ABSTRACT
OBJECTIVE: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a member of the carcinoembryonic antigen family. Although its expression has been found in chronic oral inflammatory epithelium, this study aimed to know whether CEACAM1 in oral keratinocytes participates in host immune response against Candida albicans . METHODOLOGY: We investigated CEACAM1 expression in oral keratinocytes induced by C. albicans as well as by Candida cell wall component ß-glucan particles (ß-GPs). Furthermore, the effects of CEACAM1 on ß-GPs-induced heme oxygenase-1 (HO-1) expression and its related signals were examined. RESULTS: Fluorescence staining showed CEACAM1 expression in oral keratinocytes (RT7) cells, whereas quantitative reverse transcription (RT)-PCR indicated that both live and heat-killed C. albicans increased CEACAM1 mRNA expression in RT7 cells. Examinations using quantitative RT-PCR and western blotting indicated that CEACAM1 expression was also increased by ß-GPs derived from C. albicans . Specific siRNA for CEACAM1 decreased HO-1 expression induced by ß-GPs from C. albicans as well as the budding yeast microorganism Saccharomyces cerevisiae . Moreover, knockdown of CEACAM1 decreased ß-GPs-induced ROS activity in the early phase and translocation of Nrf2 into the nucleus. CONCLUSION: CEACAM1 in oral keratinocytes may have a critical role in regulation of HO-1 for host immune defense during Candida infection.
Subject(s)
Heme Oxygenase-1 , beta-Glucans , Heme Oxygenase-1/metabolism , Heme Oxygenase-1/pharmacology , beta-Glucans/pharmacology , beta-Glucans/metabolism , Carcinoembryonic Antigen/metabolism , Carcinoembryonic Antigen/pharmacology , Cell Adhesion Molecule-1/metabolism , Glucans/metabolism , Glucans/pharmacology , Candida , Keratinocytes , Candida albicans/physiologyABSTRACT
Immune checkpoint blockade (ICB) therapy, while groundbreaking, must be improved to promote enhanced durable responses and to prevent the development of treatment-refractory disease. Cancer therapies that engage, enable, and expand the antitumor immune response will likely require rationally designed combination strategies. Targeting multiple immunosuppressive pathways simultaneously may provide additional therapeutic benefit over singular targeting. We therefore hypothesized that the use of two molecules which inhibit three independent, but overlapping, pathways (TIGIT:CD155, PD-1/PD-L1, and TGFß) would provide significant antitumor efficacy in the syngeneic ICB resistant colorectal tumor model MC38 expressing human carcinoembryonic antigen (CEA) in CEA transgenic mice. This novel combination treatment strategy has significant antitumor activity and survival benefit in two models of murine carcinomas, MC38-CEA (CRC) and TC1 (HPV+ lung carcinoma). MC38-CEA mice that responded to αTIGIT and bintrafusp alfa combination therapy generated memory responses and were protected from rechallenge. These effects were dependent on CD4+ and CD8+ T cells, as well as increased immune infiltration into the TME. This combination induced production of tumor-specific CD8+ T cells, and an increase in activation and cytotoxicity resulting in an overall activated immune landscape in the tumor. Data presented herein demonstrate the αTIGIT and bintrafusp alfa combination has efficacy across multiple tumor models, including the checkpoint-resistant model of murine colon carcinoma, MC38-CEA and the HPV+ model TC-1.
Subject(s)
Carcinoma , Colonic Neoplasms , Papillomavirus Infections , Animals , B7-H1 Antigen , CD8-Positive T-Lymphocytes , Carcinoembryonic Antigen/pharmacology , Carcinoembryonic Antigen/therapeutic use , Carcinoma/drug therapy , Colonic Neoplasms/drug therapy , Humans , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Mice , Papillomavirus Infections/drug therapy , Programmed Cell Death 1 Receptor/therapeutic use , Receptors, Immunologic/metabolism , Receptors, Immunologic/therapeutic use , Transforming Growth Factor beta/pharmacology , Transforming Growth Factor beta/therapeutic useABSTRACT
The effects of combined exposure to microplastics and contaminants are still not completely understood. To fill this gap, we assessed the effects of polyethylene terephthalate microplastic fibers (100 mg/L; 360 µm average length) on the toxicity of silver nanoparticles (AgNPs; 32 nm) and silver nitrate (AgNO3 ; 0.1-10 µg Ag/L) to Daphnia magna. Acute immobilization (median effect concentration [EC50]) and cellular energy allocation (CEA; ratio between available energy and energy consumption) were determined in neonates (<24 h old) and juveniles (7 d old), respectively. The 48-h EC50 for AgNP and AgNO3 (2.6 and 0.67 µg Ag/L, respectively) was not affected by the presence of microplastic fibers (2.2 and 0.85 µg Ag/L, respectively). No decrease in the available energy was observed: lipid, carbohydrate, and protein contents were unaffected. However, a significant increase in energy consumption was observed in animals exposed to AgNO3 (250% compared with control) and to the combination of microplastic fibers with AgNP (170%) and AgNO3 (260%). The exposure to microplastic fibers alone or in combination with both Ag forms decreased the CEA (values were 55-75% of control values). Our results show that after short-term exposure (48 h), microplastic fibers increased Ag toxicity at a subcellular level (i.e., CEA), but not at the individual level (i.e., immobilization). These results highlight the importance of combining different levels of biological organization to fully assess the ecotoxicological effects of plastics in association with environmental contaminants. Environ Toxicol Chem 2022;41:896-904. © 2021 SETAC.
