Luisa DeMarte

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.

Targeted Disruption of Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Promotes Diet-Induced Hepatic Steatosis and Insulin Resistance

Carcinoembryonic antigen-related cell adhesion molecule 1 (CC1) is a cell adhesion molecule within the Ig superfamily. The Tyr-phosphorylated isoform of CC1 (CC1-L) plays an important metabolic role in the regulation of hepatic insulin clearance. In this report, we show that CC1-deficient (Cc1(-/-)) mice are prone to hepatic steatosis, as revealed by significantly elevated hepatic triglyceride and both total and esterified cholesterol levels compared with age-matched wild-type controls. Cc1(-/-) mice were also predisposed to lipid-induced hepatic steatosis and dysfunction as indicated by their greater susceptibility to store lipids and express elevated levels of enzymatic markers of liver damage after chronic feeding of a high-fat diet. Hepatic steatosis in the Cc1(-/-) mice was linked to a significant increase in the expression of key lipogenic (fatty acid synthase, acetyl CoA carboxylase) and cholesterol synthetic (3-hydroxy-3-methylglutaryl-coenzyme A reductase) enzymes under the control of sterol regulatory element binding proteins-1c and -2 transcription factors. Cc1(-/-) mice also exhibited impaired insulin clearance, glucose intolerance, liver insulin resistance, and elevated hepatic expression of the key gluconeogenic transcriptional activators peroxisome proliferator-activated receptor-gamma coactivator-1 and Forkhead box O1. Lack of CC1 also exacerbated both glucose intolerance and hepatic insulin resistance induced by high-fat feeding, but insulin clearance was not further deteriorated in the high-fat-fed Cc1(-/-) mice. In conclusion, our data indicate that CC1 is a key regulator of hepatic lipogenesis and that Cc1(-/-) mice are predisposed to liver steatosis, leading to hepatic insulin resistance and liver damage, particularly when chronically exposed to dietary fat.

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.

Carcinoembryonic antigen-immunoglobulin Fc fusion protein (CEA-Fc) for identification and activation of anti-CEA immunoglobulin-T-cell receptor-modified T cells, representative of a new class of Ig fusion proteins

Chimeric immunoglobulin-T-cell receptor (IgTCR)-modified T cells (“designer T cells”) kill tumor cells based on antibody-redirected recognition of tumor-associated antigen. Anti-carcinoembryonic antigen (CEA) designer T cells have been prepared and applied in adoptive cellular immunotherapy regimens for CEA-positive cancers. A CEA-immunoglobulin Fc (CEA-Fc) fusion protein was created from the A3B3 region of CEA and the Fc portion of human IgG for the purposes of activation and detection of anti-CEA designer T cells. CEA-Fc was expressed at high yield in CHO cells and purified to homogeneity in a single step on a protein A affinity column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that CEA-Fc formed disulfide-linked dimers with a molecular weight of about 170 kDa and a monomer size of 85kDa. The A3B3 CEA component of the CEA-Fc bound to anti-CEA monoclonal antibody MN-14, as well as to the single-chain Fv (sFv) derived from this antibody that was expressed in the IgTCR on the surface of designer T cells. The Fc portion of CEA-Fc was recognized by anti-human IgG Fc antibody and bound by human monocyte Fc receptors. CEA-Fc activated the anti-CEA designer T cells as plate-bound or monocyte-bound form but not as soluble form, as measured by CD69 expression and T-cell proliferation. Our results indicate that the CEA-Fc fusion protein can be used to detect the expression of the anti-CEA IgTCR chimeric receptors on the modified T cells, as well as to serve as an antigen to activate the anti-CEA IgTCR modified T cells. CEA-Fc is the prototype for a new class of antigen-Fc molecules that may significantly augment the analytic and therapeutic goals of adoptive designer T-cell immunotherapies.

Abstract 5172: Role of CEACAM1 on cancer cell endothelial adhesion and liver metastasis in vivo

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: There are emerging data to suggest that tumor over-expression of the carcinoembryonic antigen-related cell adhesion molecules (CEACAM1, 5 and 6) may influence cancer metastasis; however, the mechanisms for this are unclear. CEACAM1 was shown to bind homotypically to itself and heterotypically to CEACAM5 and CEACAM6. Since CEACAM1 is the only known CEACAM family member expressed on the hepatic endothelium, we hypothesized that CEACAM1 promotes cancer metastasis by directly mediating binding between circulating tumor cells and the sinusoidal endothelium. Methods: Using intra-vital microscopy (IVM), a physiologically relevant model system to assess the early stages of liver metastasis in vivo, we have compared the hepatic endothelial adhesion of intra-splenically injected MC38 cells (mouse colon cancer cell line with minimal CEACAM1 expression) between Ceacam1+/+ and Ceacam1−/− mice. A CEACAM1-negative population of MC38 cells (MC38-null) was sorted out by FACS using specific anti-CEACAM1 antibodies. MC38-null cells were then infected with retroviruses produced from ψ2 packaging cells carrying pLXSN-CEACAM1 expression construct and infected cells were selected using neomycin-containing media. A CEACAM1-positive population of MC38 cells (MC38-CC1) was sorted out by FACS using specific anti-CEACAM1 antibodies. The migratory ability of MC38-null and MC38-CC1 cells were compared by IVM in Ceacam1+/+ mice as mentioned above. Liver metastasis assays were performed by means of intra-splenic injection of MC38 cells into the Ceacam1+/+ and Ceacam1−/− mice. Data expressed as mean ± SEM, student t-test determined significance (*p<0.05). Results: By using IVM, we observed a 3-fold decrease in hepatic sinusoidal endothelial adhesion of intra-splenically injected MC38-null cells in Ceacam1−/− mice compared to Ceacam1 +/+ mice (0.8±0.2 vs 2.5±0.3 adhered cells)*. Forced expression of CEACAM1 in MC38 cells however did not significantly increase hepatic endothelial adhesion in the Ceacam1+/+ mice (2.7±0.9 adhered MC38-CC1 cells vs 2.5±0.2 adhered MC38-null cells). Development of liver metastasis after intra-splenic injection of MC38-null cells was significantly reduced in the Ceacam1−/− mice (81% reduction in comparison to Ceacam1+/+ mice)*. Conclusion: Sinusoidal endothelial cell, but not cancer cell, CEACAM1 expression supports liver metastasis by increasing the adherence of circulating tumor cells to hepatic endothelium. Further investigation will be required to dissect the exact mechanism of how endothelial CEACAM1 influence hepatic endothelial adhesion of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5172.