Cadherin-23 Mediates Heterotypic Cell-Cell Adhesion between Breast Cancer Epithelial Cells and Fibroblasts

In the early stages of breast cancer metastasis, epithelial cells penetrate the basement membrane and invade the surrounding stroma, where they encounter fibroblasts. Paracrine signaling between fibroblasts and epithelial tumor cells contributes to the metastatic cascade, but little is known about the role of adhesive contacts between these two cell types in metastasis. Here we show that MCF-7 breast cancer epithelial cells and normal breast fibroblasts form heterotypic adhesions when grown together in co-culture, as evidenced by adhesion assays. PCR and immunoblotting show that both cell types express multiple members of the cadherin superfamily, including the atypical cadherin, cadherin-23, when grown in isolation and in co-culture. Immunocytochemistry experiments show that cadherin-23 localizes to homotypic adhesions between MCF-7 cells and also to heterotypic adhesions between the epithelial cells and fibroblasts, and antibody inhibition and RNAi experiments show that cadherin-23 plays a role in mediating these adhesive interactions. Finally, we show that cadherin-23 is upregulated in breast cancer tissue samples, and we hypothesize that heterotypic adhesions mediated by this atypical cadherin may play a role in the early stages of metastasis

GC content of early metazoan genes and its impact on gene expression levels in mammalian cell lines

With the genomes available for many animal clades, including the early-branching metazoans, one can readily study the functional conservation of genes across a diversity of animal lineages. Ectopic expression of an animal protein in, for instance, a mammalian cell line is a generally used strategy in structure–function analysis. However, this might turn out to be problematic in case of distantly related species. Here we analyzed the GC content of the coding sequences of basal animals and show its impact on gene expression levels in human cell lines, and, importantly, how this expression efficiency can be improved. Optimization of the GC3 content in the coding sequences of cadherin, alpha-catenin, and paracaspase of Trichoplax adhaerens dramatically increased the expression of these basal animal genes in human cell lines

E-Cadherin Destabilization Accounts for the Pathogenicity of Missense Mutations in Hereditary Diffuse Gastric Cancer

E-cadherin is critical for the maintenance of tissue architecture due to its role in cell-cell adhesion. E-cadherin mutations are the genetic cause of Hereditary Diffuse Gastric Cancer (HDGC) and missense mutations represent a clinical burden, due to the uncertainty of their pathogenic role. In vitro and in vivo, most mutations lead to loss-of-function, although the causal factor is unknown for the majority. We hypothesized that destabilization could account for the pathogenicity of E-cadherin missense mutations in HDGC, and tested our hypothesis using in silico and in vitro tools. FoldX algorithm was used to calculate the impact of each mutation in E-cadherin native-state stability, and the analysis was complemented with evolutionary conservation, by SIFT. Interestingly, HDGC patients harbouring germline E-cadherin destabilizing mutants present a younger age at diagnosis or death, suggesting that the loss of native-state stability of E-cadherin accounts for the disease phenotype. To elucidate the biological relevance of E-cadherin destabilization in HDGC, we investigated a group of newly identified HDGC-associated mutations (E185V, S232C and L583R), of which L583R is predicted to be destabilizing. We show that this mutation is not functional in vitro, exhibits shorter half-life and is unable to mature, due to premature proteasome-dependent degradation, a phenotype reverted by stabilization with the artificial mutation L583I (structurally tolerated). Herein we report E-cadherin structural models suitable to predict the impact of the majority of cancer-associated missense mutations and we show that E-cadherin destabilization leads to loss-of-function in vitro and increased pathogenicity in vivo

