Histol Histopathol (2000) 15: 251-260 Histology and 001: 10.14670/HH-15.251 http://www.hh.um.es Histopathology Cellular and Molecular Biology

Invited Review

Catenins and their associated protei ns in colorectal cancer

E.L. Tucker and M. Pignatelli Department of Pathology and Microbiology, University of Bristol, School of Medical Sciences, University Walk, Bristol, United Kingdom

Summary. Colorectal cancer is the second most Thomlinson , 1997). B-catenin has been shown to be common cause of cancer mortality in the western world. homologous to the armadillo protein found in Colorectal cancer has been well studied, and the genetic Drosophila, suggesting that the catenins have been steps involved in the adenoma to carcinoma sequence evolutionarily conserved (Reviewed by Ilyas and have been well elucidated. The first genetic alteration, Thomlinson, 1997). Of the catenins, y-catenin and /3- found in 85 % of adenomas, are mutations in the catenin share the greatest homology, with the y-catenin adenomatous polyposis coli (APC) gene. However, the arm repeat region sharing 85 % homology with B-catenin consequences of this and the exact function of APC in (Troyanovsky et ai., 1996). the colon is not fully understood. It has been suggested The catenins are found to associate with a large that APC could function through its regulation of /3- number of proteins (Fig. 1) namely APC, Cadherins (in catenin, an ubiquitous cytoskeletal protein with multiple adhesive junctions), Epidermal growth factor receptor binding specificities resulting in diverse functions (EGFR), C-erb2, Tyrosine Kinases (v-src) and including cell growth, adhesion, and migration. Any Phosphatases (Vandate, PTP IB and PTP.u), vinculin, (X­ change in these associations may playa role in actinin, fascin, paxillin, axin , ezrin, GSK-3/3 and the colorectaI cancer development and progression. transcription factors TCF (T cell Factor) and LEF (Leukocyte enhancing factor) (Brady-Kalnoy et ai., Key words: Cadherins, APC, EGFR, Cell adhesion, Cell 1995; Reviewed by lawhari et aI., 1997a; Reviewed by signalling Nakamura, 1997; Reviewed by Yamada and Geiger, 1997; Balsamo et ai., 1998; Hiscox et aI., 1998; Reviewed by Polakis, 1998). Catenins - An introduction /3-catenin is the most widely studied of the catenins and has been shown to have diverse binding specificities The catenins are a family of cytoplasmic proteins (Fig. lA) and as a consequence functionality, with it that are involved in a number of cellular processes being involved in cell adhesion (Via binding to members namely adhesion and signal transduction. This multi gene of the Cadherin family) (Peifer et aI., 1992; Nagafuchi et family comprises of a- (102Kda), /3- (92Kd), y­ ai.,1994; Reviewed by lawhari et aI., 1997a; Lewis et (Plakoglobin) (83Kd) catenin, and p120(CAS), and are aI. , 1997), cell signalling (via the wingless-Wnt pathway found on chromosomes 5q, 3p and 17q respectively, the (Fig. IB) and receptor and non-receptor tyrosine kinases chromosomal location of p120 is currently unknown (Fig .. lC) (Reviewed by I1yas and Thomlinson, 1997; (Peifer et aI., 1992; Liu et ai., 1997; Reviewed by Smith Reviewed by Nusse et ai., 1997; Muller et aI., 1998) and and Pignatelli, 1997). The catenins have three cell motility (B-catenin complexed with APC has been structurally distinct regions; an amino- and carboxyl­ found associated with the microtubule system (Barth et terminal domain, and a hydrophobic region comprising aI. , 1997a,b). Many of the activities of B-catenin are of arm repeats, each consisting of 42 amino acids. The regulated through the control of these functional number of these arm repeats vary between the catenins, complexes (Shibamoto et aI., 1995; Reviewed by Ilyas /3- and y-catenin having 13 arm repeats , whereas p120 and Thomlinson, 1997). has 11 (Reynolds et ai., 1994; Ruiz et aI., 1996; y-catenin, like B-catenin has also been shown to Troyanovsky et ai., 1996; Reviewed by Ilyas and associate with APC, E-Cadherin and TCF, via its ARM repeat region (Troyanosky et aI., 1996) and is Offprint requests to: Professor M. Pignatelii, Department of Pathology phosphorylated by both receptor tyrosine kinases and and Microbiology, University of Bristol, School of Medical Sciences, non-receptor tyrosine kinases (Ruiz et aI., 1996), but University of Walk, Bristol, BS8 HD, United Kingdom. e-mail: unlike B-catenin, y-catenin is also found associated with [email protected] the desmosomal proteins desmoglein and desmocollin 252 Catenins and associated proteins in colorectal cancer

