CEA-producing Cells Research Articles

The Genome-Wide Analysis of Carcinoembryonic Antigen Signaling by Colorectal Cancer Cells Using RNA Sequencing.

Сarcinoembryonic antigen (CEA, CEACAM5, CD66) is a promoter of metastasis in epithelial cancers that is widely used as a prognostic clinical marker of metastasis. The aim of this study is to identify the network of genes that are associated with CEA-induced colorectal cancer liver metastasis. We compared the genome-wide transcriptomic profiles of CEA positive (MIP101 clone 8) and CEA negative (MIP 101) colorectal cancer cell lines with different metastatic potential in vivo. The CEA-producing cells displayed quantitative changes in the level of expression for 100 genes (over-expressed or down-regulated). They were confirmed by quantitative RT-PCR. The KEGG pathway analysis identified 4 significantly enriched pathways: cytokine-cytokine receptor interaction, MAPK signaling pathway, TGF-beta signaling pathway and pyrimidine metabolism. Our results suggest that CEA production by colorectal cancer cells triggers colorectal cancer progression by inducing the epithelial- mesenchymal transition, increasing tumor cell invasiveness into the surrounding tissues and suppressing stress and apoptotic signaling. The novel gene expression distinctions establish the relationships between the existing cancer markers and implicate new potential biomarkers for colorectal cancer hepatic metastasis.

T125: Effect of carcinoembryonic antigen production by colorectal cancer cells on tumor microenvironment and cancer progression

Tumor markers play an important role in the identification of human malignancies. It has been shown that the carcinoembryonic antigen (CEA, CEACAM5) is a promoter of metastasis in epithelial cancers that is widely used as a clinical marker. The aim of this study is to elucidate the network of genes that are involved in the CEA-induced liver metastasis. Previously, we have shown that CEA is accumulated in the lungs and livers of rats by interacting with their macrophages. We identified and cloned a new gene (CEAR) for the CEA-binding protein, which is located on the surface of fixed liver macrophages, Kupffer cells (Bajenova et al, 2001). It has been shown that the interaction of CEA and CEAR proteins increases the production of IL-1, IL-10, IL-6, TNF- α cytokines (Thomas et al, 2011). This interaction changes the expression of liver adhesion molecules that enhances the survival of cancer cells to the liver. We also suggested that CEA synthesis by cancer cells may influence the E-cadherin adhesion junction complexes and have shown that CEA production violates the functional relationship between Ecadherin and its partners α -, β - and p120 catenin. A new type of interaction was discovered between the CEA and β -catenin and the increased amount of β -catenin in the nuclei of CEA producing cells. The data show that CEA production can cause the dissociation of cancer cells and trigger cancer progression. The CEA synthesis also alters splicing of p120 catenin protein and causes the release of soluble E-cadherin. Previously, CEA and epithelial E-cadherin were considered as independent tumor markers. Our data explain the correlation between the elevated levels of CEA and the increase in soluble E-cadherin in the progression of colorectal cancer (Bajenova et al, 2014). We carried out a comparative transcriptome analysis of CEA-producing cell lines. The RNA transcriptome libraries were obtained and sequenced. By pairwise comparisons of CEA producing and non-producing cell lines using Cummerband program, we selected the set of genes (90 total genes) whose expression have been changed in the CEA-producing cell lines (overexpressed or downregulated). The biological processes that are linked to this differential gene expression were identified by Gene Set Enrichment Analysis (GSEA). In total, 8 significantly enriched GO terms related to the cellular components and biological processes were identified. Using KEGG and GO databases, we also identified the signaling pathways involved in the response to CEA. These findings have direct medical application, since they allow not only to establish the relationships between the existing biomarkers but also to discover the new ones. These biomarkers can be used for diagnosis and monitoring of metastatic carcinomas and for the drug development.

Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes

Oncomarkers play important roles in the detection and management of human malignancies. Carcinoembryonic antigen (CEA, CEACAM5) and epithelial cadherin (E-cadherin) are considered as independent tumor markers in monitoring metastatic colorectal cancer. They are both expressed by cancer cells and can be detected in the blood serum. We investigated the effect of CEA production by MIP101 colorectal carcinoma cell lines on E-cadherin adherens junction (AJ) protein complexes. No direct interaction between E-cadherin and CEA was detected; however, the functional relationships between E-cadherin and its AJ partners: α-, β- and p120 catenins were impaired. We discovered a novel interaction between CEA and beta-catenin protein in the CEA producing cells. It is shown in the current study that CEA overexpression alters the splicing of p120 catenin and triggers the release of soluble E-cadherin. The influence of CEA production by colorectal cancer cells on the function of E-cadherin junction complexes may explain the link between the elevated levels of CEA and the increase in soluble E-cadherin during the progression of colorectal cancer.

Adenoviral Oncolytic Suicide Gene Therapy for a Peritoneal Dissemination Model of Gastric Cancer in Mice

Peritoneal dissemination of gastric cancer is often refractory to systemic therapies. Although adenoviral gene therapy has been reported to be a potentially useful therapeutic modality, the adenovirus itself has a dose-limiting toxicity. A novel system was constructed using adenoviral oncolytic suicide gene therapy targeting carcinoembryonic antigen (CEA), and its therapeutic effect and the possibility to reduce the total viral dose while still preserving the antitumor effect were assessed. Three types of adenoviruses were prepared for this novel system: (A) Ad/CEA-Cre, (B) Ad/lox-CD::UPRT for a Cre/loxP system, and (C) Ad/CEA-E1 for conditionally replicating adenovirus. The antitumor effect of the oncolytic suicide gene therapy (A + B + C) was then evaluated in vitro. Mice bearing peritoneal dissemination of human gastric cancer were treated with either this system (A + B + C) or with a tenfold viral dose of suicide gene therapy (A + B). The adverse effects in terms of hepatotoxicity were then evaluated between the two groups. The current system (A + B + C) demonstrated significantly better cytotoxic effect for CEA-producing cell lines than did suicide gene therapy (A + B) at the same viral dose in vitro. The effect of oncolytic suicide gene therapy was almost equal to that of the tenfold viral dose of suicide gene therapy in vivo. The hepatotoxicity of the two treated groups was also found to be equivalent. It was possible to reduce the total adenoviral dose of oncolytic suicide gene therapy while still preserving the antitumor effect.

949. Oncolytic Autonomous Parvovirus (rH1-yCD) Targeting with P4/Carcinoembryonic Antigen Chimeric Promoter for Virotherapy of Pancreatic Cancer

Background: Successful gene therapy depends on the development of safe and efficient vectors for gene expression targeting. We investigated the use of a rH1CEA-yCD recombinant parvoviral vector, bearing the yeast cytosine deaminase (yCD) suicide gene. The parvoviral P4 promoter was modified by the addition of a 319 bp Carcinoembryonic Antigen (CEA) tissue-specific promoter sequence, to improve the oncotropic and oncolytic intrinsic properties of the autonomous H-1 parvovirus. Material & Methods: To this end, we explored in vitro, the ability of this rH1CEA-yCD virus to infect CEA-producing cell line (BxPc3) and CEA-nonproducing cell lines (HA-HPC and HeLa). The suicide gene expression was evaluated by RT-PCR and western blotting. The cytotoxicity effect was measured by MTT test. For in vivo experiments, we evaluated the oncolytic efficacy of this new recombinant parvovirus on tumor growth upon subcutaneous/ peritoneal pancreatic carcinomatosis models in nude mice. Results: Our data indicate that rH1CEA-yCD infect efficiently the tumor cell lines in the same manner as its parental rH1-yCD. RT-PCR and western blot analyses showed a down-regulation of the expression of both the NS1 (viral gene) and the yCD (suicide gene) brought by rH1CEA-yCD in HA-HPC and HeLa cell lines, when compared with rH1-yCD. With regard to its cytotoxic effect, rH1CEA-yCD/ 5-FC treatment was incapable to induce cell death in those cell lines, but was as efficient as rH1-yCD to kill BxPc3 cell line. In vivo, infection with rH1CEA-yCD led to significant retardation of BxPc3 tumor growth both in subcutaneous and intraperitoneal disseminated pancreatic models. The survival rate of animals bearing CEA-producing cell lines (BxPc3) was improved (p<0.001). However, there was no effect on HA-HPC tumor models. Conclusion: In summary, we have developed a new targeted recombinant parvovirus where the parvoviral oncolytic effect and the suicide gene expression are tightly restricted to tumor cells with CEA tumor marker promoter signaling.

