GPI-anchored CEA family glycoproteins CEA and CEACAM6 mediate their biological effects through enhanced integrin α5β1-fibronectin interaction

Department of Biochemistry and McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.
Journal of Cellular Physiology (Impact Factor: 3.87). 03/2007; 210(3):757-65. DOI: 10.1002/jcp.20887
Source: PubMed

ABSTRACT Carcinoembryonic antigen (CEA) and CEA family member CEACAM6 are glycophosphatidyl inositol (GPI)-anchored, intercellular adhesion molecules that are up-regulated in a wide variety of human cancers, including colon, breast, and lung. When over-expressed in a number of cellular systems, these molecules are capable of inhibiting cellular differentiation and anoikis, as well as disrupting cell polarization and tissue architecture, thus increasing tumorigenicity. The present study shows that perturbation of the major fibronectin receptor, integrin alpha5beta1, underlies some of these biological effects. Using confocal microscopy and specific antibodies, CEA and CEACAM6 were demonstrated to co-cluster with integrin alpha5beta1 on the cell surface. The presence of CEA and CEACAM6 was shown to lead to an increase in the binding of the integrin alpha5beta1 receptor to its ligand fibronectin, without changing its cell surface levels, resulting in increased adhesion of CEA/CEACAM6-expressing cells to fibronectin. More tenacious binding of free fibronectin to cells led to enhanced fibronectin matrix assembly and the formation of a polymerized fibronectin "cocoon" around the cells. Disruption of this process with specific monoclonal antibodies against either fibronectin or integrin alpha5beta1 led to the restoration of cellular differentiation and anoikis in CEA/CEACAM6 producing cells.

