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Human Molecular Genetics, 2003, Vol. 12, No. 22 3007-3016
DOI: 10.1093/hmg/ddg316
© 2003 Oxford University Press

E-cadherin germline missense mutations and cell phenotype: evidence for the independence of cell invasion on the motile capabilities of the cells

Gianpaolo Suriano1, Maria José Oliveira2, David Huntsman3, Ana Rita Mateus1, Paulo Ferreira1, Fernando Casares4, Carla Oliveira1, Fátima Carneiro1,5,6, José Carlos Machado1,5, Marc Mareel2 and Raquel Seruca1,5,*

1Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), 4200-465 Porto,Portugal, 2Laboratory of Experimental Cancerology, Ghent University Hospital, B-9000 Ghent, Belgium, 3Department of Pathology and Laboratory Medicine, University of British Columbia Vancouver, Canada, 4Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, 4150-180, Porto, Portugal, 5Faculdade de Medicina, 4200-465 Porto, Portugal and 6Serviço de Anatomia Patológica, Hospital S. João,4200-465 Porto, Portugal

Received August 1, 2003; Revised August 22, 2003; Accepted September 10, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In Hereditary Diffuse Gastric Cancer syndrome, E-cadherin germline mutations of the missense type harbour significant functional consequences. In this study, we have characterised the effect of T340A, A617T, A634V and V832M E-cadherin germline missense mutations on cell morphology, motility and proliferation. Wild-type E-cadherin and A617T expressing cells have an epithelial-like morphology, with polarised cells migrating unidirectionally. T340A and A634V expressing cells, fibroblast-like, have a high motile phenotype. We show that this phenotype is dependent on an increased level of active RhoA. V832M expressing cells grow in piled-up structure of round cells, as an effect of the disturbance of the binding between {alpha}-catenin and ß-catenin. The destabilisation of the adhesion complex is shown to hamper the motile capabilities of these cells. We did not observe any effect of the E-cadherin mutations on cell proliferation. We show the existence of a genotype–phenotype correlation between different E-cadherin mutations and cell behaviour. However, we demonstrate that the ability of cells expressing the different E-cadherin mutations to invade is independent on their motile capabilities, providing evidence that motility is neither necessary nor sufficient for cells to invade. Our data give new insights into the understanding of the mechanisms linking invasion and E-cadherin mutations in diffuse gastric cancer.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E-cadherin is a transmembrane cell–cell adhesion receptor and major component of the adherens junction, which plays a key role in the formation and maintenance of contacts between cells and tissues.

Although cell adhesion might seem a very static process, its dynamic rearrangement underlies a variety of physiological processes, ranging from morphological changes during development (1,2), cell scattering or wound healing (3) to tumour metastasis (4,5). Adherens junctions cluster, via homophilic interactions, through the extracellular domains of calcium-dependent E-cadherin molecules on the surface of homotypic neighbour cells (6). The cytoplasmic tails of E-cadherin molecules associate with ß-catenin or plakoglobin, which directly link them to the cytoskeleton through {alpha}-catenin. p120ctn, another member of the catenin family, interacts with the protein at the level of its juxtamembrane domain (7). These interactions are essential, not only for the correct stabilisation of intercellular junctions, but also for the co-ordination of actin dynamics with direct consequences on cell shape, polarity and movement (8,9). Many different signalling molecules have been reported to interact with E-cadherin (10,11); nonetheless, such interactions appear to be strongly dependent on the cell type and cellular context. The Rho family of small-GTPases, in particular RhoA, Rac1 and Cdc42, plays a pivotal role in controlling the assembly and disassembly of actin filaments (12–14) and co-localise with the adhesion complex, at the site of cell contact. It was suggested that alteration of the Rho-GTPases-mediated signalling could enhance tumour-cell motility, invasion and metastasis, as well as promote cell proliferation (15,16). Nevertheless, a clear link between their activation and the E-cadherin function still remains to be identified.