Subject(s)
Metal Nanoparticles , Water Pollutants, Chemical , Animals , Carcinoembryonic Antigen/metabolism , Carcinoembryonic Antigen/pharmacology , Daphnia , Metal Nanoparticles/toxicity , Microplastics , Plastics/metabolism , Plastics/toxicity , Silver/metabolism , Silver/toxicity , Silver Nitrate/metabolism , Silver Nitrate/toxicity , Water Pollutants, Chemical/metabolismABSTRACT
Synthetic long peptides (SLP) are a promising vaccine modality to induce therapeutic T cell responses in patients with chronic infections and tumors. We studied different vaccine formulations in mice using SLP derived from carcinoembryonic Ag. We discovered that one of the SLP contains a linear Ab epitope in combination with a CD4 epitope. Repeated vaccination with this carcinoembryonic Ag SLP in mice shows improved T cell responses and simultaneously induced high titers of peptide-specific Abs. These Abs resulted in unexpected anaphylaxis after a third or subsequent vaccinations with the SLP when formulated in saline. Administration of low SLP doses in the slow-release vehicle IFA prevented the anaphylaxis after repeated vaccination. This study underscores both the immunogenicity of SLP vaccination, for inducing T cell as well as B cell responses, and the necessity of safe administration routes.
Subject(s)
Anaphylaxis/prevention & control , Carcinoembryonic Antigen/pharmacology , Epitopes, B-Lymphocyte/pharmacology , Immunoglobulin G/immunology , Peptides/pharmacology , Vaccines/pharmacology , Anaphylaxis/immunology , Animals , Carcinoembryonic Antigen/immunology , Delayed-Action Preparations/pharmacology , Epitopes, B-Lymphocyte/immunology , Female , Mice , Mice, Knockout , Peptides/immunology , Vaccination/methodsABSTRACT
Carcinoembryonic antigen (CEA, CD66e, CEACAM-5) is a cell-surface-bound glycoprotein overexpressed and released by many solid tumors that has an autocrine function in cancer cell survival and differentiation. Soluble CEA released by tumors is present in the circulation of patients with cancer, where it is used as a marker for cancer progression, but whether this form of CEA exerts any effects in the tumor microenvironment is unknown. Here, we present evidence that soluble CEA is sufficient to induce proangiogenic endothelial cell behaviors, including adhesion, spreading, proliferation, and migration in vitro and tumor microvascularization in vivo. CEA-induced activation of endothelial cells was dependent on integrin ß-3 signals that activate the focal-adhesion kinase and c-Src kinase and their downstream MAP-ERK kinase/extracellular signal regulated kinase and phosphoinositide 3-kinase/Akt effector pathways. Notably, while interference with VEGF signaling had no effect on CEA-induced endothelial cell activation, downregulation with the CEA receptor in endothelial cells attenuated CEA-induced signaling and tumor angiogenesis. Corroborating these results clinically, we found that tumor microvascularization was higher in patients with colorectal cancer exhibiting higher serum levels of soluble CEA. Together, our results elucidate a novel function for soluble CEA in tumor angiogenesis.
Subject(s)
Carcinoembryonic Antigen/metabolism , Neoplasms/blood supply , Neovascularization, Pathologic/metabolism , Animals , Carcinoembryonic Antigen/chemistry , Carcinoembryonic Antigen/pharmacology , Cell Differentiation/drug effects , Cell Movement/drug effects , Endothelial Cells/drug effects , Endothelial Cells/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Nude , Mice, Transgenic , Neoplasms/pathology , Solubility , Tumor Cells, CulturedABSTRACT
Colorectal cancers with metastatic potential secrete the glycoprotein carcinoembryonic antigen (CEA). CEA has been implicated in colorectal cancer metastasis by inducing Kupffer cells to produce inflammatory cytokines which, in turn, make the hepatic micro-environment ideal for tumor cell implantation. CEA binds to the heterogeneous ribonucleoprotein M (hnRNP M) which acts as a cell surface receptor in Kupffer cells. The amino acid sequence in CEA, which binds the hnRNP M receptor, is Tyr-Pro-Glu-Leu-Pro-Lys. In this study, the structure of Ac-Tyr-Pro-Glu-Leu-Pro-Lys-NH2 (YPELPK) was investigated using electronic circular dichroism, vibrational circular dichroism, and molecular dynamics simulations. The binding of the peptide to hnRNP M was also investigated using molecular docking calculations. The biological activity of YPELPK was studied using differentiated human THP-1 cells, which express hnRNP M on their surface and secrete IL-6 when stimulated by CEA. YPELPK forms a stable polyproline-II helix and stimulates IL-6 production of THP-1 cells at micromolar concentrations.
