E XP ER I ME NTAL C E LL RE S E ARCH

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Research Article

Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes Olga Bajenovaa,b,c,n, Nina Chaikac, Elena Tolkunovad, Alexander Davydov-Sinitsynd, Svetlana Gapone, Peter Thomasc, Stephen O’Briena a

Theodosius Dobzhansky Center for Genome Bioinformatics, St. Petersburg State University, St. Petersburg 199034, Russia Department of Genetics and Biotechnology, St. Petersburg State University, St. Petersburg 199034, Russia c Department of Surgery and Biomedical Sciences, Creighton University, Omaha, NE 68178, USA d Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russia e Boston Children's Hospital, Boston, MA 02115, USA b

article information

abstract

Article Chronology:

Oncomarkers play important roles in the detection and management of human malignancies.

Received 25 July 2013

Carcinoembryonic antigen (CEA, CEACAM5) and epithelial cadherin (E-cadherin) are considered as

Received in revised form

independent tumor markers in monitoring metastatic colorectal cancer. They are both expressed by

25 March 2014

cancer cells and can be detected in the blood serum. We investigated the effect of CEA production

Accepted 4 April 2014

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

Keywords: Colorectal carcinoma Carcinoembryonic antigen Metastasis CEAR RNA binding protein

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.

Adherens junction

& 2014 Elsevier Inc. All rights reserved.

E-cadherin α-catenin β-catenin p120 catenin

Abbreviations: ACTN4, actinin-4 protein; AJs, adherens junctions; CEA, carcinoembryonic antigen; E-cadherin, epithelial cadherin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; hnRNPM, heterogeneous RNA-binding protein M; PAGE, polyacrylamide gel electrophoresis; RT-PCR, reverse transcriptase-polymerase chain reaction n Corresponding author at: Theodosius Dobzhansky Center for Genome Bioinformatics, St. Petersburg State University, 41a Sredniy Prospect, St. Petersburg 199034, Russia. Tel.: þ7 8123636103. E-mail address: [email protected] (O. Bajenova).

http://dx.doi.org/10.1016/j.yexcr.2014.04.007 0014-4827/& 2014 Elsevier Inc. All rights reserved.

Please cite this article as: O. Bajenova, et al., Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.007

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Introduction CEA (CEACAM5) is one of the most widely used tumor markers and has been associated with enhanced metastatic potential in colorectal cancers [1]. CEA is a large glycoprotein ( 180 kD) and a member of a family of 29 related genes in the immunoglobulingene super family. One major function of CEA is to regulate intercellular adhesion. It is expressed on the luminal surface of the colonocytes and its expression pattern changes in the neoplastic cell so that it is also expressed on the basal and lateral membranes. CEA plays a critical role in establishing and maintaining tissue architecture and function in the colon [1]. CEA also appears to regulate a variety of cellular functions that include inhibition of cell differentiation, polarization, intercellular and matrix adhesions, signal transduction, cellular architecture, and anoikis (cell death due to the loss of cell-to-cell contacts) as well as cellular migration [1,2]. CEA has been identified as a selectin ligand in colon carcinoma cells that is important for cancer cell migration [3]. After separating from a primary tumor, metastasizing cells enter the circulation and interact with host cells before lodging in secondary organs. CEA and variant isoforms of surface glycoproteins CD44 cooperate to mediate colon carcinoma cell adhesion to E- and L-selectin in shear flow [3,4]. In preclinical models, it has been shown to have a causal effect of CEA production by cancer cells on liver metastasis. CEA injected into athymic nude mice prior to the intrasplenic injection of colorectal cancer cell lines with a low metastatic potential enhanced hepatic metastasis [5]. Anti-CEA antibodies against the NH2-terminal (MN-3) and A1B1 (MN-15) domains of CEA impeded metastasis; affected cell migration, invasion, and adhesion in vitro; and improved mouse survival in vivo [6]. In addition, weakly metastatic human colorectal carcinoma cells acquire a high metastatic potential when transfected with recombinant CEA [7,8]. To elucidate the role of CEA and CD44 in colorectal cancer metastasis the expression of these multifunctional molecules has been suppressed using siRNA in LS174T colon carcinoma cells. The ability of modified cells to metastasize was analyzed in 2 independent mouse models [9]. It has been shown that cell migration was decreased as a result of silencing CEA but was enhanced in CD44-knockdown cells. Collectively, the data indicate that CEA, but not CD44, is a viable target for therapeutics aimed at curbing colon carcinoma metastasis [9]. E-cadherin has been implicated in carcinogenesis as its expression is commonly down-regulated or lost in primary carcinomas [10] and E-cadherin is a tumor suppressor gene in colorectal carcinomas. We identified a novel CEA-binding protein – CEAR, also called heterogeneous RNA-binding protein M (hnRNPM) – in liver macrophages which is involved in the implantation and survival of tumor cells in the liver [11]. CEAR/hnRNPM belongs to a large family of heterogeneous nuclear RNA-binding proteins (hnRNPs A-U), also called “histones of RNA” [12]. The hnRNP family of proteins shares common structural domains and have central roles in regulating gene expression at both transcriptional and translational levels, alternative splicing, micro-RNA processing and stress response [13]. Individual hnRNPs are reported to act in tumor development and progression in a variety of cancers [14]. The exact role of hnRNPM in colorectal cancer progression is unknown. At present, a vast amount of data is available on molecular mechanisms of individual components of metastasis

