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Human Molecular Genetics Advance Access originally published online on April 6, 2006
Human Molecular Genetics 2006 15(10):1704-1712; doi:10.1093/hmg/ddl093
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

E-cadherin missense mutations, associated with hereditary diffuse gastric cancer (HDGC) syndrome, display distinct invasive behaviors and genetic interactions with the Wnt and Notch pathways in Drosophila epithelia

Paulo S. Pereira1,2,{dagger}, Alexandra Teixeira1,2,{dagger}, Sofia Pinho2, Paulo Ferreira3, Joana Fernandes2, Carla Oliveira3, Raquel Seruca3, Gianpaolo Suriano3 and Fernando Casares1,2,*

1Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto. Porto 4150-180, Portugal, 2Centro Andaluz de Biología del Desarrollo (CABD), CSIC-JA-Universidad Pablo de Olavide. Ctra. de Utrera km1, Seville 41013, Spain and 3Institute of Molecular Pathology and Immunology (IPATIMUP), Universidade do Porto. Porto 4200-465, Portugal

* To whom correspondence should be addressed. Tel: +34 954348947; Email: fcasfer{at}upo.es

Received March 1, 2006; Accepted March 29, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Germline mutations in the human E-cadherin (hEcad) gene, CDH1, are initiating events in cases of human hereditary diffuse gastric cancer (HDGC) indicating that hEcad is a tumor suppressor. Among the hEcad mutations identified so far, some are missense, but the pathological relevance of these missense mutants is still unclear. In vitro assays show that missense mutations result in full-length hEcad molecules that retain some distinct biological activity, but in vivo functional studies in animal models are still lacking. Here we verify the potential of a Drosophila model to in vivo characterize the functional consequences of HDGC-associated germline missense mutations and to identify signaling pathways affected by these mutations. To this end, we have generated transgenic fly strains expressing the wild-type hEcad gene or its missense mutations. Similar to the fly Ecad, expression of wild-type hEcad and missense forms in fly epithelia resulted in their localization to the subapical region. In addition, we verify a genotype–phenotype correlation associated to the specific domain affected by the mutations, because cells expressing normal or missense mutant hEcad display different migratory and invasive behaviors in fly epithelia. We show that some of these effects might be mediated through hEcad interacting with the endogenous fly ß-catenin, Armadillo, thus interfering with the Wnt signaling pathway. Therefore, the use of this simple in vivo system will contribute to characterize the effects that missense hEcad have on cell behavior in a tissue environment, and might help to understand their significance in gastric cancer onset.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The epithelial-cadherin (Ecad) is required for the formation and maintenance of epithelia. Ecad mediates cell–cell adhesion by being an essential component of the intercellular adhesion complexes. Mammalian Ecad comprises five tandem cadherin repeats in the extracellular domain, involved in Ca2+-dependent homophilic interactions. The juxtamembrane domain interacts with catenin-p120, and a highly conserved cytoplasmic domain serves as interface for the binding of ß or {gamma}-catenin, linking Ecad to the cytoskeleton (1Go). The assembly of the cadherin–catenin complex directly regulates actin cytoskeleton organization and supports the formation of molecular bridges between neighboring epithelial cells, although in a more dynamic manner than previously thought (2Go,3Go). Accordingly, human Ecad (hEcad) is an invasion suppressor as its deregulation is often found in advanced invasive carcinomas (4Go). Furthermore, the loss of Ecad-mediated cell–cell adhesion was shown to have a causal role in the transition from differentiated adenoma to invasive carcinoma in a murine pancreatic carcinogenesis model (5Go). The possibility that Ecad was a tumor suppressor, and its deregulation an initiating event in human tumorigenesis, only came out recently from the study of early hereditary diffuse gastric cancer (HDGC) lesions in carriers of germline mutations in the hEcad gene, CDH1 (6Go–8Go). It was subsequently shown that the germline mutations in CDH1 bear consequences on how those mutant hEcad proteins perform in in vitro adhesion and invasion assays. Additionally, further evidence revealed an association between the specific location of each mutation in the hEcad gene and cell phenotype. Thus, mutations in the extracellular domain of the protein (T340A and A634V) exhibited enhanced cell motility mediated by RhoA activation; whereas the intracellular V832M mutation, within the ß-catenin-binding domain, hampers cell motility by destabilizing the Ecad–catenin junctional complex (9Go,10Go). These differences might account for distinct clinical outcomes depending on the domain affected by the mutations. Still, all the aforementioned studies on missense hEcad mutations are based on the use of in vitro models.