Down-regulation of E-cadherin expression is common in several tumours and is strongly associated with poor prognosis (17,18). E-cadherin down-regulation can occur through several mechanisms, including mutation, promoter hypermethylation, alteration of regulatory pathways mediated by different transcription factors, and also failure in the post-transcriptional stabilisation of the protein (19–23). E-cadherin is well established as a tumour and metastasis suppressor in epithelial cancers (24). The demonstration of the triggering of tumorigenesis by E-cadherin has come from the identification of germline mutations of its gene (CDH1) in hereditary diffuse gastric cancer (HDGC) (25,26). In HDGC, most CDH1 germline mutations are frameshift, nonsense and splice-site mutations leading to truncated proteins, but germline missense mutations have also been identified (22,27). We and others have recently demonstrated that a proportion of these CDH1 mutations harbour significant functional consequences in vitro, supporting their effective pathogenicity in diffuse gastric cancer (27–29).

In this study, we sought to pursue the effect of four E-cadherin germline missense mutations (T340A, A617T, A634V and V832M) on cell morphology, motility and proliferation. In the CHO cell model system herein used the E-cadherin germline missense mutations strongly affect cell morphology and motility, through different mechanisms. Nonetheless, the ability of cells to invade is shown to be independent of their motile capabilities.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E-cadherin missense mutations affect cell morphology and motility
We have previously shown that four germline missense mutations of the E-cadherin gene (T340A, A617T, A634V and V832M) (Fig. 1) affect, in vitro, the ability of the protein to mediate cell–cell adhesion and to suppress invasion (Table 1) (27,29). Since migration is believed to be essential for cells to invade (30), we characterised the morphology of the cells transfected with the aforementioned mutations and studied their effect on motility. We observed three distinct morphotypes, associated with the different E-cadherin mutations here studied (Fig. 2 and Table 1). Chinese hamster ovary cells transfected with the wild-type E-cadherin or the A617T mutant showed an epithelial morphotype (e-type) with a cobblestone-like organization and extensive cell-to-cell contacts with membranous E-cadherin and ß-catenin staining (Fig. 2 and Table 1). T340A and A634V mutants revealed a fibroblastic morphotype (f-type), with spindle-shaped cells; normal membrane staining of E-cadherin and ß-catenin was observed at the areas of cell–cell contact (Fig. 2 and Table 1). V832M expressing cells showed a distinct morphotype (r-type), in which piled-up structures of round cells grow on top of a monolayer of cells with an irregular shape. All cells maintain a normal pattern of E-cadherin/ß-catenin lateral staining (Fig. 2 and Table 1). A heterogeneous cell population was characteristic of cells transfected with the empty vector (mock) (Table 1). As expected, mock cells were negative for E-cadherin, and no ß-catenin staining was observed in any cellular compartment. The proliferation studies using BrdU (5-bromodeoxyuridine) incorporation revealed that E-cadherin mutant cell lines proliferate at a similar rate as wild-type expressing cells or mock cells (Fig. 3), according to the absence of free ß-catenin in any of the cell lines studied (Fig. 2).



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  		Figure 1. Schematic of the E-cadherin protein and position of the germline missense mutations studied. EC1–5 represent the five tandemly repeated ectodomains on the extracellular region of the protein. The mutation T340A affects the third domain, while both A617T and A634V are located in the fifth repeated domain. The grey bar represents the ß-catenin binding domain, on the cytoplasmatic tail of E-cadherin. The mutation V832M affects the first residue of this domain.

 

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  		Table 1. Summary of the phenotypic features of mock cells and cells transfected with the different E-cadherin constructs
 


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  		Figure 2. E-cadherin and ß-catenin immunofluorescence staining of confluent layers of cells transfected with wild-type E-cadherin or with its mutated forms (objective 100x, ocular 10x). Transfected cells, seeded on glass coverslips were immunostained with anti-E-cadherin (R&D) and anti-ß-catenin (Transduction Laboratories) antibodies. In all E-cadherin expressing cell lines, we observed normal membranous E-cadherin-ß-catenin staining. For V832M expressing cells, the left panel represents cells at the bottom monolayer, whereas in the right panel we show the staining of cells organized in piled-up structures. No E-cadherin or ß-catenin staining was observed for mock cells.

 


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  		Figure 3. Proliferation indices of mock cells and cells expressing the different E-cadherin constructs. No effect on cell proliferation was associated to wild-type E-cadherin or its mutants, as suggested by the analysis of BrdU incorporation. Bars represent mean values of BrdU-positive cells, while flags indicate standard deviations. Differences were not statistically significant (P=0.124).