Enhanced Selective Gene Expression by Adenovirus Vector Using Cre/loxP Regulation System for Human Carcinoembryonic Antigen-Producing Carcinoma

Selective gene targeting using the carcinoembryonic antigen (CEA) promoter is useful in gene therapy for gastrointestinal cancer. However, the expression of the CEA promoter is not sufficient. In this study, we tried to enhance CEA promoter activity using the Cre/loxP system. The double infection of CEA-producing cells such as MKN45 and LoVo with AxCEANCre and AxCALNLZ at a total multiplicity of infection (MOI) of 50 achieved 7-fold higher expression level of β-galactosidase activity than single infection of those cells with AxCEALacZ at 50 MOI. On the other hand, the double infection of CEA-nonproducing cells such as MKN1 and HeLa cells showed a very low expression of β-galactosidase activity. In the subcutaneous tumor models, the administration of AxCEANCre and AxCALNLZ into the CEA-producing tumor showed stronger expression of the LacZ gene in tumor tissue than that of AxCEALacZ. In the experiment using orthotopic models of CEA-producing gastric cancer, intraperitoneal double administration of AxCEANCre and AxCALNLZ caused evident LacZ gene expression in transplanted gastric tumors, but no LacZ gene expression in the normal stomach or liver. It was confirmed that enhanced tissue-specific gene transduction under control of CEA promoter using the Cre/loxP system was useful not only in vitro, but also in vivo, especially in orthotopic models.

HSVTK gene therapy for carcinoembryonic antigen-producing human lung cancer cells

The long-term success of gene therapy for cancer relies heavily on the development of effective targeting systems. We investigate the possibility of targeted gene therapy using promoter of carcinoembryonic antigen (CEA) gene. By using luciferase reporter gene, we found that CEA promoter exhibit 16 times high activity in CEA-producing lung cancer cells, A549 than in nonproducing cells, Hela. We also constructed a recombinant expression plasmid pCEATK, in which CEA promoter drives the effector gene, thymidine kinase gene of Herpes Simplex Virus (HSVTK). A549 cells transfected with pCEATK became 865 times more sensitive to ganciclovir (GCV) than the control cells. However, Hela cells transfected with this plasmid remained resistant to GCV. These data indicate the potential for targeted gene therapy using the CEA promoter against CEA-producing tumor cells, such as lung cancer cells.

Selective gene therapy for human lung adenocarcinoma by tumor-specific expression of herpes simplex virus thymidine kinase gene

According to the fact that CEA gene expressed only in lung adenocarcinoma but not in normal lung cells, a retroviral expression vector (pCEATK) of the herpes simplex virus thymidine kinase (HSV-TK) gene regulated by CEA promoter was constructed and introduced into CEA-producing human lung adenocarcinoma cells GL and non-CEA-producing HeLa cells. The expression of pCEATK and Ganciclovir (GCV) sensitivity of the transfected cells were tested in vitro and in vivo. pCEATK expressed only in CEA-producing GL cells but not in non-CEA-producing HeLa cells. The sensitivity to GCV of pCEATK-transfected GL was 992 times higher compared with that of the parental cell line and there was obvious "bystander effect"in vitro. HeLa cells transfected wtih pCEATK were still resistant to GCV. Injection of GCV resulted in significant regression of pCEATK-transfected GL tumors in nude mice. In addition, all mice with any fraction of GL cells expressing HSV-TK exhibited a significant reduction in tumor growth, including mice with only 10% of GL cells expressing HSV-TK. These results show the possibility of HSV-TK gene-drug therapy using the tumor-specific promoter of CEA gene against CEA-producing lung cancers which was usually refractory to conventional chemotherapy.

