Clifford P. Stanners

Carcinoembryonic antigen, a human tumor marker, functions as an intercellular adhesion molecule

Carcinoembryonic antigen (CEA) is a member of a family of cell surface glycoproteins that are produced in excess in essentially all human colon carcinomas and in a high proportion of carcinomas at many other sites. The function of this widely used tumor marker and its relevance to malignant transformation is therefore of considerable interest. We demonstrate here that CEA mediates Ca2+-independent, homotypic aggregation of cultured human colon adenocarcinoma cells (LS-180) and rodent cells transfected with functional CEA cDNA. Furthermore, CEA can effect the homotypic sorting of cells in heterogeneous populations of aggregating cells. CEA can thus be considered a new addition to the family of intercellular adhesion molecules. We also show that, whereas CEA is localized mainly to epithelial cell membranes facing the lumen in normal adult intestine, it is found on adjacent cell membranes in both embryonic intestine and colonic tumors. A model for the role of CEA in the tissue architecture of adult, embryonic, and aberrant tumor intestinal epithelium is presented.

Crohn's disease adherent-invasive Escherichia coli colonize and induce strong gut inflammation in transgenic mice expressing human CEACAM

Abnormal expression of CEACAM6 is observed at the apical surface of the ileal epithelium in Crohns disease (CD) patients, and CD ileal lesions are colonized by pathogenic adherent-invasive Escherichia coli (AIEC). We investigated the ability of AIEC reference strain LF82 to colonize the intestinal mucosa and to induce inflammation in CEABAC10 transgenic mice expressing human CEACAMs. AIEC LF82 virulent bacteria, but not nonpathogenic E. coli K-12, were able to persist in the gut of CEABAC10 transgenic mice and to induce severe colitis with reduced survival rate, marked weight loss, increased rectal bleeding, presence of erosive lesions, mucosal inflammation, and increased proinflammatory cytokine expression. The colitis depended on type 1 pili expression by AIEC bacteria and on intestinal CEACAM expression because no sign of colitis was observed in transgenic mice infected with type 1 pili–negative LF82-ΔfimH isogenic mutant or in wild-type mice infected with AIEC LF82 bacteria. These findings strongly support the hypothesis that in CD patients having an abnormal intestinal expression of CEACAM6, AIEC bacteria via type 1 pili expression can colonize the intestinal mucosa and induce gut inflammation. Thus, targeting AIEC adhesion to gut mucosa represents a new strategy for clinicians to prevent and/or to treat ileal CD.

Adherent‐invasive Escherichia coli induce claudin‐2 expression and barrier defect in CEABAC10 mice and Crohn's disease patients

Background: Abnormal expression of CEACAM6 observed on the ileal epithelium in Crohns disease (CD) patients allows adherent‐invasive Escherichia coli (AIEC) to colonize gut mucosa. Since intestinal permeability is significantly increased in CD patients, we aimed at investigating whether and how AIEC alter barrier function. Methods: Tissue microarray was performed on ileal biopsies from CD patients in quiescent and active phases. CEABAC10 or wildtype mice were orally challenged with 109 bacteria. Intestinal permeability was assessed by measuring 4 kDa dextran‐FITC flux in serum, barrier integrity was analyzed using biotin tracer experiment, and claudin‐2 protein immunostaining. Bacterial translocation was analyzed in Ussing chambers. Results: Pore‐forming tight junction protein claudin‐2 is strongly expressed in the ileum of 51% patients in quiescent phase and in 49% of the patients with active CD. Infection of CEABAC10 transgenic mice expressing human CEACAMs with AIEC, but not with nonpathogenic E. coli, led to a significant 3.0‐fold increase in intestinal permeability and to disruption of mucosal integrity in a type 1 pili‐dependent mechanism. This is consistent with the claudin‐2 abnormal expression at the plasma membrane of intestinal epithelial cells observed in AIEC‐infected CEABAC10 mice. AIEC bacteria were able to translocate through CEABAC10 intestinal mucosa. Conclusions: These findings strongly support the hypothesis that AIEC type 1 pili‐mediated interaction with CEACAM6 abnormally expressed in the quiescent phase of CD may disrupt intestinal barrier integrity before the onset of inflammation. Thus, therapeutic targeting claudin‐2 induced by AIEC infection could be a new clinical strategy for preserving intestinal barrier function in CD patients. (Inflamm Bowel Dis 2011;)

Different host-cell shutoff strategies related to the matrix protein lead to persistence of vesicular stomatitis virus mutants on fibroblast cells

Acute infection of fibroblastic cell lines by the Indiana strain of vesicular stomatitis virus (VSV) usually induces dramatic cytopathic effects and shutoff of cellular gene expression. We have compared a series of independent mutants with differences in shutoff induction and found that M was mutated either in the N-terminus (M(51)R) or C-terminus (V(221)F and S(226)R). Furthermore, only double mutants (M mutation and a ts mutation related or not to M) were able to persist on fibroblast cell lines at 39 degrees C. A more detailed investigation of the infection was performed for the mutants T1026, TP3 and G31, differing in their host shutoff effects related to M protein. Viral activity in persistently infected mouse L-929 and monkey Vero cell lines was followed by viral proteins detection, RNA synthesis throughout infection and finally detection of infectious particles. All three mutants cause extensive CPE followed by emergence of persistently infected cells on Vero cells. The same thing is seen on L-929 cells except for T1026 which causes little CPE. Taken together, the results form a basis of further studies to clarify how various viral and cellular factors interact in the establishment of a persistent infection by VSV mutants.

