Oncogene (2008) 27, 6939–6957 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc REVIEW The epithelial polarity program: machineries involved and their hijacking by cancer

B Tanos1 and E Rodriguez-Boulan1,2

1Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical College, New York, NY, USA and 2Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, NY, USA

The Epithelial Polarity Program (EPP) adapts and so on) or as multilayered tissues (for example, skin, integrates three ancient cellular machineries to construct mucosae of upper digestive and upper respiratory tracts, an epithelial cell.The polarized trafficking machinery cornea). Epithelial tissues perform key functions neces- adapts the cytoskeleton and ancestral secretory and sary for homeostasis of the organism, for example, endocytic machineries to the task of sorting and delivering they transport nutrients into the internal medium and different plasma membrane (PM) to apical and eliminate waste to the outside spaces. These vectorial basolateral surface domains.The domain-identity ma- functions are carried out by a large array of transpor- chinery builds a tight junctional fence (TJ) between apical ters, channels and receptors which, in simple and and basolateral PM domains and adapts ancient polarity glandular epithelia, are dramatically polarized between proteins and polarity lipids on the cytoplasmic side of the apical and basolateral domains of the plasma membrane PM, which have evolved to perform a diversity of polarity (PM). Apical and basolateral PM domains are separated tasks across cells and species, to provide ‘identity’ to each by a junctional complex constituted by tight junctions epithelial PM domain.The 3D organization machinery (TJs) and adherent junctions (AJ). In addition, the utilizes adhesion molecules as positional sensors of other lateral domains of epithelial cells are linked by spot epithelial cells and the basement membrane and small desmosomes (D) and gap junctions. Non-vertebrate GTPases as integrators of positional information with the epithelia display a variation of the junctional complex, activities of the domain-identity and polarized trafficking with septate junctions (a form of TJ) located under AJs machineries.Cancer is a disease mainly of epithelial cells at the top of the lateral membrane. (90% of human cancers are carcinomas that derive from The organization of polarized epithelia depends on an epithelial cells) that hijacks the EPP machineries, EPP that utilizes three machineries, dynamically inter- resulting in loss of epithelial polarity, which often playing with each other, to build an epithelial cell correlates in extent with the aggressiveness of the tumor. (Figure 1). The polarized trafficking machinery is an Here, we review how the EPP integrates its three adaptation of the secretory and endocytic systems that machineries and the strategies used by cancer to hijack are found in every cell to the task of sorting and them. delivering PM proteins and lipids to apical and Oncogene (2008) 27, 6939–6957; doi:10.1038/onc.2008.345 basolateral PM domains. The domain-identity machin- ery adapts a highly conserved set of polarity proteins Keywords: epithelial polarity; vesicular trafficking; and lipids that perform a variety of polarity functions polarity proteins; polarity lipids; epithelial mesenchymal across tissues and species, to the task of generating and transition; epithelial tumors maintaining the ‘identity’ of the apical and basolateral domains. This ‘identity’ task involves two major subtasks. The first one is to control the assembly of a circumferential belt at the top of the cell, TJs (septate junctions in Drosophila), which provides a fence between The EPP: adapting three ancient cellular machineries to the apical and basolateral domains and a selective para- build an epithelial cell cellular gate between the luminal compartment and the internal medium. The second one is to organize the Epithelial tissues are located at the interface between the secretory and endocytic systems and the cytoskeleton organism and the outside world, as simple monolayers into a polarized trafficking machinery that sorts and that cover the digestive, respiratory, urinary and delivers proteins to apical and basolateral domains. This reproductive tracts, as glandular acini/alveolae (pan- involves the adaptation of ancestral endocytic signals creas, salivary glands, breast gland, prostate, liver and and decoding mechanisms to perform apical-basolateral sorting in the biosynthetic and recycling pathways. The 3D organization machinery adapts a system of small Correspondence:E Rodriguez-Boulan, Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medical GTPases that controls cell growth and cytoskeleton College, 1300 York Ave, New York, NY 10065, USA. organization in every cell to the task of coordinating E-mail:[email protected] extracellular cues sensed by cell–cell and cell–substrate The EPP: machineries involved and their hijacking by cancer B Tanos and E Rodriguez-Boulan 6940 EPITHELIAL POLARITY PROGRAM (EPP)

Polarized Trafficking Machinery Domain Identity Machinery

Polarized secretory Polarized Par1 / MT / Syntaxin 3 Apical identity and recycling routes cytoskeleton Par3 Crumbs PIP2 / Annexin 2 PIP2 Apical sorting signals Par6 PALS/Stardust PATJ/discs lost PTEN Lipid rafts Supranuclear MTOC Par1 / Apical-Basal MT aPKC Annexin 2 Apical-Basal MT Syntaxin 3 TJ Par1 Fence Centrosomal MT Par4 / Polarized TJ Ti VAMP Par4 (LKB1) function and MT cytoskeleton Basolateral sorting signals Basolateral identity Clathrin & clathrin adaptors Apical actin LGL / syntaxin 4 Scribble Syntaxin 4 Lateral actin LGL / exocyst LGL Cellubrevin Perinuclear actin DGL Exocyst Rab 8 PIP3 PI3K

Annexin 2 / 3D Organization Machinery apical Cdc42 Apical Cdc42 / Cdc42 apical actin PIP2 / Cdc42

/ Rho Par3 / TIAM 1 actin cytoskeleton JAMs TIAM 1 Rac1 Cdc42 / Rac1 / Nectins Cdc42 / Par6 lateral actin Cdc42 JAMs, Nectins / Par3 Golgi Cdc42 / E-cadherin Rac1 / TJ TGN sorting β-catenin Golgi Cdc42 / TJ ? α-catenin Cdc42 Cdc42 / Exocyst / Basolat. sorting

Rac1 / MT ? Rac1 (Apical BL axis) Integrin β 1

Figure 1 The epithelial polarity program (EPP). Establishment of a polarized epithelia requires the adaptation and coordination of three cell machineries, the polarized trafficking machinery, the domain-identity machinery and the 3D organization machinery. The polarized trafficking machinery consists of polarized secretory and recycling routes that function coordinately with the polarized cytoskeleton to accurately recognize and sort cargo molecules that contain apical and basolateral sorting signals into different transport vesicles. Rab GTPases, the exocyst and SNAREs are important for the tethering and fusion events involved in delivery of these vesicles to the plasma membrane (PM). The domain-identity machinery, consists of the tight junctional fence and of polarity proteins and lipids that establish and maintain the identity of the apical and basolateral domain. The Par and Crumb complexes establish apical identity, and the Scribble complex is responsible for basolateral identity. Par1 and Par4 are kinases that contribute to the polarized organization of the cytoskeleton and the establishment of polarized trafficking routes. The lipid phosphatase PTEN (phosphatase and tensin homologue deleted on 10)—through its product PIP2—and the lipid kinase PI3kinase—through PIP3—function in fast domain-identity regulation (see text). The 3D organization machinery consists of a network of GTPases regulated by their respective GEFs (guanine nucleotide exchange factors) and GAPs (GTPase activating proteins), that coordinate extracellular cues, sensed by cell–substrate and cell–cell contacts, with the polarized trafficking and domain-identity machineries to generate a polarized epithelial cell. Known interactions between the three EPP machineries are indicated in association with arrows that connect them.

adhesion molecules with the polarized trafficking and basolateral PM proteins are synthesized at the endo- domain-identity machineries to produce a polarized plasmic reticulum, transported to the Golgi complex epithelial cell. and sorted at the trans golgi network (TGN) into In the first half of this review, we discuss the three distinct apical and basolateral vesicular routes (Simons machineries that constitute the EPP and how the EPP and Wandinger-Ness, 1990; Rodriguez-Boulan and integrates them. In the second half of the review, Powell, 1992). After arrival at the cell surface, apical we discuss how cancer hijacks these machineries for and basolateral membrane proteins are internalized into the purpose of uncontrolled growth and metastatic separate apical sorting endosomes and basal sorting invasion. endosomes (Figure 2), (Breitfeld et al., 1989; Mostov et al., 2003; Rodriguez-Boulan et al., 2005), mixed in common recycling endosomes and sorted into distinct Polarized trafficking machinery recycling routes back to their original PM domain. The Adaptation of secretory and endocytic systems for apical recycling route includes the apical recycling polarized trafficking. The introduction three decades endosome (Apodaca et al., 1994; Brown et al., 2000). ago of the MDCK model (Cereijido et al., 1978, 2004) More recent work has shown that some newly synthe- and its extension to study polarized trafficking via sized PM proteins move from TGN to common viral envelope glycoproteins (Rodriguez-Boulan and recycling endosomes and are sorted there in not fully Sabatini, 1978; Rodriguez-Boulan and Pendergast, polarized MDCK cells (Futter et al., 1995; Leitinger 1980) allowed the mapping of the biosynthetic and et al., 1995; Orzech et al., 2000; Ang et al., 2004; recycling routes of apical and basolateral PM proteins Cancino et al., 2007; Gravotta et al., 2007; reviewed by (Figure 2) (Rodriguez-Boulan et al., 2005). Apical and Ellis et al., 2006). This ‘transendosomal route’ of PM