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[15], but the data can be quite contradictory. In this context it is especially important to have an adequate biological model, which would make it possible to explore metastasis in vitro and in vivo as a complex physiological phenomenon. A very promising field is the comparison of genetically modified cell lines of the same origin with varying CEA production and metastatic potential. One such model is the colon cancer cell line collection based on the low-differentiated, poorly metastatic, non-CEA-producing MIP101 colorectal carcinoma cell line. Poorly and highly differentiated colorectal carcinoma cells differ in morphology, adhesion, CEAproduction, motility and invasion. Poorly differentiated cells (MIP101) are highly invasive, more adherent, scattered, elongated and motile than the moderate and well-differentiated colorectal carcinomas (CX-1, DLD-2) which produce high levels of CEA [16]. We used the poorly differentiated CEA negative human colorectal carcinoma line – MIP-101 and its CEA-producing clones (clones 6 and 8) developed by transfection with the full-length CEA cDNA [7]. In this study we discovered that CEA expression by colorectal cancer cells influences the function, and the interactions between the E-cadherin junction complex proteins may explain the link between the elevated levels of CEA and the increase in soluble E-cadherin during the progression of colorectal cancer.

Materials and methods Cell lines Cells were maintained at 37 1C in a 5% CO2 atmosphere in RPMI 1640 medium containing 10% fetal bovine serum, 100 mg/ml penicillin, 100 mg/ml streptomycin, and 300 mg/ml glutamine. MIP 101 clone 6 and clone 8 cell lines produced by transfection of MIP-101with the full-length CEA cDNA [7,16] were selected on G 418. CХ1, HT-29 and CaCO2 cells are all CEA-producing aggressive colorectal cancer cells.

Semi-quantitative RT-PCR of mRNA expression The relative mRNA-expression levels of CEA, hnRNP M, and adherent junction proteins (E-cadherin, α-catenin, β-catenin) were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). All primers were custom made by Integrated DNA Technology. Primers for CEA were forward (N-domain-F) 50 -caccactgccaagctcacta; reverse (CEA-A1R) 50 -ctgggttctgggtttcacat; β-actin, forward 50 -tgagcgcggctacagctt-30 and reverse 50 -tccttaatgtcacggacgattt-30 ; human hnRNPM forward, 50 -gagcggaagaccactgaaag-30 , and reverse 50 -agaatgtctgctcggaccac-30 . The human hnRNPM primers were designed to detect two isoforms that represent the wild type (full-length) and a splice variant with a 39 amino acid deletion with the expected PCR products of 321 and 204 base pairs. E-cadherin, α-catenin, and β-catenin primer sequences have been previously published [17]. RNA was extracted from colorectal carcinoma cells using RNAsol, according to the manufacturer0 s protocol (Ambion, Inc.). Superscript™ III First Strand Synthesis system for RT-PCR (Invitrogen) was used to generate the cDNAs. We diluted synthesized cDNAs in 50 mL of diethylpyrocarbonate-treated water and used 3 mL of each reaction in each 25mL RT-PCR. DNA was amplified with the following parameters: 95 1C for 1 minute followed by 35 cycles of 95 1C for 30 s, 52–60 1C for 30 s, and 72 1C for 1 min. The gene expression was normalized with

Please cite this article as: O. Bajenova, et al., Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.007

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reference primers for the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, forward 50 -gggaaggtgaagtcggagt-30 , and reverse 50 ttgaggtcaatgaaggggtca-30 . PCR products were separated in a 2–3% agarose gel and visualized by ethidium bromide staining. To serve as a control for genomic DNA, contamination PCR reactions that included cDNA synthesis reagents except reverse transcriptase and water were set up in parallel. At least three independent amplification experiments were performed per gene.

