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.

Primary cultures of human colon cancer as a model to study cancer stem cells.

The principal cause of death in cancer involves tumor progression and metastasis. Since only a small proportion of the primary tumor cells, cancer stem cells (CSCs), which are the most aggressive, have the capacity to metastasize and display properties of stem cells, it is imperative to characterize the gene expression of diagnostic markers and to evaluate the drug sensitivity in the CSCs themselves. Here, we have examined the key genes that are involved in the progression of colorectal cancer and are expressed in cancer stem cells. Primary cultures of colorectal cancer cells from a patient's tumors were studied using the flow cytometry and cytological methods. We have evaluated the clinical and stem cell marker expression in these cells, their resistance to 5-fluorouracil and irinotecan, and the ability of cells to form tumors in mice. The data shows the role of stem cell marker Oct4 in the resistance of primary colorectal cancer tumor cells to 5-fluorouracil.

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-, ß- 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.

Carcinoembryonic antigen (CEA) and its receptor hnRNP M are mediators of metastasis and the inflammatory response in the liver.

This article discusses the role of carcinoembryonic antigen (CEA) as a facilitator of the inflammatory response and its effect on colorectal cancer hepatic metastasis. Colorectal cancer accounts for 11% of all cancers in the United States and the majority of deaths are associated with liver metastasis. If left untreated, median survival is only six to 12 months. Resection of liver metastases offers the only chance for cure. Of the small number of patients who have operable cancer most will have further tumor recurrence. The molecular mechanisms associated with colorectal cancer metastasis to the liver are largely unknown. However CEA production has been shown both clinically and experimentally to be a factor in an increased metastatic potential of colorectal cancers to the liver. CEA also has a role in protecting tumor cells from the effects of anoikis and this affords a selective advantage for tumor cell survival in the circulation. CEA acts in the liver through its interaction with its receptor (CEAR), a protein that is related to the hnRNP M family of RNA binding proteins. In the liver CEA binds with hnRNP M on Kupffer cells and causes activation and production of pro- and anti-inflammatory cytokines including IL-1, IL-10, IL-6 and TNF-α. These cytokines affect the up-regulation of adhesion molecules on the hepatic sinusoidal endothelium and protect the tumor cells against cytotoxicity by nitric oxide (NO) and other reactive oxygen radicals. HnRNP M signaling in Kupffer cells appears to be controlled by beta-adrenergic receptor activation. The cells will respond to the ß-adrenergic receptor agonist terbutaline resulting in reduced TNF-α and increased IL-10 and IL-6 production following CEA activation. This has implications for the control of tumor cell implantation and survival in the liver.

Proteomic analysis of ACTN4-interacting proteins reveals it's a putative involvement in mRNA metabolism.

Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established. In this report, we describe the identification of proteins associated with ACTN4 in the nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF mass-spectrometry revealed a large number of ACTN4-bound proteins that are involved in various aspects of mRNA processing and transport. The association of ACTN4 with different ribonucleoproteins suggests that a major function of nuclear ACTN4 may be regulation of mRNA metabolism and signaling.

Inhibition of lipopolysaccharide activation of Kupffer cells by transition metals.

BACKGROUND: Bacterial endotoxins are the principal agents causing sepsis and septic shock. Cytokine cascades produced by cellular interactions to endotoxins can cause cardiovascular failure followed by multi-organ failure and death. Endotoxin intravenously administered to mice can have fatal consequences. Previous studies have shown that the transition metals Mn2+ and Cr3+ can be protective. METHODS: The effects of Mn2+, Cr3+, Zn2+, and Cu2+ on lipopolysaccharide (LPS) binding to rat Kupffer cell extracts were analyzed using dot-blots, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western transfer. Kupffer cells were isolated from rat livers by collagenase perfusion, differential centrifugation, and adhesion to plastic. RESULTS: Five millimolar of Mn2+, Zn2+, Cr3+, and Cu2+ completely inhibited LPS binding. Isolated Kupffer cells were also exposed to Mn2+ and to LPS and tumor necrosis factor-alpha release measured. The presence of Mn2+ significantly (P < 0.05) reduced tumor necrosis factor-alpha production by Kupffer cells in response to LPS. Experiments to determine if these effects were mediated by binding to LPS-binding proteins showed this was not the case. More likely a complex occurs between the metal and LPS. We also showed significantly enhanced uptake of LPS into Kupffer cells in the presence of Mn2+. CONCLUSIONS: The data are consistent with the metals binding to LPS via its two phosphate groups and neutralizing their charge. These data also support the hypothesis that there is enhanced cellular uptake by non-receptor-mediated methods such as absorptive pinocytocis. At the same time receptor binding and activation of the cells is inhibited. This can explain the effects of transition metals on LPS toxicity.

Carcinoembryonic antigen-stimulated THP-1 macrophages activate endothelial cells and increase cell-cell adhesion of colorectal cancer cells.