Selective reversal of drug resistance in drug-resistant lung adenocarcinoma cells by tumor-specific expression of mdrl ribozyme gene mediated by retrovirus

According to the fact that CEA gene expressed only in lung adenocarcinoma and not in normal lung cells, a retroviral vector (pCEAMR) was constructed which carried the CEA promoter coupled to MDRl ribozyme gene. pCEAMR was introduced into drug-resistant lung adenocarcinoma cells GAOK with CEA expression and HeLaK without CEA expression; the expression of pCEAMR and drug resistance in the infected cells were analyzedin vitro andin vivo; pCEAMR expressed only in CEA-producing GAOK cells and not in non-CEA-producing HeLa cells. The drug resistance to doxorubicin (DOX) decreased 91.5% in the infected GAOK cells and did not change in the infected HeLa cells. In nude mice, DOX could obviously inhibit the growth of the infected GAOK tumors, and had no effect on the growth of the infected HeLa cells. These results indicated that MDRl ribozyme gene regulated by CEA promoter expressed only in human adenocarcinoma cells and reversed their drug resistance selectively. This gene-drug therapy might serve as an effective treatment method for patients with CEA-producing lung cancers which was usually refractory to conventional chemotherapy.

Accumulation of NCC-CO-411 monoclonal antibody in human colorectal adenocarcinoma through binding of the released carcinoembryonic antigen in nude mice.

NCC-CO-411, an anti-carcinoembryonic antigen (CEA) monoclonal antibody (MoAb) showed a dose-dependent reactivity with standard CEA, but did not bind to the CEA anchoring with cell membranes. When LS 174T cells, one of the CEA-producing cells, were treated with phosphatidylinositol-specific phospholipase C, NCC-CO-411 MoAb recognized the released CEA containing glycosylphosphatidylinositol as well as the standard CEA. The binding activity of NCC-CO-411 MoAb with CEA was also demonstrated by the immunostaining of the LS 174T colorectal tumor xenograft tissues. The biodistribution study showed that NCC-CO-411 MoAb was mainly taken up by tumor and kidney, while the anti-hCEA MoAb was largely concentrated in liver and spleen rather than in the tumor. These results suggest that NCC-CO-411 MoAb recognizes the released CEA and gives an excellent tumor image in LS 174T tumor-bearing mice.

Internal Image-Bearing Anti-ldiotypic Monoclonal Antibodies

Five anti-idiotypic (Id) monoclonal antibodies (MAbs) (Ab2) were prepared from a BALB/c mouse immunized with anti-carcinoembryonic antigen (CEA) MAb MA208 (Ab1) in a syngeneic system. These anti-Id MAbs appear to recognize unique idiotopes at the combining site of MAb MA208, because they were specifically reactive with MAb MA208 and showed inhibitory activity against the binding of MAb MA208 to CEA. These MAbs were divided into three groups according to the analysis of anti-anti-Id antibodies (Ab3) induced with each anti-Id MAb. Anti-anti-Id MAb M7-625 antiserum (Ab3) reacted with purified CEA in a binding assay and in Western blot analysis, and competed with Ab1 binding to CEA. Furthermore, the binding of anti-Id MAb M7-625 to MAb MA208 was inhibited with CEA, indicating that Ab2 mimics the structure of the epitope in CEA which was recognized with Ab1. These serologic findings suggest that anti-Id MAb M7-625 carries the internal image of the antigen. According to the amino acid sequences of complementarity determining region (CDR) 1, 2 and 3 of the MAb M7-625 variable region, homology of amino acid sequences exists between CDR2 in the H chain (5 amino acids of 10) and domain III of CEA (545-554). Seven anti-Id MAbs were then generated using anti-CEA synthetic peptide MAb P1-356 to analyze further the epitope structure of CEA. These anti-Id MAbs were divided into four groups. Serological analyses as described above suggested that among them, anti-Id MAb M315 had an internal image. We therefore prepared anti-anti-Id MAbs using anti-Id MAb M315. Among them, anti-anti-Id MAb 11B2 reacted directly with CEA and competed with MAb P1-356 in the competition assay. In addition, MAb 11B2 stained both cultured CEA-producing cells and colonic cancer tissues, suggesting that MAb 11B2 is Ab1 like Ab3. These MAbs (Ab1-3) will be of use for the structural analysis of the internal image.

