(2006) 25, 7538–7544 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc REVIEW Wnt signalling and the

T Akiyama and Y Kawasaki

Laboratory of Molecular and Genetic Information, Institute for Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

The tumour suppressor adenomatous polyposis coli (APC) that carry an APC truncation develop adenomas and is mutated in sporadic and familial colorectal tumours. tumours in the intestine (Oshima et al., 1997; Fodde APC binds to b-, a key component of the Wnt et al., 2001). signalling pathway, and induces its degradation. In The productof the APC is a ubiquitously addition to this role, there is increasing evidence for expressed multifunctional , and interacts with additional roles of APC, including the organization of various cellular , including b-catenin, Axin, cytoskeletal networks. APC interacts with Asef, IQGAP1, -2, end-binding protein 1 (EB1), and accumulates at their plus ends in membrane protru- microtubules and Drosophila discs large (DLG) sions. Also, it has been reported that APC is associated (Figure 1). The mostthoroughly studied function of APC with the plasma membrane in an actin-dependent manner. is its ability to induce degradation of b-catenin, a key Moreover, APC interacts with IQGAP1, an effector of Wntsignalling effector(Cadigan and Nusse, 1997; Bienz Rac1 and Cdc42, and APC-stimulated guanine and Clevers, 2000; Polakis, 2000; Fodde et al., 2001). exchange factor (Asef), a Rac1-specific guanine nucleo- The specific sites in APC to which b-catenin binds are tide exchange factor (GEF). IQGAP1 mediates associa- the three 15-amino- repeats and the more carboxy- tion of APC with cortical actin in the leading edge of proximal seven 20-amino-acid repeats, the latter of migrating and both proteins are required for cell which are responsible for the degradation of b-catenin. polarization and directional migration. APC interacts The majority of the somatic in APC found in with Asef and stimulates its activity, thereby regulating colon are confined to its central region called the the actin cytoskeletal network, cell morphology, adhesion cluster region, and they typically result in the and migration. Truncated mutant APCs present in generation of truncated gene products (Figure 1). In colorectal tumour cells activate Asef constitutively and these cancers, degradation of b-catenin is impaired, contribute to their aberrant migratory properties, which resulting in its accumulation and formation of a stable, may be important for adenoma formation as well as constitutively active complex with T cell factor (TCF), tumour progression to invasive . resulting in the activation of Wnt signalling target . Oncogene (2006) 25, 7538–7544. doi:10.1038/sj.onc.1210063 In addition, in a few percentage of colon tumours, b-catenin has been found to be mutated. Mutated Keywords: tumour suppressor; colon tumour; APC; b-catenin is resistant to APC-mediated degradation, and cytoskeleton; kinesin-2; Asef accumulates in the and constitutively trans- activates target genes. Thus, the accumulation of b-catenin per se is believed to be important for colorectal tumorigenesis. Although it is widely accepted that the ability of APC Introduction to induce degradation of b-catenin is essential for its tumour suppressor function, most mutations in colon Adenomatous polyposis coli (APC) was identified as a tumours occur in APC and only a few percentage of gene responsible for the onset of familial adenomatous mutations occur in b-catenin. In addition, it has been polyposis (FAP), an autosomal dominantly inherited reported that small adenomas with b-catenin mutations that predisposes patients to multiple colorectal do not appear to be likely to progress to larger polyps and cancers (for reviews, see Cadigan and Nusse, adenomas and invasive carcinomas (Samowitz et al., 1997; Bienz and Clevers, 2000; Polakis, 2000; Fodde 1999). It is therefore possible that inactivation of the et al., 2001). Inactivation of APC is also seen in the additional APC functions might also be important for majority of sporadic colorectal tumours and is an early colorectal tumorigenesis (Fodde et al., 2001). In this event in tumorigenesis. In mouse models, heterozygotes regard, it is interesting that APC has recently been revealed to interact with various cellular proteins other than b-catenin, including Asef, IQGAP and kinesin-2, Correspondence: Professor T Akiyama, of Molecular and and to regulate cytoskeletal networks, thereby control- Genetic Information, Institute for Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, ling and (Jimbo et al., 2002; Japan. Kawasaki et al., 2003; Watanabe et al., 2005). In this E-mail: [email protected] review, I will focus on the function of APC in the Wnt signalling and the actin cytoskeleton T Akiyama and Y Kawasaki 7539 cells and polarized cultured epithelial cells (Miyashiro a -catenin-binding sites