Western blot analysis Cell lysates were prepared from 50% to 70% confluent MIP-101 cell monolayers that were washed with PBS (pH 7.4) at 4 1C and then were lysed at 4 1C in TNE buffer (50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 40 mM β-glycerophosphate, 1 mM EGTA, 0.25% sodium deoxycholate, 1% NP40, 50 mM sodium fluoride, 20 mM sodium phosphate, 1 mM sodium orthovanadate, and protease inhibitors (2 mg/ml leupeptin, 2 mg/ml aprotinin, 1 mg/ml pepstatin, and 100 mg/ml pefabloc)). Cellular debris was removed by centrifugation at 14,000 rpm for 15 min and supernatants were analyzed for total protein content using the BCA method (Pierce, Rockford, IL). 10–30 mg of total protein were resolved on 4–12% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Bedford, MA). After blocking the membranes with 5% (w/v) nonfat dry milk in TBS (pH 7.4), containing 0.5% Tween 20 (TBS-T) for 3 hours, the membranes were incubated overnight at 4 1C with the primary antibodies. The primary antibodies were a mouse monoclonal anti-hnRNPM1-4 (sc20001, Santa Cruz, CA), anti-E-cadherin (sc8426, Santa Cruz, CA), anti-β-catenin (sc65480, Santa Cruz, CA), rabbit polyclonal anti-β-tubulin (sc5286, Santa Cruz, CA), anti-CEA (C2331, SigmaAldrich), α-catenin (sc59890, Santa Cruz, CA), GAPDH (sc-20357, Santa Cruz, CA), and β-actin (Sigma-Aldrich). Blots were visualized by enhanced chemiluminescence (Pierce, Rockford, IL) using horseradish peroxidase-linked donkey anti-rabbit or anti-mouse IgG as the secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ). The proteins were quantified by scanning the images into Adobe Photoshops and the images were analyzed using Image J version 1.30. For comparative analysis of the levels of soluble CEA and E-cadherin, cells were grown 7 days in RPMI 1640 medium containing 10% fetal bovine serum, 100 mg/ml penicillin, 100 mg/ml streptomycin, and 300 mg/ml glutamine. The cells and media were separated and evaluated by Western blotting. For immunoprecipitation, cell lysates (250 mg) were incubated for 4 h at 4 1C in TNE buffer with the corresponding mAb (antihnRNPM1-4, anti-E-cadherin, anti-β-catenin) (Santa Cruz, CA) attached to protein A/G agarose beads (Sigma-Aldrich). The pellets were washed five times with 500 ml of TNE buffer, resuspended in 20 ml of 4  SDS sample buffer followed by resolution on 4–12% SDSPAGE gels, and transferred onto PVDF membranes. Blots were treated and visualized as in the Western blotting by enhanced chemiluminescence (Pierce, Rockford, IL) with horseradish peroxidase-linked secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ).

TOPflash transfection and luciferase assay To measure the endogenous levels of β-catenin cells were transfected with TOPflash, a plasmid containing firefly luciferase gene under control of Tcf/LEF-dependent promoter, and a β-galactosidase expression vector for checking the efficiency of transfection. Transfections

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were carried out using Lipofectamine according to the manufacturer's instructions (Promega, USA). Eighteen hours prior to transfection, cells were plated in the concentration 6  105 per well in 24-well plates. One hour before transfection, the medium was renewed. If calcium phosphate was used, 12 h after the procedure the medium was renewed once more. For luciferase assay, the Promega Bright-Glo™ Luciferase Assay kit was used. 2 days after transfection, cells were lysed with Glo-Lysis buffer, and 50 μl of lysate were mixed with an equal volume of Bright-Glo luciferine solution. This started a two-step enzymatic reaction characteristic for luciferase, resulting in chemoluminescence immediately measured by a luminometer. Another part of the lysate was used for relative cell density measurement required for control. For this, 20 μl of cell lysate were mixed with 60 μl of βgalactosidase staining solution (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM Mg Cl2, 50 mM β-mercaptoethanol) and 20 μl of βgalactosidase substrate solution, ONPG (ortho-nitrophenyl-β-galactoside), 2 mg/ml. In 1 h the specimens developed a yellow staining which was measured with a spectrophotometer (420 nm wavelength). Immediately before measurement, the reaction was blocked with 50 μl of 1 M sodium carbonate per specimen. The resulting normalized values were calculated as luminometric values divided by optical density.