 
Each cell morphotype (e-, f- and r-type) is associated with distinct capability to move between the edges of an artificial wound made in a confluent monolayer of cells (Fig. 4A and B and Table 1). Cells expressing either the wild-type E-cadherin or the A617T mutant (e-type) showed a very compact front of migration, with cells unidirectionally moving between the wound edges. In contrast, cells expressing either the T340A or the A634V mutant (f-type) randomly colonized the wound 9 h after incision, showing an increase of ~25% in cell motility in comparison with wild-type expressing cells (Fig. 4B and Table 1). V832M expressing cells (r-type) and mock cells hardly moved across the wound in spite of showing a lose front of migration. As reported, differences observed in cell motility cannot be ascribed to differences in cell proliferation (Fig. 3).



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  		Figure 4. Cell motility assay. (A) The migratory properties of mock cells and cells transfected with the different E-cadherin constructs was assessed by contrast phase microscopy (20x and 40x objectives, 10x ocular), as a function of their ability to move across an artificial wound made in confluent monolayers of cells. Pictures shown were taken 9 h after wounding. (B) Percentage of wound closure. For each cell line, mean wound closure was assessed by measuring, 9 h after wounding, the distance between wound edges, in at least six regions randomly chosen. Values obtained were normalized assuming 100% wound closure for T340A expressing cells. Flags indicate standard deviations. *Statistically significant in comparison to mock cells (P<0.05).

 
E-cadherin missense mutations lead to cytoskeleton rearrangements
It is generally accepted that reorganization of the cytoskeleton is required for cell movement (31,32). To characterize the actin cytoskeleton structure of moving cells, small scrape wounds were made across a confluent monolayer of cells seeded on top of glass slips (Fig. 5). FITC-conjugated phalloidin staining, 2 h after incision, revealed a tightly packed front of migration with a well-polarized morphology for wild-type E-cadherin and A617T expressing cells (e-type cells). Cells contain abundant stress fibres, with actin-rich lamellipodial and filopodial extrusions at the leading edge (Fig. 5 and Table 1).



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  		Figure 5. FITC-conjugated phalloidin staining of F-actin in mock and E-cadherin expressing cells, at the leading edge of wounded confluent monolayers (40x) and island of cells (100x oil immersion). Fluorescence microscopy (40x and 100x objectives, 10x ocular) reveals for the e-type morphology (wt and A617T) well-polarized cells, uniformly moving across the wound. For the f-type morphology (T340A and A634V), increased lamellipodial and filopodial extrusions as well as intense stress fibres are observed in cells randomly invading the wound. V832M expressing cells are characterised by piled-up structures of round-cells growing on top of a monolayer of irregularly shaped cells. As revealed by confocal microscopy, for cells at the bottom monolayer unidirectional polarisation of the cytoskeleton is lost, with the actin randomly extruding both at the front and rear edges of cells; on the contrary, the round component of cells (r-type) display staining only of the actin cortical ring. Random organization of the actin cytoskeleton is observed for mock cells.

 
T340A and A634V expressing cells (f-type cells) exhibited a random polarization, with strong ruffling activity and lamellipodia and filopodia formation, both at the front and side edges of cells; cell junctions appear loose, with an extensive sheet of stress fibres (Fig. 5 and Table 1).

As reported above, V832M expressing cells are characterized by piled-up structures of round cells growing on top of a monolayer of irregularly shaped cells. The bottom monolayer of cells shows a reduced and random polarization of the actin at the leading front of migration, whereas the round component of cells reveals strong cortical ring staining but no polarized actin (Fig. 5, r-type). Mock cells confirm their heterogeneity, with retracted cells randomly extending filopodia and lamellipodia both into the wound and at the rear edges of the cell (Fig. 5 and Table 1). The peculiar phenotype displayed by V832M expressing cells had been previously observed in HCT-8 cells (33), a human colon cancer cell line for which aberrant expression of {alpha}-catenin, due to a homozygous nonsense mutation in its gene, was recognized as causative for the e- to r-morphotype transition. Interestingly, in the heterozygous status for the {alpha}-catenin gene, cells would keep an epithelial morphology, suggesting a dominant function played by the normal adhesion complex over the non-functional one (33). These observations together with the reduced motility of V832M expressing cells, led us to investigate the stability of the adhesion complex in this cell line by immunoprecipitation (Fig. 6B).



