dependence is indirect due to the loss of apicobasal cell when clones of mutant tissue are generated within a polarity, or is more specifically linked to interactions wild-type context highlights an important additional between Scribble, Dlg and Lgl or the other polarity factor for tumour progression, that of the interplay modules remains to be determined. Furthermore, it is between the tumour and the surrounding normal tissue, yet tobe elucidated whether the same dependency the so-called ‘tumour microenvironment’ (Radisky occurs in mammalian cells. et al., 2001). Furthermore, using clonal analysis studies in different Drosophila tissues, it has been demonstrated that the properties of the tumour are dependent on the Evidence for the involvement of Scribble, Dlg and Lgl in tissue type and that there are differences between scrib, cancer: Drosophila dlg and lgl mutants in their effect on the hallmarks of cancer. Decades of research have revealed that the generation of an epithelial tumour requires the cooperation of mutations in various oncogenes or tumour suppressor genes (Hanahan and Weinberg, 2000). For the forma- Evidence for the involvement of Scribble, Dlg and Lgl tion of an invasive tumour from a normal epithelial cell, in cancer: humans mutations affecting at least six cellular processes are required—the so-called ‘hallmarks of cancer’, they are: Targeting of Scribble/Dlg by oncoviral proteins (1) continued cell proliferation, (2) evasion of apoptosis Scribble, Dlg and Lgl have been linked tothe develop- (cell death), (3) loss of differentiation, (4) invasion/ ment of mammalian tumours, most directly by the metastasis, (5) continued telomere elongation and (6) property of being targeted by oncoviral proteins sustained angiogenesis. Moreover, although not initially (reviewed by Thomas et al., 2008 and Javier, 2008). documented in relation to the hallmarks of cancer, for Human papillomavirus (HPV) infections are associated epithelial cells toexhibit invasive/metastatic properties, with over 90% of cervical cancers. E6 and E7 they must undergoan EMT tosever cell–cell and cell– oncoproteins are the primary mediators of this malig- extracellular matrix adhesions, thereby enabling them to nancy, arising from interactions with key regulators of metastasize from the primary tumour (reviewed by growth and proliferation, most notably the tumour Thiery, 2002). EMT is related to the loss of cell polarity, suppressors p53 and pRb (Mantovani and Banks, 2001; specifically the loss of adherens junction function. In Munger et al., 2001). E6 oncoproteins from high-risk Drosophila, the first four hallmarks of cancer can be virus types (HPV-16 and HPV-18), but not from low- modelled (reviewed by Brumby and Richardson, 2005). risk virus types, also interact with a set of PDZ domain- As described above, mutations in Drosophila scrib, dlg containing proteins, including Scribble, Dlg1 and Dlg4 and lgl result in neoplastic tumours in epithelial tissues, (reviewed by Thomas et al., 2008). This interaction is characterized by a loss in apicobasal cell polarity and mediated by a short stretch of four amino acids in the C proliferation control. Strikingly, when the whole tissue terminus of E6 (Kiyono et al., 1997; Lee et al., 1997). E6 is mutant for scrib, dlg or lgl (as in homozygous mutant mutants lacking this motif retain the ability to animals), the epithelial tumours that result exhibit all target p53 for degradation, but are unable to induce four hallmarks of cancer in Drosophila (Gateff, 1978; hyperplasia or cell transformation (Kiyono et al., 1997; Woodhouse et al., 1998; Bilder et al., 2000; Brumby and Nguyen et al., 2003a). Human Scribble was isolated in a Richardson, 2005; Grzeschik et al., 2007; Zhao et al., screen for proteins targeted for ubiquination by HPV E6 2008). However, when scrib, dlg or lgl mutant patches of proteins in an E6AP-dependent manner, and over- cells (or clones, generated by clonal analysis methodo- expression of Scribble has been shown to inhibit logy; see Brumby and Richardson, 2005) are generated transformation of rodent epithelial cells by HPV E6/7 within the context of wild-type tissue, then, depending proteins (Nakagawa and Huibregtse, 2000; Nguyen on the surrounding tissue, only some of the hallmarks of et al., 2003a; Thomas et al., 2005). Data suggest either cancer are seen. For example, in scrib mutant clones a nuclear signalling pool or a membrane-localized generated in the developing eye, ectopic cell prolifera- hyperphosphorylated pool of Dlg1 is the primary target tion occurs, but clonal overgrowth is curtailed by of disruption of cellular Dlg1 by E6 (Mantovani et al., differentiation and increased cell death from signals 2001; Massimi et al., 2006). Consistent with a role of from the surrounding wild-type tissue (Brumby and polarity proteins in suppressing tumourigenesis, altera- Richardson, 2003). These mutant tissues are poorly tions of protein levels and localization of Scribble, Dlg1 invasive when tested by transplantation into wild-type and Dlg4 have been demonstrated in tumours associated adult abdomens (Pagliarini and Xu, 2003). It was only with HPV infection.

Oncogene Role of the Scribble polarity module in tumourigenesis PO Humbert et al 6891 Other oncogenic viral proteins have evolved to bind lesions to invasive carcinoma (Fuja et al., 2004). Dlg1, to and disrupt the function of polarity proteins, under- Dlg3 and Dlg4 transcript levels reportedly decrease in scoring the importance of polarity proteins in restricting gastric cancer, with Dlg4 loss associating with more tumourigenesis. In addition to HPV oncoproteins, the invasive types of gastric carcinomas (Boussioutas et al., Tax protein of human T-cell leukaemia virus-1 binds to 2003). Dlg3 decrease has alsobeen observedin human both Scribble and Dlg1, inducing the mislocalization of oesophageal cancer cell lines and in papillary thyroid Scribble in infected cells and disrupting the growth carcinomas (Hanada et al., 2000; Wasenius et al., 2003). inhibitory effects of Dlg1 (Suzuki et al., 1999; Hirata In addition to differential expression, point mutations et al., 2004; Arpin-Andre and Mesnard, 2007). and loss of heterozygosity have been described in Dlg1 E4-ORF1 of adenovirus 9 can also interact with Dlg1 in human breast cancer cases (Fuja et al., 2004). (reviewed by Javier, 2008). Intriguingly, instead of The two human homologues of Lgl have also been abrogating the cellular function of Dlg1, E4-ORF1 implicated in cancer. Intriguingly, lgl1 knockout mice recruits Dlg1 to specifically promote the activation of exhibit overproliferation of neuroblasts, leading to a phosphatidylinositol 3-kinase (PI3K) in cells, revealing phenotype reminiscent of a subset of human primitive an oncogenic role for this putative tumour suppressor in neuroectodermal tumours (Klezovitch et al., 2004). specific cellular contexts (Frese et al., 2003). Consistent with this, genomic array analysis has identified the loss of Lgl1 as an unfavourable copy number aberration associated with reduced survival in Microarray/immunohistochemical studies of Scribble/ glioblastoma (Korshunov et al., 2006). Lgl1 deletion has Dlg/Lgl in human cancer been observed in a subset of colorectal carcinomas, with Of the four human Dlg homologues, there is most particular relevance to chromosomally unstable spora- evidence for Dlg1 being causally linked to cancer. dic cases (Lassmann et al., 2007). Moreover, the loss of Indeed, Dlg1 is the only PDZ-containing substrate Lgl1 transcript and/or protein have been associated with common to the oncogenic determinants of HPV, human other solid tumours, notably melanoma, prostate, breast adenovirus 9 and HTLV-1 (human T-cell lymphotropic and lung (Grifoni et al., 2004; Schimanski et al., 2005; virus; Lee et al., 1997). It is alsonotablethat Dlg1 binds Kuphal et al., 2006). Generally, Lgl1 loss is associated to two classic tumour suppressors, adenomatous poly- with higher tumour grade, and is more pronounced in posis coli (APC) (a negative regulator of the Wnt lymph node or distant metastases. As with other pathway) and PTEN (a negative regulator of the PI3K polarity proteins, localization of Lgl1 is important for pathway), and is also associated with two proto- its putative tumour suppressor function—in human oncogenes, PI3K and Net1 (see further below) (Matsu- ovarian cancers, an apical-to-cortical redistribution of mine et al., 1996; Valiente et al., 2005; Frese et al., 2006; the aPKCz isoform is concomitant with cytoplasmic Garcia-Mata et al., 2007). Multiple studies have release of Lgl1 during carcinogenesis (Grifoni et al., investigated the association of Dlg1 with HPV-induced 2007). Given that aPKC isoforms have previously been gynaecological malignancies (Watson et al., 2002; linked with tumourigenesis, their effect on polarity may Cavatorta et al., 2004; Lin et al., 2004). An emerging be important in disease states. Studies have implicated theme is that Dlg1 protein levels are maintained or Lgl2 as having a specific role in suppressing invasion at increased in early-stage well-differentiated lesions, the margins of tumours (Aigner et al., 2007; Spaderna although its distribution extends into the cytoplasm et al., 2008). Lgl2 (but not Lgl1) is a primary negative more so than in normal tissue. In later-stage and poorly target of ZEB1 (Snail family Zn-finger transcript factor), differentiated tumours, cytoplasmic Dlg1 protein levels a recognized master regulator of the EMT (reviewed by increase, but Dlg1 is markedly decreased at the Moreno-Bueno et al., 2008). Levels of ZEB1 and Lgl2 membrane and sites of cell–cell contact. Almost are inversely correlated in human colon and breast invariably, Dlg1 is largely undetectable at invasive tumours, with the bulk of the tumour mass expressing margins of tumours. Interestingly, cDNA microarray high levels of Lgl2 (Aigner et al., 2007). At the invasive analysis identified Dlg3 as being highly upregulated in front of these tumours where cells are undergoing an serous ovarian adenocarcinomas compared with benign EMT, ZEB1 expression is high, and thus Lgl2 expres- lesions, with this expression being maintained in sion is low, consistent with a requirement to down- advanced adenocarcinomas (Tapper et al., 2001). A regulate the Scribble polarity module to enable an EMT. similar trend of early-stage Dlg1 overexpression and The single human homologue of Scribble has been mislocalization has been described in colon carcino- linked to human cancers in only a limited number of genesis (Cavatorta et al., 2004; Gardiol et al., 2006). In studies. Twostudies have analysed Scribble expression both adenomatous polyps and well- and moderately and localization in colon carcinoma progression differentiated adenocarcinomas, Dlg1 protein levels (Gardiol et al., 2006; Kamei et al., 2007). Similar to increased, but become more diffuse in localization with Dlg1, Scribble overexpression and mislocalization are increased cytoplasmic staining, and more Dlg1 was observed, with very low levels of Scribble detected in observed apically as opposed to basolaterally. In invasive projections of tumours. In addition, a link advanced poorly differentiated lesions and invasive foci, between Scribble deregulation and the cytoplasmic Dlg1 was markedly decreased or even absent. This trend accumulation of b-catenin has been proposed, implicat- was also observed in a study of breast cancer when ing Scribble in the APC/b-catenin pathway. Interest- comparing early (Ductal Carcinoma In Situ, DCIS) ingly, Scribble has been shown to bind APC (Takizawa

