Proc. Natl. Acad. Sci. USA Vol. 95, pp. 3122–3127, March 1998 Medical Sciences Intestinal trefoil factor controls the expression of the adenomatous polyposis coli–catenin and the E-cadherin–catenin complexes in human colon carcinoma cells (trefoil peptidesymigrationyadhesionyapoptosisyphosphorylation) i J. A. EFSTATHIOU*†‡,M.NODA*‡,A.ROWAN†,C.DIXON§,R.CHINERY¶,A.JAWHARI*, T. HATTORI , N. A. WRIGHT**, W. F. BODMER†, AND M. PIGNATELLI*†† *Division of Investigative Science, Imperial College of Science, Technology and Medicine, Hammersmith Campus, Du Cane Road, London W12 ONN, United Kingdom; †Cancer and Immunogenetics Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom; §Imperial Cancer Research Fund, Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom; ¶Gastrointestinal Cancer Program CC-2218 MCN, Vanderbilt University Medical Center, Nashville, TN 37232-2583; iDepartment of Pathology, Shiga University of Medical Science, Shiga 520-21, Japan; and **Histopathology Unit, Imperial Cancer Research Fund, Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom Contributed by W. F. Bodmer, December 19, 1997 ABSTRACT Intestinal trefoil factor 3 (TFF3) is a member directly to the cytoplasmic domain of E-cadherin, and a-cate- of the trefoil family of peptides, small molecules constitutively nin (102 kDa) links the bound b-org-catenin to the actin expressed in epithelial tissues, including the gastrointestinal microfilament network of the cellular cytoskeleton (6). Such tract. TFF3 has been shown to promote migration of intestinal binding is essential for the adhesive function of E-cadherin and epithelial cells in vitro and to enhance mucosal healing and for the establishment of tight physical cell–cell adhesion (7). epithelial restitution in vivo. In this study, we evaluated the A number of studies have confirmed that structural and effect of recombinant TFF3 (rTFF3) stimulation on the ex- functional integrity of the components of the E-cadherin– pression and cellular localization of the epithelial (E)- catenin complex are necessary for cell adhesion (8). We have 1 cadherin–catenin complex, a prime mediator of Ca2 depen- shown that perturbation of E-cadherin–catenin-mediated cell dent cell–cell adhesion, and the adenomatous polyposis coli adhesion is associated with cell migration and epithelial res- (APC)–catenin complex in HT29, HCT116, and SW480 colo- titution in an in vitro model of epithelial injury (9). In vivo, the 2 rectal carcinoma cell lines. Stimulation by rTFF3 (10 9 M and regenerating epithelium over ulcerated mucosa shows loss of 2 10 8 M) for 20–24 hr led to cell detachment and to a reduction membranous localization and decreased levels of E-cadherin in intercellular adhesion in HT29 and HCT116 cells. In both and a-catenin (10). Using chimeric-transgenic mice, Her- cell lines, E-cadherin expression was down-regulated. The miston and Gordon showed that disruption of cadherin me- expression of APC, a-catenin and b-catenin also was de- diated cell–cell adhesion in the crypt epithelial cells resulted creased in HT29 cells, with a translocation of APC into the in altered cell cycle, perturbed mucosal barrier function, and nucleus. No change in either cell adhesion or in the expression progressive inflammatory changes with features consistent of E-cadherin, the catenins, and APC was detected in SW480 with inflammatory bowel disease (11). cells. In addition, TFF3 induced DNA fragmentation and Loss of function or expression of any of the E-cadherin– morphological changes characteristic of apoptosis in HT29. catenin complex components also has been implicated in Tyrphostin, a competitive inhibitor of protein tyrosine ki- leading to loss of epithelial differentiation and normal archi- nases, inhibited the effects of TFF3. Our results indicate that tecture and the acquisition of a motile and invasive phenotype by perturbing the complexes between E-cadherin, b-catenin, (8). In vitro studies using human carcinoma cell lines have and associated proteins, TFF3 may modulate epithelial cell confirmed this for E-cadherin, a-catenin, and b-catenin (7, adhesion, migration, and survival. 