In addition to its structural role as a junctional component, Ecad may interact with several signaling pathways, including the Wnt pathway, as Ecad binds to ß-catenin, the major nuclear transducer of the Wnt signal. As ß-catenin levels are critical for Wnt signaling during tumorigenesis, mutations in hEcad might affect Wnt signaling (11Go).

As a first step towards the in vivo characterization of the functional consequences of HDGC-associated germline missense hEcad forms, and to identify signaling pathways specifically affected by these mutations, we have generated a hEcad overexpression model in Drosophila. The high degree of conservation in molecular, cellular and developmental processes between Drosophila and mammals, and the powerful tools developed in the fly justify its use, at least as a first approximation, for this type of studies (12Go).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Drosophila model we established is based on a binary gene targeting method, in which the gene of interest—in our case different forms of hEcad—cloned under UAS sequences, can be expressed using tissue specific GAL4 ‘driver’ lines (13Go). Thus, we generated transgenic UAS fly lines carrying cDNAs of normal or either of two missense mutant forms of hEcad, obtained from HDGC patients [hEcadA634V (extracellular) and hEcad-V832M (intracellular) (9Go); Fig. 1A].


Figure 0931
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  		Figure 1. Expression of mutant forms of hEcad disrupt Drosophila wing epithelial organization. (A) Diagram comparing the domain structure of human and Drosophila Ecad. Positions of the mutations A634V and V832M are indicated. (B) Wild-type and mutant hEcad proteins are expressed at similar levels. Western blot analysis of the expression of different forms of hEcad driven by 1096-GAL4 in Drosophila wing discs. Molecular weights (in kDa) are indicated on the right. Arrowheads mark the hEcad bands. (-) indicates 1096-GAL4/UAS-LacZ control flies. Anti-Tubulin is included as a reference for total loaded protein. (C) Cells expressing mutant hEcads sort into neighboring regions. Confocal sections across the wing pouch epithelium showing localization of wing pouch derived cells with different hEcad proteins being overexpressed with the 1096-GAL4 driver. The 1096-Gal4 expression domain is delimited by the white line. Cells invading neighboring epithelia are marked with arrow. Actin (in green) is detected with Rhodamine-phalloidin and hEcad proteins (in red) with a mouse anti-hEcad monoclonal antibody.

 
First, we checked whether the hEcad proteins might be expressed in Drosophila epithelial tissues. We analyzed the protein levels of hEcad expressed in the Drosophila developing wing epithelium (the so-called wing imaginal disc) with the wing-specific Bx1096-Gal4 driver, and observed by immunoblotting that wild-type hEcad, hEcadA634V and hEcadV832M proteins were expressed at similar levels in wing discs (Fig. 1B). The three hEcad proteins migrate in the gel with the same mobility pattern (see also Materials and Methods). To evaluate the overall consequences of expressing the three hEcad forms in the wing epithelium, we immunostained the wing discs with anti-hEcad antibody and Rhodamine-phalloidin, to detect F-actin. Cells induced to express wild-type hEcad in the wing pouch epithelium appear to maintain their cell-adhesion properties and remain confined to their normal epithelial fold (Fig. 1C). Importantly, cells from the wing pouch epithelium expressing the two mutant forms of hEcad, A634V and V832M, are observed infiltrating neighboring wing regions (arrows, Fig. 1C). Interestingly, the expression of the two mutant hEcads reveals itself in distinct patterns of tissue invasion. Cells that express hEcadA634V appear to invade as a group, suggesting that homotypic adhesion still remains, whereas hEcadV832M expressing cells invade in smaller groups or even individually. Thus, the mutation V832M might have a stronger effect on the ability of hEcad to support cell–cell adhesion.

