Human Molecular Genetics

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Human Molecular Genetics, 2003, Vol. 12, No. 5 575-582
© 2003 Oxford University Press

Identification of CDH1 germline missense mutations associated with functional inactivation of the E-cadherin protein in young gastric cancer probands

Gianpaolo Suriano^1,, Carla Oliveira^1,2,, Paulo Ferreira¹, José C. Machado^1,3, Maria C. Bordin², Olivier De Wever⁴, Erik A. Bruyneel⁴, Nicole Moguilevsky⁵, Nicola Grehan², Timothy R. Porter⁶, Frances M. Richards⁶, Ralph H. Hruban⁷, Franco Roviello⁸, David Huntsman⁹, Marc Mareel⁴, Fátima Carneiro^1,3, Carlos Caldas^2,* and Raquel Seruca^1,3,*

¹Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), 4200 Porto, Portugal, ²Cancer Genomics Program, Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Addenbrooke's Hospital, Cambridge CB2 2XZ, UK, ³Faculdade de Medicina, Hospital de S. João, 4200 Porto, Portugal, ⁴Laboratory of Experimental Cancerology, UZG, B-9000 Ghent, Belgium, ⁵Service of Applied Genetics, Institute of Biology and Molecular Medicine, B-6041 Gosselies, Belgium, ⁶Section of Medical and Molecular Genetics, Division of Reproductive and Child Health, University of Birmingham, The Medical School, Birmingham B15 2TT, UK, ⁷Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, USA, ⁸Istituto Policattedra di Scienze Chirurgiche, Universita degli Studi di Siena, 53100 Siena, Italy and ⁹Department of Pathology and Laboratory Medicine, University of British Columbia Vancouver, Canada

Received November 19, 2002; Accepted December 20, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
E-cadherin is involved in the formation of cell-junctions and the maintenance of epithelial integrity. Direct evidence of E-cadherin mutations triggering tumorigenesis has come from the finding of inactivating germline mutations of the gene (CDH1) in hereditary diffuse gastric cancer (HDGC). We screened a series of 66 young gastric cancer probands for germline CDH1 mutations, and two novel missense alterations together with an intronic variant were identified. We then analysed the functional significance of the two exonic missense variants found here as well as a third germline missense variant that we previously identified in a HGDC family. cDNAs encoding either the wild-type protein or mutant forms of E-cadherin were stably transfected into CHO (Chinese hamster ovary) E-cadherin-negative cells. Transfected cell-lines were characterized in terms of aggregation, motility and invasion. We show that a proportion of apparently sporadic early-onset diffuse gastric carcinomas are associated with germline alterations of the E-cadherin gene. We also demonstrate that a proportion of missense variants are associated with significant functional consequences, suggesting that our cell model can be used as an adjunct in deciding on the potential pathogenic role of identified E-cadherin germline alterations.

	INTRODUCTION

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E-cadherin is a 120 kDa glycoprotein localized at adherens junctions of epithelial cells, where it mediates homophilic calcium-dependent cell-adhesion (1). Its modular structure consists of five extracellular domains of roughly 110 aa with conserved calcium-binding motifs (EC1-5), a transmembrane region and a cytoplasmatic domain, which interacts with filaments of actin through catenins (2). Disruption of the E-cadherin complex is expected to induce loss of cell adhesion with a concomitant increased cell motility (3,4).

Germline truncating mutations of the CDH1 gene (EMBL/GenBank Data Libraries no. CDH1-Z13009) resulting in E-cadherin inactivation have been identified in hereditary diffuse gastric carcinoma (OMIM no. Gastric cancer-137215) (5,6). To date, 30 HDGC families harbouring CDH1 germline mutations have been described (7) and only five are associated with missense mutations. The pathogenic role of these non-truncating mutations has not yet been established.

The number of families with germline CDH1 mutations described to date is limited and the proportion of gastric cancers due to autosomal inheritance is unknown. One would predict that the proportion of gastric cancer cases related to genetic predisposition is higher in young individuals. To date, a single CDH1 truncating germline mutation in a young person with apparently sporadic diffuse gastric cancer has been reported (5).

The current study aimed at screening a series of 66 young gastric cancer probands for germline CDH1 mutations. We then analysed the functional significance of three germline sequence alterations, two identified in this study and one previously reported by us in a hereditary gastric cancer kindred.