Oncogene The EPP: machineries involved and their hijacking by cancer B Tanos and E Rodriguez-Boulan 6941

AP2 Syntaxin 3 Apical Sorting Signals: Ap E clathrin Ti VAMP Tight A2 - GPI (glycosyl PI) Junction - N- & O-glycans ASE - Specialized TM domains ARE - Cytoplasmic determinants A1 Adherent A2 AP1B Basolateral Sorting Signals: Junction - YXXφ, NPXY Syntaxin 4 clathrin Syntaxin 4 Exocyst CRE Cellubrevin ` LL B2 Lipid rafts Rab 8 - L B1 Exocyst - PXXP clathrin ? AP ? TGN A1, A2: Apical routes clathrin, AP1A B1, B2: Basolateral routes clathrin, GGAs AP1A Ap E: Apical endocytic route Bas E: Basal endocytic route BSE Ly Ly: TGN-Lysosome route TGN: Trans Golgi Network ER LE CRE: Common Recycling Endosomes Bas E nucleus ARE: Apical Recycling Endosomes ASE: Apical Sorting Endosomes AP2 clathrin Lys BSE: Basal Sorting Endosomes LE: Late Endosomes Lys: Lysosome

Figure 2 Trafficking routes and sorting signals of apical and basolateral plasma membrane proteins in polarized cells. Exocytic routes:A1 and B1 represent direct routes from trans golgi network (TGN) to apical or basolateral membranes. A2 and B2 represent recycling routes and biosynthetic routes from the TGN to the plasma membrane (PM) that use recycling endosomes as an intermediate sorting compartment. Endocytic routes:PM proteins internalized into apical sorting endosomes (Ap E) and basal sorting endosomes (Bas E) are mixed in common recycling endosomes (CRE) and recycled back to the PM, utilizing similar signals as in the biosynthetic route. Some apical proteins utilize lipid rafts for apical sorting, whereas basolateral proteins utilize clathrin and clathrin adaptors AP1B for CRE-PM routes and AP1A for TGN-PM routes. Vesicular delivery to the PM is organized by a polarized cytoskeleton and vesicular tethering, docking and fusion mechanisms regulated by rabs, exocyst and v-and t-SNAREs (syntaxins 3 and 4), specific for the apical and basolateral routes. proteins may be an arcane, high capacity mechanism for classes, found in NCAM (Le Gall et al., 1994), membrane transport, appropriate for the high transferrin receptor (Odorizzi and Trowbridge, 1997) trafficking needs of migratory cells, which is partially and polymeric IgA receptor (Aroeti and Mostov, 1994) replaced by a direct TGN-PM route when MDCK cells (Figure 2). Yeast 2 and 3 hybrid assays (Ohno et al., polarize (Gravotta et al., 2007). Different epithelial cell 1995; Janvier et al., 2003) demonstrated that Yxxf types differ in their mechanism for apical protein motifs interact with the m subunit of AP adaptors (AP1, delivery:although MDCK cells use a ‘direct’ route AP2, AP3, AP4), whereas dileucine motifs of the type from TGN/RE, liver and intestinal epithelial cells utilize [DE]XXXL[LI] bind to g and s1 subunits of AP1 and a transcytotic route through the basolateral PM. The to d and s3 subunits of AP3 (Kirchhausen, 2000; molecular basis of why some PM proteins use transcy- Bonifacino and Traub, 2003). Crystallographic studies totic and some use direct routes to the PM remains have elucidated details of some of these interactions, for largely unknown. Likewise, we know very little about example, of the AP m subunits with tyrosine motifs and the molecular mechanisms that determine that some of GGAs (Golgi-localized, g-ear containing, Arf-bind- proteins use a direct TGN to PM biosynthetic route, ing protein) with dileucine motifs in Man6PR (Bonifa- whereas others use a transendosomal route to the PM cino, 2004; Owen et al., 2004). Some basolateral signals (Gravotta et al., 2007). are also endocytic signals (for example, tyrosine and dileucine motifs), suggesting that basolateral sorting evolved as a new function of the PM endocytic Basolateral sorting mechanisms. Basolateral sorting is machinery, which involved the appearance of a family guided by basolateral sorting signals (Keller and of adaptors that recognize these signals at the PM (AP2) Simons, 1997; Rodriguez-Boulan et al., 2005), and they or at the TGN/RE (for example, AP1, AP3, AP4, include:short tyrosine motifs such as Yxx f or NPXY GGAs). (Brewer and Roth, 1991; Le Bivic et al., 1991; Matter The resemblance of basolateral and endocytic signals et al., 1992), dileucine (Hunziker and Fumey, 1994) or suggested from the start that clathrin, a major player in monoleucine motifs with nearby acidic patches (Deora endocytosis, might also be a key player in the et al., 2004), PxxP, found in EGFR (He et al., 2002) and basolateral route. Surprisingly, it took 17 years for this pleomorphic motifs that do not yet fall into established role to be directly demonstrated, when Deborde et al.