The liver is the most common site for metastasis by colorectal cancer, and numerous studies have shown a relationship between serum carcinoembryonic antigen (CEA) levels and metastasis to this site. CEA activates hepatic macrophages or Kupffer cells via binding to the CEA receptor (CEA-R), which results in the production of cytokines and the up-regulation of endothelial adhesion molecules, both of which are implicated in hepatic metastasis. Since tissue macrophages implicated in the metastatic process can often be difficult to isolate, the aim of this study was to develop an in vitro model system to study the complex mechanisms of CEA-induced macrophage activation and metastasis. Undifferentiated, human monocytic THP-1 (U-THP) cells were differentiated (D-THP) to macrophages by exposure to 200 ng/ml phorbol myristate acetate (PMA) for 18 h. Immunohistochemistry showed two CEA-R isoforms present in both U- and D-THP cells. The receptors were localized primarily to the nucleus in U-THP cells, while a significant cell-surface presence was observed following PMA-differentiation. Incubation of D-THP-1 cells with CEA resulted in a significant increase in tumor necrosis factor-alpha (TNF-alpha) release over 24 h compared to untreated D-THP-1 or U-THP controls confirming the functionality of these cell surface receptors. U-THP cells were unresponsive to CEA. Attachment of HT-29 cells to human umbilical vein endothelial cells significantly increased at 1 h after incubation with both recombinant TNF-alpha and conditioned media from CEA stimulated D-THP cells by six and eightfold, respectively. This study establishes an in vitro system utilizing a human macrophage cell line expressing functional CEA-Rs to study activation and signaling mechanisms of CEA that facilitate tumor cell attachment to activated endothelial cells. Utilization of this in vitro system may lead to a more complete understanding of the expression and function of CEA-R and facilitate the design of anti-CEA-R therapeutic modalities that may significantly diminish the metastatic potential of CEA overexpressing colorectal tumors.

Identification of two novel LPS-binding proteins in Kupffer cells: implications in TNF-alpha production.

Using a combination of gel-exclusion chromatography and ligand binding with [(125)I]-lipopolysaccharide (LPS), we discovered two novel endotoxin-binding proteins, p31(LPB) and p34(LPB), in Kupffer cells. Their molecular masses suggest that these are previously undescribed LPS-binding proteins (LBPs). Evidence from detergent-based cell extractions shows that these proteins are probably transmembrane or located on the inner leaflet of the lipid bilayer. We have partially purified the proteins from detergent extracts of Kupffer cells and proven that they bind diphosphoryl lipid A, an interaction associated with TNF-alpha production. The proteins do not bind monophosphoryl lipid A. Diphosphoryl lipid A binding occurs in the absence of serum, suggesting a mechanism of cytokine production distinct from that involving CD14 and lipopolysaccharide-binding protein (LPB). The two proteins were not detectable in resident peritoneal macrophages or in a number of other cell lines of the macrophage/monocyte lineage, suggesting specificity towards terminally differentiated macrophages such as Kupffer cells.

Surface expression and CEA binding of hnRNP M4 protein in HT29 colon cancer cells.

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.

Surface expression of heterogeneous nuclear RNA binding protein M4 on Kupffer cell relates to its function as a carcinoembryonic antigen receptor.

Elevated concentrations of carcinoembryonic antigen (CEA) in the blood are associated with the development of hepatic metastases from colorectal cancers. Clearance of circulating CEA occurs through endocytosis by liver macrophages, Kupffer cells. Previously we identified heterogeneous nuclear ribonucleoproteins M4 (hnRNP M4) as a receptor (CEAR) for CEA. HnRNP M4 has two isoform proteins (p80, p76), the full-length hnRNP M4 (CEARL) and a truncated form (CEARS) with a deletion of 39 amino acids between RNA binding domains 1 and 2, generated by alternative splicing. The present study was undertaken to clarify any isoform-specific differences in terms of their function as CEA receptor and localization. We develop anti-CEAR isoform-specific antibodies and show that both CEAR splicing isoforms are expressed on the surface of Kupffer cells and can function as CEA receptor. Alternatively, in P388D1 macrophages CEARS protein has nuclear and CEARL has cytoplasmic localization. In MIP101 colon cancer and HeLa cells the CEARS protein is localized to the nucleus and CEARL to the cytoplasm. These findings imply that different functions are assigned to CEAR isoforms depending on the cell type. The search of 39 amino acids deleted region against the Prosite data base revealed the presence of N-myristylation signal PGGPGMITIP that may be involved in protein targeting to the plasma membrane. Overall, this report demonstrates that the cellular distribution, level of expression, and relative amount of CEARL and CEARS isoforms determine specificity for CEA binding and the expression of alternative spliced forms of CEAR is regulated in a tissue-specific manner.