Subject(s)
Carcinoembryonic Antigen/chemistry , Heterogeneous-Nuclear Ribonucleoprotein Group M/agonists , Peptide Fragments/chemistry , Amino Acid Sequence , Amino Acid Substitution , Carcinoembryonic Antigen/pharmacology , Cell Line , Heterogeneous-Nuclear Ribonucleoprotein Group M/chemistry , Heterogeneous-Nuclear Ribonucleoprotein Group M/metabolism , Humans , Hydrogen Bonding , Interleukin-6/biosynthesis , Molecular Dynamics Simulation , Peptide Fragments/pharmacology , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Surface Properties , ThermodynamicsABSTRACT
Dendritic cell-based cancer immunotherapy requires tumour antigens to be delivered efficiently into dendritic cells and their migration to be monitored in vivo. Nanoparticles have been explored as carriers for antigen delivery, but applications have been limited by the toxicity of the solvents used to make nanoparticles, and by the need to use transfection agents to deliver nanoparticles into cells. Here we show that an iron oxide-zinc oxide core-shell nanoparticle can deliver carcinoembryonic antigen into dendritic cells while simultaneously acting as an imaging agent. The nanoparticle-antigen complex is efficiently taken up by dendritic cells within one hour and can be detected in vitro by confocal microscopy and in vivo by magnetic resonance imaging. Mice immunized with dendritic cells containing the nanoparticle-antigen complex showed enhanced tumour antigen specific T-cell responses, delayed tumour growth and better survival than controls.
Subject(s)
Carcinoembryonic Antigen/pharmacology , Dendritic Cells/immunology , Drug Carriers/pharmacology , Immunity, Cellular/drug effects , Immunotherapy, Adoptive/methods , Nanoparticles , Neoplasms/therapy , T-Lymphocytes/immunology , Animals , Carcinoembryonic Antigen/immunology , Dendritic Cells/transplantation , Humans , Immunity, Cellular/immunology , Mice , Neoplasms/immunologyABSTRACT
The efficacy of immunization with DNA plasmids for single truncated carcinoembryonic antigen (CEA) peptide or triple repeated CEA peptides in mice was evaluated. A DNA fragment the truncated CEA gene (nucleotide 625-667) encoding two helper T lymphocyte (HTL) epitopes was amplified by PCR and cloned for generating recombinant plasmids for single CEA(625-667) (pcDNA-CEA(625-667)) or triple CEA(625-667) (pcDNA-triCEA(625-667)), respectively. Subsequently, groups of BALB/c female mice were intramuscularly injected with pcDNA-CEA(625-667,) pcDNA-triCEA(625-667) or control pcDNA3.0 vector, respectively. Ten days after the last immunization, the frequency of splenic CD4(+) and CD8(+) T cells in the mice was determined by flow cytometry. The antigen-specific proliferation of splenic T cells and cytokine production ex vivo were analyzed by (3)H-TdR uptake and cytokine ELISA, respectively. The levels of serum antibodies against CEA in the mice were detected by Western blot and ELISA. Although immunization with plasmid for the CEA(625-667) peptide(s) did not alter the frequency of CD4(+) and CD8(+) T cells in mice, vaccination with plasmid for CEA peptide induced strong antigen-specific T cell proliferation, particularly for the plasmid encoding the triple-repeated CEA peptides, accompanied by significantly elevated levels of IFN-γ secreted by T cells from the mice immunized with triple-repeated peptides. Furthermore, immunization with the plasmid for CEA peptide stimulated higher levels of antigen-specific antibody responses in mice. Vaccination with the plasmid for the triple repeated CEA peptides induced stronger Th1 responses. Our findings may be useful for the development of effective DNA vaccine for the immunotherapy of cancer.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Carcinoembryonic Antigen/immunology , Epitopes, T-Lymphocyte/immunology , Peptides/immunology , Th1 Cells/immunology , Vaccines, DNA/immunology , Animals , Antibodies, Neoplasm/immunology , Antibody Formation/drug effects , Antibody Formation/genetics , Antibody Formation/immunology , Carcinoembryonic Antigen/genetics , Carcinoembryonic Antigen/pharmacology , Cell Proliferation/drug effects , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/pharmacology , Female , Mice , Mice, Inbred BALB C , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/therapy , Peptides/genetics , Peptides/pharmacology , Vaccination/methods , Vaccines, DNA/genetics , Vaccines, DNA/pharmacologyABSTRACT
Marked reduction in apoptosis is a hallmark of early colon tumour growth and the vast majority of these tumours exhibit a loss of expression of the glycoprotein carcinoembryonic-antigen-related cell adhesion molecule 1 (CEACAM1). We recently reported that the CEACAM1 functions as a mediator of apoptosis implicating this cell surface protein in early tumour development. However, the mechanistic involvement of CEACAM1 in cell death pathways is unclear. Here, we show that apoptosis triggers cleavage of the long form of CEACAM1 (CEACAM1-4L) at intracellular and extracellular sites in Jurkat cells and HEK293 cells. Signalling through CEACAM1 leads to caspase activation including caspase-1 and -3 and also involves non-caspase proteases. Moreover, we provide evidence that the naturally occurring CEACAM family member CEA is an inducer of CEACAM1-mediated apoptosis in HT29 colon cancer cells, an effect that depends on the abundance of CEACAM1 on the cell surface. Together, our results demonstrate that the CEACAM1-dependent cell death pathway involves dual cleavage of CEACAM1 and caspase activation and can be activated by CEA.