Oligomerization et al., 1995; Nathke et al., 1996; Rosin-Arbesfeld et al., Arm 15-a.a. 20-a.a. Basic TSV 2001; Langford et al., 2006). The association of APC repeats repeats with the plasma membrane is reported to depend on the 1 2843 actin cytoskeleton but not on microtubules: APC Axin-binding sites associated with the plasma membrane is not detected Asef Mutation Cluster Region EB1 DLG when the actin cytoskeleton is depolymerized by IQGAP1 Microtubules A in Madin–Darby canine (MDCK) Kinesin2 cells (Rosin-Arbesfeld et al., 2001; Langford et al., b 2006). On the other hand, depolymerization causes a redistribution of APC from microtubule plus ends to the plasma membrane. Furthermore, drug Figure 1 Structure of the APC protein. (a) Wild-type full-length washout experiments suggest that APC is delivered APC. (b) A typical truncated mutant APC in colorectal tumour continuously to the plasma membrane by a dynamic cells. actin-dependent process. Thus, it appears that there are two distinct peripheral APC pools that are in equili- regulation of cytoskeletal networks, the actin cyto- brium with each other in these cells: actin-dependent in particular, and discuss its relevance to membrane-associated APC and microtubule-dependent tumorigenesis. APC clusters. Interestingly, restoration of expression of full-length APC in a colorectal cell line has been shown to promote cell–cell adhesion (Faux et al., 2004). Furthermore, genetic analysis of the Drosophila homo- Clustering of APC at membrane protrusions logue of APC, E-adenomatous polyposis coli/d adeno- matous polyposis coli (E-APC/dAPC2), indicates that APC has been detected at a number of intracellular sites: Drosophila APC is also associated with the plasma the plasma membrane, cytoplasm, , kineto- membrane in an actin-dependent manner and is chores and nucleus (for a review, see Bienz, 2002). important for cellular adhesion (for a review, see Bienz Although there are a number of conflicting reports, re- and Hamada, 2004). In addition, truncated mutant evaluation of various commonly used anti-APC anti- APCs presentin colorectalcancer cells are mainly bodies has revealed that the bulk of APC resides in the cytoplasmic and are not able to associate with the cytoplasm (Brocardo et al., 2005). The moststriking plasma membrane (Polakis, 2000; Rosin-Arbesfeld feature of APC localization is its accumulation in et al., 2001; Brocardo et al., 2005), suggesting that clusters near the distal ends of microtubules at the APC associated with the plasma membrane may edges of migrating epithelial cells (Nathke et al., 1996; contribute to its tumour suppressor function. More Reilein and Nelson, 2005) (Fig. 2). Furthermore, APC recently, rather striking evidence has been provided that has been shown to move along microtubules and argues that much of the proposed cytoskeletal functions accumulate at their growing plus ends in an ATP- of APC (atleastin flies) were deduced from gain-of- dependentmanner (Mimori-Kiyosue et al., 2000a). function mutants, and may thus not be relevant in a APC interacts with microtubules directly via the basic normal physiological setting. Although complete loss of region in its carboxy terminus (Munemitsu et al., 1994; dAPC and E-APC/dAPC2 results in strong activation of Nathke et al., 1996; Zumbrunn et al., 2001) and Wingless signaling, it does not substantially alter indirectly via end-binding protein 1 (EB1), forming a -based adhesion as well as cell viability, spindle stabilizing complex at microtubule plus ends (Su et al., morphology, orientation and selection of division plane, 1995; Askham et al., 2000; Mimori-Kiyosue et al., as predicted from previous studies (McCartney et al., 2000b). Interestingly, APC and EB1 bind to mDia to 2006). In addition, truncated mutant APCs found in stabilize microtubules downstream of Rho and promote tumours have been shown to possess dominant effects (Wen et al., 2004). Also, APC interacts on cytoskeletal regulation. with kinesin-2 and this interaction is essential for transportation of APC along microtubules (see below; Jimbo et al., 2002). of APC with glycogen synthase kinase-3beta (GSK-3b) can cause The mechanism of APC trafficking to membrane APC to dissociate from microtubules (Zumbrunn et al., protrusions 2001) and prevents its accumulation at cell protrusions (Etienne-Manneville and Hall, 2003). For migration of APC along microtubules to the tips of membrane protrusions, kinesin-2 is believed to play an essential role (Jimbo et al., 2002). Kinesin-2 is one of the Actin-dependent association of APC with the mostubiquitouslyexpressed thekinesin superfamily plasma membrane proteins (KIFs) and has been implicated in the of membrane-bound It has also been reported that APC is associated with the and protein complexes in various (for reviews, lateral plasma membrane in mouse intestinal epithelial see Scholey, 1996; Miki et al., 2005). Kinesin-2 is a

