The intracellular E-cadherin germline mutation V832 M lacks ... - Nature

Oncogene (2003) 22, 5716–5719

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The intracellular E-cadherin germline mutation V832 M lacks the ability to mediate cell–cell adhesion and to suppress invasion Gianpaolo Suriano1, David Mulholland2, Olivier de Wever3, Paulo Ferreira1, Ana Rita Mateus1, Eric Bruyneel3, Colleen C Nelson2, Marc M Mareel3, Jun Yokota4, David Huntsman2 and Raquel Seruca*,1,5 1

Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), 4200 Porto, Portugal; 2Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada; 3Laboratory of Experimental Cancerology, UZG, B-9000 Ghent, Belgium; 4Biology Division, National Cancer Center Research Institute, Tokyo, Japan; 5Faculdade de Medicina, Hospital de S. Joaõ, 4200 Porto, Portugal

E-cadherin germline missense mutations have been shown to be responsible for significant loss of protein activity. A new cytoplasmic E-cadherin germline missense mutation (V832 M) was recently identified in a hereditary diffuse gastric cancer (HDGC) Japanese family. This E-cadherin mutant was cloned in a Chinese hamster ovary cell model system and functionally characterized, in terms of aggregation and invasion. Cells expressing the germline V832M mutant fail to aggregate and invade into collagen, supporting the pathogenic role of this germline missense mutation in gastric cancer. We also tested the ability of this mutation to activate the TCF–LEF trascriptional activity, in comparison with three other E-cadherin missense mutations (T340A, A634V and A617T), associated to loss of E-cadherin function. All the E-cadherin mutants reduced TCF–LEF activation to a similar extent as the wild-type protein, suggesting that the oncogenic effect of the E-cadherin mutants is unlikely to be transmitted through a b-catenin-dependent activation of the WNT pathway. Oncogene (2003) 22, 5716–5719. doi:10.1038/sj.onc.1206672 Keywords: E-cadherin; hereditary diffuse gastric cancer; mutations; cell–cell adhesion; invasion; TCF-LEF

E-cadherin is a transmembrane glycoprotein localized at the adherens junctions of epithelial cells, where it mediates homophilic, Ca2 þ -dependent cell-adhesion (Shore and Nelson, 1991; Shapiro et al., 1995; Pertz et al., 1999). The adhesion process is accomplished by homophilic interactions between extracellular E-cadherin domains, leading to the formation of zipper-like structures (Pertz et al., 1999). The E-cadherin intracellular domain interacts with catenins, assembling the celladhesion complex. b-Catenin and g-catenin compete for the same binding site at the E-cadherin cytoplasmic tail, *Correspondence: R Seruca, Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), 4200 Porto, Portugal; E-mail: [email protected] Received 12 February 2003; revised 24 March 2003

directly linking the adhesion complex to the cytoskeleton through a-catenin (Christofori and Semb, 1999). These interactions represent a prerequisite not only for cell–cell adhesion, but also for inhibition of cell motility and invasion (Chen et al., 1997). The dual role of b-catenin, as a component of the cell–cell adhesion complex as well as transcriptional coactivator in the WNT growth factor signal pathway has been extensively characterized (Cavallaro et al., 2002). In the absence of WNT, phosphorylation by glycogen synthase kinase-3b (GSK-3b) targets b-catenin for degradation in the adenomatous polyposis coli (APC)-GSK-3b complex. It was reported that APC and/or GSK-3b inactivation as well as mutations of AXIN and bcatenin itself (Bienz and Clevers, 2000) leads to an accumulation of high level of b-catenin into the cytoplasm; b-catenin could translocate into the nucleus and bind to a member of the TCF–LEF-1 family of transcription factors, activating gene expression (Polakis, 2000). Aberrant E-cadherin protein expression has been reported in several types of human cancer (Becker et al., 1994; Gayther et al., 1998; Guilford et al., 1998; Machado et al., 2001; Van Aken et al., 2001; Thiery, 2002). Despite what has been observed in other types of cancers, in sporadic diffuse gastric carcinoma as well as infiltrative lobular breast cancer E-cadherin downregulation is often associated to gene mutation (Berx et al., 1998; Becker et al., 1999). In lobular breast cancer, the most common CDH1 alterations are out-of-frame mutations; on the contrary, in stomach, most CDH1 sporadic mutations are missense and in frame deletions localized between exons 7–9 and cluster in the extracellular domain of the protein (Berx et al., 1998; Oliveira et al., 2003). In hereditary diffuse gastric carcinomas CDH1 germline mutations are dispersed all along the gene (Oliveira et al, 2003). The majority of these germline mutations are frameshift and splice site resulting in truncated nonactive proteins, though a proportion of the missense type has been also reported (Oliveira et al., 2002; Yabuta et al., 2002; Suriano and Oliveira et al., 2003). These missense mutations appear to be clustered mainly in the extracellular region of the