Fig. I A

Fig. IB

Ca 2c... ---1~"

(A)

F-acti.n F-actin

E-Cadhcrin J • (D) ~PO. olher + E-C dhcrin (fI R U~ ••••

Prolcosomc Degraded C"lenin Degraded Catcll.in Protcosomc

Fig 1. A. E-Cadherin forms calcium dependent homotypic interactions with adjacent cells. The cytoplasmic domain of E-Cadherin forms a complex with either B-catenin/a-catenin or y-catenin/a-catenin , with a-catenin linking it to the actin cytoskeleton by binding to either a-actinin, vinculin or fascin (represented as vinculin in this diagram). (B) This shows the Wnt-l pathway which leads to the inhibition of GSK-3B. GSK-3B is involved in phosphorylating the catenins which targets them for degradation. Loss of this activity leads to stabilisation and accumulation of the catenins in the cytoplasm. (C) An increase in cytoplasmic catenins results in the binding of B- or y-catenin with the transcription factors hTCF-4/hLEF-1 (in the case of colon) , which leads to translocation into the nucleus. (D) In the nucleus this transcription factor complex binds to the DNA resulting in gene expression, one such target is the c-Myc gene. (E) When the catenins are phosphorylated, they undergo ubiquitin dependent proteosomal degradation. (F) Catenins can be phosphorylated by a number of different cellular complexes including c-Src and EGFR. B. Shows the Wnt/Wg signalling pathway (A) Wnt binds to its receptor frizzled which in turn activates Dishevelled (dsh) ; (B) Activated dsh in turn inhibits GSK-3B thus preventing B- or y-catenin degradation; (C) This results in an increase in cytoplasmic catenins, which can then bind to the transcription factors TCF/LEF, enter the nucleus and bind to the DNA; (D) This results in enhanced or altered gene expression, the exact target genes have not been well elucidated, but recently it has been shown that c-Myc is one. C. Shows the role of phosphorylation within the cell; (A) APC-Catenin-GSK- 3B-Axin are all thought to form a complex resulting in the phosphorylation and subsequent degradation of catenins; (B) v-Src has been shown to phosphorylate B- and a-catenin; (C) TFF3 (Trefoil Factor 3) has been shown to phosphorylate B-catenin and the EGFR; (D) EGFR appears to phosphorylate both G- and y-catenin, and to interact with B-catenin; (E) HGF/SF has been shown to phosphorylate p120, B-catenin and y-catenin; (F) Represents the functional dissassembly of the E-Cadherin adhesive unit due to phosphorylation of the catenins. b: Beta Catenin; g: Gamma

ProICOSOl1lC Catenin; a: Alpha Catenin ; v: Vinculin. 253 Catenins and associated proteins in colorectal cancer