Oncogene Role of the Scribble polarity module in tumourigenesis PO Humbert et al 6892 et al., 2006) and, in some contexts, b-catenin (Sun et al., Patj in the establishment of apicobasal polarity (Bilder submitted). Links have alsobeen described between et al., 2003; Rolls et al., 2003; Tanentzapf and Tepass, Scribble and E-cadherin; Scribble was found to coloca- 2003) (Figure 1). Seminal studies by Betschinger et al. lize with E-cadherin in the normal cervical epithelium, (2003) revealed the mechanism of mutual antagonism but in lobular breast cancer, in which E-cadherin is between Scribble/Dlg/Lgl and the Par complex; Lgl commonly downregulated, a loss of Scribble was binds toaPKC–Par6, and aPKC acts tophosphorylate observed at high frequency (Navarro et al., 2005). and inactivate Lgl, thereby releasing it from the Scribble is also lost, but at a lower frequency, in breast membrane cortex into the cytoplasm (Betschinger ductal carcinomas, which maintain E-cadherin expres- et al., 2003). As the Par complex is localized apically, sion. An important observation which needs to be aPKC-mediated phosphorylation acts to restrict Lgl’s resolved is that the Scribble locus at 8q24.3 is frequently function to the basolateral membrane. The correct amplified in breast and ovarian tumours (Naylor et al., localization of Lgl for apicobasal cell polarity is 2005; Kim et al., 2007). This amplicon is distinct highlighted by the properties of a mutant of Lgl, which spatially from the more frequently amplified c-Myc cannot be phosphorylated by aPKC and, therefore, is locus. Similarly to Dlg1 in colon cancer and consistent not restricted to the basolateral cortex (Lgl-3a); the with amplification in ovarian cancer, our preliminary expression of lgl-3a is unable torescue the polarity evidence suggests that Scribble is found in high levels in defects of lgl mutant cells (Betschinger et al., 2003; serous ovarian cancer, although it is mislocalized to the Hutterer et al., 2004). Likewise, the binding of Lgl to cytoplasm (Galea et al., unpublished observations). Par6–aPKC acts toinhibit aPKC function, because There is currently no information available regarding when Lgl is bound, Par3 (Bazooka) cannot bind to whether there are mutations in the Scribble gene in these Par6–aPKC (Wirtz-Peitz et al., 2008), as is alsoobserved or other cancers, and it will therefore be important to in mammalian cells (Yamanaka et al., 2003, 2006). determine whether the Scribble protein in these tumours Moreover, Wirtz-Peitz et al. (2008) have delineated the is still functional. mechanism of apicobasal polarity in the regulation of asymmetric cell divisions in the Drosophila sensory organ precursor (SOP) neuroblasts. In SOPs, Lgl cortical localization is restricted basally only in pro- How does the Scribble polarity module contribute to phase of mitosis, but by the stage of nuclear envelope tumour formation? breakdown is delocalized from the cortex throughout the cell. The mislocalization of Lgl to the cytoplasm Control of apicobasal cell polarity, cell adhesion and occurs through the action of the mitotic kinase, Aurora EMT A, which through the phosphorylation of Par6 leads to Our understanding of how Scribble, Dlg and Lgl the activation of aPKC, which in turn phosphorylates function in the establishment and maintenance of Lgl and releases it from the complex and the cortex. apicobasal cell polarity has largely come from studies Once Lgl is removed from aPKC–Par6, Par3 (Bazooka) in Drosophila. In epithelial cells, distinct cellular can bind toaPKC–Par6 and confersnovel substrate domains are generated, resulting in the positioning of specificity on aPKC, allowing it to phosphorylate the the adherens junctions (composed of E-cadherin, fate determinant Numb. Phosphorylation of Numb b-catenin and a-catenin), required for cell–cell interac- restricts it tothe basal cortex,in which it becomes tions and the formation of the zonula adherens in segregated intodaughter cells destined toundergo epithelial sheets (reviewed by Tepass et al., 2001) differentiation. How aPKC activity is regulated in (Figure 1). Furthermore, apicobasal polarity is required interphase cells and whether the same mechanism occurs for the formation of tight junctions in mammalian cells, in epithelial cells is yet tobe determined. Interestingly, or septate (basolateral) junctions in Drosophila cells, recent studies have revealed two additional regulators of which establish epithelial barrier function, and for the aPKC; p32 in mammalian cells, which alsobinds toLgl1 formation of basal domains, including the localization (Bialucha et al., 2007), and DAP160 (Intersectin) in of integrins, which play an important role in the Drosophila neuroblasts (Chabu and Doe, 2008), which interaction of cells with the extracellular matrix (re- may provide the missing link in our understanding of the viewed by Tepass et al., 2001). In scrib, dlg, and lgl control of aPKC/Par3/Par6 regulation in interphase cells. mutants, apical determinants become mislocalized to Despite the use of biochemical manipulations in basolateral regions and adherens junctions are disorga- cultured mammalian cells, our understanding of the nized (Bilder and Perrimon, 2000; Bilder et al., 2000). molecular mechanisms governing the regulation of The mislocalization of apical determinants basally mammalian epithelial apicobasal cell polarity remains causes an expansion of the apical domain, which is also limited. As in Drosophila, mammalian Lgl binds Par6 observed if apical components, such as Crumbs or and is a substrate for aPKC phosphorylation, which acts aPKC, are overexpressed (reviewed by Bilder, 2004). torestrict Lgl’s activity tothe basolateral region(Plant In Drosophila epithelial cells and neural stem cells et al., 2003; Yamanaka et al., 2003, 2006; Grifoni et al., (neuroblasts), the interplay between the Scribble, Par 2007). However, it is presently unclear how Dlg and and Crumbs polarity modules is critical to defining the Scribble localization and function are regulated in apical and basal domains of cells; Scribble/Dlg/Lgl act mammalian cells. Despite this, the high degree of antagonistically to aPKC/Par6/Par3 and Crumbs/Pals/ functional conservation seen in the regulation of