We then investigated whether hEcad has a normal subcellular localization pattern in the Drosophila wing epithelium and whether the two mutations being tested affect this pattern. Confocal immunostainings revealed that both normal and missense hEcad forms localize preferentially, although not solely, to subapical regions (Fig. 2), as the fly counterpart, DE-cad, does if overexpressed in a similar manner (Fig. 2A and F). We could also observe the hEcads localizing to basolateral regions, perhaps due to overexpression levels (Fig. 2C–E). This basolateral localization results in the formation of actin foci away from their usual location in subapical region, revealing the actin nucleating capacity of hEcad proteins in Drosophila epithelial cells (Fig. 2D and E and data not shown). The overexpression of the different forms of hEcad was observed to cause a slight reduction of the endogenous DEcad at the adherens junctions (compare Fig. 2F with G). Because in our experiments DEcad is monitored using a ubiquitously expressed GFP-tagged DEcad (under a Ubiquitin-63E promoter) (14Go), the down regulation we observe most likely occurs post-transcriptionally. This can be the result of a displacement of the endogenous DE-cad from the junctional complexes by the overexpressed forms. The sequence comparison of fly and hEcad molecules reveals the absence of the so-called ‘non-cordate cadherin domain’ in the human protein (Fig. 1). This domain has been reported to be required for the correct translocation of DEcad to adheren junctions in Drosophila embryonic epithelial cells (15Go). Nevertheless, our results indicate that the lack of a non-cordate cadherin domain in hEcad does not preclude its correct subcellular localization or stable expression in Drosophila cells.


Figure 0932
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  		Figure 2. Subcellular localization of different forms of Ecad in Drosophila wing epithelial cells. (A–H) Confocal z-sections across the wing epithelium showing localization of different Ecad proteins overexpressed with the 1096-GAL4 driver. (A–C) Co-localization of sub-apical actin, DEcad (Ubi-DEcad-GFP), and hEcad, expressed under 1096-GAL4 control. Most AJ regions are stained by either DEcad or hEcad. (D and E) hEcadA634V and hEcadV832Malso show localization to subapical AJs sites, detected by actin accumulation. (F–H) Detailed comparison of the intracellular localization of hEcad and DEcad relative to actin. Actin is detected with Rhodamine-phalloidin and PKC is used as an apical cell marker. Topro3 serves as nuclear marker.

 
When transfected in CHO cells, different hEcad mutants elicit distinct migration and invasion phenotypes: hEcadV832M induces invasion but no migration, whereas hEcadA634V induces both migration and invasion (9Go). To further analyze to what extent these functions are mimicked in our in vivo model, we followed the behavior of wing epithelial cells expressing the different forms of hEcad, using the Omb-GAL4 driver that is expressed in a central domain of the wing pouch (Fig. 3A). Whereas hEcad-expressing cells remain in a compact domain and show no sign of sorting behavior (Fig. 3B), cells expressing hEcadA634V separate into several domains indicating that groups of cells have migrated collectively, away from one another (Fig. 3C). Cross-sections through the hEcad- and hEcadA634V-expressing epithelia show a normal monolayered wing epithelium, with compact nuclei arranged in a pseudo-stratified manner (Fig. 3A–C). In contrast, hEcadV832M-expressing cells lose the epithelial integrity as cell nuclei can be seen extruding through the basal epithelial side (Fig. 3D). This is better observed when hEcadV832M is expressed under the control of Ptc-GAL4 (Fig. 3E). Ptc>hEcadV832M cells are often seen exiting the epithelium through its basal side as streams of cells, reminiscent of the invasive behavior this mutant form induces in CHO-transfected cells (9Go). As the UAS lines used in this study produce similar amounts of the each hEcad form when driven by GAL4 drivers (Figs 1B and C and 5), the distinct cellular responses observed must be explained by qualitative differences among the proteins. Therefore, these in vivo experiments recapitulate the in vitro results and allow the phenotypic discrimination of different forms of hEcad in Drosophila epithelia.