Oncogene The EPP: machineries involved and their hijacking by cancer B Tanos and E Rodriguez-Boulan 6942 (2008) showed that knockdown of clathrin by RNAi considered not sufficient for apical targeting. Fifteen results in dramatic loss of polarity of a broad range of years old observations (Hannan et al., 1993) and recent basolateral PM proteins, covering the known spectrum work (Paladino et al., 2004) suggest that the ability of of basolateral sorting signals. Additional experiments glycosylphosphatidyl inositol anchors to act as apical showed that clathrin knockdown disrupted both bio- sorting signals depends on clustering through their synthetic and recycling routes to the basolateral carbohydrate or proteinaceous moieties (Rodriguez- membrane and promoted missorting of basolateral Boulan et al., 2005). It has been proposed that proteins into apical carrier vesicles at the Golgi complex. glycosylphosphatidyl inositol-protein clustering may be The polarity of different basolateral proteins was mediated by lectins of the galectin class (reviewed by affected to different extents by clathrin knockdown, Fullekrug and Simons, 2004). A second group of apical suggesting that different clathrin adaptors may be proteins that includes the envelope glycoproteins of involved in basolateral sorting and that there may be influenza virus, hemagglutinin and neuraminidase as- considerable functional overlap between them. Interest- sociates with lipid rafts through their transmembrane ingly, the polarity of Na,K-ATPase was not affected by domains (Kundu et al., 1996; Scheiffele et al., 1997; Lin clathrin knock-down, likely because of post-delivery et al., 1998). A third group of apical sorting signals surface retention mechanisms, for example, homotypic includes N-glycans (Fiedler and Simons, 1995) and O- adhesion of the beta subunits (Shoshani et al., 2005) and glycans (Yeaman et al., 1997), which may also act by interaction with the /spectrin cytoskeleton promoting association with lipid rafts (Naim et al., (Hammerton et al., 1991). To date, only one clathrin 1999; Jacob and Naim, 2001). However, not all apical adaptor has been implicated in basolateral sorting, PM proteins associate with lipid rafts; for example, the AP1B (Ohno et al., 1999). One study reported a role apical PM protein 75 neurotrophin receptor (p75) uses for AP4 in basolateral sorting (Simmen et al., 2002); O-glycans as its apical sorting signal (Yeaman et al., however, AP4 does not display typical clathrin-binding 1997) through clustering by the lectin galectin 3 sequences and its sorting role remains poorly character- (Delacour et al., 2006, 2007). Yet a fourth apical sorting ized. AP1B differs from AP1A (formerly called AP1) in mechanism, also lipid raft-independent, is that of that it possesses an epithelial-specific medium subunit rhodopsin, which binds to the microtubule (MT) motor (m1B) instead of the ubiquitous m1A. m1B is expressed by dynein by cytoplasmic determinants as an apical sorting most epithelia but is lacking in LLC-PK1 cells, a kidney strategy (Tai et al., 1999). MT motors of the kinesin type cell line believed to derive from proximal convoluted are also used by other apical proteins for apical tubule that mislocalizes basolateral proteins to the targeting, for example, p75, which requires the plus apical surface (Roush et al., 1998). Transfection of end kinesin 5B for its apical delivery (Jaulin et al., 2007) m1B into LLC-PK1 cells promotes normal localization and influenza hemagglutinin and Annexin XIIIb, which of the missorted basolateral proteins (Folsch et al., 1999, require the minus end kinesin KIFC3 (Noda et al., 2001; Gan et al., 2002). Recent studies from our 2001). laboratory have shown that AP1B localizes to recycling endosomes and sorts basolateral proteins in both biosynthetic and recycling routes (Gan et al., 2002; Adaptation of vesicular delivery mechanisms for transport Cancino et al., 2007; Gravotta et al., 2007), while AP1A to apical and basolateral domains. Transport along the participates in direct transport between TGN and the secretory and endocytic routes is mediated by transport basolateral PM (Gravotta D, Deborde S and Rodriguez- vesicles and tubules that require sophisticated cellular Boulan E, manuscript in preparation) (Figure 2). machineries for their production and transport, and for their docking and fusion with the target membrane (Rothman, 1994). This machinery involves proteins that Apical sorting mechanisms. Apical sorting is mediated mediate (i) reshaping the membrane into tubules or by mechanisms that also represent an adaptation of vesicles, for example, clathrin, epsin, epsin R, etc. (ii) ancestral endocytic mechanisms based on lipid rafts, fission of the transporter from the sorting compartment, membrane microdomains enriched in cholesterol, glyco- for example, dynamin, cortactin and syndapin (iii) sphingolipids and sphingomyelin, instead of clathrin- recognition and tethering to the target membrane, for mediated endocytosis (Perret et al., 2005; Rodriguez- example, rabs, v- and t-SNARES and exocyst. The EPP Boulan et al., 2005) (Figure 2). The concept of lipid rafts has adapted these universal trafficking mechanisms to was originally introduced to explain apical sorting, the task of generating different apical and basolateral based on the observed enrichment of lipid rafts in the transporters and delivering them to their respective PM apical PM domain and the affinity of some apical domains. Docking of apical transporters depends on the proteins for lipid rafts (van Meer et al., 1987). The first T-SNARE syntaxin 3 in MDCK cells (Low et al., 1996) apical sorting signal discovered, glycosylphosphatidyl and on a tetanus-insensitive V-SNARE (Galli et al., inositol (Lisanti et al., 1988, 1989; Brown et al., 1989; 1998; Low et al., 1998; Lafont et al., 1999). Docking of Powell et al., 1991), indeed promotes association of basolateral transporters requires syntaxin 4 (Low et al., glycosylphosphatidyl inositol-anchored proteins with 1996), rab 8 (Huber et al., 1993) and a V-SNARE, lipid rafts as they move through the Golgi complex cellubrevin, which cooperates with AP-1B in basolateral (Brown and Rose, 1992). Today, the concept of lipid membrane trafficking (Fields et al., 2007), as well as the rafts has evolved and association with lipid rafts is exocyst (Grindstaff et al., 1998; Shipitsin and Feig, 2004;

Oncogene The EPP: machineries involved and their hijacking by cancer B Tanos and E Rodriguez-Boulan 6943 Yeaman et al., 2004b; Langevin et al., 2005), a multi- cells. Non-polarized epithelial cells display a similar protein particle that is also a key regulator of yeast’s organization of the actin cytoskeleton (lamellipodia, secretory route (Potenza et al., 1992; TerBush and filopodia and stress fibers) as non-epithelial cells, for Novick, 1995; TerBush et al., 1996). Interestingly, the example, fibroblasts and mesenchymal cells. Upon exocyst interacts with the clathrin adaptor AP1B polarization, the apical actin cytoskeleton organizes (Folsch et al., 2003) and participates not only in into microvilli and an apical terminal web, directed by biosynthetic delivery, but, also, in postendocytic recy- actin organizers such as ezrin and villin (Friederich cling to both basolateral and apical membranes, the et al., 1989; Bretscher, 1999) and Cdc42 (Martin- latter in association with the small GTPase Rab11a Belmonte and Mostov, 2007, 2008; Martin-Belmonte (Oztan et al., 2007). et al., 2007). The lateral actin cytoskeleton has a Live imaging experiments using model apical or different organization, classically believed to be induced basolateral PM proteins tagged with GFP (Kreitzer by the E-cadherin complex, which includes the proteins et al., 2003) demonstrated that non polarized subcon- a-andb-catenin. Recent work by Nelson’s laboratory fluent MDCK cells fuse apical and basolateral transport has developed a new model to account for the activity of vesicles randomly with the PM, due to the non-polarized this complex in organizing the actin cytoskeleton at the distributions of syntaxins 3 and 4. Polarized MDCK lateral membrane (Drees et al., 2005; Yamada et al., cells, in contrast, dramatically reorganize their delivery 2005). According to this new model, a-catenin acts routes from the Golgi and RE, which are localized independently of E-cadherin to organize the lateral actin supranuclearly in these cells due to repositioning of the cytoskeleton, which has important consequences for the MTOC, to the PM. Apical transport carriers fuse with role of these molecules in cancer (Benjamin and Nelson, the apical PM domain, promoted by the apical 2008), discussed below. localization of syntaxin 3, whereas basolateral transport Depolymerization of the actin cytoskeleton inhibits carriers fuse with a ‘hot spot’ in the junctional region of selectively the delivery of a subset of apical proteins, the lateral membrane, guided by the restricted presence including p75 (Salvarezza et al., in press), and the of syntaxin 4 at the lateral membrane (Kreitzer et al., recycling of highly endocytic basolateral proteins such 2003) and the enrichment of the tethering particle as transferrin receptor (Sheff et al., 2002). Apical exocyst at the junctional region (Grindstaff et al., 1998). endocytosis requires polymerized actin, whereas baso- lateral endocytosis does not (Gottlieb et al., 1993). The exit of apical proteins from TGN/RE requires the actin- Adaptation of MT and actin cytoskeletons for polarized and MT-associated protein dynamin 2 (Kreitzer et al., trafficking. The dramatic reorganization of trafficking 2000; Bonazzi et al., 2005), whereas basolateral proteins routes upon polarization reflects drastic changes in the require different proteins, such as BARS (Kreitzer et al., MT cytoskeleton. In non-polarized MDCK cells, MT 2000; Bonazzi et al., 2005) and protein kinase D have the typical centrosomal arrangement found in (Yeaman et al., 2004a). other non polarized cells; upon polarization, epithelial cells organize the bulk of their MT along the apical– basal axis, with the minus end facing apically and the Domain-identity machinery plus end basolaterally (Bacallao et al., 1989; Gilbert and Tight junctions. A main task of the EPP is the creation Rodriguez-Boulan, 1991; Musch, 2004). This rearrange- of TJs at the apical–lateral border. TJs are epithelial- ment of MT is controlled by the polarity protein kinase specific structures composed of trasmembrane proteins, Par1 (Cohen et al., 2004) (Figures 1 and 3a). Recent for example, occludin and the claudin family, that work has uncovered the existence of a population of interact homotypically with molecules in the neighbor- very dynamic MT, emerging from a supranuclear ing cell and with peripheral proteins (ZO1, ZO3, MTOC, in polarized MDCK cells (Jaulin et al., 2007). cingulin) in the cytoplasmic side (see Tsukita et al., The repositioning of the MTOC to a supranuclear 2008 and Cereijido et al., 2008). TJs have the ability to position is also likely dependent on the activity of Par1 act as a fence between proteins in the apical and lateral (Cohen et al., 2004) and might be mediated by dynein, as domains. Interestingly, TJs constitute a fence for lipids in migrating cells (Etienne-Manneville and Hall, 2003). in the ectoplasmic but not in the cytoplasmic leaflet of The dual arrangement of MT in polarized MDCK cells the PM. TJs also constitute a selective paracellular accounts for the involvement of both dynein and barrier for ions and proteins (gate function). Finally, as kinesins in apical transport (Hirokawa, 1998; Tai many of the cytoplasmically associated proteins are et al., 1999; Jaulin et al., 2007). Disruption of MT with scaffolding proteins that can interact with a large nocodazole or colchicine disrupts selectively the locali- number of signaling molecules, TJs are a major site zation of apical PM proteins (van Zeijl and Matlin, for regulation of cell growth (Matter and Balda, 2003). 1990; Gilbert et al., 1991). MT disruption promotes abnormal fusion of apical transport carriers with the Fast identity: polarity lipids. Many reports have basal membrane, due to rapid depolarization of syntaxin documented the key roles of phosphatidylinositol lipids 3 upon nocodazole addition (Kreitzer et al., 2003). (PIPs) in conferring identity on different cellular The actin cytoskeleton is also drastically reorganized membranes (Di Paolo and De Camilli, 2006) and in during polarization of epithelial cells and, such as MT, the generation of anterior–posterior polarity of migrat- has very selective trafficking roles in polarized epithelial ing cells (Wang et al., 2002; Merlot and Firtel, 2003;