Oncogene Role of the Scribble polarity module in tumourigenesis PO Humbert et al 6893 adherens junction formation by the Scribble polarity Bilder et al., 2000; Bilder, 2004). Furthermore, in scrib module across different animal models supports the idea mutant clones in the developing eye, the key component that Scribble, Dlg and Lgl together have an evolutiona- of the adherens junctions, E-cadherin is downregulated rily conserved function in basal identity in epithelia. In (Pagliarini and Xu, 2003), an event that is associated zebrafish, the homologue of Lgl2, Penner, functions in with EMT, a prerequisite for the invasion and metas- the hemidesmosome formation and maintenance of the tasis of tumour cells (reviewed by Thiery, 2002). basal domain of epithelial cells (Sonawane et al., 2005). However, EMT is also associated with resistance to Similarly in mice, Lgl1, which is predominantly anoikis (cell death in response to the loss of adhesion to expressed in the brain, is required for normal apical– the extracellular matrix), thus whether loss of cell junctional complexes and proper trafficking of polarity and downregulation of E-cadherin observed in N-cadherin in neuroepithelial cells (Klezovitch et al., scrib/dlg/lgl mutants constitute a full EMT is not clear. 2004). In the worm C. elegans, mutants for either Dlg-1 Furthermore, downregulation of E-cadherin, which or Let-413 (the Scribble homologue) have defects in normally tethers the Wingless (Wnt) signalling ligand, adherens junction formation (Legouis et al., 2000; b-catenin, tothe membrane would be expected tolead to Bossinger et al., 2001). Dlg-1 is restricted tothe increased levels of cytoplasmic and nuclear b-catenin adherens junctions, where it physically interacts with that can drive cell proliferation (reviewed by Clevers, the adherens junction marker AJM-1 (Koppen et al., 2006). Moreover, in polarized epithelial cells, many 2001). The disruption to epithelial polarity seen in Let- proteins have been shown to have distinct plasma 413-deficient embryos, together with the specific effects membrane localizations, and there is accumulating seen in Dlg-1-deficient embryos in AJM-1 recruitment to evidence that protein localization to specific membrane the adherens junctions (McMahon et al., 2001), suggests domains is critical for appropriate cellular function that in contrast to Drosophila, Let-413 and Dlg-1 (reviewed by Bilder, 2004). Mislocalization of such apparently have distinct functions. In mammals, proteins may lead to inappropriate cell signalling, although knockdown of Dlg1 in the Caco-2 cell line thereby contributing to tumour formation and progres- leads to decreased accumulation of E-cadherin at sites of sion. For example, in lung epithelial cells, the epidermal cell–cell contact (Laprise et al., 2004), Dlg1 seems tobe growth factor receptors (ErbB2, 3 and 4) are normally dispensable for adherens junction formation in the few compartmentalized from the ligand, Heregulin-a,on epithelial tissues sofar examined in the mouse, including opposite sides of the tight junctions, such that the the urogenital tract (Iizuka-Kogo et al., 2007) and skin mitogenic signalling pathway will not be activated epithelium (Galea et al., in preparation). Redundancy (Vermeer et al., 2003). However, if wounding occurs, with other Dlg family members may be of relevance in and the epithelial layer is breached, the ligand is able to these situations. Further functional studies will be contact and stimulate the ErbB2–4 receptors, allowing needed toclarify the roleformammalian Scribble, Dlg cell proliferation and restoration of epithelial integrity. and Lgl in the regulation of cell polarity and cellular Thus, scrib/dlg/lgl mutants could contribute to the junctions in vivo. initiation and progression of cancer, by resulting in at As discussed above, while the Scribble polarity least a partial EMT through the loss of apicobasal cell module is important in the appropriate function of the polarity and the disruption of adherens junctions and adherens junctions, there is also evidence that adherens cell–cell adhesion, or possibly through changes in junction proteins play a role in the localization of signalling pathways. basolateral cues. Studies in Drosophila embryos have demonstrated that during the establishment of apicobasal cell polarity, the Par complex can be appropriately Control of self-renewal and differentiation localized in the absence of E-cadherin; however, As discussed earlier, in Drosophila, the epithelial basolateral determinants, such as Dlg, are mislocalized tumours and neuroblastomas (brain tumours) that (Harris and Peifer, 2004). Similarly, in mammalian cells, develop in homozygous scrib, dlg or lgl mutants are the basolateral localization of Scribble and Dlg1 is not only characterized by alterations in cell polarity but correlated with the establishment of adherens junctions also by impaired differentiation. Furthermore, in (Dow et al., 2003; Navarro et al., 2005). Thus, adherens neuroblastomas from dlg or lgl mutants, at least a junction components are important in the basolateral proportion of the tumour cells acquire an unlimited localization of Scribble and Dlg1, which in turn appear capacity for self-renewal (reviewed by Gonzalez, 2007). to be important for the localization and maturation of These brain tumours can be continually propagated the adherens junctions. through successive transplants in adult host flies, raising In summary, the evidence described above shows that the possibility that tumour growth is driven by the the Scribble polarity module is important for the deregulation of stem cell self-renewal pathways (Gateff establishment and maintenance of apicobasal cell and Schneiderman, 1974; Woodhouse et al., 1998; polarity of epithelial cells, but how is the loss of Caussinus and Gonzalez, 2005). Although it is not apicobasal cell polarity that occurs in scrib, dlg and lgl known if epithelial tumours develop from stem-like cells mutants linked tocancer? In Drosophila, the loss of (in fact, imaginal discs are not known to contain stem apicobasal polarity leads to the disruption of adherens cells, even though they are capable of unlimited self- junctions, and therefore of epithelial integrity and renewal), work in Drosophila has long implicated a stem cell–cell communication (Bilder and Perrimon, 2000; cell origin for neuroblastomas (reviewed by Januschke

Oncogene Role of the Scribble polarity module in tumourigenesis PO Humbert et al 6894 and Gonzalez, 2008). The Drosophila neural stem cell is differentiation (Klezovitch et al., 2004). The neural the neuroblast, which divides asymmetrically to self- hyperplasia of the Lgl1 knockout mice is associated with renew and generate a ganglion mother cell (GMC), increased Notch signalling, suggesting possible parallels which in turn subsequently divides togenerate differ- tothe Drosophila neuroblast tumours. entiated neuronal cells (reviewed by Gonzalez, 2007). In scrib, dlg or lgl mutants, unknown signalling pathways no longer limit neuroblast self-renewal, and excessive Control of cell proliferation numbers of neuroblasts as well as their differentiated In addition to their well-studied role in establishment progeny are generated (Caussinus and Gonzalez, 2005; and maintenance of apicobasal polarity, Drosophila lgl, Lee et al., 2006). Scribble, Dlg and Lgl are all required in dlg and scrib also play a role in the negative regulation neuroblasts to promote the asymmetric segregation of of cell proliferation (Bilder et al., 2000). Furthermore, GMC fate determinants, such as Prospero (Pros) and alleles of scrib, dlg and lgl were identified as dominant Brain-tumour (Brat), into the GMC upon neuroblast suppressors of a weak mutation in the key G1–S phase division (Albertson and Doe, 2003; Betschinger et al., cell cycle gene cyclin E(cycE) . This suppression was 2003, 2006). Indeed, loss of cell fate determinants is shown to occur by increasing the level of CycE, and sufficient tolead totumourigenesis (Caussinus and thereby the number of S-phase cells, providing a more Gonzalez, 2005; Betschinger et al., 2006). How defects in direct link between these tumour suppressors and the GMC cell fate specification result in unrestrained self- cell cycle machinery (Brumby et al., 2004). Indeed, in renewal in scrib, dlg or lgl mutant tumours has been best the developing eye, lgl and scrib mutant clones also examined with respect to the function of Lgl; in the show ectopic expression of CycE, S phases and mitoses absence of Lgl, neuroblasts not only fail to asymme- (Brumby and Richardson, 2003; Grzeschik et al., 2007). trically segregate the GMC fate determinant, Numb, but Furthermore, the E2F1 cell cycle transcription factor is also do not completely exclude the apically associated upregulated in lgl mutant clones in the developing eye neuroblast determinant, aPKC, from the daughter cell. (Grzeschik et al., in preparation). Moreover, in contrast It has been proposed that after cell division, this dual to scrib mutant clones that lose polarity and are largely defect compounds the loss of Numb from the daughter eliminated by Jun N-terminal kinase (JNK)-mediated cell because aPKC activity, nolongerrestrained by Lgl, apoptosis in the developing eye (Brumby and Richard- targets residual Numb for inactivation by phosphoryla- son, 2003), the lgl mutant tissue maintains apicobasal tion and release from the cell cortex (Wirtz-Peitz et al., polarity until later in development (Grzeschik et al., 2008). As Numb is a negative regulator of Notch 2007), illustrating that ectopic proliferation observed in signalling, and the activation of Notch signalling can in lgl mutant cells is not a consequence of loss of itself lead to tumour formation (Wang et al., 2006), apicobasal cell polarity. Thus, when Lgl levels decrease, ectopic Notch signalling may drive inappropriate cell proliferation control is lost first, and later in neuroblast-like self-renewal pathways in the neuroblast development apicobasal cell polarity defects occur. daughter cells in lgl mutants. This might be sufficient to The link between Scribble, Dlg and Lgl in control of make neuroblast-like cells refractory to signals that CycE expression is supported by a study of genes normally restrain self-renewal, but must still leave some required for basolateral junction signalling in the of the progeny with at least limited differentiation follicular epithelial cells of the Drosophila ovary (Zhao capacity. If other restraints on aPKC activity are et al., 2008). This study showed that loss of lgl, scrib removed in lgl mutant neuroblasts, by removing pins or dlg resulted in ectopic expression of CycE, and (partner of inscuteable, required for aPKC apical precocious S phases and mitoses. Thus, in Drosophila localization) function so that aPKC is no longer apically epithelial cells, Scrib, Dlg and Lgl appear toplay a rate- anchored in the neuroblast, all daughter cells remain as limiting role in regulating cell cycle progression, as well neuroblasts (Lee et al., 2006). Intriguingly, recent studies as in apicobasal cell polarity. have indicated that not all neuroblasts within the brain A role for Scribble, Dlg and Lgl in mammalian cell are equally susceptible to transformation, and lgl proliferation is less well characterized. Initial cell line tumours primarily derive from a unique neuroblast studies showed that overexpression of mammalian Dlg lineage in the brain that undergoes a transitory (Dlg1 or Dlg3) leads to the inhibition of S-phase entry amplification stage prior to the generation of GMCs and proliferation (Hanada et al., 2000; Ishidate et al., (Boone and Doe, 2008; Bowman et al., 2008). This 2000). More recent in vivo studies have indicated a suggests that expansion of a stem cell pool is a weak tissue-specific requirement for Dlg1 in proliferation. point, particularly sensitive to perturbations that pro- Loss of Dlg1 in T lymphocytes results in accelerated mote ectopic self-renewal and tumourigenesis. A similar entry intoS phase (Stephenson et al., 2007), and susceptibility may underlie mammalian carcinogenesis, epithelial cells of the lens of Dlg1gt/gt mutant mice and, although there is, to date, little evidence for show ectopic S-phase entry and increased proliferation mammalian Scribble/Dlg/Lgl proteins playing a role in (Nguyen et al., 2003b). Despite these defects, no the regulation of self-renewal, Dlg1 mutant mice exhibit widespread hyperproliferative syndromes in Dlg1-null a failure in lens cell differentiation, which is associated mice have been reported (Mahoney et al., 2006; with increased proliferation (Nguyen et al., 2003b), and Stephenson et al., 2007). Indeed, in some Dlg1-null the mouse Lgl1 knockout exhibits neural hyperplasia epithelial tissues decreased proliferation is actually due to the neural progenitor cells failing to undergo observed (Iizuka-Kogo et al., 2007). Interestingly,