Subject(s)
Antigens, CD/physiology , Apoptosis , Carcinoembryonic Antigen/pharmacology , Cell Adhesion Molecules/physiology , Caspases/physiology , Cell Line , Dose-Response Relationship, Drug , Humans , Signal Transduction/drug effectsABSTRACT
Heteroclitic peptides are used to enhance the immunogenicity of tumor-associated Ags to break T cell tolerance to these self-proteins. One such altered peptide ligand (Cap1-6D) has been derived from an epitope in human carcinoembryonic Ag, CEA(605-613) (Cap1). Clinical responses have been seen in colon cancer patients receiving a tumor vaccine comprised of this altered peptide. Whether Cap1-6D serves as a T cell agonist for Cap1-specific T cells or induces different T cells is unknown. We, therefore, examined the T cell repertoires elicited by Cap1-6D and Cap1. Human CTL lines and clones were generated with either Cap1-6D peptide (6D-CTLs) or Cap1 peptide (Cap1-CTLs). The TCR Vbeta usage and functional avidity of the T cells induced in parallel against these target peptides were assessed. The predominant CTL repertoire induced by agonist Cap1-6D is limited to TCR Vbeta1-J2 with homogenous CDR3 lengths. In contrast, the majority of Cap1-CTLs use different Vbeta1 genes and also had diverse CDR3 lengths. 6D-CTLs produce IFN-gamma in response to Cap1-6D peptide with high avidity, but respond with lower avidity to the native Cap1 peptide when compared with the Cap1-CTLs. Nevertheless, 6D-CTLs could still lyse targets bearing the native epitope. Consistent with these functional results, 6D-CTLs possess TCRs that bind Cap-1 peptide/MHC tetramer with higher intensity than Cap1-CTLs but form less stable interactions with peptide/MHC as measured by tetramer decay. These results demonstrate that priming with this CEA-derived altered peptide ligand can induce distinct carcinoembryonic Ag-reactive T cells with different functional capacities.
Subject(s)
Antigens, Neoplasm/immunology , Carcinoembryonic Antigen/immunology , Cross-Priming , Oligopeptides/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/pharmacology , CD8-Positive T-Lymphocytes/immunology , Carcinoembryonic Antigen/chemistry , Carcinoembryonic Antigen/pharmacology , Cell Line , Cell Line, Tumor , Epitopes/immunology , Humans , Molecular Sequence Data , Oligopeptides/pharmacology , Receptors, Antigen, T-Cell, alpha-beta/analysis , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocytes, Cytotoxic/drug effectsABSTRACT
The carcinoembryonic antigen (CEA)-derived peptide CAP1 and heteroclitic peptide CAP1-6D are stimulators of HLA-A*A0201 restricted CEA-specific T cells in vivo and in vitro. The goal of this study was to evaluate differences in T cell responses to peptide and modified peptide antigens from CEA. The heterogeneity of responses among individuals is potentially important for the design of future CEA-directed immunotherapy trials. Peripheral blood mononuclear cells from blood donors were stimulated with peptide, IL-2, and IL-7. Weekly, microcultures were restimulated with irradiated, autologous peptide-loaded peripheral blood mononuclear cells and expanded in IL-2. Established T cell lines were tested by cytokine release assays using peptide-loaded T2 targets. T cell avidity was measured by cytokine release using targets expressing diminishing concentrations of peptide. Fine specificities were measured using targets loaded with alanine-substituted CAP1 peptide. Tumor recognition was measured using HLA-A*A0201/CAP1-transduced COS tumor targets. Varied responses to CAP1 and CAP1-6D were seen among individuals. The immunogenicity of CAP1 or CAP1-6D was donor dependent. Many T cells recognized one peptide but did not cross-recognize the altered peptide. The avidities of T cell lines were moderate to low, and fine specificities were consistent with a narrow antigen-specific repertoire. CAP1-6D-based immune therapy may not be optimal in some patients with CAP1-specific precursors. The T cell repertoire may be a central contributor to the limited responses seen with CEA-directed immunotherapy to date. Treatment strategies designed to alter or expand the T cell repertoire against CEA should be considered for trials.