Results mRNA and protein expression of adherens junction proteins in colorectal carcinoma cell lines and supernatants To study the effect of CEA on AJ complexes first we evaluated the comparative levels of AJs mRNA (Fig. 1A) and proteins (Fig. 1B) in CEA-over expressing versus CEA-negative CRC cells. The levels of soluble E-cadherin and CEA were also detected in the media (Fig. 1C). The data confirm that MIP-101 cells do not produce CEA. MIP-101 clone 6 and clone 8 cell lines expressed high levels of CEA and excreted CEA in the medium (Fig. 1C). Soluble E-cadherin is a degradation product of the cellular molecules and reflects the cleavage of the ectodomain from the mature molecule. Our data show that CEA production by clone 6, clone 8 and CX1 colorectal cancer cell lines increases the cleavage of E-cadherin in the cell lysates (Fig. 1C: lanes 2–4) and the amount of soluble E-cadherin in the medium. The CEA production did not change the level of expression of β-catenin in the cell lines (Fig. 1A and B). The level of α-catenin was increased in clone 6 cell line relative to MIP101 and clone 8 cells (Fig. 1A and B). A possible explanation can be that the genomic localization of transfected CEA gene can be different between these cell lines and may result in the genetic differences between clones 6 and 8. The hnRNPM protein is expressed in all cell lines with two known alternatively spliced mRNA isoforms 1 and 2 (full-length and truncated form, respectively). Their sequences and reading frames are similar, except for a 39 amino-acid deletion in the linker region between the RNAbinding domains 1 and 2 [11]. The hnRNPM family of proteins consists of four 75–85 kD isoforms – M1, M2, M3, and M4 – defined by 2-D gel electrophoresis. The smallest is hnRNPM1 and hnRNPM4 is the largest protein in this group [12]. The sequences of other hnRNPM family members and the significance of these isoforms in metastasis are not known.

Please cite this article as: O. Bajenova, et al., Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.007

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Fig. 1 – mRNA and protein expression in colorectal carcinoma cell lines and cell supernatants. (A) Using RT-PCR, we studied mRNA expression in six human colorectal cell lines: MIP-101, clone 6, clone 8, HT-29, CX-1, and CaCO2. Comparative mRNA expression of CEA, CEAR/hnRNP M, and adherens junction proteins in colorectal carcinoma cell lines shows that parental MIP-101 cells do not produce CEA (lane 2). The highest levels of CEA were detected in clone 6 (lane 3), clone 8 (lane 4), and CX-1 (lane 5). HT-29 and CaCO2 cells produce lower levels of CEA. The mRNA expression of E-cadherin is lower in CX-1 and HT-29 cells versus MIP-101 derivatives and CaCO2 cells. The data show no differences in the level of β-catenin expression in all cell lines. The α-catenin mRNA expression is up regulated in MIP clone 6 cells versus MIP-101. Primers designed for hnRNPM can recognize two hnRNPM splicing isoforms, the full-length protein, and truncated delta-hnRNPM, which carry a deletion of 39 amino-acids between RNA-binding domains 1 and 2. (B) Comparative protein analysis of CEA, hnRNPM and adherens junction proteins in the cell lysates of colorectal carcinoma cells indicates correlations between mRNA and protein expression in the examined cell lines. Cleavage of E-cadherin is increased in CEA-producing cell lines (lanes 2–4). (C) The analysis of soluble CEA and E-cadherin in the culture medium revealed an increase in the soluble E-cadherin in the supernatants of CEA-producing clone 6, clone 8 and CX1 cells versus parental MIP-101 cells. Colorectal carcinoma cells were cultured in vitro in RPMI 1640 medium containing 10% fetal bovine serum for 1 week, and the supernatants were evaluated by Western blotting for soluble CEA and E-cadherin proteins. The relative intensity of the soluble cadherin bands are as follows (C): MIP101–28.971.1; clone 6–41.770.6; clone 8–59.471.5; CX1–67.670.3 relative units (n¼ 3). There are significant differences between the CEA-producing (clones 6 and 8) and СЕА-deficient -parental MIP 101 cells, both (Po0.01). (D) The anti-p120 antibody can recognize four p120 isoforms in non-CEA-producing MIP101 cell line. In CEA-producing cells, the amount of isoforms decreases to two (clone 6) or three (clone 8).