Figure 0933
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  		Figure 3. Changes in cell behavior elicited by in vivo expression of hEcad forms in the wing epithelium. Wing disc expression of GFP (A) and different forms of hEcad (B, C and E) driven by Omb-GAL4 (A–D), or Ptc-GAL4 (E). Confocal images of whole discs (left column) and z-sections, through the green line (right column) are shown. Apical is up in the right column. (A) Omb-GAL4 directs expression in a solid domain in the wing primordium. (B) Expression of wild type hEcad does not disrupt the epithelial structure. (C) Expression of hEcadA634V results in the fragmentation of the OMB domain (arrowheads). (D) Expression of hEcadV832M within the Omb domain induces hEcad-expressing cells to invade the basal region of the epithelium (arrowheads). (E) hEcadV832M expression driven by Ptc-GAL4 (expressed in an anterior stripe abutting the anterior-posterior border of the wing primordium) cause cells to exit the basal side of the epithelium forming cords of cells (arrowhead).

 

Figure 0935
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  		Figure 5. Drosophila Arm interacts in vivo with both wild-type and V832M hEcad proteins. Hs-GAL4 driven overexpression of wild-type hEcad and of hEcadV832M results in high levels of hEcads protein in total third-instar larvae protein extracts, as detected with anti-hEcad (lower panel). In anti-Arm immunoprecipitates (IPP:arm) of these extracts, we detect both wild-type and V832M hEcad proteins (upper panel) interacting with Arm (middle panel). The interaction of Arm with hEcadV832M is weaker (35% reduction) than the one with wild-type hEcad. WB: western blot.

 
We next analyzed whether the human protein could recapitulate a fundamental signaling property of Ecads in fly tissues: its interaction with Armadillo (Arm), the fly ß-catenin homolog, through its intracellular domain (16Go). To test this point, we induced clones of marked cells overexpressing different Ecad forms in the wing epithelium (driven by an actin-GAL4 promoter; see materials and methods), and analyzed the effects on the amount and localization of Arm (Fig. 4). Overexpression of hEcad results in the membrane accumulation of Arm relative to the wild-type neighboring cells (Fig. 4C), similar to that induced by overexpression of the fly DEcad (Fig. 4A) or of DEcad-Intra5 (Fig. 4B), a truncated form for the extracellular region that maintains the transmembrane and the intracellular domains, the latter harboring the Arm-interacting region (17Go,18Go). Interestingly, though the expression of the extracellular mutation hEcadA634V strongly accumulates Arm (Fig. 4D), this accumulation is much less evident in hEcadV832M-expressing cells (Fig. 4E). Immunoblotting analysis of total Arm levels in wing discs expressing the three distinct hEcad protein forms shows that this effect is not due to significant changes in Arm levels (data not shown), indicating that the accumulation of Arm is due to the stabilization and/or relocation of the protein, rather than due to a transcriptional activation. The hEcadV832M mutant is reported not to affect the ß-catenin/Ecad complex in CHO cells, but the ß-catenin–alpha-catenin interaction instead, without affecting Wnt signaling (9Go). Our in vivo results indicate, though, that the hEcadV832M mutant protein could have a reduced ability to stably interact with Arm at the plasma membrane, at least in Drosophila epithelial cells. To further investigate whether that was the case or not, we compared the ability of the hEcadV832M mutant and of wild-type hEcad to interact in vivo with Drosophila Arm in third-instar larvae (Fig. 5). Overexpression of wild-type hEcad and of hEcadV832M in whole larvae of UAS lines was achieved by heat-shock driven expression of Gal4 (13Go), resulting in high level production of hEcad proteins from UAS-constructs, as detected with anti-hEcad immunoblotting of total protein lysates (Fig. 5, lower panel). Co-immunoprecipitation experiments show that both wild-type and hEcadV832M interact with Arm (Fig. 5). Nevertheless, the quantification of the western blot bands indicates that Arm pulls down ~35% less hEcadV832M than wild-type hEcad (Fig. 5 and data not shown). Therefore, hEcad protein carrying the intracellular V832M mutation interacts more weakly with Arm than the wild-type hEcad form in biochemical assays.