Cea expression of colorectal adenocarcinomas is correlated with their resistance against lak‐cell lysis

We have investigated the in vitro interaction of LAK cells with 20 different human tumor cell lines freshly isolated from colorectal carcinoma (CRC) specimens. Three steps of LAK cell/tumor cell interaction, namely adherence, infiltration and lysis, have been studied. All are important for the cytotoxic effect of LAK cells against solid tumors: LAK-cell adherence was studied on tumor-cell monolayer cultures, the infiltration capacity of LAK cells using tumor-cell spheroids and the resulting cytotoxic effects of LAK cells against CRC cells grown as spheroids or monolayers. Finally, we correlated the degree of lysis of the CRC cells with their carcinoembryonic antigen (CEA) production, the secretion of which varied in a broad range from not detectable to 2,800 ng/day in culture medium. Cytotoxicity experiments showed a good correlation between CEA expression of CRC cells and their resistance against allogeneic LAK cells. Spheroids of CEA-producing cells prevented LAK-cell infiltration resulting in high resistance against LAK-cell lysis. Adhesion of LAK cells on monolayers of CEA-expressing CRC cells was minimal. Our results indicate that CEA expression may be an escape mechanism protecting colon carcinoma cells from an attack by cytotoxic cells.

Polysaccharide-Coated Immunoliposomes Bearing Anti-CEA Fab' Fragment and Their Internalization by CEA-Producing Tumor Cells

Immunoliposomes, bearing Fab' fragments of anti-carcinoembryonic antigen (anti-CEA) monoclonal antibody, have been prepared according to a new method developed in our laboratory. First, egg phosphatidylcholine liposomes were coated with pullulan derivatives. Then, SH-bearing Fab' fragments of anti-CEA were conjugated to the pullulan derivatives attached to the liposomal surface. These immunoliposomes showed specific binding to CEA-producing cells, BM314, which was approximately 20-fold greater than to CEA-non- producing cells, RPMI #4788. In addition, these immunoliposomes were endo cytosed by colon cancer cells, which was confirmed by fluorescence microscopy using liposomes labelled by a hydrophobic fluorescent probe, terbium trisace tylacetonate.

Ultrastructural differentiation and CEA expression of butyrate-treated human pancreatic carcinoma cells.

The effects of butyrate (a biological response modifier) on cellular morphologic features and carcinoembryonic antigen (CEA) expression of human pancreatic carcinoma cells were studied and compared in a well-differentiated, CEA-producing cell line (CAPAN-1), and a poorly differentiated cell line (PANC-1). Butyrate treatment resulted in the acquisition of phenotypic traits commonly attributed to increased "differentiation," including a twofold increase in doubling time, decreased saturation densities, and approximately 50% reduction in colony forming efficiency in both cell lines. Elongation and flattening of cells with extending cellular processes were seen by light microscopy. Significant ultrastructural changes were seen only in the PANC-1 cells, including an increased number of intercellular desmosomes, tonofilaments, and lipid droplets. In contrast, to the coarsely clumped nuclear chromatin (heterochromatin) of untreated PANC-1 cells, the nuclei of the butyrate-treated cells consisted of finely dispersed chromatin (euchromatin). CAPAN-1 cells responded to butyrate with increased CEA synthesis and release. This effect was greatest in the stationary growth phase. Butyrate had no effect on the already low rate of CEA synthesis by PANC-1 cells. These studies suggest that CEA synthesis and state of differentiation are affected independently by butyrate treatment and that the original tumor phenotype plays an important role in response to such treatment.

Production kinetics and immunochemical properties of carcinoembryonic antigen and nonspecific cross-reacting antigen synthesized by various human tumor cell lines.