Oncogene Role of the Scribble polarity module in tumourigenesis PO Humbert et al 6895 Dlg1 can be phosphorylated in a cell cycle-dependent component of the apoptotic machinery, the Drosophila manner by the cell cycle protein kinases, CDK1 and inhibitor of apoptosis-1 (DIAP1) (Grzeschik et al., 2007; CDK2 (L Banks, personal communication). Similarly to Zhao et al., 2008; Grzeschik et al., in preparation). Zhao Dlg, overexpression of mammalian Scribble can inhibit and colleagues showed that DIAP1 protein levels are cell proliferation in a variety of cell lines by inhibiting elevated in lgl, dlg and scrib mutant ovarian follicle cell cell-cycle entry from G1 to S phase, whereas the loss of clones, which presumably allows their survival. In the Scribble by RNAi in Caco-2 cells leads to a correspond- developing eye, Lgl is also necessary for normal ing accumulation of cells in S phase (Nagasaka et al., developmental cell death (Grzeschik et al., 2007). In lgl 2006). In vivo studies are now required to validate the mutant clones in the eye disc, upregulation of DIAP1 physiological significance of these findings. With both transcription and protein leads to a suppression of Dlg1 and Scribble, the reported physical association of developmental cell death within the lgl mutant tissue these proteins with APC has been proposed to be (Grzeschik et al., in preparation). The effect of Lgl on important for mediating these cell cycle effects (Ishidate cell death in the developing eye also appears to be dose et al., 2000; Nagasaka et al., 2006). dependent, as halving the dose of lgl is able tosuppress The few studies examining mammalian Lgl in cell the ablated eye phenotypes due to ectopic expression proliferation control have implicated a role for Lgl in of Rpr, Hid or Grim in the eye (Grzeschik et al., in the regulation of chromosome segregation; overexpres- preparation). Thus, in different tissues, mutations in lgl, sion or knockdown of Lgl (Lgl1 and Lgl2) in HEK293 scrib or dlg not only lead to a failure in cell cycle exit but cells induced disorganization of the mitotic spindle, alsoact toprotectthe proliferatingtissue against resulting in a multinucleate phenotype (Yasumi et al., apoptosis, by either downregulating expression of the 2005). Interestingly, Lgl2 was foundtobind toLGN, cell death inducers, rpr, hid or grim in salivary glands or the mammalian homologue of Drosophila pins, a key upregulating DIAP1 in follicle cells and the developing regulator of asymmetric division, and loss of LGN eye. Whether a common mechanism occurs in all tissues phenocopied the loss of Lgl in these experiments in lgl, scrib or dlg mutants remains tobe determined; (Yasumi et al., 2005). In addition, as described above, that is, in the salivary glands is DIAP1 alsoupregulated, Lgl1-knockout mice display defective asymmetric divi- and in follicle or the developing eye cells are rpr, hid and sions with impaired cell cycle exit and differentiation. grim downregulated in scrib/dlg/lgl mutants? Despite these defects in cell cycle exit, Lgl1-knockout Data is currently limited on the effect of loss of mice did not show an overall increase in neural Scribble, Dlg or Lgl on apoptosis in mammalian progenitor cell number, as many of these cells die by systems. Together with cell cycle withdrawal defects, apoptosis (Klezovitch et al., 2004). Any proliferative lgl1-null neural progenitor cells show a concurrent advantage conferred by loss of function of Scribble/Dlg/ increase in apoptosis in vivo (Klezovitch et al., 2004). Lgl during tumour development could therefore be In addition, recent work has revealed a role for masked by apoptosis, as discussed below. mammalian Scribble in the control of apoptosis during three-dimensional acini formation in MCF10A normal breast epithelial cells (S Muthuswamy, personal com- Control of cell survival munication). Indeed, loss of Scribble by RNAi can In Drosophila, ectopic expression of cell cycle genes, override Myc-induced apoptosis in MCF10A cells and such as cycE, has been shown not only to promote cooperate with Myc to give large tumours in mammary ectopic proliferation but also leads to increased cell fat pad transplant experiments (S Muthuswamy, perso- death, perhaps due to tissue-intrinsic compensatory nal communication). The study of spontaneous cancer mechanisms that prevent overgrowth (reviewed by models in genetically modified mice mutant for Scribble, Brumby and Richardson, 2005). In order for clonal Dlg and Lgl, should help establish what contribution the overgrowth to occur upon upregulation of key cell cycle anti-apoptotic and other functions of these genes have regulators, cells must escape the constraints of apopto- on tumour progression. sis. There is growing evidence that lgl, scrib and dlg,in certain contexts at least, are able to negatively regulate apoptosis, thereby allowing clonal survival. Control of invasion and metastasis In Drosophila salivary gland development, Lgl plays a Cell migration is a fundamental process required during role in the implementation of apoptosis/hydrolysis development. The mechanisms regulating migration are (Farkas and Mechler, 2000). In a dose-dependent disrupted in pathological situations, including tumour manner, overexpression of lgl promoted expression of cell invasion/metastasis (reviewed by Yang and Wein- the ecdysone-triggered cell death genes, reaper (rpr), berg, 2008). A significant body of evidence implicates head involution defective (hid) and grim, accelerating the Scribble/Dlg/Lgl as regulators of migration in different hydrolysis of the salivary glands. Conversely, loss of lgl cells, organisms and processes; an emerging paradigm is led to delay in the expression of these cell death genes that Scribble, Dlg and Lgl can either promote or restrict in the salivary glands. Thus, through an unidentified cell migration dependent upon cell context (reviewed mechanism, Lgl regulates the transcription of the key by Humbert et al., 2006; Dow and Humbert, 2007) cell death genes, rpr, hid and grim. In other Drosophila (see Etienne-Manneville, 2008). tissues, however, scrib/dlg/lgl mutants lead tothe A role for the Scribble polarity module in regulating suppression of cell death through effects on another migration in Drosophila has been largely defined in three