The anti-p120 catenin (p120) antibody can recognize four p120 isoforms in non-CEA-producing MIP-101 cells. In CEA-producing cells, the number of isoforms decreases to two in clone 6 cells and to three in clone 8 cells (Fig. 1D).

Identification of novel interactions between CEA and β-catenin To examine if CEA is involved in complex with α- and β-catenins, colorectal carcinoma cell lysates were immunoprecipitated with the anti-α- and anti-β-catenin antibodies, and tested by western blot with anti-CEA antibodies. Our data revealed that CEA co-

immunoprecipitates with β-catenin in clone 8 CEA producing cells (Fig. 2: lane 3).

CEA production suppresses the interactions of α- and β-catenins with E-cadherin in colorectal carcinoma cells To investigate the influence of CEA on E-cadherin adherens junction complexes, we incubated colorectal carcinoma cell lysates with antiα-catenin (Fig. 3A: lanes 4–6) and anti-β-catenin antibodies (Fig. 3A: lanes 7–9) and then tested for interaction with E-cadherin. The immunoprecipitation with N-cadherin served as a negative control (Fig. 3A: lane 10). The data indicate that, in CEA-negative

Please cite this article as: O. Bajenova, et al., Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.007

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Protein interaction of CEAR with α-catenin is suppressed in CEA-producing carcinoma cells

CEA

To further study the function of CEA/CEAR complexes, the wholecell lysates from parental MIP 101 and CEA-producing clones were immunoprecipitated with CEAR/hnRNPM1-4 antibody, ran on SDS-PAGE, and probed with anti-α- and β-catenin antibodies (Fig. 5). Our experiments show that CEAR strongly interacts with α-catenin in MIP101 cells (Fig. 5: lane 3). These interactions did not occur in the CEA-producing clone 6 (Fig. 5: lane 1) and clone 8 (Fig. 5: lane 2) cells.

Changes in p120 catenin binding due to CEA-production by MIP101 cells.

-catenin IP α-catenin 1

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Fig. 2 – Novel interactions of CEA with β-catenin in CEA-producing MIP-101 cells. The data show co-immunoprecipitation of CEA and of β-catenin (lane 3) in clone 8 cells.

MIP-101 cells, E-cadherin bound to β-catenin (Fig. 3A: lane 9). This interaction was impaired in CEA-producing clone 6 and 8 cells (Fig. 3A: lanes 7 and 8). To test for the interaction between CEAR/hnRNPM and E-cadherin, we immunoprecipitated cell lysates from CEA-producing and CEAnegative MIP-101 cell lines with anti-hnRNPM1-4 antibody and tested with anti-E-cadherin antibodies. As a negative control, immunoprecipitation was used with anti-N-cadherin antibody (Fig. 3B: lane 1). A large amount of CEAR was present in the cell lysates of all three cell lines, as shown in Fig. 3B: lanes 5–7. No interaction between E-cadherin and CEAR/hnRNPM (Fig. 3B: lanes 2–4) was identified. To test for potential interactions between E-cadherin and CEA, the cell lysates were immunoprecipitated with anti-CEA antibodies and tested with E-cadherin antibodies, and we also immunoprecipitated with anti-E-cadherin antibodies and tested for CEA. No interactions between CEA and E-cadherin were detected (not shown).

The interactions between α- and β-catenins are disrupted in CEA-producing colorectal carcinoma cells. It has been reported that cytoplasmic domain of E-cadherin is linked to the actin cytoskeleton via AJs catenin group of proteins, including α-, β-, and p120 catenins [17]. To study the effect of CEA on the interaction between α- and β-catenins leading to cytoskeleton signaling, we immunoprecipitated cell lysates from CEA-producing and CEA-negative colorectal carcinoma cells with anti-α-catenin antibody and then tested for β-catenin (Fig. 4: lanes 4–6). These experiments identified co-immunoprecipitation of α- and β-catenins in MIP101 cells (Fig. 4: lane 6) that is absent in the CEAproducing clones 6 and 8 (Fig. 4A: lanes 4 and 5, respectively). Equally high amounts of β-catenin were present for immunoprecipitation in cell lysates (Fig. 4: lanes 1–3) and on the Western blot probed with anti-β-catenin antibody (Fig. 4: lanes 7–9).