(Ruiz et aI., 1996; Troyanosky et aI., 1996). adhesive functions (Reviewed by Smith and Pignatelli, a-catenin has also been shown to bind directly to a 1997). number of proteins, including y-catenin and B-catenin, B-catenin has been shown to mediate epithelial cell vinculin, a-actinin and v-src. It has also been shown to adhesion by binding to the cytoplasmic domain of E­ bind indirectly to E-cadherin via y-catenin and B-catenin, Cadherin, and then linking E-Cadherin to the actin having an essential role in adhesion (Oyama et aI., 1994; cytoskeleton by binding to a-catenin via its amino Reviewed by Barth et aI., 1997b; Reviewed by terminal, or by associating directly with the actin Hirohashi, 1998). Sequence analysis of cloned a-catenin cytoskeleton (Reviewed by Smith and Pignatelli, 1997; has revealed two isoforms of the protein, a-E which is Reviewed by Polak is, 1998). This E-cadherin/B-catenin found in epithelial tissue and a-N which is found in complex has been shown to also interact with the a2- neuronal tissues (Hirano et aI., 1992; Hoschuetzky et aI., integrin, a collagen receptor, an interaction influenced by 1994). stimulation with TGF-a (Transforming growth factor p120 (Cas, cadherin-associated src substrate) was alpha) and hSP (Human spasmolytic polypeptide), which originally identified as one of several substrates of the alters cell migration and cell matrix adhesion (Jawhari et tyrosine kinase pp60src (Reynolds et aI., 1994; Liu et aI., aI., 1997a,b; Reviewed by Pignatelli, 1998). B-catenin is 1997). Subsequently it was shown to associate with /3- also found in association with APC, where APC together catenin and E-Cadherin (Liu et aI., 1997). Unlike f3- and with GSK-3B is thought to regulate B-catenin by y-catenins, p120 has been shown to have at least 4 controlling its free cytoplasmic levels (Reviewed by different isoforms, which appear to be expressed Gordon, 1998; Reviewed by Polakis, 1998). E-Cadherin differentially in a variety of cell types (Reynolds et aI., is never found in association with these APC/B-catenin 1994). complexes and it has been shown that APC and E­ Cadherin both compete for the binding to B-catenin in Catenins in cell adhesion transient expression assays (Hulsken et aI., 1994; Reviewed by Barth et aI., 1997b). Cell adhesion to neighbouring cells (via cadherin­ y-catenin has also been shown to bind to E-cadherin catenin) and the extracellular matrix (via Integrins) plays at adherens junctions in epithelial cells (Knudsen and an important role in cell motility, growth, differentiation Wheelock, 1992; Troyanovsky et aI., 1996; Reviewed by and survival (Watabe et aI., 1994; Reviewed by Ben Ze­ Barth et aI., 1997b), with evidence pointing to B-catenin ev and Geiger, 1998; Reviewed by Efstathiou and and y-catenin forming mutually exclusive complexes Pignatelli, 1998). Perturbation of any of these (Nagafuchi et aI., 1994; Reviewed by Barth et aI., interactions results in changes in intercellular adhesion 1997b). The domains of y-catenin and B-catenin which and cell transformation (Valizadeh et aI., 1997; Guilford associate with cadherins and a-catenin have been et aI. , 1998). mapped. It was found that while the central arm repeats The cadherins are a family of transmembrane of catenins interact with cadherins, the amino terminal glycoproteins that mediate calcium dependent adhesion domain, as well as the first arm repeat bind to a-catenin and share common amino acid sequences. They have (Ruiz et aI., 1996). Unlike B-catenin, y-catenin is also been divided up into more than 10 subclasses dependent found associated with desmosomal cadherins, mainly on their tissue distribution, including E- (Epithelial), N­ desmoglein (Dsg) and desmocollin (Dsc) (Troyanovsky (Neuronal) and P-cadherin (Gagliardi et aI., 1995; et ai, 1996; Reviewed by Barth et aI., 1997b). In Shiozaki et aI. , 1996; Reviewed by Smith and Pignatelli, desmosomes, plakoglobin has been shown to specifically 1997). A novel member of the family, Fat, found in interact with Dsg and Dsc, but no association with a­ Drosophila suggests that cadherins have been catenin or p120{Cas) in these complexes have been evolutionary conserved (Mahoney et aI., 1991). observed (Troyanovsky et aI., 1996). Ruiz et al. (1996) The E-Cadherin gene (CDH 1), found on chromo­ generated a null mutation of the plakoglobin gene in some 16q22.1, was first isolated by Berx et al. (1995), mice to he lp elucidate the role of plakoglobin in and was found to span 100Kb consisting of 16 exons development and consequently cell adhesion. They (Berx et a I. , 1995). E-Cadherin (also known as found that homozygous null mice died between days 12- uvomorulin, L-Cam, Arc 1 and cell-CAM 120/ 80 16 of embryogenesis due to defects in heart function. (Gagliardi et aI., 1995) is found at adherens junctions These defects revealed loss of desmosome formation, forming homophilic interactions between cells in with increased adherens junction formation, Thus epithelial tissues (Gagliardi et aI., 1995; Shiozaki et aI., suggesting that plakoglobin is an essential component of 1996; Reviewed by Smith and Pignatelli, 1997). It is myocardial desmosomes, playing a crucial role in the linked to the actin cytoskeleton via its interactions with sorting of desmosomal and adherens junction the cytoplasmic proteins the catenins (a-, B-, y-catenins, components, and as a consequence tissue architecture of p120), a-actinin and vinculin (Barth et al.,1997a; the heart (Ruiz et aI., 1996; Reviewed by Barth et aI., Hulsken et aI., 1994). Changes in the expression levels 1997b; Lewis et aI., 1997). Recent observations of of cadherins and catenins have been shown to affect cell­ Troyanovsky et al. (1996) suggest that it is the arm cell ad hesion (Reviewed by Adams and Nelson, 1998), repeat region of plakoglobin that mediates these thus suggesting that catenins mediate E-cadherin functions. The Arm re peat region was shown to 254 Catenins and associated proteins in colorectal cancer