Oncogene The EPP: machineries involved and their hijacking by cancer B Tanos and E Rodriguez-Boulan 6944 a

Syntaxin 3 Crumbs 3 Annexin 2 _ PIP2 PATJ PALS Cdc42 PTEN FERM Apical TJ Apical actin TJ Rho aPKC Route Par 1 + JAM Par6 Par3 Nectin Cdc42 Kinesin PI3K TIAM 1 Rac1 Par 4 ARE Dynein ? PIP3 E-cadherin Lateral AMPK actin RE Exo cyst MTOC LGL Scribble Cdc42 Golgi DLG Peri- Golgi actin Basolateral route MT Syntaxin 4 Exo cyst Nucleus + Apical-Basal Axis

Rac1 Integrin

Laminin

b 1 2 3 4

VACs Formation of loose Homotipic VACs cell-cell contacts VAC fusion exocytosed Golgi Lumen Nucleus

Single cell Cyst Figure 3 (a) The epithelial polarity program (EPP) at work. Snapshot depicting the coordinate interactions between the polarized trafficking machinery, the domain-identity machinery and the 3D organization machinery. Extracellular cues sensed by cell–cell and cell–substrate adhesion molecules control the proper localization of the Par complex and the basolateral Scribble complex (Scribble, DLG, LGL) in cooperation with small GTPases Cdc42 and Rac1. Phosphorylation events maintain them segregated into different domains. The Crumbs complex (Crumbs1/PATJ/PALS) interacts with the Par complex (Par3/Par6/aPKC) to control tight junction (TJ) formation and apical domain identity. The Scribble complex controls basolateral membrane identity and contributes to the formation of TJ. The identity of the apical and basolateral membrane also depends on the presence of highly dynamic basolateral PIP3, synthesized by PI3K and apical PIP2, produced from PIP3 by the phosphatase PTEN. Par1 controls the arrangement of the bulk of microtubules (MT) (double red lines) with the minus (À) end towards the apical side and the plus ( þ ) end towards the basolateral side. A small subset of dynamic MT with the þ end towards the apical side is constantly generated from the MTOC (double red lines, small). The kinase Par4 (LKB1) controls the polarity of the cell’s cytoskeleton by the kinase AMPK independently of extracellular cues. The identity of apical and basolateral domains is the result of the installation of TJ as a fence and of the establishment of polarized trafficking routes to the PM as a result of cytoskeletal organization. (b) VACs and lumen formation. Epithelial and endothelial cells often generate their luminal domains intracellularly, as a vacuolar apical compartment (VAC). VACs grow into larger vacuoles by homotypic fusion and eventually fuse with the PM to generate the lumen.

Oncogene The EPP: machineries involved and their hijacking by cancer B Tanos and E Rodriguez-Boulan 6945 Stephens et al., 2008). Two remarkable papers by the that provide ‘identity’ to apical and basolateral Mostov laboratory have recently demonstrated a key domains, and which were originally identified by genetic role for PIs in the development of epithelial polarity experiments in Drosophila and Caenorhabditis elegans. (Gassama-Diagne et al., 2006; Martin-Belmonte et al., Polarity proteins are organized as three major com- 2007). Gain and loss of function experiments showed that plexes, Crumbs (Crumbs, PALS/Stardust and PATJ/ PtdIns(3,4,5)P3 (PIP3) confers its typical identity on the Discs lost), Par (Par3, Par6/Bazooka, and atypical basolateral membrane, whereas PtdIns(4,5)P2 (PIP2) protein kinase C (aPKC)) and Scribble (Scribble, confers identity on the apical membrane. Addition of LGL, DLG) (Figures 1 and 3a) (see Aranda et al., each PI to the opposite membrane resulted in a very rapid Humbert et al., this issue). Polarity proteins specify the ‘identity shift’ of that compartment into the other one. identity and size of the apical and basolateral PM This identity shift occurred within minutes and depended compartments through mechanisms that are still largely on the rapid endocytosis and transcytosis of molecules unknown. Crumbs and Par cooperate in establishing from the opposite PM domain, as shown by use of the apical domain and in the assembly of TJ, whereas inhibitors of endocytosis such as GTPase-deficient dyna- Scribble plays a key role in the definition of the min. The polarization of PIs was reinforced by the basolateral PM domain. They interact with each other recruitment of PI3K (synthesizes PIP3) to the basolateral through PDZ domains and key phosphorylation events domain and the exclusion of the phosphatase PTEN that result in exclusion of Scribble from the apical (degrades PIP3 to PIP2) from the basolateral domain and domain and of Crumbs from the basolateral domain its enrichment at the apical domain (Figure 3a). Because (reviewed by Schock and Perrimon, 2002; Gibson and PIPs localize to the cytoplasmic side of the PM and Perrimon, 2003; Nelson, 2003; Shin et al., 2006; Gold- because TJs do not block lipid diffusion on this side of the stein and Macara, 2007). aPKC is a key player in the PM, the continuous segregation of PIP2 and PIP3 to exclusion process, as its phosphorylation of LGL apical and basolateral domains depends on the polarized promotes its exclusion from the apical domain and its distribution of PI3K and PTEN. PTEN is recruited to the phosphorylation of Crumbs promotes the localization of PM by the PDZ domain-containing TJ proteins Magi-1-3 this complex to the apical domain (Figure 3a). (Wu et al., 2000a, b; Kotelevets et al., 2005). Magi-2 has Another Par protein that plays an important role in beenshowntopromotePTENstability(Wuet al., 2000a). epithelial morphogenesis is Par1, a protein kinase that In addition, Par3 has been shown to be required for PTEN phosphorylates MT-associated proteins and other pro- localization to the tight junction in MDCK cells (Feng teins and exists as several isoforms in mammals. In et al., 2008). PTEN is believed to act as a gatekeeper at the polarized MDCK cells, Par1b (also called EMK1) level of the TJ, promoting accumulation of PIP2 at the localizes to the lateral membrane; knockdown of Par1b apical PM and PIP3 and the basolateral PM (Martin- or expression of kinase-dead Par1b during polarization Belmonte and Mostov, 2008). of MDCK cells in collagen gels prevents them from How does the polarized distribution of PIP2 and PIP3 localizing the MTOC supranuclearly and from shifting determine polarity? The apical accumulation of PIP2 to a non-centrosomal arrangement of MT (Cohen et al., recruites Annexin 2, a protein shown to be involved in 2004). Overexpression of Par1b leads to a change in the the trafficking of the apical protein sucrase isomaltase polarity axis of MDCK cells, which adopt a liver like (Jacob et al., 2004). Annexin in turn recruits the small morphology, with the apical domain located at the GTPase Cdc42 and the polarity proteins Par6 and lateral membrane, resembling bile canaliculi. Experi- aPKC (see below). How PIP3 accumulation leads to ments in Saccharomyces cerevisiae by the Brennwald basolateral identity is less clear. Belmonte and Mostov laboratory have shown that the normal function of Par1 in also showed that knockdown of any of these proteins yeast involves genetic and physical interactions with resulted in anomalous cytoplasmic accumulation of exocyst and PM SNAREs (Lehman et al., 1999). Identify- large intracellular vacuoles with the characteristics of ing the downstream substrates of Par1b is necessary to apical membranes or VACs (vacuolar apical compart- understand how Par1b regulates lumen formation and to ments) (Gassama-Diagne et al., 2006; Martin-Belmonte elucidate possible trafficking roles of Par1b. et al., 2007). Past studies have shown that VACs Yet another Par protein, Par4 (LKB1), plays funda- accumulate in MDCK cells deprived of cell–cell contacts mental organizational roles in epithelial cells. Par4 is an by Ca chelation (Vega-Salas et al., 1987). Recent studies established tumor suppressor, a substrate of aPKC and have confirmed in both epithelial and endothelial cells a master kinase, which phosphorylates Par1 and AMP (Figure 3b) (Martin-Belmonte and Mostov, 2008) the activated protein kinase (AMPK/PKA), involved in a original hypothesis that VACs constitute a normal parallel polarity pathway important under conditions of intermediate in the formation of the apical lumen energetic stress (Lee et al., 2007; Mirouse et al., 2007) (Vega-Salas et al., 1987). VAC-like structures have been (see Hezel and Bardeesy, 2008). Activation of Par4 described as ‘intracellular lumens’ in a variety of promotes the polarization of intestinal epithelial cells in transformed cells, suggesting a disruption of apical the absence of cell–cell contacts (Baas et al., 2004a). The trafficking in cancer (Remy et al., 1990). underlying mechanism is unclear but is independent of Par1 and involves the remodeling of both actin and MT cytoskeletons (Baas et al., 2004a, b). Sustained identity: polarity proteins. The polarization A key question is how the polarity complexes process is guided by highly conserved polarity proteins contribute to generate the identity and size of the apical