Oncogene Role of the Scribble polarity module in tumourigenesis PO Humbert et al 6896 major model systems; embryonic dorsal closure, post- Richardson, 2005; Gonzalez, 2007). Dlg and Lgl have mitotic ovarian follicular epithelia and in clonal patches been described as fundamental suppressors of epithelial of eye imaginal discs (reviewed by Brumby and invasion in the developing Drosophila ovary follicular Richardson, 2005; Jang et al., 2007). Using zygotic cells (Zhao et al., 2008). Indeed, loss of Dlg and Lgl mutations, Bilder et al. (2000) have shown that loss of causes follicle cells to overproliferate, change polarity either Scribble or Dlg alone is not sufficient to disrupt (partial EMT), delaminate from the epithelium and migration associated with embryonic dorsal closure; invade between germ cells (Goode et al., 2005; however, halving the dosage of dlg in a scrib mutant Szafranski and Goode, 2007; Zhao et al., 2008), background results in a complete failure of dorsal properties similar to human tumour cells. In this closure. This implies a genetic interaction between process, genetic interactions between a weak lgl mutant Scribble and Dlg in positively regulating epithelial sheet combination and dlg or scrib mutants have been migration. Furthermore, in the migration of ovarian described (Zhao et al., 2008). Furthermore, transplanta- follicular border cells (which delaminate from the tion studies of lgl or dlg mutant imaginal disc epithelial follicular epithelium and migrate through the germ-line or brain tumours into wild-type adult hosts have shown nurse cells in the oocyte), scrib mutants act toreduce how tumour cells can continue to proliferate and migration (Zhao et al., 2008). However, in this context, metastasize, degrading basement membranes and pene- dlg and lgl mutants have the opposite effect and border trating muscle layers to infiltrate the host ovary cell migration is increased when dlg and lgl are depleted. (Woodhouse et al., 1994, 1998; Caussinus and Gonzalez, Thus in this situation, differences in the roles between 2005; Beaucher et al., 2006, 2007). The effects of scrib Scribble and Dlg and Lgl can be discerned, with Lgl and mutant tissues in invasion/metastasis have not been as Dlg normally acting to repress migration and Scribble well characterized as dlg or lgl mutant tissues. From acting to promote it. studies of scrib mutants in ovarian follicular epithelial There is a growing body of evidence that Scribble/ cells it appears that although they show loss of polarity, Dlg/Lgl also function as migration regulators in they show less invasive behaviour than dlg or lgl mammalian systems. Consistent with dorsal closure mutants (Zhao et al., 2008). Thus, again there are defects in Drosophila, the circletail and rumpelstiltzchen differences in the properties of scrib mutants as (rumz or Line90) mouse strains, which are independent compared with dlg or lgl mutants. Scribble mutant alleles, display major defects in As discussed above scrib, dlg or lgl mutant cells show embryonic fusion events related to epithelial sheet different migratory properties in different contexts (for migration (Murdoch et al., 2001; Zarbalis et al., 2004). example, sheet migration during dorsal closure versus These defects include failure of neural tube closure border cell migration). How can these different migra- (craniorachischisis), abdominal wall defects (gastroschi- tory behaviours be reconciled? The pathways promoting sis) and an eyes-open-at-birth phenotype. It was further invasion in homozygous mutants are not known; shown that rumz mice exhibit an impaired embryonic however, considerable work has investigated the path- wound response associated with total failure of sheet ways controlling tumour invasion in a Drosophila cancer migration of keratinocytes (Dow et al., 2007). In model that relies upon the expression of oncogenic addition, Dlg1 mutant mice exhibit a cleft palate (activated) Ras (RasACT) in clonal patches of scrib (Caruana and Bernstein, 2001) and an eyes-open-at- mutant tissue in the eye epithelium (Brumby and birth phenotype (Galea et al., in preparation), and we Richardson, 2003; Pagliarini and Xu, 2003). Normally, have now shown that similar to Scribble, Dlg1 is also scrib mutant clones of tissue are eliminated by JNK- required for embryonic wound response in vivo (Dow dependent cell death; however, the expression of RasACT et al., 2007; Galea et al., in preparation). Interestingly, in the mutant tissue inhibits apoptosis and cooperates similarly to Drosophila, mice heterozygote for both with the loss of scrib to produce overgrown and highly Scribble and Dlg1 mutations showed defective embryo- invasive tumours (Brumby and Richardson, 2003; nic wound closure, indicating genetic interaction in this Uhlirova et al., 2005; Igaki et al., 2006). In some cases, system (Galea et al., in preparation). Members of the tumour cells become invasive, moving locally into the Scribble polarity module have been shown to regulate ventral nerve cord and then forming secondary tumours migration in vitro in human mammary cell lines, at distant sites (Pagliarini and Xu, 2003). Moreover, MDCK, T cells, astrocytes and fibroblasts (Plant when transplanted intowild-type adult abdomens, the et al., 2003; Ludford-Menting et al., 2005; Qin et al., scrib mutant RasACT eye epithelial tumours are highly 2005; Osmani et al., 2006; Dow et al., 2007). Notably, aggressive and invasive (Pagliarini and Xu, 2003). Scribble has been shown to be required for b-Pix, Rac1 The mechanism by which oncogenic Ras cooperates and Cdc42 recruitment tothe leading edge and also with scrib mutants requires the cell survival function of for reorientation associated with directed migration the Ras signalling pathway; activated Ras abrogates (reviewed by Humbert et al., 2006; Dow and Humbert, the effects on the cell death machinery by the 2007; Etienne-Manneville, 2008). JNK signalling pathway subverting it from a pro- In contrast to the requirement for the Scribble apoptotic role to pro-migratory role through JNK- polarity module in directed cell migration in Drosophila dependent expression of genes, such as the matrix and mammals, studies in Drosophila suggest that dlg or metalloproteinase, Mmp1 (Uhlirova and Bohmann, lgl mutant tumours are characterized by inappropriate 2006). Interestingly, the activation of JNK signalling migration and invasion (reviewed by Brumby and in Drosophila scrib mutant clones is more likely to be a

Oncogene Role of the Scribble polarity module in tumourigenesis PO Humbert et al 6897 tissue-intrinsic response to remove aberrant cells, rather observed in scrib mutant mosaic eye discs (Leong et al., than reflecting a direct role for Scribble in JNK in preparation). This may be due to the phenomenon of repression (Leong et al., in preparation) (see below). morphogenetic apoptosis, where alterations in signalling Furthermore, although dlg and lgl mutant clones are pathways lead to the upregulation of JNK and to JNK- alsosusceptible toRas ACT-mediated transformation, mediated cell death (Adachi-Yamada and O’Connor, JNK signalling is sufficient with RasACT todrive tumour 2004). Intriguingly, however, the normal pattern of growth and invasion (Uhlirova and Bohmann, 2006), developmental cell death is also blocked non-autono- and many cell polarity mutants, including cdc42, sdt and mously throughout the wild-type tissue in lgl mutant baz, alsomediate Ras ACT-induced tumourigenesis in a mosaic eye discs (Grzeschik et al., 2007). How this JNK-dependent manner (Igaki et al., 2006). Whether non-autonomous block in apoptosis is achieved is loss of the Scribble polarity module plays any other role unknown—signalling pathways affected by the loss of in Ras-induced tumourigenesis, independent of JNK, lgl are one possibility, compensatory effects, caused by and whether JNK signalling plays an essential role in the the elimination of cells along the clonal borders another. invasive capabilities of the tumours in homozygous In another tissue, the Drosophila ovarian follicle cells, scrib, dlg or lgl mutants, is presently not known. In lgl mutant clones survive, but wild-type cells adjacent to summary, although some forms of cell migration may be these cells undergo apoptosis (Froldi et al., in prepara- compromised by the loss of the Scribble polarity tion). These lgl mutant follicle cells not only show module, the mutant cells may be induced to migrate if upregulation of DIAP1 (Zhao et al., 2008) but alsoshow subjected toan environment where cell intrinsic and higher levels of expression of the Drosophila homologue extrinsic signals can synergize todrive invasion. This of Myc, a key molecule in cell competition, a process may be similar to the response of tumour cells to where less ‘fit’ cells are eliminated by apoptosis (Moreno cytokine expression in mammals, and in fact the and Basler, 2004; de la Cova et al., 2004; Gallant, 2005). Drosophila TNFR (tumour necrosis factor receptor), Thus, the juxtaposition of high dMyc expressing cells Wengen, has alsobeen implicated in mediating JNK next tolowdMyc expressing cells leads tothe death of activation in the fly tumours (Igaki et al., 2006). the low dMyc expressing cells. How dMyc is upregu- Consistent with Drosophila studies, Scribble functions lated in the lgl mutant follicle cells is unknown. to restrict Ras-mediated invasion of MCF10A normal In the developing wing, lgl mutant clones are largely breast epithelial cells (Dow et al., 2008). These studies eliminated by cell death, which is associated with lower have further indicated that Scribble regulates phosphor- levels of dMyc and DIAP1 expression in the lgl mutant ylation of Erk and suppresses invasion through a Raf– cells, but higher levels of dMyc are observed in the wild- MEK–ERK pathway. It is interesting tonotethat the type tissue immediately flanking the lgl mutant clones switch between the positive regulation of migration (Froldi et al., in preparation). In this case, it is likely that (sheet migration) and negative regulation (invasion) dMyc is upregulated in the wild-type cells surrounding occurs by the same polarity module in the same cell the dying lgl mutant tissue in order to compensate for types. Dissecting the signalling role of the Scribble the missing cells by stimulating cell growth and polarity module should provide insight into the func- proliferation. Indeed, upregulation of dMyc is also tional importance of Scribble, Dlg and Lgl in human implicated in the phenomenon of compensatory pro- tumourigenesis. liferation, a mechanism by which the loss of cells by apoptosis is compensated for by increased proliferation of surrounding cells (Gallant, 2005). Compensatory Control of the tumour microenvironment proliferation has also been shown to require the Mutations in some tumour suppressor genes not only secretion of the morphogens, Wingless, Dpp or Hedge- leads tochanges within the mutant tissue itself hog by the dying cells (Huh et al., 2004; Perez-Garijo (proliferation, suppression of death, differentiation et al., 2004; Ryoo et al., 2004; Fan and Bergmann, 2008). defects and/or cell polarity changes affecting migration), Indeed, in a study by Arquier et al. (2001), Lgl has been but in many instances they can, by affecting signalling implicated in the control of the Dpp signalling pathway. pathways, also have non-autonomous effects on the In the developing wing, lgl was found to be required surrounding wild-type tissue (reviewed by Brumby and only in dpp-expressing cells downstream of dpp expres- Richardson, 2005; Vidal and Cagan, 2006). Moreover, sion and upstream of its receptor Thickveins (Tkv) for the surrounding normal tissue (the tumour microenvir- the expression of dpp signalling pathway targets. This is onment) can affect the development of the tumour most likely due to an effect on the secretion of Dpp in (Radisky et al., 2001). the Dpp-producing lgl mutant cells, which leads tonon- In the analysis of Drosophila lgl mutant mosaics, cell autonomous effects on the surrounding wild-type effects on the surrounding wild-type tissue have been cells. As Dpp is a morphogen required for wing tissue observed in several situations. In lgl mosaic developing growth and survival, it is unlikely that this effect of lgl eyes at the larval stage, despite the blockage of cell death mutants on reducing Dpp secretion would contribute to in lgl mutant clones, cells at the border of the mutant tumour progression; however, similar effects on the clone show upregulation of the JNK pathway and production of negative growth factors in lgl mutant undergo apoptosis (Grzeschik et al., 2007; Grzeschik tissue could contribute to the non-cell autonomous et al., in preparation). Upregulation of the JNK effects on cell survival observed in the Drosophila pathway, particularly at the clonal borders, is also developing eye (Grzeschik et al., 2007).