comprise of two functionally distinct regions, with the Nelson, 1998). Alternatively as p120 is a major src first 5 arm repeats involved in specific binding of E­ substrate and is phosphorylated in response to ligand cadherin and desmoglein, with the remaining arm stimulation of receptor tyrosine kinases (Reynolds et a!., repeats being involved in targeting plakoglobin/cadherin 1994; Liu et aI., 1997; Valizadeh et a!., 1997), it may act complexes to junctional structures (Troyanovsky et aI., to mediate the regulation of cadherin adhesion by these 1996). signalling pathways (Hoschuetzky et a!., 1994; Reynolds a-catenin, an actin bundling protein in vitro, has et al,. 1994; Reviewed by Barth et al.,1997b). been shown to be involved in adhesion by linking A number of other proteins have been shown to be cadherin to the actin cytoskeleton (Reviewed by Adams associated with the E-Cadherin/catenin complex (Liu et and Nelson, 1998; Weiss et aI., 1998). Biochemical aI., 1997). Non-receptor and receptor tyrosine kinases, evidence suggests that a-catenin does not directly including EGFR, and receptor tyrosine phosphatase, associate with cadherins but binds to the cytoplasmic PTP.u and PTP 1B, alter the phosphorylation status domain of cadherins via J3-catenin or y-catenin, of components in the cadherin-catenin complex mediating the connection between the cadherin-catenin (Hoschvetzky et aI., 1994; Brady-Kalnoy et aI., 1995; complex with the actin cytoskeleton (Hoschuetzky et aI., Liu et aI., 1997; Balsamo et aI., 1998; Soler et aI., 1998). 1994; Oyama et al., 1994; Reviewed by Efstashiou and The phosphorylation of tyrosine residues in the 13-catenin Pignatelli, 1998; Reviewed by Hirohashi, 1998). Simcha protein was observed in mammalian fibroblasts et al (1998) showed that a-catenin, along with cadherins, transduced with v-src, where src was shown to were able to sequester 13-catenin in the cytoplasm, thus accumulate at adherens junctions and increase tyrosine inhibiting its transcriptional activity. Amino acid phophorylation levels (Tsukita et aI., 1991). Each of the sequencing revealed that a-catenin is homologous to different kinases/phosphatases have different specificity vinculin, a protein found localised at adherens junctions for cadherin/catenin complex components (Fig. 1C). For and focal contacts where it functions as a cytoplasmic example TFF3 (Trefoil factor 3) has been shown to anchorage for receptors of extracellular matrix proteins cause rapid and specific tyrosine phosphorylation of 13- (Nagafuchi et aI., 1994; Reviewed by Shiozaki et aI., catenin and epidermal growth factor receptor, but not E­ 1996; Weiss et aI, 1998). Adams and Nelson (1998) have Cadherin or a-catenin (Liu et aI., 1997; Efstathiou et aI., thus suggested that vinculin may bind to the Cadherin/13- 1998). Hepatocyte growth factor/scatter factor (HGF/SF) catenin or Cadherin/y-catenin complex, thereby has been shown to induce the phosphorylation of p120, compensating for the lack of a-catenin. Furthermore aE­ 13- and y- catenin (Shibamoto et aI., 1995). Hoschuetzky catenin has been