Figure 0934
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  		Figure 4. Stabilization of Arm by different forms of Ecad. (A–E) Confocal images of clones expressing different Ecad forms in the wing disc. Ecad-expressing cells are labeled positively by ß-galactosidase (lacZ). Expression of (A) DEcad, (B) DEcad-Intra5, (C) hEcad and (D) hEcadA634V stabilizes Arm, detected by an increased Arm signal compared with neighboring cells (dashed lines mark the boundaries of the clones). Expression of hEcadV832M (E), by contrast, does not increase the Arm levels significantly. Arrows mark the epithelial folds in the normal epithelium where Arm stain appears specially strong.

 
The overexpression of Arm-interacting forms of fly Ecad (DEcad and DEcad-Intra5) have been shown to phenocopy the effects of the loss of wingless (wg), the fly Wnt-1 homolog, during the development of the wing, as they sequester Arm away from the wg signaling cascade (17Go,18Go). Accordingly, the wing-specific expression of hEcad and hEcadA634V, using the Omb-GAL4 driver, results in a severe wing reduction (Fig. 6A, C and E) comparable to that induced by similar overexpression of DEcad (data not shown). This is also reflected in the loss of expression in wing epithelia of the wg target gene Distal-less (Dll; 19Go)(Fig. 6J, L, N and P). The co-expression of a non-degradable form of Arm, ArmS10 (Arm*), along with these hEcad forms significantly, but not totally, rescues the wing phenotype (Fig. 6D, F and H), confirming that a major outcome of expressing hEcad and hEcadA634V is the reduction of Arm available for Wnt signaling. This functional reduction of signaling Arm must result from its re-location from the cytoplasmic/nuclear pool to the membrane, and not from changes in total levels of Arm, as mentioned previously (Fig. 4 and data not shown). By contrast, expression of hEcadV832M results in a milder wing phenotype (notching of the wing margin; Fig. 6G), and almost no reduction of Dll expression in wing discs (Fig. 6R). Again, these results indicate that, in our in vivo model, different hEcad variants can be discriminated by their functional properties. The fact that the phenotype produced by overexpression of hEcad forms can be significantly rescued by co-expression of Arm indicates that the overexpression of hEcad molecules is not deleterious per se, but produced through specific interactions with signaling pathway components. We also noticed that hEcadV832M+Arm* wings show a phenotype reminiscent of the loss of Notch signal (compare Fig. 6H and I). In fact, when the Notch target gene cut (20Go) is examined in wing discs overexpressing normal hEcad or either of the mutant forms, cut expression is lost (Fig. 6K, M, O, Q, S and U). This result is interesting as the Notch pathway has been associated with cancer (21Go). Nevertheless, the expression of a reporter of E(Spl) mß, a member of the HES family and direct target of the Notch signaling pathway (22Go,23Go), is not significantly altered in hECad overexpressing wing discs (Fig. 6T and U). These results indicate that hEcad cannot be regarded as a general repressor of the Notch pathway in our Drosophila model.