Oncogene Role of the Scribble polarity module in tumourigenesis PO Humbert et al 6898 The few examples of non-cell autonomous loss-of- correlates with the upregulation of CycE and poor function phenotypes for Scribble/Dlg/Lgl in vertebrates prognosis (Eder et al., 2005). Furthermore, aPKCl/i is indicate that effects on the microenvironment will also upregulated in invasive ductal carcinoma of the breast need tobe consideredin these analyses. Forexample, (Kojima et al., 2008). As tothe Crumbs complex in the landlocked (Scribble) mutation in zebrafish leads to mammalian cells, Crumbs3 is important in tight junc- non-cell autonomous migration defects in facial motor tion establishment, and overexpression leads to a delay neurons (Wada et al., 2005), and ectopic expression of in tight junction assembly and loss of cell polarity in Lgl/aPKC can act in a cell non-autonomous manner to MDCK epithelial cells (Roh et al., 2003). Conversely, regulate convergent extension in the development of the functional downregulation of Crumbs3 can lead to the frog, Xenopus (Ninomiya and Winklbauer, 2008). loss of tight junctions, disruption in cell adhesion and contact inhibition, and is associated with increased migration and metastasis in a mouse kidney cell line model (Karp et al., 2008). Consistent with this, the Snail General mechanism of action transcription factor, a key driver of the EMT, acts to repress Crumbs3 expression (Whiteman et al., 2008). How Scribble, Dlg and Lgl act as tumour suppressors in These findings show that misregulation of components Drosophila and mammals may pertain totheir regula- of the Crumbs and aPKC complexes contribute to tion of (1) other polarity complexes, (2) Myosin II cancer progression and could therefore contribute to activity and the actin cytoskeleton, (3) signalling path- tumourigenesis when Scribble, Dlg or Lgl are down- ways or (4) vesicle trafficking. Evidence for the role of regulated (see alsoAranda et al., 2008). These studies, Scribble/Dlg/Lgl in these processes in epithelial cells taken together with studies in Drosophila implicating will now be discussed (see also Hawkins and Russell, polarity complexes in tumourigenesis (Pagliarini and 2008 for a review of the Scribble polarity module in Xu, 2003), suggest that loss of cell polarity, rather than T-cell leukaemia). the hierarchical relationship between the Scribble, Par and Crumbs polarity modules, is the most important Scribble/Dlg/Lgl and other polarity complexes factor in tumourigenesis. The relationship between the Scribble polarity module and the Crumbs and Par complexes is critical for the establishment and maintenance of apicobasal cell Effects on Myosin II activity and the actin cytoskeleton polarity. Moreover, the antagonistic relationship be- Part of the ability of mutants in the Scribble polarity tween the Scribble polarity module and the Crumbs and module to result in changes in cell shape and cell Par complexes, highlighted by increased Crumbs or Par behaviour may relate to effects on the actin cytoskele- complex activity observed in loss-of-function mutations ton. Indeed, Lgl has been shown to bind to the non- in scrib/dlg/lgl in Drosophila and mammalian cells muscle myosin, Myosin II, both in Drosophila and (Bilder et al., 2003; Rolls et al., 2003; Tanentzapf and human cells (Strand et al., 1994, 1995; Betschinger et al., Tepass, 2003; Yamanaka et al., 2003, 2006), may be key 2005). Furthermore, Lgl and Myosin II (Zipper) to tumourigenesis. Indeed, overexpression of Crumbs or genetically interact; in lgl mutant neuroblasts, the active forms of aPKC phenocopy the scrib/dlg/lgl mislocalization of basal determinants (Miranda) can mutant phenotype (Bilder et al., 2003; Lu and Bilder, be rescued by reducing the dose of zipper (Ohshiro et al., 2005). However, Crumbs upregulation does not appear 2000; Peng et al., 2000). Thus, Lgl and Myosin II act to be a prerequisite for the apical domain expansion antagonistically in the basal targeting of cell fate seen in mutants of the Scribble polarity module (Bilder determinants. However, these studies also revealed that et al., 2003), suggesting that the activity of the Par there was another unidentified Myosin that works complex may be more relevant. Indeed, upregulation of synergistically with Lgl in the basal localization of cell aPKC activity is necessary for the Crumbs overexpres- fate determinants in larval neuroblasts. It was proposed sion phenotype (Sotillos et al., 2004). Recent studies on that Lgl acts to restrict Myosin II to the apical cortex of the molecular action of Lgl (Wirtz-Peitz et al., 2008) neuroblasts during prometaphase and metaphase of suggest that it can act upstream as well as downstream mitosis, where it acts to exclude cell fate determinants of aPKC, which is supported by genetic evidence (Barros et al., 2003), but given that Lgl has been shown showing that the polarity and proliferation defects of to be excluded from the cortex at prometaphase in lgl mutant larvae can be rescued by a loss-of-function neuroblast cells of the sensory organ precursors (Wirtz- mutant in aPKC (Rolls et al., 2003), whereas the Peitz et al., 2008), it is unclear how Lgl can direct the overgrowth phenotype due to expression of an activated localization of Myosin II to the apical region. Interest- allele of aPKC can be rescued by expression of a non- ingly, in mammalian cells, aPKC activity seems tobe phosphorylatable lgl mutant (Lee et al., 2006). This required to phosphorylate and regulate Myosin IIB inverse relationship between Lgl and aPKC is supported filament formation and subcellular localization (Even- by studies showing that in some human cancers, a Faitelson and Ravid, 2006; Rosenberg and Ravid, reduction in cortical Lgl is associated with higher 2006). However, in Drosophila, the aPKC phosphoryla- cortical aPKC (Grifoni et al., 2007). Indeed, upregulation sites on Myosin II are not essential for viability tion of aPKC is observed in non-small-cell lung cancers (Su and Kiehart, 2001), although it is possible that (Regala et al., 2005) and in ovarian cancers, where it there may be more subtle roles for this regulation. The