shown, in association with vinculin, to et a!. (1994) clearly demonstrated that EGF results in the playa role in the assembly of apical junctional complex tyrosine phosphorylation of 13- and y- catenin, whereas in epithelia (Watabe-Uchida et aI., 1998). with v-src the primary targets are a-, (3- catenin and p120, like 13- and y-catenin, has also been shown to p120(Cas) (Hoschuetzky et a!., 1994; Liu et aI., 1997; bind directly to E-Cadherin, and is found associated with Valizadeh et aI., 1997). Hoschuetzky et al. (1994) also both the E-Cadherin/13 -catenin and the E-Cadherin/y­ demonstrated that this is also cell type specific. The Catenin complexes (Reynolds et aI., 1994; Jou et aI. , precise biological consequence of this tyrosine 1995; Shibamoto et aI., 1995; Troyanovsky et aI., 1996; phosphorylation of catenins is currently unknown. It has Reviewed by Barth et aI., 1997b). But unlike (3- and y­ been suggested that it inactivates cadherin-mediated catenin p120 does not bind a-catenin (Jou et aI., 1995). adhesion, this is supported by the fact that The binding site of p120 on E-Cadherin, although in phosphorylated 13-catenin is exclusively found in close proximity, is distinct from that of its structural detergent soluble fractions, suggesting that tyrosine homologues 13- and y-catenin (Valizadeh et aI., 1997), phosphorylation induces disassembly of the cadherin­ although other groups have suggested otherwise catenin complex from the actin filament network, (Reynolds et aI., 1994; Shibamoto et aI., 1995). The although other explanations are possible (Hoschuetzky et exact role of p120 catenin in cell to cell adhesion a!., 1994; Reviewed by Barth et aI., 1997b; Liu et a!., remains a matter of debate (Reviewed by Adams and 1997). This loss of cad herin-mediated adhesion, through Nelson, 1998). Skoudy et a!. (1996) results suggest that tyrosine phosphorylation, resulting in destabilisation of p120 catenin plays a role in regulating E-Cadherin. the adherens junctions, was shown to induce cell Cadherin-p120 complexes maybe formed initially to dispersion/migration (Liu et aI., 1997). increase the rates of homotypic recognition and binding between cadherin molecules on two adjacent cells Catenins in cell signalling (Reviewed by Adams and Nelson, 1998). Subsequently a-catenin, 13-catenin and the actin cytoskeleton may Studies in Drosophila have revealed the role of the associate with and stabilize the cadherin-p120 cluster armadillo protein in the wingless-wnt pathway that and, thereby, strengthen the cell-cell adhesion. This mediates cell fate determination (Reviewed by Barth et interpretation of their results would explain why cells aI., 1997b). As 13-catenin is highly homologous to the that overexpress cadherin, which have an intact 13- armadillo protein it was proposed that l3-catenin would catenin binding region, but lacks the p120 binding also playa part in the vertebrate wingless-wnt pathway region, adhere very slowly (Reviewed Adams and (Reviewed by Ilyas and Thomlinson, 1997; Reviewed by 255 Catenins and associated proteins in colorectal cancer