Subject(s)
Carcinoembryonic Antigen/immunology , Neoplasms/immunology , Oligopeptides/immunology , T-Lymphocytes/immunology , Carcinoembryonic Antigen/analysis , Carcinoembryonic Antigen/pharmacology , Cross Reactions , Humans , Immunotherapy/methods , Interleukin-2/pharmacology , Interleukin-7/pharmacology , Neoplasms/chemistry , Neoplasms/therapy , Oligopeptides/analysis , Oligopeptides/pharmacology , Receptors, Antigen, T-Cell/analysis , T-Lymphocytes/drug effectsABSTRACT
This study was conducted to test if tumour drug uptake could be increased in experimental colorectal cancer peritoneal metastases, by using pretreatment with peritoneal vasoconstriction or radioimmunotherapy. A total of 29 nude rats with peritoneal metastases were injected intraperitoneally (i.p.) with 14C-labelled 5-FU. The animals were randomly allocated to 5 groups. Six days prior to 5-FU, group I (control) received i.p. NaCl, group II was subjected to i.p. radioimmunotherapy (RIT) 131I-labelled anti-CEA monoclonal antibody (150 MBq) and group III received i.p. Norbormide 10 minutes before 5-FU. Two days prior to 5-FU group IV and V received i.p. NaCl (control) and RIT, respectively. 5-FU uptake was visualised with autoradiography and quantified by computer-based image analysis. Tumours in group III showed a higher uptake (mean+/-SD, 21.4+/-17) than in group I (11.8+/-10, p=0.04). This was also true when the analysis was restricted to larger tumours (> or = median 627 pixels) group III (23.2+/-19) vs. group I (11.8+/-7, p=0.002). Peritoneal tumours in group II were of smaller size (median area 308 pixels) than in group I (619 pixels), in group III (901 pixels), in group IV (769 pixels) and in group V (808 pixels). RIT decreased the tumour size whereas it did not affect 5-FU uptake. The uptake of 5-FU was potentiated by pretreating the animals with Norbormide. These results demonstrate that 5-FU uptake in experimental peritoneal metastases is increased when the peritoneal absorption of the drug is blocked using pretreatment with a vasoconstrictive agent. This principle may also be relevant when treating patients with colorectal cancer peritoneal metastases.
Subject(s)
Adenocarcinoma/metabolism , Adenocarcinoma/secondary , Antimetabolites, Antineoplastic/pharmacokinetics , Fluorouracil/pharmacokinetics , Immunotoxins/pharmacology , Norbornanes/pharmacology , Peritoneal Cavity/blood supply , Peritoneal Neoplasms/metabolism , Peritoneal Neoplasms/secondary , Adenocarcinoma/drug therapy , Adenocarcinoma/radiotherapy , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Autoradiography , Carbon Radioisotopes , Carcinoembryonic Antigen/immunology , Carcinoembryonic Antigen/pharmacology , Colonic Neoplasms/pathology , Female , Humans , Iodine Radioisotopes/therapeutic use , Male , Peritoneal Neoplasms/drug therapy , Peritoneal Neoplasms/radiotherapy , Radioimmunotherapy , Random Allocation , Rats , Vasoconstriction/drug effectsABSTRACT
Human peripheral blood lymphocytes can be transduced to express antigen-dependent CD3zeta chimeric immune receptors (CIRs), which function independently of the T-cell receptor (TCR). Although the exact function of these domains is unclear, previous studies imply that an extracellular spacer region is required for optimal CIR activity. In this study, four scFvs (in the context of CIRs with or without extracellular spacer regions) were used to target the human tumor-associated antigens carcinoembryonic antigen (CEA), neural cell adhesion molecule (NCAM), the oncofetal antigen 5T4, and the B-cell antigen CD19. In all cases human T-cell populations expressing the CIRs were functionally active against their respective targets, but the anti-5T4 and anti-NCAM CIRs showed enhanced specific cytokine release and cytotoxicity only when possessing an extracellular spacer region. In contrast, the anti-CEA and anti-CD19 CIRs displayed optimal cytokine release activity only in the absence of an extracellular spacer. Interestingly, mapping of the scFv epitopes has revealed that the anti-CEA scFv binds close to the amino-terminal of CEA, which is easily accessible to the CIR. In contrast, CIRs enhanced by a spacer domain appear to bind to epitopes residing closer to the cell membrane, suggesting that a more flexible extracellular domain may be required to permit the efficient binding of such epitopes. These results show that a spacer is not necessary for optimal activity of CIRs but that the optimal design varies.