Figure 0936
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  		Figure 6. Analysis of interactions between different hEcad forms and the Wnt and Notch pathways. (A–I) are micrographs of adult wings. (J–U) show wing imaginal discs. Wild-type (A) and Omb-driven Arm* (B) wings are shown for comparison. (D, F and H) Coexpression of Arm* along with hEcad (D), hEcadA634V (F) and hEcadV832M (H) significantly rescues the phenotypes produced by the expression of each of the hEcad forms alone (C, E and G). (H) Wings of Omb>hEcadV832M+Arm* flies show distal vein thickenings reminiscent of those produced by a down regulation of the Notch pathway in Omb> NotchDN (I) (Thickenings are marked by the bracket). Extra vein material seen in (H) (asterisk) is typical of Arm* overexpression. (J–S) Distal-less (Dll; J, L, N, P and R) and cut (K, M, O, Q and S) expression in wing discs (genotypes as marked). Arrows point to the wing disc domains where the normal expression of these genes should be. Dll expression is severely reduced in all cases but when hEcadV832M is expressed, whereas cut expression is almost completely lost in all overexpressing genotypes. (T and U) E(spl)mß expression is present in 1096>UAS-hEcad discs (U; arrow), despite the reduction of the pouch, when compared to 1096 discs (T).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Germ line loss-of-function mutations in the CDH1 gene have been shown to underlie ~30% of HDGC cases. Therefore these mutations could be used for the presymptomatic identification of individuals at risk and their effective treatment, which might include prophylactic gastrectomy (24Go). Recent work shows that up to 30–40% of the CDH1 mutations are missense (8Go). Whereas the loss of Ecad function can be easily explained in cases involving truncating CDH1 mutations in one allele by the inactivation of the other allele (by loss of heterozygosity or promoter hypermethylation), the molecular, cellular and pathological significance of carrying missense CDH1 alleles is still unclear. Model systems aimed at characterizing the behavior of cells carrying these missense mutant forms and the molecular mechanisms affected might therefore contribute for the understanding of the lesions and in aiding in their prognosis. In vitro models where it is possible to assay for the cellular and molecular function of different missense hEcad have been produced (9Go,25Go). Nevertheless, in vivo models are required to study the biological function of these molecules in a tissue context. In addition, such models may allow for functional screens to identify the molecular pathways affected by specific mutations in this tissue environment.

In this report, we have established a Drosophila model to study the function of hEcad missense mutations associated with HDGC. One of the reasons to establish this model is the expectation that the powerful genetic tools available for Drosophila would allow the identification of signaling pathways affected by hEcad expression, and to test for mutation-specific modifier effects of hEcad function. As a first step, we show that human wild-type hEcad, as well as the missense mutations tested, can be effectively expressed in Drosophila epithelial cells, where the human proteins localize to intercellular junctions. Wing-primordium epithelial cells expressing wild-type hEcad were able to maintain a normal epithelial arrangement, but crucially that was not observed when cells were induced to express the HDGC-associated missense forms, A634V and V832M. In the latter conditions, expressing cells lose adhesion and invade neighboring non-expressing region of the epithelium, and manifest a ‘sorting out’ behavior. During normal animal development, groups of cells can sort out, even in morphologically homogenous tissues. This can result in the formation of tissue boundaries where distinct groups of cells are prevented from intermingling. In particular, classic cadherins have been shown to mediate cell sorting in vivo. For example, the formation of a boundary between the cerebral cortex and striatum primordia in the mouse brain is accompanied by the establishment of a complementary expression pattern of R-cadherin and cadherin-6 (26Go). This complementarity is thought to stabilize the formation of the boundary, because if cells near the boundary are induced to express one of the cadherins, these cells will sort out into the compartment expressing that cadherin. In this context, it is interesting to compare the invasion profiles of wing-primordia epithelial cells expressing the two hEcad mutations. The cells that express the A634V hEcad leave the expression domain (wing pouch) and invade the adjacent hinge region as tight groups of cells, so we infer that these cells gain either motile or invasive properties without completely losing the ability to bind between them in a homophilic way. The mutation A634V locates to the fifth extracellular cadherin-repeat domain, the repeat most proximal to the plasma membrane. In in vitro CHO cell assays, this mutation induced a fibroblastic morphotype and increased motility (by ~25%) and conferred an aggressive invasive behavior. However, in contrast to our results, in CHO cells hEcadA634V has no detectable cell–cell adhesion ability in aggregation assays, unlike wild-type hEcad. This discrepancy could either result from the the fact that, in our in vivo assay, cells are in a tissular environment, or from the fact that the invasive Drosophila wing epithelial cells still express endogenous cadherins. Importantly, the pattern of invasion of cells expressing the hEcadV832M is significantly different from the one described for hEcadA634V, as the former cells invade as small roundish groups of cells or individually. The invasion of the wing epithelium by hEcadV832M cells is even more dramatic, as cells appear to leave the wing pouch through the basal side. This result correlates well with the previous observation that hEcadV832M-expressing CHO cells grow as piled-up structures of round cells on top of a monolayer of irregularly shaped cells. These round cells show no polarized actin, whereas the A634V-expressing CHO cells exhibited random polarization with lamellipodia and filopodia formation (9Go). This difference might underline the different invasion patterns observed in the Drosophila wing primordia. The in vivo behavior we show for different missense mutations might be relevant during the onset of cancers associated to these mutations because, as soon as the wild-type CDH1 allele becomes inactivated, the missense form will be the only Ecad molecule expressed by the mutant cells. If the different behaviors exhibited by Drosophila cells carrying each of the missense hEcads were recapitulating the early stages of cancer lesions in situ, this knowledge could contribute to predict the behavior of mutant cells in humans and help in the prognosis of the lession.

