The coxsackie adenovirus receptor inhibits cancer cell migration
Introduction
Cancer cell migration resulting in cancer spread is the major cause of cancer-related death. Loss or reduced expression of cell adhesion proteins such as cadherins facilitates detachment of single cancer cells from the tumor bulk. Recently, we found the coxsackie and adenovirus receptor (CAR) to be enriched at cell–cell contacts of human cancer cells with significant differences in expression levels.
CAR is a 46-kDa transmembrane glycoprotein, originally identified as the cellular receptor for coxsackie B viruses and adenoviruses [1], [2], [3]. CAR sequence analysis revealed two immunoglobulin-like extracellular domains [1], [2], [3]. Physiologically, immunoglobulin superfamily members include cell surface receptors as well as cell adhesion proteins [4]. The physiological cellular function of CAR is still unclear. Tissue expression pattern of CAR varies significantly [2], [5] and is regulated during embryonic development and tissue differentiation [6], [7]. In differentiated human or canine epithelial cells, CAR was found to be localized at basolateral cell–cell adhesions [8] and tight junctions [9]. In human ovarian and cervical cancer cells, we recently observed enrichment of CAR at the cell–cell contact sites as viewed by immunofluorescence analysis [10]. These findings suggested, but did not prove, a potential role for CAR in formation or stabilization of cell–cell contacts.
As for cancer research, CAR has become of interest as the primary receptor for recombinant adenoviruses applied in gene therapy. Several studies have been performed to analyze the CAR expression levels of cancer cells in cell culture with relation to the efficiency of adenoviral gene transfer [11], [12], [13], [14]. When studying the expression level of CAR in human cancer tissues, it has repeatedly been observed that CAR expression was reduced in less differentiated cancer tissues [15], [16], [17]. CAR expression in the U-118 glioblastoma cell line reduced volume of tumor xenografts [18]. Reduced expression of CAR during cancer progression suggests a potential role for the differential CAR expression in cancer progression.
To study the role of differential CAR expression in human cancer cells, we established CAR-expressing cell clones of an ovarian and a cervical cancer cell line. This study shows the impact of CAR on cellular morphology and migratory potential.
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Cell lines and cell culture
Cervical epidermoid carcinoma cell line CaSki (ATCC CRL-1550), cervical adenocarcinoma cell line HeLa (ATCC CCL2), ovarian carcinoma cell line A2780 (ECACC 93112519), hepatoblastoma cell line HepG2 (ATCC HB-8065), and human embryonic kidney cell line 293 (ATCC CRL 1573) are widely distributed, commercially available cell lines. Cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum, 100 μg/ml streptomycin, 100 U/ml penicillin G, and 2 mM glutamine at
CAR expression induced cell–cell aggregations in A2780 ovarian cancer cells
We established stable CAR-expressing cell clones from the ovarian cancer cell line A2780 and the cervical cancer cell line CaSki, using the V5-tagged CAR expression plasmid pcDNA3.1CARV5. Expression of CAR mRNA as determined by RT–PCR analysis of A2780 cells, CaSki cells, and the derived CAR-V5-expressing cell clones A2780CAR6 and CaSkiCAR1 is shown in Fig. 1. Both A2780 and CaSki cells were selected for establishing CAR-expressing cell clones because these cell lines as well as the derived
Discussion
This study shows the effect of CAR expression on cell adhesion and migration of ovarian and cervical cancer cells. Most ovarian and cervical cancers are of epithelial origin. In untransformed, nonmalignant epithelia, several localization studies for CAR revealed expression and enrichment of CAR at basolateral cell–cell adhesions and tight junctions of epithelia cells [21], [22]. In ovarian and cervical carcinoma cells, which in general lose epithelial organization during cancer progression,
Acknowledgements
We thank Lioba Walz for excellent technical assistance and Dr. Hendrik Rohlder for cloning CAR into pcDNA3.1. We thank Inge Engels (Institute of Environmental Medicine, University Hospital Freiburg) for her kind help in FACScan analysis. The adenoviral vector AdGal was kindly provided by Dr Daniel Maneval (Canji Inc., San Diego, CA, USA). This work was supported by funds of the Deutsche Forschungsgemeinschaft (RU 476/5) granted to I.B.R.
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