Oncogene The EPP: machineries involved and their hijacking by cancer B Tanos and E Rodriguez-Boulan 6946 and basolateral domains. Many studies show that the the apical–basolateral axis (O’Brien et al., 2001) three complexes facilitate the assembly of TJ (Figures 1 and 3a). Cdc42 is recruited to the apical (Figure 3a), which is essential to maintain the identity membrane by Annexin 2 and PIP2 (Figures 1 and 3a), of apical and basolateral domains. However, this would leading to the recruitment of Par6/aPKC, which is be insufficient, by itself, to generate identity or regulate required for the formation of the apical lumen (Martin- the size of the PM domains. A second mechanism, Belmonte et al., 2007). Downregulation of Cdc42 or currently more speculative, is that the polarity com- expression of dominant negative mutants of Cdc42 plexes regulate key steps in vesicular trafficking to results in aberrant lumen formation and accumulation basolateral and apical membranes. Indeed, it has been of intracellular lumens or VACs (Martin-Belmonte shown that LGL interacts with the basolateral T- et al., 2007) described earlier in the review (Figure 3b). SNARE syntaxin 4 (Musch et al., 2002) and with the Cdc42 also has important functions in polarized exocyst (Zhang et al., 2005), which are components of trafficking from the Golgi complex; it regulates differ- the basolateral trafficking machinery. This concept is ently the exit of apical and basolateral proteins from the supported by recent work by Bilder’s laboratory, which TGN (Kroschewski et al., 1999; Cohen et al., 2001; implicates two components of the apical endocytic Musch et al., 2001), presumably by organizing the local machinery in Drosophila as downstream effectors of actin cytoskeleton at the perinuclear region (Kroschews- polarity and as a source of transformation when ki et al., 1999; Cohen et al., 2001; Musch et al., 2001) mutated (Lu and Bilder, 2005) (discussed later in the and through interactions with the exocyst (Zhang et al., review). 2001; Yeaman et al., 2004b) (Figures 1 and 3a).

3D organization machinery A key event in the polarization of epithelial cells is the How cancer hijacks the EPP machineries establishment of cell–cell junctions, mediated by Ca- dependent (E-cadherin) and Ca-independent (JAM, Cancer can arise from the unregulated activity of Nectins) adhesion molecules. Upon establishment of oncogenic proteins, such as the epidermal growth factor cell–cell contacts, epithelial cells establish adherent receptor (EGFR) family and transforming growth junctions, a required step for the subsequent formation factor (TGF) receptor, or from the decreased expression of TJ and for the reorganization of the cytoskeleton and or activity of tumor-suppressor proteins, such as those of the polarized trafficking routes of epithelial cells in the E-cadherin or Scribble complexes. In mammals, (Cereijido et al., 2008; Miyoshi and Takai, 2005; just one mutation event is usually not sufficient for Yeaman et al., 2004b) (Figure 1). The small GTPases cancer to develop. By contrast, in Drosophila, single Cdc42 and Rac1 are recruited and activated at the inactivating mutations of individual members of the junctional region, where they coordinate the extracel- Scribble group result in the development of lular cues provided by adhesion molecules (E-cadherin, unregulated growth and loss of polarity of the epithelial JAM and Nectins) with the activities of the polarity cells involved (Bilder, 2004; Dow and Humbert, 2007; proteins (Figures 1 and 3a). Rho also has a role in Wodarz and Nathke, 2007). Early alterations in stabilizing TJ (and is the target for activated TGFbR epithelial polarity are a hallmark of epithelial cell (transforming growth factor receptor), which promotes cancers or carcinomas and contribute to their develop- its degradation as a transforming mechanism (see ment as carcinomas in situ or their progression to below). JAM and Nectin recruit Par3, which recruits invasive adenocarcinomas. the Rac GEF TIAM1, which in turn activates Rac1, Although alterations in polarity are difficult to identify leading to increased actin dynamics in the junctional in carcinomas in situ, which may be fully polarized, they area; this acts as a negative regulator of TJ assembly are obvious in invasive carcinomas with sarcomatous (Chen and Macara, 2005). Par3 also recruits Par6, a characteristics. Metastatic carcinomas are usually char- scaffolding protein with a CRIB domain that binds acterized by low expression levels or function-inactivating Cdc42 and has an additional binding site, octicosapep- mutations of E-cadherin (or its associated proteins, the tide repeat, for aPKC (Gao et al., 2002). How exactly catenins) that promote transformation of the epithelial Cdc42 and the Par complex contribute to TJ formation phenotype into that of a migratory mesenchymal cell has not been fully elucidated, but it appears to be a (epithelial–mesenchymal transition, EMT) (reviewed by facilitating role, as TJs are formed but slower if the Thiery, 2002; see Moreno-Bueno et al., 2008). The complex is disrupted (Joberty et al., 2000; Lin et al., disassembly of the junctional complex by oncogenes 2000; Rojas et al., 2001; Etienne-Manneville and Hall, unleashes a variety of cell growth signaling pathways that 2003). The mechanism involves interactions of Par6 with promote proliferation. Significant advance has been made Crumbs 3, (which is found in most epithelial cells, unlike in the characterization of how the oncogenic process Crumbs 1, which localizes to brain and eye) by its promotes disassembly of the junctional complex, but large associated proteins PALS and PATJ (Lemmers et al., gaps remain on the specific roles of polarity proteins in 2002; Hurd et al., 2003; Roh et al., 2003). normal and transformed epithelial cells. Here we analyse In addition to their roles at the TJ, Rac1 and Cdc42 the various strategies that cancer utilizes to hijack the have further roles in the generation of epithelial polarity. three machineries that compose the EPP, resulting in a Rac1 cooperates with integrin-b1 in the establishment of motile, invasive cell.

Oncogene The EPP: machineries involved and their hijacking by cancer B Tanos and E Rodriguez-Boulan 6947 Hijacking of the polarized trafficking machinery localized EGFR could remain for longer times at the Prolonged signaling from activated growth factor (GF) PM resulting in increased EGFR signaling and higher receptors, such as EGFR, TGFbR and Hepatocyte tendency to transformation (additional discussion be- growth factor receptor (HGFR/Met), has been shown to low). Recently, both human EGFR (HER1) and human promote EMT and is a mechanism underlying several HER2 were shown to interact with a PDZ domain in oncogenic phenotypes as well as transformation. HGF- Erbin, an interaction required for their basolateral induced EMT has been well studied in MDCK cells, localization (Borg et al., 2000) (Figure 6). Interestingly, where it was initially named Scatter factor because of Erbin downregulation reduced the number of metastases the increased cellular separation and motility observed of colon carcinoma (Dardousis et al., 2007), probably upon its binding to HGFR (Balkovetz, 2006). reflecting an effect in PM localization of the EGFR either in the absence or overexpression of this protein. Recent work by Bilder’s laboratory has reported that EGFR sorting and localization. The EGFR, a critical mutations of two core components of the endocytic protooncogene, contains two basolateral sorting signals, trafficking machinery, the syntaxin Avalanche (avl) namely PXXP and LL-motifs (He et al., 2002), and has and Rab5, cause an expansion of the apical membrane been shown to localize to the basolateral side in domain of Drosophila melanogaster epithelia; this polarized epithelia (Borg et al., 2000) and to the leading polarity defect is coupled with overproliferation to form edge in EGF stimulated and migrating cells (Diakonova neoplastic tumors (Lu and Bilder, 2005). Loss of avl et al., 1995; Zhao et al., 1999; Sorkin et al., 2000; Lynch results in the accumulation of Notch and Crumbs, due et al., 2003; Orth et al., 2006). A critical feature of to decrease in lysosomal degradation of these proteins. polarized epithelia is the different compartmentalization These results uncovered a role for endocytic trafficking of GFs to the basolateral side and receptors to the apical in the control of both apical–basolateral polarity and side, which prevents receptor activation. In one striking cell proliferation. case, the loss of polarity of epithelial cells was shown to allow the intermixing of basolaterally localized HER2/ ErbB2, a member of the EGFR family and its activator Decreased EGFR downregulation as a mechanism for cell heregulin, normally secreted to the apical side (Vermeer transformation. A mechanism by which signals from et al., 2003), leading to cell proliferation (Figure 4). activated GF receptors are downregulated involves Although this mechanism is responsible for wound receptor internalization and is termed ‘desensitization’. healing of the respiratory epithelium in response to Internalized receptors continue signaling for some time various types of aggression, it is not difficult to imagine in endosomes (Sorkin, 2001; Miaczynska et al., 2004) the involvement of similar mechanisms in neoplastic where they can undergo further targeting to lysosomes transformation of epithelial cells. for degradation or recycle back to the PM (Sorkin, 2000; Overexpression has been shown to promote misloca- Yarden, 2001b; Yarden and Shilo, 2007). Failure to lization of the EGFR to the apical side in polarized internalize activated GF receptors results in cellular epithelia (Borg et al., 2000), likely through saturation of transformation (Yarden, 2001b). Twelve years ago, the basolateral targeting machinery. As apical endocy- work in Sandra Schmid laboratory identified the tosis is slower than basolateral endocytosis (Perret E GTPase dynamin as a key endocytic regulator (Vieira and Rodriguez-Boulan E, unpublished data), apically et al., 1996). This study found not only that expression