We also show, biochemically and genetically, that normal and missense hEcad products functionally interact with the endogenous Drosophila ß-catenin homolog, Arm, although different forms do so with significantly different strengths. Thus, the V832M mutation appears to compromise partially the interaction with Arm in vivo, in agreement with biochemical data that show a weaker hEcadV832M-Arm interaction.

In addition, our results suggest a novel interaction between Ecad and the Notch signaling pathway. All the Ecad forms tested were able to inhibit expression of cut, a Notch target gene in the wing disc. Ecad interference with the Notch signaling pathway appears to happen in a branched path downstream of the Notch receptor itself, as expression of a distinct Notch target, the E(Spl) mß gene, is not significantly affected. An alternative explanation is that Ecad-mediated inhibition of Notch signaling is context-dependent and spatially restricted to the dorsal-ventral boundary of the wing disc, where other signals would be present and cooperate with Ecad. We believe that the results presented here, together with identification of a hEcad-Notch putative genetic interaction, validate this Drosophila system and show its potential to study the molecular and cellular functions, both common and specific, to different germline hEcad missense mutations.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of UAS-hECad transgenic strains
cDNAs from wild-type A634V and V832M Ecads were cloned into pcDNA3 vector and amplified using primers Fwd: 5' AGC GGC CGC TAC CAT GGG CCC TTG GAG C 3' and Rev: 5' AAA CTC GAG CTA GTC GTC CTC GCC GCC 3'. These fragments were cloned into the NotI-XhoI site of pUAST vector. Constructs were germ-line transformed using standard procedures. At least three transgenic lines were established per hEcad form. Transgenic lines driving comparable amounts of the different hEcad forms were selected for the experiments.

Genotypes and genetic manipulations
Fly cultures were kept at 18°C, unless otherwise noted. The GAL4 driver lines used were Bx1096-GAL4 (called 1096) (27Go), Omb-GAL4 (gift from M. Calleja), Ptc-GAL4 (28Go) and hs-Gal4 (13Go); UAS-DEcad and UAS-DEcad-Intra5 (18Go); UAS-ArmS10 (29Go); UAS-DN-Notch serves to drive the expression of a dominant-negative Notch receptor (30Go); UAS-LacZ (13Go). The E(spl) m ß-CD2 reporter is described in (23Go). The Ubi-DEcad-GFP is a transgene expressing a GFP tagged DEcad under the control of the ubiquitous promoter Ubiquitin-63E which localizes as the endogenous DECad does to the subapical adherens junctions (14Go).