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  		Figure 6. Western blot and immunoprecipitation studies suggesting reduced stability of the interaction between {alpha}-catenin and ß-catenin in V832M expressing cells. (A) {alpha}-catenin immunostaining in both wild-type and V832M expressing cells. The {alpha}-catenin antibody was used at 1 : 1000 dilution. (B) Immunoprecipitation of {alpha}-catenin and ß-catenin or of ß-catenin and E-cadherin in the V832M cells was compared with the wild type E-cadherin expressing cells. Experiments were performed by using the Immunoprecipitation Starter Pack (Amersham Bioscience).

 
Our results are suggestive of a reduced affinity of {alpha}-catenin for ß-catenin, but an apparently intact binding of ß-catenin to E-cadherin. These results were analysed in comparison to what was observed in cells expressing the wild-type protein (Fig. 6B). The presence of {alpha}-catenin in both cell lines, independently on the correct formation of the adhesion complex, was demonstrated by western blot analysis (Fig. 6A), thus supporting the hypothesis of a reduced stability of the adhesion complex in V832M expressing cells.

The immunoprecipitation analysis was extended to the other E-cadherin mutants including mock cells. We analysed the E-cadherin/ß-catenin/{alpha}-catenin complex, together with the phosphorylation status of p120ctn and its interaction with E-cadherin. No differences were observed in comparison to cells expressing the wild-type protein for the mutants T340A, A617T, A634V either for the adhesion complex or the phosphorylation status of p120ctn. No positive immunostaining was obtained for mock cells after immunoprecipitation, confirming the absence of the E-cadherin adhesion complex in the parental cell line.

E-cadherin missense mutations influence cell migration through Rho-GTPases activity
The Rho family of small GTPases, and in particular RhoA, Rac1 and Cdc42, play a pivotal role in the reorganization of the actin cytoskeleton, thereby regulating cell migration (31,34). The establishment of E-cadherin mediated cell–cell adhesion requires changes in the activity of Rho GTPases. To gain further insights into the molecular mechanisms responsible for the distinct motility behaviour associated with the different E-cadherin mutations, we have analysed the levels of active RhoA, Rac1 and Cdc42 in our stable cell lines. For this analysis, an artificial wound was made in confluent monolayers of cells and total protein was extracted 4 h after incision. In the E-cadherin expressing cells, an increase in active RhoA was observed in comparison to mock cells (Fig. 7). Within the E-cadherin mutants, the level of active RhoA was more pronounced for T340A and A634V expressing cells (Fig. 7). Both cell lines (f-type) showed higher motility in wound healing and increased stress fibre formation, when compared to the wild-type and to the other E-cadherin mutants (Fig. 4B and Table 1). After incubating T340A and A634V expressing cells (f-type) with the RhoA inhibitor C3T exoenzyme (Clostridium botulinum exoenzyme C3 transferase) or the specific ROCK (Rho-associated coil-coiled kinase) inhibitor Y27632, ~30% and ~25% reduction of cell motility was respectively observed, in comparison to the untreated cells. Cells (f-type) appeared to migrate in a more unidirectional fashion, resembling the wild-type and A617T expressing cells (e-type cells) (Fig. 8 and Table 1). Interestingly, treatment with the same inhibitors did not affect the ability of T340A and A634V expressing cells to invade into collagen type I (Fig. 9 and Table 1). The effect of both inhibitors was milder on wild-type or A617T expressing cells (10%), while no effect was observed for mock cells or V832M expressing cells.



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  		Figure 7. Rho GTPases activity in function of the E-cadherin mutants. The level of active RhoA, Rac1 and Cdc42 (GTP status) was assessed in all cell lines and compared with the level of total protein. The intensity of the bands was normalized in function of loading and the fold of increase calculated in comparison with mock cells.