Cell migration Polarized epithelia EGF leaks through open junctions Hyperproliferation to distant tissues

EGF EGF EGF EGF EGF Increased EGF EGF EGF EGF EGF EGF cell proliferation Injury EGF EGF TJ TJ TJ TJ EGF EGF EGF

EGF EGF Tight junction EGF Neoplastic dissasembly EGF EGF EGF EGF transformation EGF EGF EGF EGFR Activated EGFR Cellular dysplasia, tumor formation, EMT Direction of migration

12 34 Figure 4 Disruption of epithelial polarity activates EGF receptor. (1) EGFR localizes basolaterally and its ligand localizes apically in epithelial cells. (2) Disruption of tight junction (TJ) by injury of the epithelium allows intermixing of receptor and ligand and activation of EGFR, leading to proliferation and closing of the wound; oncogenic dissolution of TJ may promote similar effects. (3) Prolongued EGFR activation can promote an increase in cell proliferation and neoplastic transformation followed by epithelial–mesenchymal transition (EMT). (4) Motile, invasive metastatic cells invade distant tissues. Alternatively, missorting of EGFR to the apical membrane may lead to similar results.

Oncogene The EPP: machineries involved and their hijacking by cancer B Tanos and E Rodriguez-Boulan 6948 of a dominant negative dynamin K44A mutant inhibited ErbB2 EGFR endocytosis, but, importantly, that this effect Activated also altered the phosphorylation pattern of several ErbB2 Par6 proteins downstream of EGFR, including Shc and aPKC PLC, and increased cell proliferation downstream of TJ the GF EGF. From that time on, several papers have disassembly uncovered how failure to downregulate EGFR pro- Occludin motes transformation and how several mutations in the TGFβR1 Activated P EGFR and its family members (that is, kinase activating Par6 mutations) result in increased receptor stability. Thus, TGFβR2 the deficient downregulation or ‘desensitization’ of the RhoA EGFR may be responsible for malignancy in a high SMURF degradation number of cancers, including HER2 expressing cancers (Yarden, 2001b), such as breast cancer. Several sorting Figure 5 Oncogenic ErbB2 and TGFbR hijack the domain- routes regulate the amount of EGFR (and other identity machinery. Activated ErbB2 and TGFbR interact with receptors) at the surface. These include internalization Par genes and disrupt tight junction (TJ), leading to epithelial– rates; sorting from early endosomes to late endosomes mesenchymal transition (EMT) (see text for details). Activated and multivesicular bodies (MVB), or recycling back to ErbB2 interacts with Par6, disrupting the Par3/Par6/aPKC complex and leading to disruption of apical–basal polarity, TJ the PM; sorting within MVB; exocytosis and receptor dissolution and EMT. Par6 also interacts constitutively with biosynthesis. Rab GTPases have been shown to localize TGFbR1 (which also binds occludin). Upon TGFbR2 activation, and regulate cargo targeting to these compartments Par6 interacts and is phosphorylated by TGFbR2; phosphorylated and are important for the sorting pathways that define Par6 recruits Smurf (E-3 ligase), which targets RhoA for polarized cells, Rab 4 and 5 regulate basolateral degradation, leading to TJ dissolution. targeting and localization to early endosomes, Rab 3, 11 and 25 regulate apical recycling endosomes, and Rab 4 and 17 regulate the common recycling endosomes (van Hijacking of the domain-identity machinery Ijzendoorn et al., 2003). Interestingly, Rab GTPases Oncogenes promote dissolution of TJ. Several GF regulate EGFR trafficking (Ceresa, 2006) and Rab signaling pathways relevant for cancer regulate polarity expression and activation are deregulated in cancer and growth in a coordinated manner. Examples are the situations (Stein et al., 2003). ErbB2 and TGFb receptor pathways, which regulate Accurate EGFR sorting has also been shown to be TJ by interaction with the Par complex and which dependent on ubiquitination by the E3 ligase Cbl, and, when activated oncogenically, promote dissolution of the efficacy of targeted antitumor therapies, such as TJ (Figure 5). The ErbB2 receptor pathway normally herceptin, may depend on the targeting of HER2 for regulates the development of the breast gland; however, Cbl-mediated internalization and degradation (Yarden, ErbB2 overexpression is observed in a third of breast 2001a). Impairment in Cbl E3 ligase function has been cancers, as well as in ovary, prostate and pancreatic shown to promote transformation (Dikic and Schmidt, cancers (Wodarz and Nathke, 2007). Interestingly, 2007), and oncogenic Cbl was originally identified as ErbB2 activation disrupts apico–basal polarity in lacking E3 ligase function (Langdon et al., 1989). MDCK cells (Aranda et al., 2006) and EGFR activation Oncogenes, such as Src or Abl, hijack the EGFR promotes EMT and polarity disruption in cancer cells trafficking machinery and uncouple the Cbl E3 ligase (Lo et al., 2007). In MDCK cells, ErbB2 associates with from the EGFR downregulation pathway (Bao et al., Par6/aPKC (Aranda et al., 2006), and sequesters this 2003; Tanos and Pendergast, 2006). Thus, several complex away from Par3, thus disrupting the Par3/Par6/ mechanisms that impair Cbl targeting to the EGFR aPKC complex and compromising tight junction integ- and inhibit its downregulation could promote transfor- rity. It would be interesting to know whether the ErbB2 mation by generating an increase in total levels and effect involves activated Cdc42, as this Rho GTPase is surface levels of the EGFR. New data by Sorkin and co- an effector of EGFR that, when constitutively activated, workers show that EGFR ubiquitination is not required may mediate epithelial depolarization (Musch et al., for internalization but plays a role in the targeting of the 2001). EGFR to late endosomes and multivesicular bodies for The TGFb receptor promotes invasion and metastasis degradation, providing additional mechanistic detail on in late-stage carcinomas; recent data have established a the role of Cbl in EGFR downregulation (Huang et al., mechanism through which oncogenic TGFb receptor 2007). Interestingly, activated Cdc42 sequesters Cbl promotes disassembly of TJ (Figure 5). TGFb type 1 away from the EGFR and inhibits its downregulation receptor binds constitutively to Par6 (Ozdamar et al., (Wu et al., 2003). However, there is no evidence 2005) and to the TJ component occludin (Barrios- linking the effect of activated Cdc42 in EGFR down- Rodiles et al., 2005). Upon ligand binding, TGFb regulation and signaling in the context of polarized receptor 2 associates with this complex and phosphor- epithelia, and it is not known whether the depolarization ylates Par6, an event required for EMT and for of epithelia observed upon expression of activated recruitment of the E3 ubiquitin ligase SMURF1, which Cdc42 is related to an increase in EGFR-mediated mediates degradation of RhoA, required for the signaling. maintenance of TJ (Wang et al., 2006b). TGFb signaling