	RESULTS

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The histological characteristics of the carcinomas in the 66 early-onset gastric cancer patients studied were: 37/66 (56.1%) diffuse/isolated cell type gastric carcinomas, 17/66 (25.8%) atypical/mixed type carcinomas and 12/66 (18.2%) intestinal/glandular type carcinomas. Patients with diffuse/isolated cell type gastric cancer were significantly younger (41.6±5.8 years) than those with intestinal gastric carcinoma (45.2±3.8 years; P=0.05). The same holds true when the age of patients with atypical/mixed carcinomas (39.9±7.0 years) was compared with that of patients with intestinal/glandular carcinomas (P=0.03). No significant difference was observed between the age of patients with diffuse/isolated cell and atypical/mixed carcinomas. Patients with diffuse/isolated cell type gastric carcinomas younger than 40 years showed a nearly equal male:female ratio (1.3:1), whereas patients older than 40 showed a male preponderance (2.3:1). Atypical/mixed carcinoma patients showed a male:female ratio of 2:1 in younger than 40 and 2.7:1 in older than 40 years. Patients with intestinal/glandular type tumours had a male:female ratio of 2:1. These results confirm previous reports that intestinal carcinomas are more frequent in older patients, while diffuse/isolated cell type cancers predominate in young patients. Furthermore, the diffuse type of gastric carcinoma in young patients demonstrates a nearly equal sex ratio, compared with a male preponderance in the intestinal form.

The mutation screening identified five cases (7.6%) with potentially deleterious germline sequence variants. Four cases were diffuse/isolated cell type carcinomas and one was an atypical/mixed-type gastric carcinoma. Details of patients, tumours and CDH1 putative deleterious sequence alterations in the five cases are presented in Table 1. No germline CDH1 sequence alterations were found in intestinal gastric cancer patients.

View this table: [in this window] [in a new window]   Table 1. Details of patients, tumours and CDH1 putative deleterious sequence alterations in five cases of early-onset gastric cancer

 
Three of the five patients with germline sequence alterations in CDH1 harboured missense variants in exon 12. Two African-American female patients, 293 and 294, had the identical transition mutation in codon 617 (1849 G to A), leading to an amino acid substitution (Ala to Thr) [Fig. 1A, lane 2, B(2)]. Laboratory contamination was excluded by repeating the mutation analysis with new samples obtained separately from both patients, and a familial relationship between them was excluded by microsatellite analysis (data not shown). We screened exon 12 in 100 Portuguese blood donors and none of these controls showed the alteration. We also screened 93 African (sub-Saharans) DNA samples for this alteration and two showed the same 1849 G to A change (allele frequency 1%). E-cadherin immunohistochemistry in the two cancer cases revealed membranous staining in neoplastic cells forming microglandular/trabecular structures, and heterogeneous membranous staining in signet ring cells.

View larger version (48K): [in this window] [in a new window]   Figure 1. Germline missense mutations in the CDH1 gene. (A) CDH1 exon 12 PCR/SSCP analysis, showing a band pattern in normal DNA (N) and band shifts in DNA from patients with early-onset diffuse gastric cancer (1, J68; and 2, 293). (B) Sequence chromatograms. (1) Missense mutation in codon 634 (1901 C to T) in patient J68; (2) Missense mutation in codon 617 (1849 G to A) in patient 293 (patient 294 had an identical mutation).

 
The second missense alteration (Table 1) was identified in individual J68, with a signet ring cell carcinoma of the stomach at the age of 30. The alteration, a transition at codon 634 (1901 C to T), leads to an amino acid substitution (Ala to Val) [Fig. 1A, lane 1, B(1)]. This missense variant was not present in 100 Portuguese normal controls. Expression of E-cadherin protein in the cancer was not determined because of lack of tumour tissue.

The remaining germline sequence alteration (Table 1) was a transition in intron 4, at position 532-18 (C to T transition) found in two individuals. This alteration was not found in a series of 100 Portuguese normal controls. The RT–PCR as well as the immunohistochemical analysis, in the cancer cells of one of these individuals for whom tumour material was available (CE 137), revealed lack of E-cadherin mRNA and protein expression, respectively (data not shown). MS-PCR of the CpG island 3 of the CDH1 promoter in this case showed the presence of both methylated (M) and unmethylated (U) alleles in the tumoural DNA extracted from frozen material and analysis of tumour DNA did not reveal any somatic CDH1 mutation or LOH (data not shown).

