Molecular basis for effects of carcinogenic heavy metals on inducible gene expression.

Certain forms of the heavy metals arsenic and chromium are considered human carcinogens, although they are believed to act through very different mechanisms. Chromium(VI) is believed to act as a classic and mutagenic agent, and DNA/chromatin appears to be the principal target for its effects. In contrast, arsenic(III) is considered nongenotoxic, but is able to target specific cellular proteins, principally through sulfhydryl interactions. We had previously shown that various genotoxic chemical carcinogens, including chromium (VI), preferentially altered expression of several inducible genes but had little or no effect on constitutive gene expression. We were therefore interested in whether these carcinogenic heavy metals might target specific but distinct sites within cells, leading to alterations in gene expression that might contribute to the carcinogenic process. Arsenic(III) and chromium(VI) each significantly altered both basal and hormone-inducible expression of a model inducible gene, phosphoenolpyruvate carboxykinase (PEPCK), at nonovertly toxic doses in the chick embryo in vivo and rat hepatoma H411E cells in culture. We have recently developed two parallel cell culture approaches for examining the molecular basis for these effects. First, we are examining the effects of heavy metals on expression and activation of specific transcription factors known to be involved in regulation of susceptible inducible genes, and have recently observed significant but different effects of arsenic(III) and chromium(VI) on nuclear transcription factor binding. Second, we have developed cell lines with stably integrated PEPCK promoter-luciferase reporter gene constructs to examine effects of heavy metals on promoter function, and have also recently seen profound effects induced by both chromium(VI) and arsenic(III) in this system. These model systems should enable us to be able to identify the critical cis (DNA) and trans (protein) cellular targets of heavy metal exposure leading to alterations in expression of specific susceptible genes. It is anticipated that such information will provide valuable insight into the mechanistic basis for these effects as well as provide sensitive molecular biomarkers for evaluating human exposure

Genetic Factors Involved in Human Colorectal Cancer Metastasis

Experimental and clinical studies have shown that one of the most accurate clinical markers of metastasis in epithelial cancers is the increasing level of carcinoembryonic antigen (CEA, CEACAM-5, CD66e) in the patient’s serum. However, the signaling mechanism determining the ability of cells to produce CEA and to stimulate metastasis has been insufficiently studied. We first cloned a gene of the receptor protein on the surface of liver macrophages - Kupffer cells, which interacts with CEA. This receptor protein has been named CEAR. The CEA/CEAR interaction on the surface of Kupffer cells enhances survival of metastatic cancer cells in the liver through stimulation of cytokine production and influences cell adhesion molecules in the liver. To elucidate CEAR function, its expression was inhibited by short interfering RNAs (siRNA) in MIP-101, a colorectal cancer cell line. Comparative analysis of cell lines with different levels of CEAR and CEA production showed that the inhibition of CEAR expression decreases the expression level of CEA, Ecadherin and integrin alpha 3, and also affects the ability of colorectal cancer cells to adhesion and invasiveness. In vivo experiments showed the catalytic role of CEAR in the formation of tumors in immunodeficient mice. These data revealed the CEAR as a potential new target molecule to prevent the development of CEA-dependent malignancies in humans. The comparative analysis of transcriptomes of four genetically-related colorectal cancer cell lines with different expression levels of CEA and CEAR was carried out using RNA sequencing technology. We discovered 3 genes that are down-regulated in colorectal cancer cells as a result of the CEA production. These genes are major protein-coding cell regulators, including transcription factors that are involved in the response to the DNA-damage, stress, cell cycle and apoptosis. Also we discovered 5 genes that are up-regulated in response to CEA production. These genes are all protein coding genes for the cell surface complexes and signaling proteins that interact with the cytoskeleton and regulate intracellular junctions, cell proliferation and signaling pathways. The analysis of RNAseq data, obtained by Trinity and Tophat pipelines, was performed using the KEGG pathway and GO enrichment analysis

T125

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

Comparative 3 ' UTR Analysis Allows Identification of Regulatory Clusters that Drive Eph/ephrin Expression in Cancer Cell Lines

Eph receptors are the largest family of receptor tyrosine kinases. Together with their ligands, the ephrins, they fulfill multiple biological functions. Aberrant expression of Ephs/ephrins leading to increased Eph receptor to ephrin ligand ratios is a critical factor in tumorigenesis, indicating that tight regulation of Eph and ephrin expression is essential for normal cell behavior. The 3'-untranslated regions (3'UTRs) of transcripts play an important yet widely underappreciated role in the control of protein expression. Based on the assumption that paralogues of large gene families might exhibit a conserved organization of regulatory elements in their 3'UTRs we applied a novel bioinformatics/molecular biology approach to the 3'UTR sequences of Eph/ephrin transcripts. We identified clusters of motifs consisting of cytoplasmic polyadenylation elements (CPEs), AU-rich elements (AREs) and HuR binding sites. These clusters bind multiple RNA-stabilizing and destabilizing factors, including HuR. Surprisingly, despite its widely accepted role as an mRNA-stabilizing protein, we further show that binding of HuR to these clusters actually destabilizes Eph/ephrin transcripts in tumor cell lines. Consequently, knockdown of HuR greatly modulates expression of multiple Ephs/ephrins at both the mRNA and protein levels. Together our studies suggest that overexpression of HuR as found in many progressive tumors could be causative for disarranged Eph receptor to ephrin ligand ratios leading to a higher degree of tissue invasiveness