To study p120 interactions in AJ protein complexes, cell extracts were immuno-precipitated with E-cadherin, α-catenin, and β- catenin antibodies and tested with anti-p120 antibodies by Western blot. The analysis revealed that, in the parental MIP101 colorectal carcinoma cells, the interactions between p120 and E-cadherin, α-catenin, β-catenins were impaired (Fig. 6В: lanes 1, 4, 7). In the CEA-producing clone 6 cells, we identified two p120 isoforms that interacted with E-cadherin, α-catenin, and β-catenins (Fig. 6В: lanes 3, 5, 9). In clone 8 cells only one low-molecularweight p120 isoform interacted with E-cadherin (Fig. 6B: lane 2) with no specificity to α-catenin (Fig. 6B: lane 6) and β-catenin (Fig. 6B: lane 8). These data show that CEA production influences the splicing isoforms of p120 and its complexes with other AJ proteins. This may affect cell-signaling pathways that regulate cell adhesion and invasion and the function of AJ complexes.

CEA production by colorectal cancer cells increases the endogenous level of β-catenin To elucidate the effect of elevated CEA expression on the Wnt signaling pathway which is one of the key regulators in gut epithelium and carcinogenesis, we compared the levels of endogenous β-catenin activity in the cells with different levels of CEA. For this assay, a plasmid vector TOPflash was used that expresses firefly luciferase gene under a Tcf/LEF-dependent promoter. The luciferase activity measurements (Fig. 7) showed that the level of endogenous β-catenin in the CEA producing cells of Clone 8 is significantly (1.5  ) higher (Po0.01) than in the parental MIP101 cells. Thus, there is a positive correlation between CEA production and the level of endogenous β-catenin that is consistent with the idea that CEA production can affect the Wnt signaling pathway. β-catenin, the Wnt pathway effector, is the pivotal factor in the balance between self-renewal and differentiation. The elevated level of endogenous β-catenin in Clone 8 also correlates with its increased metastatic and tumorigenic potential of these cells [7,8]. These findings support the hypothesis that Wnt/β-catenin pathway plays an important role in CEA-induced progression of colorectal carcinoma.

Discussion Intercellular adhesion molecules comprise a broad class of linker proteins that are crucial for the development of multicellular organism and its homeostasis. The deregulation of cellular

Please cite this article as: O. Bajenova, et al., Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.007

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Fig. 3 – Interactions of E-cadherin with β-catenin are suppressed in CEA-producing colorectal carcinoma cells. (A) Cell lysates of CEA-producing (lanes 1, 2, 4, 5, 7, and 8) and deficient (lanes 3, 6, 9, and 10) cells were immunoprecipitated with anti-α-catenin (lanes 4–6) and anti-β-catenin (lanes 7–9) antibodies. Whole cell lysates (lanes 1–3) and immunoprecipitation with N-cadherin (lane 10) were controls. A co-immunoprecipitation of β-catenin and E-cadherin was detected in CEA-negative MIP-101 cells (lane 9) and is significantly suppressed in CEA-producing clones 6 and 8 (lanes 7 and 8). (B) HnRNPM does not directly interact with E-cadherin. MIP101 cell lysates were immunoprecipitated with anti-E-cadherin antibody (lanes 2–7) and tested for binding with hnRNP M. Immunoprecipitation with anti-N-cadherin antibodies (lane 1) was our control. The data show no interaction between hnRNP M and E-cadherin proteins in MIP-101 cells (lanes 2–4). Equally large amounts of hnRNP M protein are present in the cell lysates (lanes 5–7).

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Fig. 4 – Interactions between α- and β-catenins are suppressed in CEA-producing MIP-101 cells. Cell lysates of CEA-producing (lanes 1, 2, 4, 5, 7, and 8) and deficient MIP-101 (lanes 3, 6, and 9) cells were immunoprecipitated with anti-α-catenin antibody (lanes 4–6) and tested for β-catenin. Whole cell lysates (lanes 1–3) and immunoprecipitation with anti-βcatenin antibodies (lanes 7–9) were our controls. The data show that the interaction between α- and β-catenins in MIP101 cells (lane 6) significantly decreases (lane 4) and is absent (lane 5) in CEA-producing cells.