We selected for analysis the two distinct germline missense CDH1 variants here described (Ala634Val; Ala617Thr), together with a germline missense CDH1 variant previously described by us in a HDGC kindred (Thr340Ala) (8). CHO (Chinese hamster ovary) cells do not express E-cadherin and fail to aggregate homotypically, and were therefore chosen as a model. For each mutation, two independent transfected clones were analysed. Using western blotting, the expected 120 kDa E-cadherin band was detected in the transfected clones and not in the native CHO cells. Only clones expressing comparable levels of E-cadherin were chosen for the functional characterisation. For example of selected clones see Figure 2.

View larger version (45K): [in this window] [in a new window]   Figure 2. Western blot analysis of E-cadherin transfected cell lines using the specific antibody HECD1. Equal amounts of protein were loaded in each lane.

 
CHO cells failed to aggregate in both the fast and slow aggregation assays. Cells transfected with the wild-type CDH1 construct displayed cell-to-cell aggregation in both assays [Table 2; Fig. 3A(1) and B(1)]. The dependence of aggregation on the presence of E-cadherin was demonstrated by its inhibition with the MB2 antibody [Table 2; Fig. 3A(1) and B(2)]. Cells expressing Ala634Val or Thr340Ala E-cadherin mutants failed to aggregate in both assays [Table 2; Fig. 3A(2) and B(3) and (4)]. Cells expressing the mutant Ala617Thr displayed an intermediate phenotype. In the fast assay a 10-fold decreased aggregation was observed in comparison to the wild-type protein [Table 2; Fig. 3A(1)], and this reduced aggregation was shown to be dependent on residual E-cadherin function, since it was blocked by the MB2 antibody [Table 2; Fig. 3A(1)]. Cells expressing this mutant showed normal aggregation when tested in the slow assay.

View this table: [in this window] [in a new window]   Table 2. Median values of particle diameters in fast aggregation assay

 

View larger version (38K): [in this window] [in a new window]   Figure 3. (A) Fast aggregation assay. Panel 1—aggregate formation after 30 min (T30). Cells transfected with wild-type E-cadherin (red solid line); Ala617Thr (blue solid line); incubated with the E-cadherin-blocking antibody MB2 (red dashed line, wild type; blue dashed line, Ala617Thr). Panel 2—aggregate formation for mutant Thr340Ala (red solid line, T0; red dashed line, T30) and for mutant Ala634Val (blue solid line, T0; blue dashed line, T30). *Difference statistically significant. (B) Slow aggregation assay. Typical examples are shown for wild-type (panel 1) and Thr340Ala (panel 2).

 
Cells expressing the wild-type E-cadherin protein were not invasive when tested in a collagen invasion assay (Fig. 4). These cells, when incubated with the E-cadherin blocking antibody MB2, acquired invasive properties and were able to penetrate the collagen matrix. Cells expressing the Ala634Val and Thr340Ala E-cadherin mutants showed an invasive phenotype in this assay (see Fig. 4). Cells expressing the Ala617Thr mutation showed the same non-invasive behaviour as cells expressing wild-type protein (see Fig. 4).

View larger version (10K): [in this window] [in a new window]   Figure 4. Collagen invasion assay. Bars represent the percentage of invasion for each of the experimental conditions.

 
In a wound closure assay (Fig. 5), cells expressing wild-type E-cadherin showed a conserved epithelial-like architecture and a compact front of migration, with cells moving unidirectionally. Cells expressing the Ala634Val and Thr340Val mutants appeared spindle-shaped, with a non-uniform front of migration and loose cells. Cells expressing the Ala617Thr mutation behaved similarly to cells expressing wild-type protein.

View larger version (43K): [in this window] [in a new window]   Figure 5. Wound closure assay. (A) Results of the wound closure assay graphically represented as distance migrated between wound edges (µm in function of time after wound). Typical representative wounds after 5 h (20x) are shown for Ala617Thr (B), and Ala634Val (C).

 

	DISCUSSION

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Our findings of sequence variants in the germline of young patients with apparently sporadic gastric cancer contrast with those published by Stone et al. (9) (5/66 versus 0/96 with sequence variants, P<0.05), but the two series differed significantly. First, the mean age of the patients reported here was lower (41.7±6.0 versus 62.0±10.3 years) and secondly it included a higher proportion of diffuse and atypical/mixed tumours (82 versus 27%). As was observed in HDGC (10), sporadic tumours in carriers of CDH1 germline sequence variants are diffuse or mixed (one case) and never of the intestinal type.