 


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  		Figure 8. Effect of the C3T and of Y27632 on the migration of mock and E-cadherin expressing cells. Wounded monolayers of cells were incubated for 9 h, in the presence of the RhoA inhibitor C3T (6.6 µM) or of the ROCK inhibitor Y27632 (20 µM). As example, we show the effect of Y27632 at 9 h in all cell lines in comparison to the untreated cells (contrast phase, 40x objective, 10x ocular). For T340A and A634V expressing cells we observed ~30% (C3T) and ~25% (Y27632) decrease of cell motility, in comparison to the untreated cells. A milder effect was obtained for wild-type and A617T expressing cells (10% with both inhibitors), while the motility of V832M and mock cells was not altered by the treatment (Table 1).

 


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  		Figure 9. Effect of the C3T and of Y27632 on the ability of T340A and A634V expressing cells to invade into collagen type I. No differences were observed between treated and untreated mutant cells. Wild-type E-cadherin expressing cells were chosen as negative control. We did not extend the analysis to A617T expressing cells, since as the wild-type, these cells are not invasive into collagen type I. Mock cells and V832M expressing cells were not tested, since for these cell lines both inhibitors had no effect on cell motility. Flags indicate standard deviations.

 
Altogether, these results suggest that increased levels of active RhoA play a pivotal role for the higher and random cell motility observed for T340A and A634V mutants, without influencing cell invasion. Although the increased level of active RhoA is expected to result in decreased levels of active Rac1/Cdc42 (35), nonetheless, no significant differences in the activation level of these small GTPases was observed between all cell lines, including CHO mock cells (Fig. 7).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
E-cadherin is involved in a wide range of processes, such as tissue morphogenesis and cohesion, synaptogenesis or wound healing (1), in which cell–cell adhesion is constantly rearranged by the remodelling of the adhesion complex itself. Conversely, E-cadherin deregulation is the major determinant of tumour progression and invasion in epithelial cancers (24). E-cadherin exerts its function not only contributing to cell–cell adhesion but also by acting as cell membrane receptor, and a consistent body of evidence has been produced supporting a direct E-cadherin-activated signalling pathway (5,14). Particular attention has been given to the implication of E-cadherin as a part of a protein complex, and to its direct link to the cytoskeleton through catenins (36). Many different signalling molecules have been reported to interact with E-cadherin, and many different signalling pathways are linked to this protein, nonetheless, their effects seem to be dependent on the specific cell type and context.

In this study, we have used an E-cadherin negative epithelial cell line and distinct germline missense mutations of the E-cadherin gene (T340A, A617T, A634V and V832M) as a sensitive in vitro system to assess their effect on cell proliferation, morphology and motility. This cell model has been previously used to successfully address the pathogenicity of the aforementioned germline mutations in diffuse gastric cancer. The effect of such mutations on the ability of E-cadherin to mediate cell–cell adhesion and to suppress cell invasion was investigated using both cell aggregation and collagen invasion assays (27,29).

Transfection of wild-type E-cadherin into CHO cells did not result in a reduction of proliferation. Moreover, none of the E-cadherin mutants studied affect cell proliferation rates. This data together with the absence of cytoplasmic or nuclear ß-catenin in all cell lines under study does not support a direct correlation between loss of E-cadherin and proliferation, through an up-regulation of the ß-catenin signalling in the WNT pathway. A possible role of E-cadherin in the WNT pathway, as a consequence of its interaction with ß-catenin, has been claimed by several authors (37,38). As observed for mutant APC, ß-catenin or axin cancer cells (39,40), it was suggested that mutations of E-cadherin could cause constitutive activation of the WNT pathway, leading to an increase of cell proliferation. The present results are in accordance to our previous report showing that none of the studied germline E-cadherin mutations is responsible for an increase in the ß-catenin dependent TCF-LEF transcriptional activity (29), and to what has been described for breast and prostate cancer cell lines (41,42).

Transfection of the different E-cadherin constructs into CHO cells resulted in an increased level of active RhoA, according to the existence of a bidirectional cross-talk between E-cadherin and Rho-GTPases implicated in the maintenance of cell junctions (16,43). Rac1 and Cdc42 have been reported to control membrane protrusion and focal complexes formation, being negatively regulated by the activity of RhoA as well as by its downstream target ROCK (44,45). In the cell model system herein used, we did not observe this inverse correlation between active levels of Rac1/Cdc42 and RhoA.