Nusse et al.,1997), which subsequently revealed the group sex determining (sry) family of transcription importance of B-catenin in embryogenesis (Reviewed by factors, that induce bends in the DNA helix on binding. Barth et aI., 1997b). These transcription factors, LEF and TCF, by themselves Catenins have already been shown to be involved in are poor promoters of transcription. However when /3- the tyrosine kinases a nd phosphatase signalling catenin is complexed with these transcription factors the pathways that regulate cell adhesion (Barth et aI., DN A bend produced by these transcription factors 1997a), with evidence pointing to cellular adhesion itself (Behrens et aI., 1996) is altered, which is thought to mediating signalling pathways (Benze've, 1997). 13- enhance and alter gene transcription (Reviewed by catenin was also found to be a major player in the Moon and Miller, 1997; Reviewed by Nusse et aI., Wnt/wingless signalling pathway. Hinck et al. (1994a,b) 1997). The target genes of these transcription factors showed that Wnt-1 expression resulted in the have long been sort after, but are thought to be involved accumulation of l3-catenin and y- catenin into the in inhibiting apoptosis or promoting cellular proli­ cytoplasm, coupled with an increase in cellular adhesion feration. Recently, He et al. (1998) have shown that one due to the increased stability between the cadherins and of the target genes of Tcf-4 is c-myc, which is a well catenins (Bradley et aI., 1993; Hinck et aI., 1994a). An known oncogene over expressed in colon cancer. y­ increase in the stability between APC and the catenins catenin has also been shown to enter the nucleus and has also been observed (Papkoff et aI., 1996). The Wnt-1 interact with these transcription factors, thus suggesting pathway has been fairly well elucidated and is shown in some functional homology between the two proteins, /3- fig. 1B, with Cook et al. (1996) using 10Tl/2 fibroblasts catenin and y-catenin. Simcha et al. (1998) have recently showing th at Wnt acted through the Wingless pathway shown that y-catenin and /3-catenin differ in their nuclear to inhibit GSK-3B. Fig. IB shows that Wnt-l binds to its translocation and their association with the transcription receptor frizzled which in turn activates the vertebrate factor Lef-I. They found that Lef-1 dependent trans­ homologue of dishevelled. Dishevelled in turn inhibits cription is preferentially driven by l3-catenin, with 13- the vertebrate homologue of ZW-3 known as GSK-3B. catenin binding to Lef-l in association with vinculin. GSK-3/3 is a serine/threonine kinase that negatively The l3-catenin-Lef complex has also been shown to bind regulates B-catenin, when B-catenin levels increase to to the 5' end of E-cadherin gene (Ben-Ze've, 1997). excess. In Xenopus and Drosophila GSK-3B serves to Whether tyrosine phosphorylation of B-catenin is downregulate both B-catenin and y-catenin, through involved in the wingless-Wnt signalling pathway has not phosphorylation (Rubinfeld et aI., 1996; Reviewed by been clarified (Reviewed by Nakamura, 1997). Jankowski et ai, 1997; Morin et aI., 1997a). Subsequent studies revealed the involvement of GSK-3B in the Catenins in cancer phosphorylation of both APC (increasing APCs affinity for y-catenin) and B-catenin (on the N-terminal region) Colorectal cancer is the second most common cause resulting in the increased degradation of B-catenin of cancer death, causing 17000 deaths in England and (Rubinfeld et aI., 1996; Reviewed by Moon and Miller, Wales alone each year (Dept. of Health, 1998). The 1997) via the ubiquitin dependent proteosomal pathway development of colorectal cancer has been well studied, (Aberle et aI., 1997; Muller et aI., 1998). GSK-3/3 and and the genetic steps involved in the adenoma to APC may also regulate /3-catenin independently of each carcinoma sequence have been well elucidated (Fearon other (Reviewed by Moon and Miller, 1997; Sakanaka et and Voge\stein, 1990; White, 1998). The first genetic aI., 1998), or work in a complex which also consists of alteration in the multistep process, in inherited and th e Axin (Conductin) molecule (Reviewed by Gordon, sporadic tumours, is the loss of APC (Wasan et aI., 1998) 1998; Ikeda et aI., 1998). Axin was shown to bind both with 80-85% of adenomas carrying an APC mutation B-catenin and GSK-3/3 in colorectal cancer cells that (Reviewed by Nakamura,1997). The APC gene is contained mutant APC, blocking B-catenin transcription located on chromosome 5q21-22 and codes for a 310 Kd (Sakanaka et aI., 1998). Thus inhibition of GSK-3B protein (Mulkens et aI., 1998). APC has been described results in increased stability of /3-catenin. This increase as a 'gatekeeper' gene in that it directly regulates the in cytoplasmic B-catenin leads to translocation of 13- growth of tumours by inhibiting growth and/ or catenin into the nucleus where it associates with the promoting death (Reviewed by Kinzler and Vogelstein, transcription factors LEF and TCF (Behrens et aI., 1996; 1996; Reviewed by Barth et aI., 1997b; Reviewed by Reviewed by Barth et aI., 1997b; Reviewed by Morin et aI., 1997b; Reviewed by Kinzler and Jankowski et al.,1997; Korineket aI., 1997; Morin et aI., Vogelstein, 1998: Mulkens et aI., 1998). APC is a 1997a; Reviewed by Nusse, 1997; Muller et aI., 1998). cytoplasmic protein with multiple functional domains There is some discrepancy over whether B-catenin enters (Reviewed by Moon and Miller, 1997). Both y-catenin the nucleus of its own accord (but it is a large molecule and B-catenin have been found to directly associate with with no signal peptide), or more likely whether it APC (Rubinfeld et aI., 1993; Su et aI., 1993; Reviewed associates with Lef or Tcf in the cytoplasm and these Jawhari et aI., 1997a), whereas a-catenin indirectly molecules carry l3-catenin into the nucleus (Behrens et associates with APC via /3-catenin (Su et aI., 1993). APC aI., 1996). is thought to function by controlling the cytoplasmic LEF and TCF are members of the high mobility levels of B-catenin, with it having two B-catenin binding 256 Catenins and associated proteins in colorectal cancer