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

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 α5β1, underlies some of these biological effects. Using confocal microscopy and specific antibodies, CEA and CEACAM6 were demonstrated to co‐cluster with integrin α5β1 on the cell surface. The presence of CEA and CEACAM6 was shown to lead to an increase in the binding of the integrin α5β1 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 α5β1 led to the restoration of cellular differentiation and anoikis in CEA/CEACAM6 producing cells. J. Cell. Physiol. 210: 757–765, 2007.

A co-clustering model involving α5β1 integrin for the biological effects of GPI-anchored human carcinoembryonic antigen (CEA)

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 (ΔNCEA), normally incapable of self binding and clustering, led to the co‐localization of integrin α5β1 with patches of ΔNCEA on the cell surface. Activation of α5, as defined by an anti‐α5 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 α5β1 integrin activation. Conversely, anti‐α5 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 α5β1 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. J. Cell. Physiol. 211: 791–802, 2007.

Tyrosine phosphorylation of biliary glycoprotein, a cell adhesion molecule related to carcinoembryonic antigen

Biliary-glycoprotein (BGP), a cell adhesion molecule related to carcinoembryonic antigen (CEA), has been shown to exist as several alternatively spliced isoforms. Here we show that BGPa and BGPb are phosphorylated in the chronic myelogenous leukaemia cell line KG-1, which constitutively expresses several BGP isoforms, and Chinese hamster LR-73 cells transfected with the cDNAs encoding BGPa and BGPb. The phosphorylation can be augmented with the protein tyrosine phosphatase inhibitor ammonium vanadate and with TPA (an activator of protein kinase C). Phospho-amino acid analysis of phosphorylated BGPs demonstrated that phosphorylation occurs on serine, threonine and tyrosine residues. Phosphorylation reactions carried out in in vitro membrane preparations from KG-1 cells revealed a close association of BGP proteins with membrane associated protein tyrosine kinases. These observations suggest an association of BGP proteins with signal transduction molecules which is regulated by alternative splicing of the cytoplasmic domain.

Specific inhibition of GPI-anchored protein function by homing and self-association of specific GPI anchors

The functional specificity conferred by glycophosphatidylinositol (GPI) anchors on certain membrane proteins may arise from their occupancy of specific membrane microdomains. We show that membrane proteins with noninteractive external domains attached to the same carcinoembryonic antigen (CEA) GPI anchor, but not to unrelated neural cell adhesion molecule GPI anchors, colocalize on the cell surface, confirming that the GPI anchor mediates association with specific membrane domains and providing a mechanism for specific signaling. This directed targeting was exploited by coexpressing an external domain-defective protein with a functional protein, both with the CEA GPI anchor. The result was a complete loss of signaling capabilities (through integrin–ECM interaction) and cellular effect (differentiation blockage) of the active protein, which involved an alteration of the size of the microdomains occupied by the active protein. This work clarifies how the GPI anchor can determine protein function, while offering a novel method for its modulation.

Opposite Functions for Two Classes of Genes of the Human Carcinoembryonic Antigen Family

The human carcinoembryonic antigen (CEA) family can be divided into two subgroups according to the means of anchorage of member glycoproteins to the cell membrane: glycophosphatidyl inositol (GPI) linkage and transmembrane linkage. The GPI-linked members tend to be up-regulated in human tumours, whereas the transmembrane-linked members tend to be down-regulated. Thus the question as to whether the GPI members could be formally considered to function as oncogenes and the transmembrane members as tumour suppressors deserves consideration. Members of both subgroups function in vitro as intercellular adhesion molecules, but the characteristics of this adhesion, including temperature and divalent-cation dependence, differ markedly between the groups. Even the mechanism of intermolecular adhesion appears to differ fundamentally in that GPI-linked CEA-CEA binding involves a double reciprocal bonding between two domains, whereas transmembrane-linked biliary glycoprotein (BGP)-BGP binding requires only one domain. Finally, the ectopic expression of CEA in myoblasts can block myogenic differentiation leaving the cells with the ability to divide, while expression of BGP does not affect or may even accelerate myogenic differentiation. These differences in phenotypic effects in vitro thus mirror the differences observed in expression in tumours and support the view that the GPI and transmembrane groups have opposite effects on cells in relation to the malignant phenotype.

T-cell killing of heterogenous tumor or viral targets with bispecific chimeric immune receptors

We have previously described several novel chimeric immune receptors (CIRs) that redirect human T cells to kill malignant or HIV-infected cells. These CIRs comprise a cancer- or virus-specific ligand or single-chain antibody fused to the signaling domain of the T-cell receptor CD3-ζ subunit. Binding of the ligand- or antibody-based CIR to the target antigen (Ag) triggers T-cell-mediated cytolysis of the tumor- or virus-infected cell independent of target cell major histocompatibility complex class I expression. A new type of CIR was developed to mediate the lysis of cells that expressed one or more distinct viral or tumor Ags; three bispecific CIRs (BCIRs) were generated that recognized the carcinoembryonic Ag (CEA) and TAG-72 tumor Ags or, alternatively, distinct epitopes in the HIV envelope (HIVenv). T cells expressing the antitumoral Ag BCIR lysed both CEA- and TAG-72-expressing targets and did not kill Ag-negative targets or target cells expressing other members of the CEA family. Similarly, T cells expressing the anti-HIVenv BCIR lysed target cells expressing both the wild-type HIVenv and a mutant HIVenv that lacked the epitopes recognized by the monospecific CIRs. This approach permits the generation of T cells with a broader spectrum of activity capable of killing virus-infected cells and malignant cells and reduces the potential of progression of disease due to Ag loss variants.