Oncogene Wnt signalling and the actin cytoskeleton T Akiyama and Y Kawasaki 7540 heterotrimeric complex composed of a KIF3A/3B It should be noted here that ‘migration in the intestinal heterodimer and an adaptor protein, the kinesin super- ’ refers to the upward displacement of a whole family-associated protein 3 (KAP3). The KIF3A/3B adhesive cell sheet with stable and well-developed heterodimer possesses a plus end-directed microtubule adherens junctions, which is different from the move- sliding activity that uses derived from ATP ment of individual cells on a in cell culture. . APC interacts with KIF3A/3B via an association with KAP3 (Jimbo et al., 2002). Dominant- negative experiments show that this interaction of APC Role of APC in polarization of migrating astrocytes with kinsein-2 is required for its accumulation in clusters. During scratch-induced migration of astrocytes, Cdc42- Interestingly, mutant APCs derived from cancer cells mediated activation of par, protease-activated are unable to accumulate efficiently in clusters, although 6-protein kinase Cz (Par6-PKCz) leads to phosphoryla- these mutant proteins possess the tion and inactivation of GSK-3b, which causes APC to that is responsible for the interaction with associate with microtubule plus ends at the leading edge kinesin-2 (Jimbo et al., 2002). Thus, the interaction of (Etienne-Manneville and Hall, 2003). RNA interference APC with KAP3 via the armadillo repeat domain (RNAi) and dominant-negative experiments show that appears not to be sufficient for cluster formation, APC clustering at microtubule ends is required for whereas the downstream region that is frequently microtubule network polarization in migrating astro- mutated in colorectal cancers is required for the cytes. Furthermore, in migrating astrocytes, APC is accumulation of APC. It is possible that APC requires induced to associate with DLG, an orthologue of the interaction with another via this region for Drosophila tumour suppressor discs large protein functional movement along the microtubules. Indeed, it (Etienne-Manneville et al., 2005). The carboxy-terminal has recently been shown that the interaction of APC S/T-X-V (S, Ser; T, Thr; X, any ; V, Val) of with b-catenin is required for its efficient targeting to APC is responsible for binding to the PDZ domain membrane clusters (Sharma et al., 2006). Systematic of DLG (Matsumine et al., 1996). The APC–DLG deletion mapping shows that a minimal core sequence interaction appears to link the basal plasma membrane comprising amino 1–2226 determines optimal and microtubule plus ends to promote microtubule targeting of APC to membrane clusters. Thus, the anchoring. interaction of APC with DLG or EB1 is not required for APC membrane targeting. Also, although the basic region was reported to interact with microtubules, this region is dispensable for the accumulation of APC Role of APC–IQGAP complex in cell polarization (Mimori-Kiyosue et al., 2000a; Zumbrunn et al., 2001). It remains to be determined how b-catenin regulates It has recently been shown that the accumulation of APC targeting to membrane clusters. APC at lamellipodia is mediated through a direct The function of the APC clusters may be dynamically interaction of APC with IQGAP1, and the APC– regulated in response to extracellular signals. For IQGAP complex is implicated in cell polarization and example, Wnt-3a induces redistribution of b-catenin migration (Watanabe et al., 2005). On the other hand, not only to the nucleus but also to the APC clusters in IQGAP1 is not involved in the accumulation of APC at membrane protrusions (Jimbo et al., 2002). Ithas been the membrane clusters. IQGAP1 interacts with Rac1 reported that membranes containing APC clusters are and Cdc42 and is considered to act as an effector of involved in cell migration in response to wounding of these guanosine 50-triphosphatases () (Briggs epithelial monolayers, as well as to the addition of the and Sacks, 2003; Mateer et al., 2003). IQGAP1 motorgen , suggesting that possesses a region homologous to GTPase-activating APC may function in cell migration (Nathke et al., 1996; proteins (GAPs), butdoes notactas a GAP. IQGAP1 McCartney and Peifer, 2000). Indeed, in migrating directly binds and crosslink actin filaments in the astrocytes, the association of APC with the plus ends presence of activated Rac1 and Cdc42 (Bashour et al., of microtubules at the leading edge has been shown to 1997). Also, IQGAP1 interacts with cytoplasmic linker be essential for cell polarization (see below, Etienne- protein 170 (CLIP-170), a microtubule-stabilizing pro- Manneville and Hall, 2003). Moreover, itis possible that tein (Fukuta et al., 2002). APC is involved in the upward migration of intestinal APC interacts directly via its armadillo repeat domain epithelial cells from the crypt to the villus tip. Consistent to IQGAP1 (Watanabe et al., 2005). Moreover, with this notion, it was reported that forced expression IQGAP1 and APC interact with activated forms of of APC induces disordered cell migration in the Rac1 and Cdc42 and these four proteins accumulate at (Wong et al., 1996). Also, inactiva- the leading edge in Vero fibroblast cells during migra- tion of APC completely abrogates migration along the tion into a wound. RNAi and dominant-negative crypt-villus axis (Sansom et al., 2004). In addition, the experiments show that IQGAP1 and APC localize to fact that mutated APCs in cancer cells are deficient in the leading edge interdependently; knockdown of either their ability to accumulate in clusters suggests that the protein inhibits the accumulation of the other protein. absence of the APC clusters in colorectal tumour cells Furthermore, depletion of IQGAP1 or APC prevents might be related to their aberrant migratory properties. the formation of the actin meshwork at the leading edge