The T340A and A634V expressing cells (f-type) are very motile when tested in wound healing assay. The RhoA/ROCK signalling was recently shown to promote migration in THP-1 monocytes by restricting integrin activity and membrane protrusion to the leading edge (46). Moreover, it was previously reported that RhoA activation is absolutely required for actin stress fibres formation and migration of rat intestinal epithelial cells (47,48). We propose that the higher levels of active RhoA displayed by T340A and A634V expressing cells are a consequence of the specific type of E-cadherin missense mutation, likely responsible for their pronounced motile phenotype. In favour of this hypothesis, cells treated with specific inhibitors of the RhoA/ROCK pathway resulted in the reversion of their motile phenotype. Both cell lines were previously shown to be also very invasive when tested in collagen invasion assay (27), suggestive of a very aggressive behaviour inferred by the E-cadherin mutations to the cells. Interestingly, this in vitro data mirrors the aggressive behaviours of the primary tumours of patients carrying both germline missense mutations. In particular, for the patient carrying the A634V mutation the neoplasia had extensively involved the peritoneoum (peritonial carcinomatosis) immediately one month after diagnosis. The patient harbouring the T340A mutation had positive lymph node metastases, despite being an early gastric cancer.

V832M expressing cells show very low cell motility, despite having an increased level of active RhoA. Immunoprecipitation studies revealed that, in these cells, the adhesion complex is destabilised, likely due to a reduced affinity of {alpha}-catenin for ß-catenin. V832M mutation affects the first residue of the binding site of ß-catenin to E-cadherin, but the E-cadherin/ß-catenin interaction is not perturbed. Based on this observation, we hypothesize that the mutant V832M E-cadherin/ß-catenin complex results in an unfavourable conformation for the correct binding of ß-catenin to {alpha}-catenin, thus justifying the abnormal actin polarization displayed by these cells. In this respect, the disturbance of the adhesion complex in these cells appears to hamper cell motility, in spite of having increased levels of active RhoA. It was previously reported that a homozygous deletion of the {alpha}-catenin gene was responsible for loss of cell–cell adhesion in a human lung cancer cell line (49). Additionally, it was shown that a deletion of {alpha}-catenin abolished the binding to ß-catenin and was associated with the inhibition of adhesion junctions formation in an ovarian carcinoma cell line (50). In agreement with these observations, the V832M expressing cells, beside low cell motility, also show loss of cell–cell adhesion, strengthening the hypothesis of a disturbance in the interaction between ß-catenin and {alpha}-catenin due to the mutation.

The A617T and wild-type E-cadherin expressing cells share the same epithelial morphology, and behaved similarly in wound healing. This data supports our previous report showing that in the CHO in vitro system the A617T E-cadherin mutant has only mild effect on the protein activity.

Our results show that the correct formation of the adhesion complex at the cell surface and its interaction with the cytoskeleton are required for an efficient cell migration. According to this, CHO cells negative for E-cadherin and V832M cells, with a reduced stability of the adhesion complex, are the only cell lines that fail to migrate unidirectional, as a result of the low polarization. This data supports previous reports showing the implication of the adhesion complex in the direction of cell migration during epithelial wound healing (51) or intercellular motility during epithelial morphogenesis (52).

Nevertheless, in our experimental conditions, invasion and motility appear to be distinct properties since no positive correlation was found between the motile behaviour and the invasion ability of these cells. The motile phenotype of T340A and A634V expressing cells was reverted after incubation with the specific RhoA and ROCK inhibitors, whereas no effect was observed on their ability to invade into collagen type I. CHO parental cells and V832M expressing cells despite their ability to invade, display very low cell motility when tested in wound healing assay. On the contrary, cells expressing either the wild-type E-cadherin or the A617T mutant show a more consistent unidirectional migration, despite their inability to invade into collagen. Altogether this data suggests that the different effects in cell motility mediated by the E-cadherin mutations are not pivotal for invasion. This assumption is in accordance to what has been recently shown by Wong and Gumbiner (42) who demonstrated a role of E-cadherin as invasion suppressor independent on its specific effects on cytoskeletal organization or cell motility.