Intracellular E-cadherin germline mutation V832M G Suriano et al

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protein, where a hot spot region spanning the second and third E-cadherin conserved calcium binding domains was identified (Berx et al., 1998; Oliveira et al., 2002; Suriano et al., 2003). Functional studies have shown that germline missense mutations cause impaired or reduced cell–cell adhesion, increased cell motility and invasion resulting in the scattered cell morphology and invasive phenotype often observed in diffuse gastric carcinoma (Huber and Weis, 2000; Handschuh et al., 2001; Većsey-Semjeń et al., 2002; Suriano and Oliveira et al., 2003). Recently, a new E-cadherin germline missense mutation (V832M) has been identified in a HGDC Japanese family with three affected members, all carriers of the same alteration (Yabuta et al., 2002). The residue V832 represents the first amino acid of the E-cadherin cytoplasmc domain involved in the recognition and binding of b-catenin (Huber and Weis, 2001). The mutation was shown to segregate with the disease, supporting its effective pathogenic role, although immunohistochemical studies revealed normal E-cadherin and b-catenin staining both in normal and tumoral cells (Yabuta et al., 2002). Basing on these intriguing findings, and considering that all the functional studies reported to date have been based on the characterization of truncating or missense mutations affecting always the extracellular E-cadherin domain, we performed functional characterization of the V832 M E-cadherin mutant. A cell-line expressing the V832 M E-cadherin mutant was established by stably transfecting Chinese hamster ovary (CHO) cells. These cells are E-cadherin negative, fail to aggregate homotypically and invade into collagen, thus representing a good in vitro model for the functional characterization of E-cadherin mutants. The functional studies were carried out in comparison with cells expressing the wild-type protein and the parental cells. Stable expression of E-cadherin in CHO cells (Figure 1) showed a full-length E-cadherin band (120 kDa) for both the wild-type and V832 M mutant. Support for functional significance of the V832 M mutant was derived from a fast cell aggregation assay. As previously reported (Suriano and Oliveira et al., 2003), cells expressing the wild-type protein showed extensive cell–cell adhesion, whose E-cadherin dependence was demonstrated by inhibition with the MB2 E-cadherin blocking antibody (Figure 2). Cells expressing the V832 M mutant did not show aggregates formation, as observed for the parental cells (Figure 2). In a collagen invasion assay (Figure 3), cell expressing the V832 M E-cadherin mutant showed a strong ability to penetrate the collagen matrix, in a similar extent as the parental cells, or as the cells expressing the wild-type protein incubated with the E-cadherin blocking antibody MB2. Altogether these results demonstrate that the germline V832 M mutant affects both the ability of E-cadherin to mediate cell-adhesion and to suppress cell invasion. As the V832 residue localizes within the binding site of b-catenin to E-cadherin and E-cadherin downregula-

Figure 1 Western blotting analysis of E-cadherin-transfected cell lines, expressing wild-type or V832 M mutant. Several independent clones were used for the functional characterization, in order to exclude clonal dependence. Each measurement was repeated in duplicate. The cDNA encoding the V832 M mutation was kindly given by Prof. Yokota. The mutated DNA was cloned in a pcDNA3 mammalian expression vector and the resulting plasmid was stably transfected into CHO E-cadherin negative cells (cells were kindly given by Dr Nicole Moguilevsky, Service of Applied Genetics, ULB Belgium). Cells were cultured at 371C under 5% CO2 in humidified air, in a-MEM ( þ ) medium (GIBCO-BRL) supplemented with 5% foetal bovine serum (HYCLONE), 2 mm lglutamine (GIBCO-BRL), 1% penicillin/streptomycin (GIBCOBRL) and 1000 mg/ml geneticin (GIBCO-BRL) for the selection. Single-cell clones were selected and analysed for E-cadherin expression. A total of 5 104 cells were lysed with 50 ml Triton 114 buffer (EDTA 2 mm, PBS Mg2 þ , Ca2 þ free 1 , Triton 114 1%, DTT 1 mm, Protease inhibitors) and the extracted protein quantified by following the Bradford dye-binding procedure (Bradford, 1976). A measure of 10 mg of protein was separated on a 7.5% SDS–polyacrylamide gel electrophoresis, followed by transfer onto a nitrocellulose membrane (C-bond, Millipore). Human E-cadherin monoclonal antibody HECD1 (R&D System, 1/3500 dilution) was used for the immunostaining. Only clones expressing comparable levels of E-cadherin were chosen for the functional characterization