12). In vivo studies of a variety of human malignancies, including oesophageal, gastric, and colonic adenocarcinomas, Interference with cellular attachment and increased migration have shown that reduced E-cadherin–catenin expression is has been implicated during the neoplastic process (1) and is associated with tumor dedifferentiation, infiltrative growth, fundamental during the early reparative response of epithelial and lymph node involvement (13, 14). restitution in mucosal healing (2). Spatial and temporal The cadherins and catenins, however, are more than just changes in cell–cell and cell–matrix interactions occur during intercellular glue. b-catenin seems to participate in signal migration and several families of adhesion molecules that transduction pathways independently from its role in cell–cell mediate these interactions have been shown to regulate cell adhesion and thus links the cell surface to downstream cyto- motility (3). plasmic and nuclear events. For instance, b-catenin and its In epithelial cells, cell–cell adhesion is mediated primarily by Drosophila homolog, Armadillo, are involved in a Wnt– epithelial (E)-cadherin, a 120-kDa transmembrane glycopro- Wingless signaling pathway critical for developmental pattern- tein localized at the adherens junctions (4). In the presence of ing at least in Drosophila and Xenopus (15). It also has been Ca21, E-cadherin’s extracellular domain interacts homotypi- discovered that b-catenin can bind not only to E-cadherin but cally with the E-cadherin molecules of neighboring cells to also to other molecules such as the epidermal growth factor maintain intercellular adhesion, and its cytoplasmic carboxy receptor (EGFR) (16) and the adenomatous polyposis coli tail associates with a group of closely related but distinct (APC) gene product (17). membrane undercoat proteins, termed the catenins (a, b, and g b g ) (5). Both -catenin (92 kDa) and -catenin (83 kDa) bind Abbreviations: TFF3, intestinal trefoil factor 3; rTFF3, recombinant TFF3; E, epithelial; EGF, epidermal growth factor; EGFR, EGF The publication costs of this article were defrayed in part by page charge receptor; APC, adenomatous polyposis coli; MBP, maltose binding protein; TBST, Tris buffered saline Tween 20; FAP, familial adeno- payment. This article must therefore be hereby marked ‘‘advertisement’’ in matous polyposis; PI; propidium iodide. accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡J.A.E. and M.N. contributed equally to this study. © 1998 by The National Academy of Sciences 0027-8424y98y953122-6$2.00y0 ††To whom reprint requests should be addressed. e-mail: m.pignatelli@ PNAS is available online at http:yywww.pnas.org. rpms.ac.uk. 3122 Downloaded by guest on September 25, 2021 Medical Sciences: Efstathiou et al. Proc. Natl. Acad. Sci. USA 95 (1998) 3123 The APC gene encodes a 310-kDa cytoplasmic protein (18, standard techniques (48). The fusion protein was composed of 19) and the C-terminal portion of the APC protein is deleted the first 1.3 kb of APC which includes the first 433 amino acids in most of the mutations that are found commonly in inherited and the first nine exons of the gene. The hybridoma was familial adenomatous polyposis (FAP) patients and in spo- selected by differential b-galactosidaseyanti-b-galactosidase radic colorectal cancers (20, 21). Such mutations appear to be ELISA (GAG-ELISA) (49) screening using the APC–MBP an early event during colorectal tumorigenesis (18, 19, 22, 23). fusion protein and the MBP alone. The antibody is an IgG1 and The APC protein competes directly with E-cadherin for is called ALI-12–28. binding to b-catenin and can form distinct complexes contain- mAbs Against E-Cadherin, Catenins, and Phosphotyrosine ing combinations of a-catenin, b-catenin, and g-catenin that Residues. A mouse mAb against human E-cadherin (HECD-1) are independent from the cadherin–catenin complexes and was kindly provided by M. Takeichi (Kyoto University, Kyoto, through them bind to the cytoskeleton (24, 25). Mutant APC Japan). Commercially available purified mouse mAbs (1 mgy protein may bind more avidly to b-catenin than wild-type ml) against human a-, b- and g-catenins (Transduction Lab- APC, and it appears that APC and b-catenin regulate their oratories, Lexington, KY) were used. Mouse anti-phosphoty- mutual expressions (26). Phosphorylation of APC by glycogen rosine mAb, 4G10 (1 mgyml), was purchased from Upstate synthase kinase-3b regulates the interaction of APC with Biotechnology (Lake Placid, NY). b-catenin and may lead to b-catenin degradation (27). Free rTFF3 and Tyrphostin. Recombinant rat intestinal trefoil b-catenin seems to accumulate in the nucleus (28) and there factor (rTFF3) was purified as described (47). Immunoblot and associate with the DNA binding proteins Tcf-Lef, triggering laser desorption time of flight mass-spectrometric analyses gene expression leading to proliferation or inhibition of apo- confirmed the identity of the purified material which was ptosis (29). It recently has been suggested that APC may act assessed to be '99% pure as judged by SDSyPAGE and as part of the Wnt–b-catenin signaling cascade in Xenopus (30) Coomassie Brilliant blue staining (50). Tyrphostin A25 was and that APC may alter transformation properties, decrease obtained from Calbiochem-Novabiochem. growth rate, or induce