In conclusion, our results demonstrate that different germline E-cadherin mutations are associated with well-defined cellular phenotypes and biological behaviours. Mutations like T340A and A634V lead to enhanced cell motility mediated by RhoA activation, and to the establishment of a fibroblastic morphotype. In contrast, the mutation V832M in the ß-catenin binding domain hampers cell motility, by promoting a reduced affinity between ß-catenin and {alpha}-catenin, hence destabilizing the E-cadherin adhesion complex. We therefore conclude that E-cadherin should be considered as a cell-signalling receptor, capable of interfering with specific signalling pathways whenever mutated, thus leading to distinct functional consequences. We have identified three distinct morphotypes associated to the specific set of germline mutations here considered; nevertheless, different missense mutations of the E-cadherin gene are likely to result in a broader spectrum of cell behaviours and phenotypes. For, the specific effects mediated by E-cadherin mutations seem to be not pivotal for tumorigenesis, since the absolute correlation between loss of cell–cell adhesion and invasion is the only common denominator to all the germline E-cadherin mutations studied. In other words, the disturbance of the adhesion complex is the key event for the promotion of cell invasion, independently on the specific cell phenotype, which likely reflects the cellular type and context used. Our data thus gives new insigths into the understanding of the mechanisms linking E-cadherin mutations to the process of cell invasion in diffuse gastric cancer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell culture
CHO-K1 (Chinese hamster ovary) cells transfected with the pcDNA3 vector alone (mock cells) or stably expressing the E-cadherin cDNA mutants (wild-type, T340A, A634V, A617T and V832M) were established as previously described (27,29). Cells were grown at 37°C under 5% CO2 in humidified air, in {alpha}-MEM (+) medium (Gibco-BRL) supplemented with 5% foetal bovine serum, 2 mM L-glutamine, 1% penicillin/streptomycin and 1 mg/ml geneticin. At least two independent clones for each cell line were used in each experiment, to exclude clonal dependence of the results.

Cell motility assay
The migratory/motility behaviour of the transfected cells was analysed in an in vitro wound healing assay, as previously described (53). An artificial wound was created in monolayers of confluent cells with a Gilson pipette tip (1 mm diameter). After washing each culture to remove detached cells, migration was assessed by measuring the distance between wound edges at time intervals (0, 2, 5 and 9 h). The migratory capability of cells was also characterised in the presence of the RhoA inhibitor C3T (6.6 µg/ml) (kindly provided by Dr C. Gespach, INSERM U482, France) and of the ROCK inhibitor Y27632 (20 µM) (Calbiochem).

Immunofluorescence staining
For immunofluorescence staining of E-cadherin and ß-catenin, cells were grown on top of glass coverslips at a density of 1x104 and fixed in methanol (4°C, 10 min) when confluent. For E-cadherin and its mutants, the anti-human E-cadherin monoclonal antibody HECD1 (R&D System) was used at 1 : 100 dilution and an anti-human ß-catenin monoclonal antibody (Transduction Laboratories) was used at 1 : 200 dilution. An anti-mouse FITC-coupled antibody (DAKO) was applied as secondary antibody at 1 : 200 dilution. To assess the cell morphology of moving cells, small scrape wounds were made across confluent monolayers of cells on glass coverslips as previously described (31). After 2 h, cells were fixed in 4% paraformaldehyde (4°C, 30 min) and FITC-conjugated phalloidin (Sigma) was used at 1 : 500 dilution to label F-actin filaments. Actin cytoskeleton structure was also assessed in subconfluent cultures. Coverslips were mounted on slides using an antifading, DAPI containing reagent (Vectashield, Vector) and examined on a LeicaDM IRE2 Fluorescence Microscope using Leica, 20x, 40x and 100x oil immersion objectives, or on a BioRad MRC-600 confocal microscope. Images were taken with a DC-300F Leica camera and processed with LeicaFW4000 software and Adobe PhotoShop.