The production kinetics and immunochemical properties of carcinoembryonic antigen (CEA) and nonspecific cross-reacting antigen (NCA) in various human tumor cell lines were studied. By radioimmunoassay (RIA), five CEA-producing tumor cell lines tested--2 derived from colonic (M7609 and CCK-81), one from pancreatic (QGP-1) and 2 from lung (HLC-1 and KNS-62) carcinomas--were found to produce NCA simultaneously. The cellular contents of CEA and NCA and the amounts of both antigens released into the culture medium were highly variable among the cell lines. It was a distinct contrast that one cell line (CCK-81) released very large amounts of CEA and NCA into the medium while having the smallest amounts of both antigens in the cells, whereas the others contained much larger amounts of the antigens in the cells as compared with the amounts released into the medium. For most of the cell lines, the production of both CEA and NCA increased in the stationary phase of growth as compared with the exponential phase. The production kinetics of both CEA and NCA appeared to be parallel with each other in all the cell lines, though the amount ratio of CEA to NCA produced was variable. By means of a double immunodiffusion test with polyclonal antibodies, antigenic uniformity with no unique organ-specificity was confirmed for all the CEA preparations from spent media of the cell lines, though some differences in the sugar moiety of CEA were detected by RIA using monoclonal antibodies. No antigenic differences among NCA preparations were observed. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), molecular heterogeneity was observed among CEA or NCA preparations isolated from cell lysates.

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: a systematic approach for the determination of epitope specificities of monoclonal antibodies.

A systematic approach for the determination of epitope specificities of monoclonal antibodies to a complex antigen system is described. After initial screening to identify antigen-binding monoclonal antibodies, one or more of the clones are isolated by limiting dilution cloning, grown in ascites, and the resulting antibodies secreted into the ascitic fluid are affinity purified on Sepharose-bound protein A, radiolabeled, and cross-compared with antibodies from other clones by a solid-phase competitive immunoassay. In this work, BALB/c mice were immunized with either purified carcinoembryonic antigen (CEA) or the CEA-producing cell line HC 84S. Spleen cells were fused with the mouse myeloma cell line Sp2/0-Ag14. The supernatants from 25 hybrids showed a significant binding of 125I-CEA (greater than or equal to 15%). Nine hybrids were cloned, resulting in 33 different clones. The antibodies produced by the different cloned hybrids and the remaining uncloned hybrids recognized a total of five different epitopes on CEA. All of the epitopes reside on the protein moiety of the molecule as determined by antibody binding to deglycosylated CEA. The monoclonal antibodies with five different epitope specificities were reacted with tissue sections of normal and cancerous tissues and with peripheral blood smears. Each of the five monoclonal antibodies reacted with tissue sections from colonic, gastric, lung, and mammary carcinomas, as well as from a benign colonic polyp and a resection margin from a colonic carcinoma. Four monoclonals reacted with normal liver tissue. Granulocytes in peripheral blood smears bound three antibodies strongly and one antibody weakly, and one antibody was not bound. One monoclonal antibody that reacted with normal liver tissue was not bound by granulocytes. The ability of these five monoclonal antibodies to differentially detect three different CEA-related antigens in normal and malignant tissues may have clinical utility.

Detection of new cross-reacting carcinoembryonic antigen(s) on cultured tumor cells by mixed hemadsorption assay.

Antisera highly specific for carcinoembryonic antigen (CEA) from New Zealand White rabbits and a goat reacted strongly in antibody binding tests with cultured tumor cell lines, irrespective of the ability of the cell lines to produce CEA. The most reactive were colon carcinoma and melanoma cell lines, the former known to produce CEA and the latter not associated with CEA production. The reactivity was not diminished by absorption with perchloric acid extracts of normal lung or spleen, whereas absoprtion with purified CEA preparations abolished the reactivity. Quantitative absorption studies indicated that reactivity against CEA-producing cell lines could be totally removed by absorption with other CEA-producing lines but not with melanoma cell lines. Reactivity against melanoma cell lines could be completely removed by colon carcinoma cells as well as by melanoma cells. Antisera raised against purified CEA, after absorption with extracts of normal lung, still contained two populations of antibodies, one that binds a newly described antigen cross-reacting with CEA which is present on melanoma cells.