Fig. 5 – Interaction of hnRNPM with α-catenin is suppressed in MIP-101 cells. CEA-producing and deficient MIP-101 cell lines were immunoprecipitated with anti-α-catenin (lanes 1–3) and anti-β-catenin (lanes 4–6) antibodies. The data show that CEAR/hnRNP M strongly interacts with α-catenin in MIP101 cells (lane 3) that is absent in CEA-producing cells (lanes 1 and 2). Total cell lysates (lanes 11–13), immunoprecipitations with anti-hnRNP M (lanes 8–10), and anti-N-cadherin (lane 7) proteins were our controls.

adhesion is linked to the progression of most solid tumors [18,19]. Cell–cell adhesion molecule E-cadherin regulates cell polarity, architecture, differentiation, proliferation and migration through its intimate association to the actin cytoskeletal network [20,21].

Our data first show that CEA production by MIP101 colorectal cancer cells increases the amount of excreted soluble E-cadherin (Fig. 1C) and alters the binding preferences between E-cadherin and its partners – α-, β- and p120 catenin proteins (Figs. 2–6). CEA

Please cite this article as: O. Bajenova, et al., Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.007

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Fig. 6 – Binding preferences of P120 catenin are altered in CEAproducing MIP-101 cells. Cells were immunoprecipitated with anti-E-cadherin, α-catenin, and β-catenin antibodies and tested for direct interactions with p120 catenin. There is no interaction between p120 catenin and tested AJ proteins (Ecadherin, α- and β-catenins) in MIP101 cells. In CEA-producing clones, we identified the binding of two p120 isoforms (isoforms 2 and 4) with E-cadherin, α-catenin, and β-catenins in clone 6 cells. In clone 8 cells, only one low-molecularweight p120 isoform (isoform 4) interacted with E-cadherin.

Fig. 7 – CEA production by colorectal cancer cells increases the endogenous level of β-catenin. For the assay, a plasmid vector TOPflash was used that expresses firefly luciferase gene under a Tcf/LEF-dependent promoter. The results of luciferase activity measurement demonstrate the significant difference in the level of endogenous β-catenin in the CEA producing cells of MIP101 clone 8 (1.97 times higher) than in the parental MIP101 cells (Po0.01) (n¼ 3). There is a positive correlation between CEA production and endogenous β-catenin level in CEAproducing cancer cells.

overexpression changes functional interactions between AJ proteins that are crucial for maintaining the function of adherens junction complexes, their signaling and epithelial tissue architecture. Clinically there is an interest in the use of soluble E-cadherin as an independent prognostic oncomarker, mostly in the setting of metastatic disease [22–24]. The soluble ectodomain of E-cadherin accumulates in human serum and is also associated with colorectal cancer progression. De Wever et al. showed that soluble E-cadherin present in cell-culture medium stimulates epithelial cancer-cell dispersion and promotes cell-to-cell junction disruption [10]. This study shows the effect of CEA over-expression on the function of E-cadherin junction complexes that may explain the link between the elevated levels of CEA and the increase in soluble E-cadherin during the progression of colorectal cancer.