The potentially deleterious germline sequence variants found in the present study included two distinct missense coding sequence alterations and an intronic transition. One of the missense alterations identified was a transition at codon 634 (1901 C to T) leading to an amino acid substitution (Ala to Val). This variant was not present in 100 normal controls and was previously reported as a mutation in a colon carcinoma cell line and the corresponding primary tumour (11). The second missense variant, present in two African-American individuals, was a transition in codon 617 (1849 G to A) leading to an amino acid substitution (Ala to Thr). It occurs in the fifth extracellular repeat (EC5) of E-cadherin, affecting a conserved sequence encoding one of the calcium binding motifs (12). As known, cadherins are functional only in the presence of calcium ions, which stabilize the protein active conformation. Adhesion is promoted in a process of cis-dimerization between neighbour E-cadherin molecules, for which a key role played by repeats 1 and 2 (EC1 and EC2) was demonstrated (13–15). Although less information is available for the other repeats, a comparable activity could be predicted on the basis of their sequence homology, suggesting a putative pathogenic effect for the mutation we found. Besides, this variant was also previously reported as a somatic mutation in an endometrial cancer in association with loss of heterozygosity (16), fulfilling the classic two-hit model of tumour suppressor gene inactivation. E-cadherin immunohistochemistry in these two cases revealed a pattern similar to what is commonly seen in sporadic tumours with somatic missense mutations. The Ala617Thr variant has an allele frequency of 1% in Africans, suggesting it may be an uncommon polymorphism occurring in populations of African origin. Nevertheless, it would be a very improbable occurrence (less than 1:10 000 chance) that both cases of diffuse gastric carcinoma in young African-Americans would also be carriers of the same rare polymorphism, unless such polymorphism is associated with predisposition to stomach cancer. We note with interest that a third case of the identical germline sequence variant in an African-American with gastric cancer has now been reported, further supporting a pathogenic association (17). The remaining germline sequence alteration was a transition in intron 4, at position 532-18 (C to T transition), present in two individuals. The results obtained in one of these individuals, CE 137, were consistent with disruption of gene expression as a result of inactivation of one allele by the germline alteration (first hit) and of the second (wild-type) allele by somatic methylation of the CDH1 promoter in tumour cells (second hit), although this could not be formally proven. One can only speculate why the germline sequence variant would not be expressed, although its location near the two splicing branch sites where the splicing lariat loop could potentially form (‘A’ nucleotides at positions -13 and -27 upstream of the 3' splice acceptor site) could somehow disrupt transcript stability.

The pathogenic significance of the two distinct missense variants (Ala634Val and Ala617Thr) identified in these apparently sporadic diffuse gastric cancer cases was assessed by analysing the functional consequences in a cell model system. We also tested a missense variant previously identified by our group in a HDGC family (Thr340Ala) (8). Interestingly this variant was shown by computer modelling to be located in a newly characterized E-cadherin sequence motif, likely to be involved in the stabilization of the active protein conformation. Two of the E-cadherin missense variants (Thr340Ala and Ala634Val) studied resulted in dramatic functional consequences: cell adhesion was disrupted, and the cells acquired a highly invasive potential associated with enhanced cell motility and loss of epithelial structure. One of these, Ala634Val, is the result of a nucleotide substitution that was shown in a colon carcinoma cell line to result in activation of a cryptic splicing site leading to a truncated protein (11). Our results showed that the sequence variant also results in an amino acid alteration that disrupts the function of the full length E-cadherin protein, which would be expressed when abnormal splicing is not complete. Loss of epithelioid organization in cell lines has been shown to be characteristic of malignant transformation and has been associated to the acquisition of invasiveness and loss of differentiation (18,19). The aggressive phenotype observed for Thr340Ala and Ala634Val is clearly mediated by the E-cadherin mutations introduced, since untransfected CHO cells fail to aggregate, but show low motility and reduced ability to invade (data not shown) when compared with clones transfected with the two mutants. Based on this functional data, we propose that these two missense variants are pathogenic and should be considered as mutations.