Oncogene Role of the Scribble polarity module in tumourigenesis PO Humbert et al 6899 importance of Lgl in Myosin II regulation and thereby interaction with the PCP mouse mutant, Vangl2/looptail F-actin filament contractility in cell shape changes and (Montcouquiol et al., 2003, 2006; Murdoch et al., 2003; cellular function is therefore still an unresolved issue. Zarbalis et al., 2004; Klein and Mlodzik, 2005). In zebrafish, a similar genetic interaction has been described between the scribble and vangl2 genes (Wada General function as signalling adaptors et al., 2005) and between scribble and the zyxin Scribble, Dlg and Lgl have been implicated in the homologue LPP (Vervenne et al., 2008) in the control regulation of several signalling pathways in various of the PCP-mediated convergence extension movements systems (see alsoHawkins and Russell, 2008), the best during gastrulation. Together, these data implicate studied being the Wnt, G-protein-coupled receptor Scribble, Dlg and Lgl in the regulation of both canonical (GPCR), Notch, Ras and Salvador–Warts–Hippo path- and non-canonical Wnt signalling during development ways, which have well-established roles in cancer, as in a variety of tissues and organisms. How this discussed below. functional interaction relates to the role of these polarity proteins in tumour development and invasion at this Wnt pathway. Misregulation of the Wingless/Wnt stage remains unclear, although given these strong links signalling pathway is a major factor in colorectal it is likely tobe important. cancers as well as other human tumours (reviewed by Clevers, 2006). Multiple lines of evidence strongly link GPCR signalling. In addition to binding to the Frizzled Scribble/Dlg/Lgl toWnt signalling. Dlg was first receptors, mammalian Dlgs can control the clustering implicated in Wnt (Wingless) signalling from the finding and trafficking (and hence the function) of a number of that Dlg1 could bind to a negative regulator of Wnt, the other GPCRs, including the serotonin 2A receptor, adenomatous polyposis coli, APC tumour suppressor mGlu receptors and the tumour vasculature marker (Matsumine et al., 1996). This APC–Dlg interaction has TEM5 (Xia et al., 2000; Yamamoto et al., 2004; Funke since been shown to be functionally important for the et al., 2005). Similarly, mammalian Scribble can regulate effects of Dlg1 on the cell cycle (Ishidate et al., 2000) and thyroid-stimulating hormone receptor signalling through polarized cell migration (Etienne-Manneville et al., receptor endocytosis and recycling (Lahuna et al., 2005). 2005). Interestingly, Dlg family members can alsobind This is achieved through direct binding of Scribble to this tothe Wnt receptors,Frizzled-1, -2, -4 and -7 (Hering GPCR and requires signalling from Scribble through and Sheng, 2002). Dlg1 and Frizzled-2 form a ternary b-Pix/GIT1/ARF6. Downstream from GPCRs, mam- complex with APC, suggesting that Dlg proteins can act malian Lgl2 and Dlg family members, have alsobeen torecruit intracellular Wnt signalling pathway proteins shown to interact with the regulator of GPCR signalling, tothe receptor. mPins (Sans et al., 2005; Yasumi et al., 2005 and see In Xenopus and Drosophila, Lgl can act downstream alsoJanuschke and Gonzalez, 2008). The genetic of the Frizzled receptor through the binding of Lgl to interaction of mPins and Scribble/Dlg/Lgl is conserved Dishevelled (Dvl), a key regulator of canonical and non- in Drosophila, with pins itself being a potent tumour canonical planar cell polarity (PCP) Wnt signalling suppressor in Drosophila brain neuroblasts (Lee et al., pathways (Dollar et al., 2005). In this study, Dvl was 2006). Despite the obvious cancer connection of the required for Lgl localization at the cortex in Drosophila Scribble polarity module with Pins, how the interaction and Xenopus cells, and therefore for Lgl function in of Scribble/Dlg/Lgl with GPCRs is associated with apicobasal cell polarity. Furthermore, this study showed tumourigenesis is currently unknown. that the expression of Frizzled-8 receptor in Xenopus resulted in the dissociation of Lgl from the cortex. Wnt signalling can therefore impact on the location and Notch pathway. Upregulation of the Notch signalling consequently the function of Lgl; however, it is not pathway is a causative agent in T-cell leukemia and known whether the converse occurs; that is whether loss perhaps other cancers (Clark et al., 2007; Farnie and of Lgl from the cortex leads to effects on Dvl Clarke, 2007; Roy et al., 2007) (Hawkins and Russell, localization and on Wnt signalling. Pertinent to this, 2008). In Drosophila neuroblasts of the SOPs, Lgl is however, are the recent findings that Dvl acts to regulate important in the asymmetric distribution of Numb aPKC activity in cultured neural cells (Zhang et al., (Betschinger et al., 2003; Wirtz-Peitz et al., 2008), an 2007) and that aPKC signalling is required toregulate inhibitor of Notch signalling. Indeed, SOP cells have Wnt-mediated anterior–posterior axon guidance in ectopic Notch signalling (Langevin et al., 2005; Roegiers mouse brain explants (Wolf et al., 2008). How this et al., 2005). Similarly, the neural tube of lgl1À/À mice relates to aPKC’s role in apicobasal cell polarity and displays hyperplasia that is associated with increased proliferation control or tumourigenesis is currently not Notch signalling (Klezovitch et al., 2004). Whether known. regulation of the Notch pathway by Lgl (or Scribble/ Finally, the PCP phenotype of Scribble mutant mice Dlg) also occurs in other Drosophila or mammalian provides a further link to the non-canonical Wnt tissues is unknown. However, an analysis of factors pathway. The circletail and rumz Scribble mutant mice affecting Notch signalling in Drosophila ovarian follicle show a phenotype characteristic of Wnt/PCP signalling epithelial cells showed that lgl mutant cells do not show mutants, including neural tube closure defects, disrup- ectopic activation of Notch signalling (Vaccari et al., tion of cochlear hair cell orientation and genetic 2008).

Oncogene Role of the Scribble polarity module in tumourigenesis PO Humbert et al 6900 Ras pathway. Similarly to Drosophila, a recent study negatively regulating cell proliferation and promoting has shown that loss of mammalian Scribble can cell death. Indeed, further analysis indicated that Mats cooperate with activated Ha-Ras or Raf to induce and Yorkie also showed a strong involvement in invasive behaviour of mammary epithelial cells (Dow basolateral junction signalling (S Goode, personal et al., 2008). This study showed that the effects of communication), suggesting that at least some of the mammalian Scribble on invasion appear to be through other core components of the SWH pathway are also direct regulation of Ras–MAPK signalling by Scribble; involved. This study is of particular interest with respect loss of Scribble in MCF10A and 293T epithelial cells toa recent paper by Menut et al. (2007) where it was gives rise to hyperactivation of MAPK pathway and shown that strong mutations in warts lead tomassive activation of Ras-inducible genes such as IL-8. Con- neoplastic overgrowth of eye imaginal discs, including versely, overexpression of Scribble can potently suppress loss of differentiation and altered apicobasal cell the effects of Ha–Ras V12 on mammary epithelial cells, polarity. However, eye discs mutant for hippo, salvador including MAPK signalling, loss of polarity, induction or mats do not show defects in differentiation or of EMT, anchorage-independent growth, cytokine apicobasal cell polarity, suggesting that there may be production and invasion in three-dimensional matrices specialized roles for Warts, independent of Hippo, (Dow et al., 2008). These effects on Ras–MAPK Salvador and Mats. The similarity with the phenotype signalling are a highly conserved function of Scribble, of strong warts mutants and lgl, scrib or dlg mutants in as Drosophila Scribble could also prevent Ras and the follicle cells and eye imaginal discs suggest that they Raf-mediated defects in Drosophila wing development. may act similarly. Moreover, in polarized mammalian Although p38 signalling did not seem to be regulated by epithelial cells, expression of the Yorkie homologue, Scribble, loss of Scribble also leads to a hyperactivation YAP, induces an EMT, suppresses apoptosis and of JNK, as has been seen in Drosophila (see above). The promotes growth factor-independent proliferation and functional significance of this hyperactivation of JNK anchorage-independent growth in soft agar (Overholtzer remains unclear as the effects of loss of Scribble on et al., 2006). Thus, at least in mammalian cells, YAP invasion appeared to be refractory to chemical inhibi- (Yorkie) has a strong disruptive effect on apicobasal cell tion of JNK (Dow et al., 2008). In addition to MAPK, polarity, as well as promoting cell survival and cell the other conserved key mediators of Ras action, Ral proliferation. This is more potent than knockdown of and PI3K signalling, need tobe considered. With regard Scribble in polarized epithelial cells (Dow et al., 2007), to PI3K, it is of interest that the constitutive activation suggesting either additional pathways stimulated by of PI3K by the adenovirus 9 E4ORF1 oncoprotein YAP or some level of redundancy between Scribble, Dlg appears tobe Dlg1-dependent (Frese et al., 2006) and and Lgl. As discussed previously, in scrib or lgl mutant that Dlg1 itself has been showntobe able tobind to eye, imaginal epithelia or scrib, dlg or lgl mutant ovarian PTEN, a negative regulator of the PI3K pathway (Adey follicular epithelial cells, CycE and DIAP1, are upregu- et al., 2000). Furthermore, it remains to be established lated, and E2F1 is upregulated in lgl mutant cells in the whether Dlg and Lgl can alsoregulate Ras signalling, developing eye, as also occurs in SWH pathway and what the functional significance of the regulation of mutants. The relationship between the SWH pathway PI3K and JNK by Scribble/Dlg/Lgl may be in tumour and scrib/dlg/lgl is an important issue to clarify in the development. understanding of the regulation of apicobasal cell polarity, and how basolateral junction proteins affect the cell proliferation and apoptosis machinery. The Salvador/Warts/Hippo pathway. The Salvador/ Warts/Hippo(SWH) pathway is a newly described pathway in organ size control and cancer (Harvey Scribble/Dlg/Lgl and vesicle trafficking and Tapon, 2007; Zeng and Hong, 2008). The core One attractive mechanism of how apicobasal polarity is components of this pathway are the protein kinase regulated and how the loss of function of Scribble/Dlg/ Hippo, which together with its adaptor, Salvador, Lgl can lead tochanges in signalling pathways is vesicle phosphorylates the Warts protein kinase and its adaptor trafficking. The junctions of mammalian epithelial cells Mats, which in turn phosphorylates and inhibits the have been proposed to promote the correct spatial activity of the co-transcription factor Yorkie/YAP. organization of cellular components by acting as sorting Yorkie/YAP when unphosphorylated enters the nu- sites for a subset of vesicles (Yeaman et al., 1999). In cleus, and together with the transcription factor addition, PDZ domains, which are frequently found in Scalloped/TEAD (TEF), leads to the transcription of polarity proteins, have been implicated to act at several key targets, including the G1–S-phase cell cycle reg- different sites in various protein trafficking pathways ulator, CycE; the cell cycle transcription factor, E2F1 (Cao et al., 1999; Wenthold et al., 2003; Franklin et al., (Goulev et al., 2008); and the cell death inhibitor, 2005). There are several studies showing that members DIAP1. Recently, mutants in Drosophila warts were of the Scribble polarity module associate with compo- identified as enhancers of dlg and lgl mutants in ovarian nents of vesicle trafficking pathways. In MDCK follicle cell tumourigenesis (Zhao et al., 2008). Zhao epithelial cells, Lgl2 has been shown to interact with et al. (2008) propose a model whereby basolateral syntaxin 4, a component of the basolateral exocyst junction proteins (Scribble/Dlg/Lgl) utilize Warts sig- machinery (Musch et al., 2002). While in Drosophila, the nalling torepress CycE and DIAP1 expression, thereby Scribble polarity module has been shown to genetically