Oncogene The EPP: machineries involved and their hijacking by cancer B Tanos and E Rodriguez-Boulan 6949 also promotes a second aspect of EMT through Par- amplification and elevated constitutive activity of aPKC independent transcriptional activation of mesenchymal has been correlated with poor prognosis in ovarian, genes (, metalloproteases) (Miettinen et al., lung and colon cancer, suggesting that this protein 1994; Ozdamar et al., 2005), required for cell intravasa- may behave as an oncogene (Murray et al., 2004; tion and invasion of distant tissues. Eder et al., 2005; Regala et al., 2005a, b). Tumors Cancer promoting viruses, such as human papilloma with elevated levels of aPKC display loss of polarity, virus (HPV), also utilize the Scribble and Par complexes in agreement with the known role of this kinase in for oncogenic purposes (see Thomas et al., 2008). HPV regulating epithelial polarity at normal expression levels. destabilizes the Crumbs 3-Par complex, required for In addition, aPKC functions downstream of Ras and tight junction formation (Roh et al., 2003) by interacting upstream of Rac in cell transformation (Murray et al., with PATJ and targeting this protein for degradation 2004; Regala et al., 2005a, b). The Par complex (Storrs and Silverstein, 2007). The disassembly of TJ associates with the Von Hippel-Lindau tumor suppres- induces epithelial depolarization and increases the sor protein, an E3 ubiquitin ligase required for correct predisposition of the infected epithelia to cancer MT orientation in kidney epithelial cells (Schermer (Latorre et al., 2005). HPV oncoproteins also target et al., 2006), which promotes ubiquitination of activated Magi-1 and Magi-3, TJ proteins that recruit PTEN to aPKC (Okuda et al., 2001) as well as degradation of the TJ; this may also contribute to their transforming hypoxia-inducible factor, a transcriptional activator of ability (Massimi et al., 2008). several GF receptors. Mutations in the tumor suppressor Par4 (LKB1), a kinase for AMPK, and a major inducer of cell polarity Ral GTPases. Ral GTPases are Ras effectors that when activated, lead to the hereditable Peutz–Jeghers promote cell proliferation (Bodemann and White, 2008), cancer syndrome, characterized by predisposition to a and have been shown to play a role in polarized variety of rare intestinal cancer and other types of trafficking (Shipitsin and Feig, 2004). The GTPase RalA cancer (Wodarz and Nathke, 2007) (see Hezel and has been shown to cooperate with the EGFR to Bardeesy, 2008). The lipid phosphatase PTEN is promote transformation (Lu et al., 2000). Activated also considered a tumor suppressor and controls the RalA interacts with the TJ protein ZONAB, which is recruitment of the Par complex to the apical PM either also a transcriptional repressor for the ErbB2 promoter. directly or through the generation of PIP2 (Martin- Interestingly, RalA activation through expression of Belmonte et al., 2007). Both AMPK and PTEN are either a constitutively active mutant or oncogenic Ras upstream regulators of mammalian target of rapamycin can sequester ZONAB away from the ErbB2 promoter (mTOR kinase), a central regulator of cell growth and and allow ErbB2 transcription to take place (Frankel proliferation. Interestingly, inhibition of AMPK basal et al., 2005). activity in murine neuroblastoma N2a cells inhibits internalization and degradation of EGF-EGFR con- Polarity proteins, polarity lipids and cancer. The first taining complexes, and promotes accumulation of hint that polarity proteins might contribute to cancer inactive EGFR in early endosomal structures (in a was provided by experiments in Drosophila, which clathrin-dependent manner), where the EGFR avoids showed that knockout of any of the members of the targeting to the lysosomal/degradative pathway and Scribble group contributes to loss of polarity and is probably recycled back to the PM (Salazar and hyperproliferation of the affected epithelial cells (Dow Gonzalez, 2002). and Humbert, 2007; Wodarz and Nathke, 2007). In mammalian cells, the contribution of these genes to epithelial polarity is well established, but their contribu- Hijacking the 3D organization machinery tion to human carcinogenesis is just starting to emerge. Advanced cancers are characterized by a highly invasive Several reports describe a strong correlation between phenotype. Transformation of polarized epithelial cells decreased expression of Scribble, Lgl and Dlg and tumor into invasive migratory cells requires loss of epithelial progression (Wodarz and Nathke, 2007; Humbert et al., junctions and changes in the MT and actin cytoskele- 2008). Decreased expression of Scribble results in tons, a process known as epithelial–mesenchymal abnormal TJ, as genes of the Scribble group are known transition (EMT) (for review see Thiery, 2002; Wodarz to cooperate with the Par group in the assembly of TJ and Nathke, 2007; Turley et al., 2008; Moreno-Bueno and are needed for normal function of E-cadherin (Qin et al., 2008) (Figure 6). EMT is normally observed et al., 2005). Finally, experiments in Drosophila have during the embryonic development of tissues and organs shown that decreased expression of Scribble genes but it may be a transient feature in the progression of potentiates the metastatic potential of cells expressing many carcinomas and a permanent feature of highly oncogenic Ras, likely through the JNK pathway invasive tumors with a mixed carcinoma/sarcoma (Pagliarini and Xu, 2003; Igaki et al., 2006). appearance. A hallmark of EMT is the loss of surface The members of the Par complex are central expression of E-cadherin, a central component and a components of signaling pathways that control polarity major organizer of cell–cell junctions and of apical– and proliferation (Figures 1 and 3a) and hence, it is not basolateral polarity (Halbleib and Nelson, 2006; Jeanes surprising that recent data implicate the Par complex in et al., 2008). Decreased expression of E-cadherin often human carcinogenesis (see Aranda et al., 2008). Gene correlates pathologically with tumor grade and stage,

Oncogene The EPP: machineries involved and their hijacking by cancer B Tanos and E Rodriguez-Boulan 6950 Integrin Focal adhesion Numb

EGF Par3 6 Cdc42 P Par aPKC PI3K AQP5 Syntaxin 3 Numb PIP3 Crumbs 3 Annexin 2 PATJ PALS PIP2 Cdc42 Apical E-Cadherin ABP PTEN PI3K TJ FERM actin Dynein TJ loss EGFR Rho aPKC Apical Rho EGF Par6 Par 1 Route PTEN JAM Cdc42 Actin Par3 Rac1 PI3K Nectin Kinesin Dynamics TIAM 1 Par4 ARE Cdc42 Dynein ? EMT Synt Lateral PIP3 E-cadherin AMPK PIP2 Nucleus GIT1 axin 3 actin MTOC Rac1 RE βpix Pax Direction Exo Golgi of migration Ral Exo Focal cyst MTOC PTEN Basolateral route ? cyst LGL adhesion Scribble Cdc42 Synta Golgi DLG Peri- xin 3

Golgi ? 4 Syntaxin EGFR Apical route ? Basolateral route actin Syntaxin 4 Erbin AQP5 Exo Nucleus ErbB2 ? EGFR activation P cyst signal transduction downstream Apical-Basal P Axis AQP3 P Erbin ? Rac1 AQP3 AQP3 Integrin ErbB2 Laminin Apical-basal polarity Anterior/posterior polarity

Numb Lamellipodia EGFR PIP2 Apical route Exocyst EGF PIP3 Basolateral route ErbB2 GIT1 Clathrin βpix coat Actin Integrin binding Paxillin proteins

Figure 6 E-cadherin loss leads to epithelial–mesenchymal transition (EMT) and conversion of apical–basal polarity into anterior– posterior polarity. A variety of mechanisms lead to loss of the tumor-suppressor E-cadherin, which results in EMT. During EMT, the cell acquires the ability to migrate, through reorganization of polarized trafficking, domain identification and 3D organization machinery. In the migrating cell, integrin at focal adhesion complexes signals to reorganize the microtubules (MT) cytoskeleton and to localize the MTOC anterior to the nucleus by Cdc42. Tissue EGF acts as a cytokine through EGFR, organizing the anterior–posterior axis through Cdc42 and Rac. The polarized trafficking machinery reorganizes into an anterior domain, which has some characteristics of the basolateral domain and a posterior domain, with characteristics of the apical domain of epithelial cells.