The Molecular Evolution of the p120-Catenin Subfamily and Its Functional Associations

p120-catenin (p120) is the prototypical member of a subclass of armadillo-related proteins that includes δ-catenin/NPRAP, ARVCF, p0071, and the more distantly related plakophilins 1–3. In vertebrates, p120 is essential in regulating surface expression and stability of all classical cadherins, and directly interacts with Kaiso, a BTB/ZF family transcription factor.To clarify functional relationships between these proteins and how they relate to the classical cadherins, we have examined the proteomes of 14 diverse vertebrate and metazoan species. The data reveal a single ancient δ-catenin-like p120 family member present in the earliest metazoans and conserved throughout metazoan evolution. This single p120 family protein is present in all protostomes, and in certain early-branching chordate lineages. Phylogenetic analyses suggest that gene duplication and functional diversification into “p120-like” and “δ-catenin-like” proteins occurred in the urochordate-vertebrate ancestor. Additional gene duplications during early vertebrate evolution gave rise to the seven vertebrate p120 family members. Kaiso family members (i.e., Kaiso, ZBTB38 and ZBTB4) are found only in vertebrates, their origin following that of the p120-like gene lineage and coinciding with the evolution of vertebrate-specific mechanisms of epigenetic gene regulation by CpG island methylation.The p120 protein family evolved from a common δ-catenin-like ancestor present in all metazoans. Through several rounds of gene duplication and diversification, however, p120 evolved in vertebrates into an essential, ubiquitously expressed protein, whereas loss of the more selectively expressed δ-catenin, p0071 and ARVCF are tolerated in most species. Together with phylogenetic studies of the vertebrate cadherins, our data suggest that the p120-like and δ-catenin-like genes co-evolved separately with non-neural (E- and P-cadherin) and neural (N- and R-cadherin) cadherin lineages, respectively. The expansion of p120 relative to δ-catenin during vertebrate evolution may reflect the pivotal and largely disproportionate role of the non-neural cadherins with respect to evolution of the wide range of somatic morphology present in vertebrates today

Convention-cadre relative au programme de gestion durable de l'azote en agriculture wallonne

Rapport d'activités annuel intermédiaire 2005 de GRENeR

Ancient Origin of the CARD–Coiled Coil/Bcl10/MALT1-Like Paracaspase Signaling Complex Indicates Unknown Critical Functions

The CARD–coiled coil (CC)/Bcl10/MALT1-like paracaspase (CBM) signaling complexes composed of a CARD–CC family member (CARD-9, -10, -11, or -14), Bcl10, and the type 1 paracaspase MALT1 (PCASP1) play a pivotal role in immunity, inflammation, and cancer. Targeting MALT1 proteolytic activity is of potential therapeutic interest. However, little is known about the evolutionary origin and the original functions of the CBM complex. Type 1 paracaspases originated before the last common ancestor of planulozoa (bilaterians and cnidarians). Notably in bilaterians, Ecdysozoa (e.g., nematodes and insects) lacks Bcl10, whereas other lineages have a Bcl10 homolog. A survey of invertebrate CARD–CC homologs revealed such homologs only in species with Bcl10, indicating an ancient common origin of the entire CBM complex. Furthermore, vertebrate-like Syk/Zap70 tyrosine kinase homologs with the ITAM-binding SH2 domain were only found in invertebrate organisms with CARD–CC/Bcl10, indicating that this pathway might be related to the original function of the CBM complex. Moreover, the type 1 paracaspase sequences from invertebrate organisms that have CARD–CC/Bcl10 are more similar to vertebrate paracaspases. Functional analysis of protein–protein interactions, NF-κB signaling, and CYLD cleavage for selected invertebrate type 1 paracaspase and Bcl10 homologs supports this scenario and indicates an ancient origin of the CARD–CC/Bcl10/paracaspase signaling complex. By contrast, many of the known MALT1-associated activities evolved fairly recently, indicating that unknown functions are at the basis of the protein conservation. As a proof-of-concept, we provide initial evidence for a CBM- and NF-κB-independent neuronal function of the Caenorhabditis elegans type 1 paracaspase malt-1. In conclusion, this study shows how evolutionary insights may point at alternative functions of MALT1