The third missense variant (Ala617Thr) did not result in increased motility or invasiveness but was associated with a significant reduction of cellular adhesion on the fast aggregation assay. These mild functional consequences, disruption of adhesion without an effect on motility, have been previously noted when single amino acid substitutions in conserved extracellular domains of E-cadherin were tested (20), and concur with data showing that adhesion and motility are mediated by different E-cadherin-dependent mechanisms (21). The occurrence of this missense substitution as a somatic mutation associated with LOH and as a germline variant in three African-American diffuse gastric cancer patients constitutes strong circumstantial evidence for its association with carcinogenesis. In the assays used here this mutation resulted only in mild functional consequences and therefore we could not conclusively determine its pathogenicity.

In conclusion, we report that a small proportion of apparently sporadic early-onset diffuse gastric carcinomas are associated with CDH1 germline variants. It has previously been reported that some families with hereditary diffuse gastric cancer are also associated with germline CDH1 missense variants (8,22–24). Here we were able to demonstrate that there is a proportion of missense variants associated with significant functional consequences, suggesting they are pathogenic mutations. We propose that functional assays can be used as an adjunct in deciding on the potential pathogenic role of germline variants with significant potential to help clinical counselling of gene carriers.

	MATERIALS AND METHODS

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Patients
Constitutional DNA was isolated from 66 gastric carcinoma patients with no familial history of stomach cancer diagnosed before the age of 51 (65 patients were 50 or younger). Cases originated from the UK (n=26), Portugal (n=22), Italy (n=11), USA (n=6) and Greece (n=1). Genomic DNA was isolated, using standard methods, either from peripheral blood or from fresh frozen normal gastric mucosa. The gastric carcinomas were classified according to Laurén and Carneiro (25,26).

PCR–SSCP
All 16 exons including intron–exon boundaries and the promoter region of the CDH1 gene were amplified by PCR. Primer sequences and PCR conditions were based on those reported previously (5,8,27). Genomic DNA (25–100 ng) was amplified by PCR using as cycling conditions: 30 s at 94°C, 30 s at the appropriate annealing temperature, and 45 s at 72°C for 35 cycles. Reaction products were subsequently diluted 1:1 with denaturing buffer (formamide with 0.025% xylene cyanol and 0.025% bromophenol blue) and heated up to 99°C for 10 min prior to loading onto 0.6x and 0.8x mutation detection enhancement (MDE; Flowgen, Rockland, ME, USA) gels. Three gel conditions were used: 0.6x MDE/10% glycerol, 8°C; 0.8x MDE, 8°C; 0.8x MDE, 20°C. Gels were run at constant temperature for 12–18 h and stained with silver nitrate.

Sequencing analysis
Abnormal bands as well as corresponding normal bands, detected by SSCP, were recovered from the gels and submitted to PCR re-amplification with the original primer sets. Re-amplified products were purified and sequenced on both strands on an ABI Prism 377 automated sequencer (Perkin-Elmer) using the ABI Prism Dye Terminator Cycle Sequencing Kit (Perkin-Elmer, Foster City, CA, USA) and the original primers. The CDH1 amplicon including exon 1 was directly sequenced as reported by Oliveira et al. (8). All sequence alterations detected were confirmed in a second independent PCR.

Polymorphism analysis
The germline mutations detected and polymorphisms were tested in at least 100 Caucasian blood donors using the same primer sets as described before. In addition to the group of Caucasian controls, 93 DNA samples from black Africans (sub-Saharans), without history of neoplasia, were screened for an exon 12 missense alteration (1849 G to A).

Immunohistochemical analysis
Formalin-fixed paraffin embedded tissues were used for the study of E-cadherin immunohistochemical (IHC) expression using a monoclonal antibody HECD-1 (diluted 1:200; Zymed, San Francisco, CA, USA). A modification of the avidin–biotin–peroxidase complex method was used.

Methylation assay
CDH1 promoter methylation analysis was performed using methylation-specific PCR (MS–PCR) and primers described by Graff et al. (28) for CpG island 3 of the CDH1 promoter. This assay entails initial modification of DNA by sodium bisulphite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA.

Statistical analysis
The statistical analysis was performed using the Student's t-test and ² test. Differences were taken to be significant at P<0.05. The Kolmogrov–Smirnov test was used for the statistical analysis of the aggregation curves in fast aggregation assay.