Download full-text


Available from: Maria del Pilar Camacho Leal, May 13, 2015
1 Follower
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Human carcinoembryonic antigen (CEA) is a cell surface adhesion molecule member of the Immunoglobulin Superfamily (IgSF). Aberrant upregulation of CEA is a common feature found in a wide variety of human cancers such as colon, breast and lung. Previous in vitro and in vivo results have demonstrated that CEA can have tumorigenic effects including the inhibition of cell differentiation and anoikis, a specific type of apoptosis triggered by the absence of extracellular matrix-cell contacts. In the present work, we investigate the involvement of the caspase cascade in CEA-mediated inhibition of anoikis and the structural requirements for this signal. Expression of CEA and/or a chimeric protein consisting of the NCAM extracellular domain attached to the CEA-GPI anchor correlates with an early inactivation of caspase-9 and activation of the PI3-K/Akt survival pathway, and at later times, inactivation of caspase-8. The CEA-mediated caspase inactivation as well as activation of Akt was not observed by expression of a CEA molecule incapable of self-binding (DeltaNCEA). These results suggest that the intrinsic caspase pathway is involved in the inhibitory effects of anoikis by CEA and this signal is dependent on the presence of self-adhesive extracellular domains and a CEA-GPI anchor.
    Oncogene 04/2008; 27(11):1545-53. DOI:10.1038/sj.onc.1210789 · 8.56 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: CEA functions as an intercellular adhesion molecule and is up-regulated in a wide variety of human cancers, including colon, breast and lung. Its over-expression inhibits cellular differentiation, blocks cell polarization, distorts tissue architecture, and inhibits anoikis of many different cell types. Here we report results concerning the molecular mechanism involved in these biological effects, where relatively rapid molecular changes not requiring alterations in gene expression were emphasized. Confocal microscopy experiments showed that antibody-mediated clustering of a deletion mutant of CEA (DeltaNCEA), normally incapable of self binding and clustering, led to the co-localization of integrin alpha5beta1 with patches of DeltaNCEA on the cell surface. Activation of alpha5, as defined by an anti-alpha5 mAb-sensitive increase in cell adhesion to immobilized fibronectin, and an increased binding of soluble fibronectin to cells, was also observed. This was accompanied by the recruitment of integrin-linked kinase (ILK), protein kinase B (PKB/Akt), and the mitogen-activated protein kinase (MAPK) to membrane microdomains and the phosphorylation of Akt and MAPK. Inhibition of PI3-K and ILK, but not MAPK, prevented the alpha5beta1 integrin activation. Conversely, anti-alpha5 antibody inhibited the PI3-K-mediated activation of Akt, implying the involvement of outside-in and inside-out signaling in integrin activation. Therefore we propose that CEA-mediated signaling involves clustering of CEA and co-clustering and activation of the alpha5beta1 and associated specific signaling elements on the internal surfaces of membrane microdomains. These changes may represent a molecular mechanism for the biological effects of CEA.
    Journal of Cellular Physiology 06/2007; 211(3):791-802. DOI:10.1002/jcp.20989 · 3.87 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Carcinoembryonic antigen (CEA) is a cell surface protein attached to the membrane by a glycophosphatidylinositol (GPI) anchor, a common modification of cell surface proteins. CEA is overexpressed in many human cancers, and plays a role in tumor progression through its ability to activate certain integrins, thereby blocking cellular differentiation, inhibiting anoikis, and disrupting normal tissue architecture. Previous work established that the CEA GPI anchor contains important and specific information directing these functions, which served as the basis for an investigation of the underlying mechanisms involved. The ability of the GPI anchor to determine protein function was examined using a chimera that consisted of the CEA GPI anchor attached to neural cell adhesion molecule (NCAM) self-adhesive external domains; this chimera, NCB, possessed CEA-like, rather than NCAM-like, functions. The CEA anchor targets the protein to specific domains on the cell surface, resulting in an association with specific signaling molecules. This targeting was employed to modify CEA function, as the presence of a protein with non-functional external domains but the same anchor led to a complete and specific loss of biological function of the CEA-like protein. GPI anchor addition is determined by a specific carboxy-terminal signal sequence, which we hypothesized contained information directing the addition of a particular GPI anchor with functional specificity. To identify this signal, chimeras were generated exchanging amino acids in this signal sequence between CEA and NCAM, a protein with different functional properties. A stretch of 6 amino acids within the signal sequence was found to be necessary and sufficient for the addition of the CEA-specific anchor. Since this region is well conserved, but not identical, in the CEA family members CC6 and CC7, we examined whether these proteins were also attached to the same GPI anchors. Surprisingly, while the anchors of these proteins are functionally equivalent to that of CEA, they are not completely identical. This work therefore explores the molecular basis for functional specificity of GPI anchors, demonstrating how specificity of GPI-anchored proteins is determined and the resulting functional consequences, while offering a novel method to inhibit the function of proteins with this type of anchorage. L'antigène carcinoembryonnaire humain (CEA) est un membre d'une famille de protéines de surface cellulaire fixées à la membrane par un ancrage glycophosphatidylinositol (GPI), une modification commune des protéines de la membrane plasmique. CEA est surexprimé dans plusieurs cancers humains et joue un rôle dans la progression tumorale par sa capacité à activer certaines intégrines, bloquant de ce fait la différentiation cellulaire et l'anoikis, et perturbant l'architecture tissulaire normale. Plusieurs recherches antérieures ont établi que l'ancre GPI de CEA contient de l'information importante et spécifique dirigeant ces fonctions. Ces travaux ont servi de base pour l'étude biologique et moléculaire des mécanismes impliqués. Afin de démontrer les fonctions biologiques de CEA, nous avons étudié les capacités de l'ancre GPI à modifier la fonction des protéines en utilisant une protéine hybride (NCB) composée de l'ancre GPI de CEA attachée aux domaines externes auto-adhésifs de la molécule d'adhésion cellulaire neuronale (NCAM). La protéine chimérique NCB possède des fonctions similaires à CEA. L'ancre de CEA cible la protéine à des domaines spécifiques de la surface cellulaire, ce qui mène à l'association de la protéine avec des éléments de signalisation spécifiques. Ce ciblage a été utilisé pour modifier la fonction de CEA, car la présence d'une protéine dont les domaines extracellulaires sont non fonctionnels, mais dont l'ancre est la même, a causé la perte complète et spécifique des fonctions biologiques de NCB. L'ajout d'ancres GPI est déterminé par une séquence spécifique située à l'extrémité carboxyle-terminale. Nous avons présumé que cette séquence contiendrait l'information nécessaire à l'addition sélective d'une ancre GPI de fonction spécifique. Afin d'identifier ce signal, des protéines hybrides ont été produites en échangeant des acides aminés entre CEA et NCAM, deux protéines de fonctions distinctes. La caractérisation de ces hybrides a démontré que l'ajout de l'ancre spécifique de CEA est déterminé par une séquence nécessaire et suffisante de 6 acides aminés. Puisque cette région est bien conservée, mais non identique, chez les membres CC6 et CC7 de la famille CEA, nous avons mené des recherches pour déterminer si ces protéines sont également fixées à la même ancre GPI. Quoique les ancres de ces protéines soient équivalentes à celle de CEA en termes de fonction, elles ne sont pas complètement identiques. Cette thèse présente de nouvelles informations sur la spécificité fonctionnelle des ancres GPI, en démontrant comment leur spécificité est déterminée. Ce travail discute les conséquences fonctionnelles des ancres GPI et présente une nouvelle méthode pour altérer la fonction de protéines associées à ce type d'ancrage.