Proliferation assay
The proliferative index of each cell line was obtained as a function of 5-bromodeoxyuridine (BrdU) incorporation (54). Briefly, subconfluent cells were incubated with 10 µM BrdU (Sigma) for 1 h, rinsed with PBS and fixed in 4% paraformaldehyde, for 30 min at 4°C before immunostaining. The percentage of BrdU (+) cells was determined for ~500 cells in multiple fields in three independent experiments.

Immunoprecipitation and immunoblotting
Immunoprecipitation studies were performed by using the Immunoprecipitation Starter Pack (Amersham Bioscience). Confluent cells were lysed in cold PBS containing 1% Triton X-100, 1% NP40, protease Inhibitor Cocktail (Roche 1 tablet/50 ml buffer) and phosphatase Inhibitor Cocktail (Sigma, 1 : 100 dilution). Total protein was quantified by following the Bradford dye-binding procedure (55). An aliquot of 500 µg of protein was immunoprecipitated with protein G, according to the manufacturer's instructions; 1–10 µg of monoclonal antibodies against {alpha}-catenin, ß-catenin, p120 catenin or the PY20 antibody against tyrosine phosphorylated proteins (Transduction Laboratories) were used. Immunoprecipitated proteins were separated on a 7.5% SDS-polyacrylamide gel by electrophoresis, followed by transfer onto a nitrocellulose membrane (Hybond C-extra, Amersham Bioscience). Immunoblotting was performed with antibodies against E-cadherin (R&D System, 1 : 3500), {alpha}-catenin, ß-catenin or p120 catenin (Transduction Laboratories, 1 : 1000), with the ECL western blotting detection kit (Amersham Bioscience).

RhoA, Rac1 and Cdc42 activity assays
Small GTPases activity assays were performed as previously reported (56). To take into account the effect of wound healing on the activation level of the small GTPases, confluent monolayers of cells in 6-well plates were wounded with a Gilson tip, 4 h prior to the assay. Equal amount of proteins were then engaged in the pull-down assays. GTP-bound Rac1 or GTP-bound Cdc42 were precipitated by PAK167-150 while GTP-bound RhoA was precipitated by the C21 domain of Rhotekin, each fused to GST and pre-coupled to glutathione-sepharose beads (Upstate Biotechnology). Precipitated complexes were analysed on western blot using monoclonal antibodies against RhoA (Santa Cruz Biotechnology), Rac1 (Upstate Biotechnology) and Cdc42 (Santa Cruz Biotechnology). For each transfection, immunoblots were quantified with the Quantity One Software (Bio-Rad), taking as 1 the ratio between RhoA-GTP and Rhotekin bands, Rac1- or Cdc42-GTP and PAK167-150 bands for mock cells. Using the same antibodies, the total amount of small GTPases in whole-cell lysates (2.5% of input) was analysed in parallel.

Collagen invasion assay
Collagen invasion assays were performed as described (27). Briefly, collagen gels, with a minimum thickness of 250 µm, were prepared in a 6-well plate (Nunc), from a collagen type I solution (Upstate Biotechnology), and polymerized overnight at 37°C. T340A or A634V expressing cells (1x105) were incubated on top of the collagen gels, for 24 h at 37°C, with or without the ROCK inhibitor Y27632 (20 µM) or the RhoA inhibitor C3T (6.6 µg/ml). Invasion indices (%) are ratios between the number of invasive cells inside the gel and the total number of cells, counted in at least 12 microscopic fields and determined with a computer-assisted-inverted-microscope.

Statistical analysis
The statistical analysis was performed using the Student's t-test. Differences were taken to be significant at P<0.05.


   ACKNOWLEDGEMENTS
 
The authors thank Professor Carlos Caldas and Dr Jun Yokota for providing information about the germline mutations A617T and V832M, respectively. This study was funded by grants from the Fundação para a Ciência e a Tecnologia, Portugal (BD 15980, Project: POCTI/35374/CBO/2000 and POCTI/CBO/40820/2001), FORTIS Verzekerngen and the Fund for Scientific Research, Flanders (FWO), Brussels, Belgium.


   FOOTNOTES
 
* To whom correspondence should be addressed at: IPATIMUP, Rua Dr Roberto Frias s/n, 4200-465 Porto, Potugal. Tel: +351 225570764; Fax: +351 225570799; Email: rseruca{at}ipatimup.pt Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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