Subject(s)
Antigens, Neoplasm/immunology , CD3 Complex/genetics , Immunoglobulin Variable Region/genetics , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/immunology , Antigens, CD19/immunology , Antigens, CD19/pharmacology , CD3 Complex/immunology , Carcinoembryonic Antigen/immunology , Carcinoembryonic Antigen/pharmacology , Extracellular Space , Humans , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/immunology , Membrane Glycoproteins/immunology , Membrane Glycoproteins/pharmacology , Neural Cell Adhesion Molecules/immunology , Neural Cell Adhesion Molecules/pharmacology , Protein Structure, Tertiary , Receptors, Antigen, T-Cell/immunology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , T-Lymphocytes/drug effects , TransfectionABSTRACT
Carcinoembryonic antigen (CEA) has been shown to participate in the progression and metastatic growth of colorectal cancer. However, its biological function remains elusive. Recently, we found that CEA protects colon cancer cells from undergoing apoptosis, suggesting a complex role that includes signal transduction activity. Additionally, it was reported that CEA binds to Kupffer cells and macrophages to a membrane-anchored homolog of heterogeneous nuclear protein M4 (hnRNP M4), which subsequently was named CEA-receptor (CEAR). Cytoplasmatic and membranous expression of CEAR in CEA-positive colon cancer tissues prompted us to analyze the CEA-CEAR interaction in HT29 colon cancer cells. Both, CEA and CEAR were found on the cell surface of HT29 cells, as demonstrated by confocal microscopy. Imaging analysis suggested co-localization and, thus, interaction of both molecules. To confirm this observation, immunoprecipitation experiments and Western blot analysis were performed and indicated binding of CEA and CEAR. Immunoprecipitation of CEA resulted in a pull down of CEAR. The pull down of CEAR correlated with the amount of CEA as demonstrated by ribozyme targeting of CEA. Finally, external treatment of HT29 cells with soluble CEA induced tyrosine phosphorylation of CEAR, suggesting a CEA-dependent role of CEAR in signal transduction. Future experiments will elucidate whether the CEA-CEAR interaction is involved in CEA's antiapoptotic role and mediates the prometastatic properties of CEA in colon cancer cells.
Subject(s)
Carcinoembryonic Antigen/metabolism , Colonic Neoplasms/metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group M/metabolism , Receptors, N-Acetylglucosamine/metabolism , Animals , Carcinoembryonic Antigen/biosynthesis , Carcinoembryonic Antigen/pharmacology , Cell Membrane/immunology , Cell Membrane/metabolism , Colonic Neoplasms/immunology , HT29 Cells , Heterogeneous-Nuclear Ribonucleoprotein Group M/biosynthesis , Humans , Immunohistochemistry , Immunoprecipitation , Macrophages, Alveolar/immunology , Macrophages, Alveolar/metabolism , Mice , Phosphorylation/drug effects , Receptors, N-Acetylglucosamine/biosynthesisABSTRACT
Tumor cells cause ischemia/reperfusion (I/R) injury as they arrest within the hepatic microvasculature with the production of nitric oxide (NO) and reactive oxygen species (ROS) that kill both host liver and implanting tumor cells. Carcinoembryonic antigen (CEA) both facilitates the survival of experimental metastasis to nude mouse liver by weakly metastatic human colorectal carcinomas (CRCs) and induces the release of the proinflammatory cytokine IL-6. We hypothesized that CEA also stimulates the release of the antiinflammatory cytokine IL-10 causing inhibition of the toxicity of hepatic I/R injury and indirect stimulation of tumor cell colonization of the liver. Intravenous injection of CEA produced more than 1 ng/ml of IL-10 in the systemic circulation within 1 hr which subsided by 8 hr. The IL-10 response is specific to CEA since the pentapeptide sequence in CEA that binds to the CEA receptor stimulated isolated Kupffer cells to produce IL-10. IL-10, but not IL-6, increased the survival of weakly metastatic CRC cocultured with ischemic-reoxygenated liver fragments but did not affect the survival of CRC exposed to oxidative stress in the absence of any host cells. CEA, IL-6 and IL-10 pretreatment reduced expression of iNOS but only CEA and IL-10 strongly inhibited NO and total reactive species production by ischemic-rexoygenated liver. IL-6 was toxic to CRC exposed to oxidative stress while IL-10 did not have a direct effect on CRC. Thus, CEA stimulates production of IL-10 that may enhance metastasis by promoting the ability of circulating CRC cells to survive the I/R injury of implantation.