For clonal induction of the different Ecads, larvae form the cross of yw hs-Flp122; act> y+> GAL4, UAS-lacZ/CyO females to UAS-Ecad/UAS-Ecad males (at 25°C) were heat-shocked during second larval stage to induce Flp expression, as in (31Go). Wing discs were dissected from wandering third instar larval stage and clones were marked positively with anti-ß-galactosidase.

For rescue experiments, the wings of females 1096/UAS-LacZ; UAS-Ecad were compared to wings from 1096; UAS-Ecad; UAS-ArmS10. The UAS-LacZ is a neutral UAS introduced in the genotypes to equal the number of UAS sites in the genotypes in order to avoid quantitative differences in hEcad expression among genotypes.

For immunoprecipitation assays, third-instar larvae from the crosses between hs-Gal4 and UAS-hEcads were heat-shocked for 1 h at 37°C, and allowed to recover and induce hEcads expression for 5 h at 25°C.

Immunostainings
Stainings were done as in (32Go); Anti-hEcad (1/300) was from R&D System; Anti-DEcad (33Go); anti-PKC (1/1000) (34Go); Anti-Arm (N2 7A1) (1/100) was from Developmental Studies Hybridoma Bank; rabbit anti-ß-galactosidase (1/2000) from Cappel; mouse anti-CD2 (1/500) from Serotec. Secondary antibodies were from Molecular Probes. Rhodamine-phalloidin (1:400) (Sigma) was used to detect actin. Topro3 serves as nuclear marker. Confocal imaging was performed in a Leica TCS SP2 system.

Western blotting and immunoprecipitation
For western blot analysis of 1096-Gal4 driven expression of human cadherins in the wing pouch region, wing discs were dissected from L3 larvae in 1xPBS, lysed for 10 min at 4°C in lysis buffer (1% Triton X-100, 1% NP40, 150 mM NaCl, 50 mM Tris-Cl at pH 8) containing a complete protease inhibitor cocktail (Roche). Lysates were clarified by centrifugation for 10 min at 4°C, boiled in 1xLaemmli buffer, and separated in 7% SDS-PAGE gels. For immunoprecipitation analysis, 35 third-instar larvae were homogenized using a Dounce tissue homogenizer in lysis buffer (as above), and clarified by two centrifugations (10 min, at 4°C). Lysates were precleared by incubation with protein A-Sepharose beads (Amersham Bioscience) for 30 min at 4°C. Following centrifugation, cleared supernatants were incubated with anti-arm (1:200) for 2 h at 4°C, then protein A-Sepharose beads added for a further 1 h incubation. Immunoprecipitates were washed three times with 1xPBS and then resuspended in 1xLaemmli buffer. Immunoblotting was performed using anti-Arm (N2 7A1, 1:200), anti-hEcad (R&D System, 1:2500) and anti {alpha}-tubulin (Sigma, T5168) antibodies. We have observed that depending on the strength of the induction and to which larval tissue hEcad is targeted, two or three distinct bands can be detected. These bands could be due to a protein modification of unknown nature or, more likely, the result of incomplete processing and cleavage of signal and propeptide sequences. Immunoblots were quantified using the ImageJ image processing tool.


   ACKNOWLEDGEMENTS
 
We thank M.D. Martín-Bermudo for reagents and M.D. Martín-Bermudo and J. Castelli-Gair Hombría for comments on the manuscript. This work has been funded by grants POCTI/CBO/44770/2002 and POCI/SAU-OBS/57111/2004 from Fundação para a Ciência e a Tecnologia, co-funded by FEDER funds, to FC.

Conflict of Interest statement. The authors declare no conflict of interests.


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


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TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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