tion might result in the activation of the WNT signalling pathway through b-catenin during tumour progression, we decided to evaluate the effect of this mutation on the TCF–LEF activity. To accomplish this aim, a TCF-luciferase reporter assay was performed, using human colon cancer SW480 cells transiently expressing both the wild-type and V832 M E-cadherin. These cells express endogenous E-cadherin, but they carry a truncating mutation of the APC gene, which results in constitutive activation of the TCF reporter (Figure 4). Overexpression of the V832 M mutant resulted in the inhibition of TCF transcriptional activity, in similar levels to what observed for the wild-type E-cadherin protein. As a control, we transfected a deleted E-cadherin mutant lacking the entire b-catenin binding site (DbCat), which is unable to repress the constitutive activation of the reporter in SW480 cells (Figure 4). To further test whether the blockage of b-catenin interaction with the transcription factors LEF-1 and TCF-4 was specific of the V832M Oncogene


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Figure 2 Aggregate formation for cells transfected with wild-type E-cadherin after 30 min (——T30) and when incubated with the Ecadherin-blocking antibody MB2 (- - - - -). Aggregate formation for mutant V832M at time 0 and after 30 min (—— T0; - - - - - T30). *Statistically significant difference between the two populations. Method: The fast cell aggregation assay was performed as previously reported (Boterberg et al., 2000). Single cell-suspensions were incubated in an isotonic buffer containing 1.25 mm Ca2 þ . Particle diameters were measured at the start (T0) and after 30 min (T30), using a particle size counter LS200 (Coulter, Electronics). Results were plotted against the percentage of volume distribution. Cells were incubated with the E-cadherin-blocking antibody MB2 (1/20 dilution) as control for inhibition of aggregation

Figure 3 Percentage invasion into collagen matrix of parental CHO cells, cells expressing wild-type E-cadherin and cells expressing the V832 M mutant. The invasion into collagen matrix was performed as described by Bracke et al. (2000). Invasion was expressed as function of the cell ability to penetrate a matrix of collagen (type I solution, Seromed, Bichrom KG, Berlin, Germany) and evaluated using a microscope interfaced with a computercontrolled step motor.

mutant, we examined three other E-cadherin missense mutations (T340A, A634V and A617T), which were previously described to be associated with significant loss of protein functions (Suriano and Oliveira et al., 2003). Similar to the V832 M mutant, none of the other mutants had an increase in the TOPFLASH luciferase activity (Figure 4). Our results reinforce the hypothesis that the oncogenic effect of the V832 M E-cadherin mutant is unlikely to be transmitted through a b-catenin-dependent activation of the WNT pathway. Oncogene

Figure 4 TCF luciferase reporter assay (TOPFLASH) indicating that the V832 M mutation has a similar capacity to wild-type (Wt) E-cadherin for inhibition of TOPFLASH activity. An E-cadherin mutant for b-catenin binding (Db-Cat) was used as a control for the assay and appears as a lower molecular weight (B90 kDa) than wild-type E-cadherin when stained for E-Cadherin (Transduction Lab.). A total of 10 mg of DNA was transiently transfected into SW480 cells for 16 h followed by 24 h of full serum treatment. Relative luciferase units (RLU) were evaluated using the Dual Promega Luciferase kit (Promega). Other germline mutations previously reported (Suriano and Oliveira et al., 2003) showed a reporter activity similar to the V832M mutation.

In favour of this hypothesis and in analogy to what was previously observed in the tumour cells (Yabuta et al., 2002), immunocytochemical studies revealed normal E-cadherin/b-catenin costaining at the lateral cell–cell contact, suggesting an intact activity of the b-catenin binding to E-cadherin (data not shown). Other mechanisms independent on b-catenin might lead to the activation of the WNT pathway; although alternative putative pathways might also be considered to explain the oncogenic effect of the E-cadherin mutants. Van de Wetering et al. (2001) reported that in breast cancer, cells carrying mutant E-cadherin do not show constitutive WNT signalling. The invasive phenotype has been associated to dramatic cytoskeletal rearrangements (Behrens et al., 1989; Gumbiner, 2000); cellular factors connecting the E-cadherin activity to the cytoskeleton organization (i.e. small GTPases of the Rho family and p120ctn) represent pointing these pathways as good candidates for further investigations (Anastasiadis et al., 2000; Chausovsky et al., 2000), towards the understanding of the mechanism linking Ecadherin downregulation and tumorigenesis in diffuse gastric cancer. Acknowledgements This study was funded by grants from: Fundaçaõ para a Cieˆncia e a Tecnologia, Portugal (Project: POCTI/35374/CBO/ 2000 and POCTI/CBO/40820/2001); FORTIS Verzekerngen and the Fund for Scientific Research – Flanders (FWO), Brussels, Belgium.


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