Oncogene Wnt signalling and the actin cytoskeleton T Akiyama and Y Kawasaki 7541 and inhibits cell migration. Constitutively active IQ- Inactive form Active form GAP1 induces multiple leading edges and provides the Rac Rac anomalous accumulation sites of APC in an actin filament-dependent manner and inihibits proper cell migration. Thus, both APC and IQGAP1 appear to be required for the stabilization and polarized organization ABR of the actin meshwork at the leading edge for migration APC Asef Asef of Vero cells. In addition, depletion of IQGAP1 or APC ABR DH PH disrupts localization of CLIP-170. Hence, APC, IQ- DH PH GAP1 and CLIP-170 may form a complex that links the actin cytoskeleton and microtubule dynamics during cell Figure 3 Structure and function of Asef. In the absence of APC, polarization and directional migration. IQGAP1 inter- the GEF activity of Asef is negatively regulated by its NH2- terminal region. APC may activate the GEF activity of Asef by acts with truncated mutant APC in colon cancer cells. binding to its NH2-terminal region and relieving negative regula- Functional significance of this interaction remains to be tion. A mutant form of Asef lacking the NH2-terminal region investigated. shows strong GEF activity even in the absence of APC. ABR, APC-binding region; DH, Dbl domain; PH, Pleckstrin domain.

Role of APC–Asef complex in aberrant migration of tumour cells

APC is also involved in the regulation of actin dynamics via its interaction with Asef (Kawasaki et al., 2000, 2003). Asef was identified as a guanine-nucleotide exchange factor (GEF) specific for Rac1 that interacts with APC. Asef contains the Dbl homology (DH), Pleckstrin (PH) and Src homology 3 (SH3) domains (Figure 3). The APC-binding region resides in the NH2- terminal region upstream of the SH3 domain. APC binds to Asef via its armadillo repeat domain and

Figure 4 APC organizes cytoskeletal networks and regulate cell migration. (a) The APC–Asef complex and the APC–IQGAP1 complex activated by external signals regulate the actin networks Figure 2 APC (red) localizes to clusters near the distal ends of and stimulate cell migration. (b) Truncated mutant APCs present in microtubules (green) at the edges of migrating epithelial MDCK colorectal tumour cells activate Asef constitutively and contribute cells. to their aberrant migratory properties.