Subject(s)
Carcinoembryonic Antigen/pharmacology , Colorectal Neoplasms/pathology , Interleukin-10/pharmacology , Interleukin-6/pharmacology , Liver Circulation/drug effects , Liver Neoplasms/secondary , Reperfusion Injury/prevention & control , Animals , Cell Line, Tumor , Coculture Techniques , Humans , Interleukin-10/physiology , Interleukin-6/physiology , Kinetics , Mice , Mice, Nude , Nitric Oxide/metabolism , Oxidative Stress/drug effects , Rats , Rats, Sprague-Dawley , Transplantation, HeterologousABSTRACT
Elevated Carcinoembryonic antigen (CEA) levels in the serum indicate a poor prognosis for colorectal cancer patients. Induction of proinflammatory cytokines by CEA interaction with Kupffer cells has been proposed as a mechanism for hepatic metastasis formation. Studies show that the cytokine response in circulating and peritoneal macrophages is regulated by beta-adrenergic receptor signals, though little information is available regarding Kupffer cells. We investigated the relationship between beta-adrenergic receptor stimulation and the response of Kupffer cells to CEA. Comparisons between unstimulated and CEA stimulated rat Kupffer cells, using cDNA arrays, showed up-regulation (>4 fold) of the beta2-adrenergic receptor mRNA. Peak up-regulation occurred after 30 min with a decline at 1 h. We examined the effects of the specific beta2-adrenergic receptor agonist terbutaline on cytokine production by CEA stimulated rat Kupffer cells. Pre-treatment of Kupffer cells with terbutaline followed by CEA caused a significant increase in IL-6 and IL-10 production, but a significant reduction in TNF-alpha production (>3 fold). mRNA levels reflected those of the ELISA assays for IL-6 and IL-10 but not for TNF-alpha. For IL-6 and TNF-alpha, these changes were serum independent, while IL-10 was serum dependent. This response is different from LPS treated Kupffer cells where all three cytokines showed serum dependency. Overall, these data suggest that Kupffer cell stimulation by CEA is under beta-adrenergic receptor control and induction of the beta-receptor is an early event following CEA binding to its receptor. Control of TNF-alpha production is negatively affected by terbutaline, while that of IL-6 and IL-10 is positively controlled suggesting that very different beta-adrenergic receptor signaling pathways are involved.
Subject(s)
Carcinoembryonic Antigen/pharmacology , Cytokines/biosynthesis , Gene Expression Profiling , Liver Neoplasms/secondary , Receptors, Adrenergic, beta-2/metabolism , Adrenergic beta-Agonists/pharmacology , Animals , Carcinoembryonic Antigen/chemistry , Cytokines/blood , Cytokines/genetics , Interleukin-10/genetics , Interleukin-10/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Kupffer Cells/cytology , Kupffer Cells/drug effects , Kupffer Cells/metabolism , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Terbutaline/pharmacology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Most circulating tumor cells die within 24 h of entering the hepatic microvasculature because their arrest initiates an ischemia-reperfusion (I/R) injury that is cytotoxic. Human colorectal carcinomas (CRC) produce the glycoprotein Carcinoembryonic Antigen (CEA) that increases experimental liver metastasis in nude mice. Since CEA induces release of IL-6 and IL-10, we hypothesized that CEA inhibits the I/R injury through a Kupffer cell-mediated cytokine-dependent pathway. We assessed cytokine effects in CRC co-cultured with liver and in vivo. Human CRC prelabeled with fluorescent dyes were incubated with a reoxygenated suspension of ischemic nude mouse liver fragments in a bioreactor. CEA, rhIL-6 or rhIL-10 were either administered to the donor mice prior to hepatic ischemia or during co-culture. Liver donors were athymic nude or iNOS, IL-6 or IL-10 knock out mice. Ischemic-reoxygenated liver kills Clone A CRC through production of nitric oxide (NO) and superoxide anion. Treatment of liver donors with CEA prior to hepatic ischemia inhibited this in vitro cytotoxicity through an IL-10 and Kupffer cell dependent pathway that inhibited NF-kappaB activation, NO production and iNOS upregulation. IL-10 but not IL-6 enhanced CRC survival in nude mouse liver in vivo. Thus, CEA enhanced metastasis by inducing IL-10 to inhibit iNOS upregulation in host liver.
Subject(s)
Carcinoembryonic Antigen/pharmacology , Colorectal Neoplasms/pathology , Interleukin-10/metabolism , Liver Neoplasms/secondary , Animals , Cell Survival/drug effects , Coculture Techniques , Colorectal Neoplasms/metabolism , Fluorescent Dyes , Humans , Interleukin-6/genetics , Interleukin-6/pharmacology , Interleukin-6/physiology , Kupffer Cells/drug effects , Kupffer Cells/metabolism , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , NF-kappa B/genetics , NF-kappa B/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Oxidative Stress/drug effects , Oxygen/metabolism , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Signal Transduction , Superoxides/metabolismABSTRACT
There is increasing evidence that tumors elicit specific T-cell responses in a substantial proportion of patients. Recently, we have shown that in patients with colorectal cancer specific T cells against the tumor-associated antigens (TAA) Ep-CAM, her-2/neu or CEA can be detected in peripheral blood using IFNgamma-ELISPOT assay. In our study, we have analyzed T-cell responses against HLA-A*0201-restricted epitopes of these TAA in peripheral blood of patients with breast cancer and colorectal cancer. Surprisingly, a complete absence of ex vivo T-cell responses against these TAA was found in 20 patients with breast cancer. In contrast, specific T cells were detectable in 12 of 49 patients with colorectal cancer against at least 1 of these TAA, confirming our previous results. T-cell responses against influenza-derived peptides were similar in both malignancies. The results of our study indicate a difference either of tumor immunogenicity or of the migratory pattern of tumor-specific T cells between breast cancer and colorectal cancer patients. The findings reported here have implications for the development of antigen-specific T-cell therapies.