sites, and in association with GSK-3/3 and axin, results in the small intestine, suggesting that when APC or /3- 8-catenin degradation (Reviewed by Moon and Miller, catenin are mutated in human epithelial cells, these cells 1997 ; Reviewed by Ben Ze-ev and Geiger, 1998; may retain stem cell characteristics thus resulting in Reviewed by Gordon, 1998; Reviewed by Ikeda et aI., malignant transformation (Korinek et aI. , 1998). y­ 1998). catenin has also been shown to associate with APC, Tcf Familial Adenomatous Poliposis (FAP) is an and Lef and also to enter the nucleus, suggesting there inherited autosomal dominant disease, resulting in the may be further functional homology. Mutations in APC inheritence of a mutant APC gene leading to the early and /3-catenin are thought not to be the only reasons for onset of colorectal cancer. FAP has provided a useful loss of the regulation of /3-catenin levels. Recent data model for studying CRC and is characterised by the have also revealed a number of other complexes development of thousands of adenomatous polyps at a involved in /3-catenin degradation, namely Axin which is young age (Reviewed by Kinzler and Vogelstein, 1996). thought to form a complex with APC and GSK-3/3 Stabilization and accumulation of cytoplasmic /3-catenin, leading to phosphorylation and subsequent 8-catenin which result from mutations in either the APC or /3- degradation (Reviewed by Gordon, 1998; Ikeda et aI., catenin genes are causitively associated with colon 1998), thus potentially mutations in Axin could also pl ay carcinogenesis (Takahashi et aI., 1998; Rubinfeld et aI., a role in the fundamental disruption of this multiprotein 1996). APC mutations usually involve at least one of the complex (Reviewed by Gordon, 1998; Reviewed by /3-catenin binding sites, thus resulting in increased Pennisi, 1998). Mutations in GSK-3/3 could also be a stability of the 8-catenin protein (Reviewed by Moon significant factor, suggested by the fact that mutations in and Miller, 1997). This is shown in cells expressing the GSK-3B consensus sequence of /3-catenin has been mutant APC, as they possess an abnormally large pool of shown to result in its increased stability and increased monomeric /3-catenin (Reviewed by Moon and Miller, activity (Reviewed by Moon and Miller, 1997; 1997). Using immunohistochemical techniques. Hao et Takahashi et aI., 1998). A further possible candidate is al. (1997) showed that in normal tissue 8-catenin was the ubiquitin dependent proteosomal degrad ation found associated with the cell membranes, but in the (Aberle et aI., 1997), any abnormality in the proteins adenoma and carcinoma tissues there was reduced 13- involved in this pathway could also have a significant catenin membranous staining which was accompanied effect on 8-catenin degradation, and function. with an increase in cytoplasmic and nuclear staining As early as 1989, Behrens et al. suggested that the (Hao et aI., 1997; Sheng et aI., 1998; Takahashi et aI., loss of adhesive function of uvomorulin (E-Cadherin) 1998), this was also found to correlate with the was a critical step in the promotion of the malignant progression from adenomas to carcinomas (Takayama et phenotype of epithelial cells. This is coupled with the aI., 1996; Hao et aI., 1997). However, where there are no emerging concept that this loss of adhesion leads to an APC mutations observed, there are a number of other increase in genomic instability, thus increasing the risk possible candidates which could be involved in of further mutations (Tisty, 1998). E-Cadherin is colorectal carcinogenesis, namely the catenins. essential for the formation and maintenance of epithelia, Takahashi et al. (1998) showed that in AMO and any perturbation of the adhesive complex, i.e. any (azoxymethane) treated rats there was an increase in mutation in any of the components results in a disruption both cytoplasmic and nuclear 8-catenin. Further analysis in E-Cadherin function leading to increased invasiveness of 8-catenin revealed that in this model a mutation in the and decreased differentiation (Reviewed by Barth et aI., GSK-38 consense motif for 8-catenin resulted in its 1997a,b; Reviewed by Jankowski et aI., 1997; Guilford increased stability. There are a number of other possible et aI., 1998). Loss of E-cadherin mediated adhesion mutation sites in B-catenin, it has been shown that in appears to be a fundamental aspect of the neoplastic epithelial cells /3-catenin may undergo tyrosine process (Jawhari et aI., 1997b). Mutations in E-cadherin phosphorylation due to a mutation in the coding itself, /3-catenin, a-catenin and y-catenin have all been sequences of the serine residues (Reviewed by shown to result in the perturbation of cellular adhesion Jankowski et aI., 1997). Amino terminal deletion in /3- (Gagliardi et aI., 1995). In vitro non invasive, catenin also results in the stabilisation of the protein differentiated cell lines tend to express E-cadherin, along with hyperphosphorylation of APC (Munemitsu whereas the invasive, undifferentiated cell lines had et aI., 1996). Alterations in cell signalling, as in reduced E-cadherin expression, suggesting that E­ inappropriate Wnt-l signalling, can also result in cadherin is an invasion suppressor molecule (Frixen et increased free cytoplasmic /3-catenin (Hinck et aI., aI., 1991). Gagliardi et al. (1995) used immunohisto­ 1994). As mentioned previously an increase of free /3- chemical techniques to evaluate the expression of E­ catenin results in increased and inappropriate cadherin in normal and cancerous tissues. They found transcription of hLEF-l and hTCF-4 (Reviewed by that in normal colorectal epithelial cells there was typical Moon and Miller, 1997), which has recently been shown membranous staining at the adherens junctions. But in by He et al. (1998), to result in c-Myc expression, thus the adenoma and carcinoma tissues there were changes leading to a possible understanding of why c-Myc is in the immuno-reactivity and cellular localisation, and overexpressed in colorectal tumours. Tcf-4 has also these changes correlated with tumour size, tumor type, recently been shown to maintain the crypt stem cells of growth patterns and degree of dysplasia (Dorudi et aI. , ------