Construction of the plasmids encoding wild-type and mutant E-cadherins
According to the human E-cadherin nucleotide sequence reported in literature (29), oligonucleotide primers were designed and purchased from GIBCO-BRL (forward: 5'-AAA GCT TAC CAT GGG CCC TTG GA-3'; reverse: 5'-AAA CTC GAG CTA GTC GTC CTC GC-3'). A human colon cDNA library (human colon 5'-stretch plus cDNA library, 1 ng/µl, colon cells pooled from 50 Caucasian males) was purchased from Clontech (Clontech Laboratories Paolo Alto, CA, USA) and the full-length E-cadherin cDNA (2649 bp) amplified by using the Hot Start Method (30). Amplification was performed for 30 cycles using the following settings: denaturation at 95°C for 1 min, annealing at 58°C for 1 min and extension at 72°C for 1 min and 30 s per cycle. Sequencing was performed as aforementioned. The cDNA was inserted into the mammalian expression vector pcDNA3 (Invitrogen) as a HindIII–XhoI cassette, leading to the plasmid pECAD1. Mutant plasmids were obtained by nested PCR, by using specific primers carrying the desired mutations (Thr340Ala: Ecad1012F, 5'-TTC CCT GCG TAT ACC CTG G-3' and Ecad1031R, 5'-ACC AGG GTA TAC GCA GGG AA-3'; Ala634Val: Ecad1893F, 5'-ACA CGG GGT GAG TGC CAA C-3' and Ecad1911R, 5'-GTT GGC ACT CAC CCC GTG T-3'; Ala 617Thr: Ecad1841F, 5'-TCA TTG ATA CAG ACC TTC CTC-3' and Ecad1859R, 5'-GGA AGG TCT GTA TCA ATG ATG-3') and pECAD1 as DNA template.

Cell culture and cDNA transfection
CHO (Chinese hamster ovary) DG44 dhfr^- cells were chosen for stable transfection. Cells were tested to exclude presence of E-cadherin expression before transfection, and cultured at 37°C under 5% CO₂ in humidified air, in -MEM (+) medium (GIBCO-BRL) supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 1% penicillin/streptomycin. For the selection of clones 1000 µg/ml geneticin was added to the culture medium.

Cells (5x10⁶) were stably transfected with the different cDNA constructs and with the plasmid alone (25 µg each) by electropermeabilization, and neomycin selection was carried on for the following 3 weeks. Single cell clones were selected and analysed for E-cadherin expression by western blotting.

SDS–PAGE and western immunoblotting
A 5x 10⁴ cell sample was lysed with 50 µl TRITON 114 buffer (EDTA 2 mM, PBS Mg²⁺, Ca²⁺-free 1x, Triton 114 1%, DTT 1 mM, Protease Inhibitor Cocktail Roche 1 tablet/50 ml buffer) and the extracted protein quantified by following the Bradford dye-binding procedure (31). Ten micrograms of protein were then separated on a 7.5% SDS–PAGE, followed by transfer to a nitrocellulose membrane (C-bond, Millipore). Human E-cadherin monoclonal antibody HECD1 (R&D System, 1/3500 dilution) was used.

Cell aggregation assays
Fast cell aggregation assays were done as reported (32). Briefly, single cell-suspensions were prepared and incubated in an isotonic buffer containing 1.25 mM Ca²⁺. 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.

For the slow aggregation assay (32), cells were first trypsinized and then transferred to an agar gel (0.66% w/v) in a 96-well plate. Aggregates formation was evaluated after 24 h using an inverted microscope. For both assays, cells were incubated with the E-cadherin-blocking antibody MB2 (1/20 dilution) to control for inhibition of aggregation.

Collagen invasion and wound closure assays
The invasion assay was performed as previously described (33). 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 computer-controlled step motor.

For the wound closure assay (34), cells were cultivated to confluence in six-well plates. An artificial wound was then created with a plastic pipette tip (1 mm diameter) and migration assessed by measuring the distance between wound edges as a function of time (0, 2, 5 and 9 h).

	ACKNOWLEDGEMENTS

 
This study was funded by grants from: Fundação para a Ciência e a Tecnologia, Portugal (projects: POCTI/35374/CBO/2000 and POCTI/CBO/40820/2001); FORTIS Verzekerngen and the Fund for Scientific Research, Flanders (FWO), Brussels, Belgium; and by Cancer Research UK.

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: +0044 1223 3319 89; Fax: +0044 1223 3317 53; Email: cc234{at}cam.au.uk and Tel: +351 225570764; Fax: +351 225570799; Email: rseruca{at}ipatimup.pt

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

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