Subject(s)
Antigens, Neoplasm/immunology , Breast Neoplasms/immunology , Cell Adhesion Molecules/immunology , Colorectal Neoplasms/immunology , Lymphocyte Activation/immunology , Peptide Fragments/immunology , T-Lymphocytes/immunology , Antibodies, Monoclonal/immunology , Breast Neoplasms/blood , Breast Neoplasms/pathology , Carcinoembryonic Antigen/immunology , Carcinoembryonic Antigen/pharmacology , Case-Control Studies , Colorectal Neoplasms/blood , Colorectal Neoplasms/pathology , Enzyme-Linked Immunosorbent Assay/methods , Epithelial Cell Adhesion Molecule , Epitopes, T-Lymphocyte/immunology , Female , Flow Cytometry , HLA-A Antigens/immunology , HLA-A2 Antigen/immunology , Humans , Immunity, Cellular , Interferon-gamma/metabolism , Neoplasm Staging , Peptide Fragments/pharmacology , Pokeweed Mitogens/immunology , Pokeweed Mitogens/pharmacology , Receptor, ErbB-2/immunology , T-Lymphocytes, Cytotoxic/immunology , Viral Matrix Proteins/immunologyABSTRACT
Biliary glycoprotein (BGP, CD66a, CEACAM1) is a member of the carcinoembryonic antigen family (CEA, CD66), a group of transmembrane proteins belonging to the immunoglobulin superfamily. The structural features surrounding the tyrosine residues in the cytoplasmic domain of BGP share similarity with the consensus sequence of the immunoreceptor tyrosine-based inhibition motif (ITIM), the docking site for SHIP, SHP-1, and SHP-2 molecules. Using the well-characterized inhibitory receptor, FcgammaRIIB, we constructed a FcgammaRIIB-BGPa chimeric molecule that contained the extracellular and transmembrane domain of FcgammaRIIB and the cytoplasmic tail of BGPa and expressed it in DT40 B cells. Our results showed that FcgammaRIIB-BGPa, just like the unmodified FcgammaRIIB molecule, inhibited calcium influx in activated DT40 B cells. Substitution of tyrosine with phenylalanine (Y459F) in FcgammaRIIB-BGPa completely abrogated its ability to inhibit calcium influx, indicating that the motif surrounding Y459 is ITIM. The presence of ITIM was also supported by showing that the FcgammaRIIB-BGPa-mediated inhibitory effect was reduced in SHP-1and SHP-2 mutant DT40 B cells and further diminished in a SHP-1/-2 double-deficient mutant line. The results suggest that SHP-1 and SHP-2 are required for the FcgammaRIIB-BGPa-mediated inhibitory signals.
Subject(s)
Antigens, CD/physiology , Glycoproteins/pharmacology , Receptors, IgG/physiology , Signal Transduction/drug effects , Animals , Antigens, CD/pharmacology , Antigens, Differentiation/pharmacology , Antigens, Differentiation/physiology , Calcium Signaling/drug effects , Carcinoembryonic Antigen/pharmacology , Carcinoembryonic Antigen/physiology , Cell Adhesion Molecules , Cell Line , Chickens , Glycoproteins/genetics , Glycoproteins/physiology , Humans , Mutagenesis, Site-Directed , Protein Structure, Tertiary , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/physiology , Transfection , src Homology Domains/physiologyABSTRACT
BACKGROUND: Carcinoembryonic antigen (CEA) has been suggested to promote colon cancer progression. In this study we analyzed the prognostic impact of CEA expression on intraperitoneally detected single colon cancer cells. METHODS: Peritoneal lavage samples of 135 colorectal cancer patients were immunocytologically analyzed, including a staining of cellular CEA; serum CEA levels were measured; and 5-year survival rates were calculated according to immunocytological findings and CEA expression. RESULTS: The worst survival rate of 20% was found in patients suffering from CEA-expressing intraperitoneal tumor cells (P = 0.0006). The prognostic impact of an intraperitoneal tumor cell finding significantly increased when serum CEA levels were elevated: only 23% survived 5 years in contrast to a 85% 5-year survival rate of patients who neither had signs of dissemination nor showed elevated serum CEA values (P = 0.0010). CONCLUSIONS: This study shows that the determination of CEA expression improves the prognostic impact of an intraperitoneal tumor cell finding.