257 Catenins and associated proteins in c%recta/ cancer

Table 1. Examples of genetic and epigenetic changes in catenin associated proteins in colorectal cancer.

MOLECULE MUTATION TYPE MUTATION CODON PHENOTYPE

APC Deletion AA MCR 1310 Truncated protein, Moderate dysplasia, Tubular adenoma (Mulkens et al. , 1998) APC Missense AAA>TAA MCR 1370 Truncated protein, Moderate dysplasia, Tubular adenoma (Mulkens et al. , 1998) APC Insertion A MCR 1384 Truncated protein, Moderate dysplasia, Tubulovillous adenoma (Mulkens et aI., 1998) APC Deletion G 1399 Found in 2/84 ACF (Otori et aI. , 1998 ) APC Missense Gln--Stop 1338 Most common mutation found resulting in a truncated protein (Smith and Pignatelli, 1997) APC Deletion 5bp 1309 Germline mutation found (Muller et aI. , 1998) B-catenin G:C to AT GGA>GAA Found in GSK-3B consense motif (Takahashi et aI. , 1998) B-catenin Deletion 234-760 Stabilisation of B-catenin protein. found in 7/222 patients with CRC (Iwao et aI. , 1998) B-catenin Deletion 3bp codon 45 Stabilised protein, found in the RER+ cell line HCT116 (llyas et aI., 1997; Muller et aI. , 1998) B-catenin Missense Stabilised protein, found in RER+ cell line SW48 (llyas et aI. , 1997; Muller et aI., 1998 ) B-catenin Point Mutation Loss of phosphorylation siles (Polakis, 1998) or Intestital deletion E-cadherin Methylation Promoter region Intron 1? Only been found in breast and gynaecological cancers (Yoshiura et al.,1995) E-cadherin Phosphorylation Post translational modification, resulting in non-functional E-cadherin (Jawahari et aI. , 1997a,b) E-cadherin Missense G--T Exon 7 Found as a germline mutation in gastric carcinomas (Guilford et aI. , 1998) Catenins Tyrosine Reduced cellular adhesion, resulting in increased invasion, with Band y Phosphorylation decreased differentiation.

MCR: Mutational Clusler Region ; ACF: Aberrent Crypt Foci; RER +: Replication Error Positive.

1993; Gagliardi et aI., 1995). It has been suggested that tumour invasion and metastasis through E-Cadherin loss of E-cadherin mediated cell-cell adhesion could dysfunction. They found that normal epithelium allow cells to escape normal growth control signals expressed u-catenin strongly, without exception. resulting in cellular proliferation. Alternatively Guilford However, u-catenin was reduced or absent in a number suggests that the cytoplasmic domain of E-cadherin may of primary tumours of the esophagus, stomach and modulate the Wnt signalling pathway by inhibiting the colon, thus suggesting that E-Cadherin mediated availability of free B-catenin, similarily to APC adhesion may be abrogated by downregulation of u­ (Guilford et aI., 1998). catenin (Shiozaki et aI., 1994). Skoudy et al. (1996) also It has also been shown that there is an increase in the reported altered u-catenin expression in colorectal activation of several receptor tyrosine kinases, i.e. EGFR cancer. and c-MET in cancer cells, resulting in tyrosine Skoudy et al. (1996) demonstrated that in the normal phosphorylation of the catenin molecules, thus colon, p120-catenin was present in the crypt and surface disrupting cellular adhesion (Behrens et aI., 1993; epithelium, the cells showed reactivity in both the Reviewed by Jankowski et aI., 1997). The activation of membrane and cytosol, with Valizadeh et al. (1997) the tyrosine kinase Src has also been shown to be an showing that the staining intensity was greatest in the early event in colorectal cancer, resulting in enhanced proliferating crypt cells. Reduced expression of p120 anchorage independence, although this is thought not to was observed in 20% of adenomatous polyps, with be cadherin mediated (Pories et aI. , 1998). Other loss of membranous p120 expression correlating mechanisms of E-Cadherin inactivation have also been with reduced E-Cadherin expression. Decreased described by Yoshiura et al. (1995) who showed that expression of p120 was also found to correlate methylation on CpG islands near the promoter region with the larger size tumour (Skoudy et aI., 1996). These also inactivated E-Cadherin at least in ovarian, breast findings are difficult to explain as the physiological and prostate carcinomas. role of p120 is unknown, but Skoudy et al. (1996) results Shiozaki et al. (1994) looked at whether the possible suggest that changes in p120 catenin levels are a down regulation of u-catenin expression played a role in common event in colorectal tumours, and suggest that 258 Catenins and associated proteins in c%recta/ cancer

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