Oncogene Wnt signalling and the actin cytoskeleton T Akiyama and Y Kawasaki 7542 enhances its GEF activity and thereby stimulates Fodde et al., 2001). In addition, the armadillo repeat Asef-mediated cell flattening, membrane ruffling and domain is the most highly conserved feature in APC, lamellipodia formation in MDCK cells (Figure 4). In suggesting that it is also essential for the function of the absence of APC, Asef exhibits very weak GEF wild-type APC. activity. On the other hand, a mutant form of Asef Intriguingly, Asef is activated in colorectal tumour lacking the NH2-terminal region shows strong GEF cells containing truncated mutant APC but not in those activity even in the absence of APC. Thus, the containing intact APC (Kawasaki et al., 2003). Also, GEF activity of Asef is negatively regulated by its unlike truncated mutant APC, full-length APC does not NH2-terminal region. APC may activate the GEF enhance Asef-induced cell migration when overex- activity of Asef by binding to its NH2-terminal region pressed exogenously in MDCK cells. Thus, wild-type and relieving negative regulation. full-length APC appears not to be an efficient activator Asef decreases E-cadherin-mediated cell–cell adhesion of Asef unless it is activated by some signal. when overexpressed in MDCK cells (Kawasaki et al., 2003). Asef decreases amounts of E-cadherin and b-catenin localized at the sites of cell–cell contact and increases amounts of E-cadherin and b-catenin localized Conclusion in the cytoplasm. This is consistent with previous reports showing that Rac1 causes the disruption of E-cadherin- APC has been characterized most extensively in the mediated cell–cell contacts (Keely et al., 1997; Sander context of the Wnt signalling pathway. However, recent et al., 1998). However, Rac1 has also been reported to studies have revealed that APC is involved in the increase E-cadherin-mediated cell–cell contacts (Hordijk regulation of cytoskeletal networks (Figure 4). APC et al., 1997; Takaishi et al., 1997; Braga et al., 2000), and is transported along microtubules in a kinesin-2- and these opposing effects of Rac1 have been shown to be b-catenin-dependent manner and accumulates in clusters dependent on the cell substrate (Sander et al., 1998). at microtubule plus ends at the edges of migrating Thus, it is possible that the effects of Asef are influenced epithelial cells. APC in clusters may be involved in by the matrix upon which cells are plated. microtubule stabilization, polarization and attachment Overexpression of Asef promotes migration of epithe- of microtubules to membrane cortex. APC also activates lial MDCK cells. This activity is stimulated by truncated Asef and regulates the actin cytoskeletal networks, cell mutant APCs expressed in colorectal tumour cells morphology, adhesion and migration. The accumu- (Kawasaki et al., 2003). RNAi and dominant-negative lation of APC at lamellipodia in migrating cell is experiments show that both mutated APC and Asef are IQGAP1-dependent, and the APC–IQGAP1 complex required for migration of colorectal tumour cells is involved in cell polarization and migration. The expressing truncated APC. Thus, the truncated mutant relationship between the APC–Asef complex and the APCs present in colorectal tumour cells appear to APC–IQGAP complex remains to be determined. For activate Asef constitutively and cause decreased cell–cell example, the simplest possibility is that the APC– adhesion and aberrantmigratory properties(Figure 4). IQGAP1 complex functions downstream of the APC– Interestingly, it has been reported that the proliferation Asef complex. Furthermore, truncated mutant APCs rate of early adenoma cells in APC knockout mice is present in colorectal tumour cells activate Asef consti- similar to that of the normal crypt epithelium, but tutively and contribute to their aberrant migratory adenoma cells lack directed migration along the crypt- properties. Thus, compounds that target the APC–Asef villus axis (Oshima et al., 1997). Thus, aberrant complex might be good candidates for novel antitumour migratory behaviour due to Asef activation by truncated reagents. Understanding the role of APC in the mutant APC may be important for adenoma formation regulation of cytoskeletal networks would give new as well as tumour progression to invasive malignancy. insights into the strategy to generate novel antitumour This hypothesis appears to be consistent with the fact reagents. that the Asef-binding region, that is, the armadillo repeat domain, is invariably retained in truncated Acknowledgements mutant APCs in colorectal tumours (Cadigan and Nusse, 1997; Polakis, 2000; Bienz and Clevers, 2000; We thank M Bienz for valuable comments on this manuscript.

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