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We discovered CEA to be a β-catenin-binding protein in clone 8 colorectal cancer cells (Fig. 2). This can lead to the competition between CEA and E-cadherin for the functional interactions with catenin proteins and alter E-cadherin-mediated cell-to-cell adhesion at sites of CEA expression. In normal epithelial cells, E-cadherin interacts with β-catenin. β-catenin also binds to α-catenin to regulate cell-to-cell adhesion and connection with actin cytoskeleton [17,19,20]. If E-cadherin-mediated cell-to-cell adhesion is altered, the interacting proteins – such as β-catenin and α-catenin – are released from a membrane-bound state into the cytoplasm. Released β-catenin is either rapidly degraded or acts as an oncogenic transcription cofactor under the activation of Wnt and Notch signaling pathways and may contribute to colorectal cancer progression [24]. Wnt signaling is mediated by secreted proteins that interact with specific cell-membrane receptors and is involved in the growth and differentiation of cells during development. Uncontrolled activation of this signaling pathway may induce inappropriate proliferation of target cells, including cancer stem cells, and may contribute to the development of colorectal cancer metastasis [25]. β-catenin is a proto-oncogene and a downstream molecule in the Wnt signaling pathway and is regulated by the adenomatous polyposis coli (APC) protein. The APC, besides its established role in the β catenin/Wnt signaling, can coordinate microtubule and actin organization during cell migration [24]. More than 80% of colorectal cancers contain mutations in apc or β-catenin genes [17,19]. Wnt pathway dysregulation has also been implicated in colorectal cancer dissemination and metastasis [24,25]. In addition, interaction of CEA with β-catenin may have a significant effect on cell cycle and cell proliferation. This process is unlikely to happen in normal polarized colon epithelium where CEA resides on the apical surface, while β-catenin resides on the lateral surface. Loss of polarity during transformation can create a permissive environment for CEA and β-catenin interaction. This study shows that dysfunction of this regulatory pathways by CEA may result in the accumulation of a hypo-phosphorylated stable form of β-catenin in the nucleus, where it can bind to the high mobility group domain factor Tcf/LEF, and stimulates the transcription of target genes such as c-myc and cyclin D1 [17,19,20]. This study shows that CEA production by colorectal cancer cells can modulate splicing and binding partners for p120 catenin protein isoforms (Fig. 1D). Depending on its cellular localization and splicing, p120 catenin protein can participate in various processes, such as cadherin-dependent cell-to-cell adhesion, actin cytoskeleton remodeling, intracellular trafficking, cell proliferation, and contact inhibition [17,26]. Evidences indicate that complete loss, down-regulation or mis-localization of p120 correlates with progression of different types of human cancers [17,26]. In epithelial cells certain splicing isoforms of p120 catenin interact with the cytoplasmic tail of classical cadherins to mediate strong cell-to-cell adhesions by facilitating cadherin clustering [17,26]. Extensive alternative mRNA splicing and multiple phosphorylation sites generate additional complexity. Our previous research identified a specific receptor for soluble CEA named as CEAR/hnRNPM that is involved in CEA signaling in macrophages [11,27]. We have shown that CEA–CEAR interaction on the surface of liver macrophages, Kupffer cells, leads to colorectal cancer metastasis by inducing cytokine production and altering the hepatic microenvironment [27]. Unlike the interactions of intracellular CEA/CEAR described here this activity occurs only at the distant metastatic site (liver and lungs) and

Please cite this article as: O. Bajenova, et al., Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.007

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involves multiple cell types. Disruption of AJ complexes would be most important for invasion at the primary site. In human epidermoid carcinoma A431 cells using a combination of two-dimensional gel electrophoresis and MALDI-TOF mass-spectrometry we have shown the association of hnRNP M with actinin-4 protein [28]. Actinin-4 (ACTN4) is an actin-binding protein and in the cytoplasm which participates in structural organization of the cytoskeleton via cross-linking of actin filaments. Cellular localization of ACTN4 depends on the cell environment. Our data suggest involvement of nuclear complexes hnRNPM/ACTN4 in mRNA metabolism, transcription and cytoskeletal cell signaling. In this study, we identified CEAR/hnrnp M protein as a binding partner for α-catenin protein in MIP101 colorectal carcinoma cells. In support of our data Sato et al. [29] showed that CEAR/hnRNP M protein can physically interact with β-catenin in DLD-1 colorectal carcinoma cells. Sato et al. also demonstrated that the hnRNPM/β-catenin/FUS/TLS complex participates to regulate pre-mRNA splicing and that activation of the Wnt signaling pathway may induce certain mRNA-splicing abbreviations seen in human cancers [29]. The involvement of FUS in the cell spreading also suggests a potential role for hnRNPs in cytoskeletal signaling [29]. Shivley and his group discovered that the CEA family member CEACAM-1 and the Wnt signaling pathway are associated. CEACAM1 and the cytoskeletal molecules actin and tropomyosin are known to interact [30]. CEACAM1 regulates Fasmediated apoptosis in Jurkat T-cells via its interaction with β-catenin protein [31]. Our study shows that CEA over-expression by colorectal cancer cells disrupts the normal function of the core intercellular adhesion–E-cadherin complexes and the interactions between catenin AJ proteins. This may influence the cytoskeletal cell signaling and promote invasion and metastasis. Future studies should clarify the details of CEAs role in these signaling mechanisms.

Conflicts of interest The investigators have no conflicts of interest.

Acknowledgments This work was financially supported by the Russian Foundation for Fundamental Research, Grant RFFI # 11-04-01711 to O.B., by the Government of the Russian Federation mega Grant 11G34.31.0068 to Dr. S. O’Brien and by Creighton University Health and Future Foundation grants to P.T. and O.B.

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Please cite this article as: O. Bajenova, et al., Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.007

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 cadheri...
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