and E-cadherin inactivating mutations are found in a novel model for the function of E-cadherin and a- variety of breast and gastric carcinoma, supporting its catenin in organizing lateral actin (see ‘Adaptation of denomination as a tumor suppressor (Benjamin and MT and actin cytoskeletons for polarized trafficking’- Nelson, 2008). Tumors may downregulate E-cadherin section). by promoter hypermethylation or by oncogenic activa- Repression of E-cadherin expression is not sufficient tion of the E-cadherin repressors Snail, Slug, Twist, for EMT; the cells must also acquire the ability to SIP1 and ZEB (see reviews by Kang and Massague, migrate in response to cytokines and the capacity to 2004 and Peinado et al., 2007) or, alternatively, through secrete a variety of proteases that digest the extracellular phosphorylation, ubiquitination and degradation of matrix. During EMT, epithelial cells replace their E-cadherin. E-Cadherin has been shown to be ubiqui- apical-basolateral polarity by antero-posterior polarity tinated by the E-3 ligase Hakai, and Hakai over- (Figure 6). In professional chemotaxing cells, such as expression disrupts cell–cell contacts and promotes neutrophils and slime mold, a key event in response to E-cadherin ubiquitination and degradation (Fujita cytokines is the recruitment of PI(3)kinase (PI3K) to et al., 2002). Oncogenes such as Src and the EGFR the leading edge, which results in the synthesis and promote E-cadherin internalization, ubiquitination and accumulation of PIP3; in turn, this leads to the degradation (Palacios et al., 2005; Shen et al., 2008). recruitment of proteins with pleckstrin-homology do- Mutations of a-catenin, a protein that interacts with mains, such as WAVE2, Rac GEFs (Sos, PIX), the Rho the cytoplasmic domain of E-cadherin and contributes GEF Vav1 and Ezrin, which increase actin dynamics to the organization of cortical actin at the lateral and the exclusion of the phosphatase PTEN from the membrane, also increase the risk for cancer but anterior pole (see Merlot and Firtel, 2003; Comer and interestingly, this increase appears to be independent Parent, 2007; Kolsch et al., 2008; Stephens et al., 2008 from E-cadherin (Benjamin and Nelson, 2008). The for comprehensive reviews) (Figure 6). PTEN, which explanation for this observation may be provided by converts PIP3 into PIP2, accumulates at the posterior recent results by Nelson’s laboratory that demonstrate a pole (or uropod), as it binds with higher affinity to PIP2

Oncogene The EPP: machineries involved and their hijacking by cancer B Tanos and E Rodriguez-Boulan 6951 than to PIP3. This phenomenon has also been observed metastatic prostate cancer cells capture the exocyst in cancer cells with the cytokine in this case being EGF through RalGTPases to promote cell migration and produced by macrophages associated with invaded invasion (Spiczka and Yeaman, 2008). blood vessels or invaded tissues (Condeelis et al., If we interpret the lamellipodia of a migrating cell, as 2005). EGFR may also be released from cancer cells an extending basolateral side, we can hypothesize that to the medium in exosome-like vesicles, and taken up by the ‘basolateral’ pathway of polarized epithelial cells is other cells, with the resultant growth activation of the conserved in migrating cancer cells as the route to the recipient cells (Al-Nedawi et al., 2008). Cancer cells leading edge (Figure 6). polarize in response to EGF (Segall et al., 1996; To metastasize, migrating, invasive tumor cells have Condeelis et al., 2005), and show activation of PI3K to leave the primary tumor, cross the extracellular and PIP3 accumulation at the leading edge (Condeelis matrix, and intravasate blood vessels; these events take et al., 2005; Yip et al., 2007). This suggests that the place with the help of matrix metalloproteases, which determinants of the apical–basal organization of polar- direct the disruption of the extracellular matrix and the ized epithelia acquire a new anterior–posterior polarity basement membrane (Condeelis et al., 2005). Matrix generating function in migrating cancer cells (Etienne- metalloprotease accumulation at specific points of Manneville, 2008). contact may require polarized vesicle trafficking. Re- Transformed epithelial cells may partially recover cently, Chavrier and collaborators showed that the their epithelial cytoarchitecture through expression of exocyst regulates the exocytosis of matrix metallopro- E-cadherin (Rajasekaran et al., 1996), integrins or other teases in invasive breast carcinoma cells in combination molecules downregulated in tumors (for example, with the Cdc42 effector IQGAP1, the expression of ) (Weaver et al., 1997; Wang et al., 1998; which is highly increased in a variety of cancers Anders et al., 2003). Recent work indicates that (Rittmeyer et al., 2008; Sakurai-Yageta et al., 2008). interference with the activity of Na,K-ATPase may be a useful strategy to treat some forms of breast cancer Aquaporins and cancer cell migration. Aquaporins (Chen et al., 2006). Interestingly, overexpression of (AQP) have recently emerged as potential regulators of PTEN can revert the migratory phenotype in glioblas- cancer and metastasis (Hansel et al., 2004; Hara- toma cells (Raftopoulou et al., 2004), although in this Chikuma and Verkman, 2008b). AQP are a family of case it is the protein phosphatase (and not the lipid water transporting proteins that can also transport small phosphatase) function of PTEN that is responsible for neutral solutes such as glycerol and urea; their role is this change. essential for the function of a variety of epithelial organs (King et al., 2004; Wang et al., 2006a). In polarized epithelial cells, AQP3 is sorted to the basolateral domain Exocyst, focal adhesions and directional migration. Fo- due to an NH2 YRLL sorting signal (Rai et al., 2006). In cal adhesions are sites where the actin cytoskeleton contrast, AQP5 is retained in the apical surface by a connects with the extracellular matrix through integrins, COOH terminal signal (Wellner et al., 2005) (Figure 6). in the lamellipodia of migrating cells, and are essential AQP3 colocalizes with E-cadherin in early stages of cell– for cell movement and metastatic cell invasion. Integrin cell contact formation (Nejsum and Nelson, 2007) and its trafficking through clathrin-coated pits is regulated by overexpression promotes cell movement (Cao et al., 2006; the endocytic adaptor Numb, which, interestingly, Hara-Chikuma and Verkman, 2008a), probably by interacts with Par3 and is a substrate for aPKC promoting water influx into lamellipodia and transforma- (Nishimura and Kaibuchi, 2007) (Figure 6). Focal tion (Cao et al., 2006; Ji et al., 2008; Verkman et al., 2008). adhesion assembly at the cell front and dissolution at Aquaporin 3 recruitment to E-cadherin-rich cell–cell the rear end is critical for migration in many adherent contacts is impaired upon functional disruption of the cells. Recent work has shown that in metastatic prostate exocyst complex, SNAREs and MT (Nejsum and Nelson, tumor cells, which have lost E-cadherin, the exocyst 2007). AQP3 downregulation decreases the onset of skin localizes to protrusive cell extensions where it colocalizes cancer in mouse models (Hara-Chikuma and Verkman, with syntaxins 3 and 4 (Spiczka and Yeaman, 2008) and 2008b) and could be a key regulator for EMT. Interest- with focal adhesion components (for example, paxillin) ingly, AQP3 is overexpressed in metastatic neoplasms and regulates the polarized delivery of biosynthetic (Hansel et al., 2004) downstream of the EGFR in a PI3 cargo, a5 integrin in particular. Knockdown of the kinase and ERK-dependent manner (Cao et al., 2006). exocyst components Sec5 and Sec6 inhibited this effect. Thus, AQP are attractive proteins to study in the onset of In polarized cells, Ral GTPases regulate basolateral trafficking and cancer and are currently under study as targeting through an interaction with the exocyst potential drug targets for a number of disorders (King complex (Moskalenko et al., 2003). Interestingly, et al., 2004; Wang et al., 2006a). exocyst recruitment to focal complexes and exocyst- mediated migration and invasion requires Ral GTPases, as restoring small interfering RNA knockdown cells Conclusions with a Sec5 construct impaired for interaction with RalA did not restore these effects. In addition, RalA Important advances in our knowledge of the three basic knockdown impaired exocyst localization to protrusive mechanisms underlying the EPP:the polarized traffick- cell extensions and its association with paxillin. Thus, ing, domain identity and 3D organization mechanisms,

Oncogene The EPP: machineries involved and their hijacking by cancer B Tanos and E Rodriguez-Boulan 6952 allow a better integration between these research areas, Acknowledgements which have been studied independently from each other for many years. This opens the possibility of a more We thank Aparna Lakkaraju for her artistic support and mechanistic understanding of epithelial cancers. Future Marcelino Cereijido for useful comments on the manuscript. research is expected to further clarify how these This study was supported by NIH Grants GM 34107 and mechanisms are interconnected, which should result in EY08538 to ERB, by the Dyson Foundation and by the Research to Prevent Blindness Foundation. more and better targets for cancer therapy.

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