Oncogene Role of the Scribble polarity module in tumourigenesis PO Humbert et al 6901 interact with mutants in a core component of the genesis, and how they impact on each of the hallmarks exocytic machinery, exo84 (Blankenship et al., 2007), of cancer (see Figure 2). Accumulated data on the which parallels with studies in yeast showing that the expression of Scribble/Dlg/Lgl in human tumours all Lgl homologues, Sro7 and Sro77, can interact with the point to a role late in tumourigenesis for loss of function exocytic machinery (Gangar et al., 2005; Zhang et al., of these genes in tumour progression. However, despite 2005). Furthermore, Scribble has been shown to have an many reports, no clear clinical correlations have yet important role in regulating exocytosis in neuroendo- been gleaned from such studies. A comprehensive crine cells through its association with the b-Pix–GIT1 analysis of Scribble, Dlg1–4 and Lgl1–2 expressions in complex (Audebert et al., 2004). Scribble was alsofound tumours from patients with a well-annotated clinical to inhibit basal receptor endocytosis and promote history now needs to be performed to determine whether recycling of the thyroid-stimulating hormone receptor loss or changes in the expression of members of the in PC12 cells (Lahuna et al., 2005). Taken together, Scribble polarity module correlate with survival, stage these data suggest that members of the Scribble polarity or aggressiveness of these cancers. Moreover, definitive module may have key functions in vesicle trafficking. proof of the tumour-suppressive function of mammalian How general this phenomenon is and whether this Lgl, Dlg and Scribble will require the generation of mechanism is critical in the establishment of apicobasal appropriate conditional knockouts in mice and the use polarity are intriguing questions that have not yet been of defined mouse models of cancer. Such experiments comprehensively addressed. Moreover, as many signal- are underway for Scribble, Dlg and Lgl in a number of ling pathways involve vesicle trafficking to promote laboratories, including our own, and will provide signalling or to downregulate receptors and thereby important information on this matter. One issue that dampen signalling (reviewed by Giebel and Wodarz, will need tobe addressed is the possibleredundancy 2006), this role of Scribble/Dlg/Lgl in vesicle trafficking between the various Dlg and Lgl family members, as may be fundamental to the regulation of signalling well as any redundancy between Lgl, Dlg and Scribble. pathways by Scribble polarity module. Moreover, as discussed herein, evidence is accumulating, primarily from studies in Drosophila, that Lgl, Dlg and Scribble have distinct functions, rather than working Conclusions and future directions simply in a common pathway, and therefore in mammalian systems these will undoubtably impact Despite the established roles of Scribble/Dlg/Lgl as in different ways upon the hallmarks of cancer, cell tumour suppressors in Drosophila, strong evidence is proliferation, survival, differentiation and invasion/ still lacking in mouse or humans to prove a causative metastasis. Finally, in view of the number of signalling role for Lgl, Dlg or Scribble mutations in tumouri- pathways that are regulated by Scribble, Dlg and Lgl

Subapical Crumbs aPKC region Hallmarks of Cancer: ? - Proliferation (CycE, E2F1) ? Lgl - Survival (DIAP1, RHG) Signalling Pathways Basolateral ? ? - Inhibition of Differentiation Scrib (WNT, GPCR, Notch, Junctions (Pros, Brat) Ras, SWH) Dlg - EMT (E-Cadherin) and Invasion / Metastasis (JNK, MMPs, Rac1, β-Pix, Cdc42)

Figure 2 Regulation of signalling pathways by Scribble/Dlg/Lgl and their impact on the hallmarks of cancer. From genetic and physical interactions, Scribble, Dlg and Lgl have been associated with the regulation of several signalling pathways, including the Wnt, G-protein-coupled receptor (GPCR), Notch, Ras and Salvador/Warts/Hippo (SWH) pathways. Owing to the mutually repressive relationship between the Scribble polarity module and the Crumbs and Par complex (of which Crumbs and aPKC have been documented to play an active role), it is possible that these polarity modules may also play an important role in the effect of mutants in Scribble, Dlg or Lgl on signalling pathways. Mutants of Scribble, Dlg and Lgl give rise to the hallmarks of cancer (due to their effects on the key genes listed—see text for details). Remaining questions of concern are the following: (1) what are the extrinsic or instrisic cues that trigger the action of the polarity modules to mediate their effects on signalling pathways and prevent tumour formation; (2) what is the precise mechanism by which the polarity regulators control signalling pathways; and (3) how misregulation of signalling pathways affect the hallmarks of cancer—cell proliferation, survival, differentiation, the epithelial-to-mesenchymal transition (EMT), leading to invasion/metastasis. Key regulators of these processes shown to be affected by mutants of Scribble, Dlg or Lgl are listed (see text for details). The function of Scribble, Dlg or Lgl in vesicle trafﬁcking or on Myosin II activity and the actin cytoskeleton may also impact on signalling pathways and on cell polarity regulation (not shown). In addition, mutations in the polarity module components, by their effects on the loss of apicobasal polarity and cell adhesion, are also likely to directly mediate EMT and thereby invasion/ metastasis (not shown).

Oncogene Role of the Scribble polarity module in tumourigenesis PO Humbert et al 6902 (see Figure 2), the next challenge will be toestablish Acknowledgements whether and how any of these pathways can modulate tumourigenesis and how this is manifested in human We thank Linda Parsons and Sarah Russell for critical reading of tumours. The precise mechanism by which Scribble, the manuscript and numerous discussions. We also thank Shernaz Dlg and Lgl regulate these signalling pathways and Bamji, Lawrence Banks, Chris Doe, Scott Goode, Daniela how their documented functions in vesicle trafﬁcking Grifoni, Juergen Knoblich and Senthil Muthuswamy for commu- nicating results before publication. We apologize to those authors and Myosin II regulation may be related to this are key whose work was not cited directly owing to space limitations. areas for future research. Genetic analysis in model POH was supported by a Career Development Award, HER by a organisms such as Drosophila will continue to be the Senior Research Fellowship from the Australian NHMRC and IE cornerstone of our ability to study in vivo the interac- by a Postgraduate Cancer Research Scholarship from the Cancer tions between Scribble, Dlg and Lgl, and other polarity Council Victoria. This work was supported by grants from the complexes, and to delineate the processes they regulate Australian NHMRC (POH, AMB, NAG and HER), Association in normal cells and how these are deregulated during for International Cancer Research UK (POH) and Australian tumourigenesis. Cancer Council Victoria (POH and HER).

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