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. 2011 Jan 31:11:45.
doi: 10.1186/1471-2407-11-45.

Effects of EpCAM overexpression on human breast cancer cell lines

Johanna M Gostner  1 , Dominic Fong, Oliver A Wrulich, Florian Lehne, Marion Zitt, Martin Hermann, Sylvia Krobitsch, Agnieszka Martowicz, Guenther Gastl, Gilbert Spizzo
Affiliations

Effects of EpCAM overexpression on human breast cancer cell lines

Johanna M Gostner et al. BMC Cancer. .

Abstract

Background: Recently, EpCAM has attracted major interest as a target for antibody- and vaccine-based cancer immunotherapies. In breast cancer, the EpCAM antigen is overexpressed in 30-40% of all cases and this increased expression correlates with poor prognosis. The use of EpCAM-specific monoclonal antibodies is a promising treatment approach in these patients.

Methods: In order to explore molecular changes following EpCAM overexpression, we investigated changes of the transcriptome upon EpCAM gene expression in commercially available human breast cancer cells lines Hs578T and MDA-MB-231. To assess cell proliferation, a tetrazolium salt based assay was performed. A TCF/LEF Reporter Kit was used to measure the transcriptional activity of the Wnt/β-catenin pathway. To evaluate the accumulation of β-catenin in the nucleus, a subcellular fractionation assay was performed.

Results: For the first time we could show that expression profiling data of EpCAM transfected cell lines Hs578TEpCAM and MDA-MB-231EpCAM indicate an association of EpCAM overexpression with the downregulation of the Wnt signaling inhibitors SFRP1 and TCF7L2. Confirmation of increased Wnt signaling was provided by a TCF/LEF reporter kit and by the finding of the nuclear accumulation of ß-catenin for MDA-MB-231 EpCAM but not Hs578T EpCAM cells. In Hs578T cells, an increase of proliferation and chemosensitivity to Docetaxel was associated with EpCAM overexpression.

Conclusions: These data show a cell type dependent modification of Wnt signaling components after EpCAM overexpression in breast cancer cell lines, which results in marginal functional changes. Further investigations on the interaction of EpCAM with SFRP1 and TCF7L2 and on additional factors, which may be causal for changes upon EpCAM overexpression, will help to characterize unique molecular properties of EpCAM-positive breast cancer cells.

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Figures

Figure 1
Figure 1
EpCAM protein expression. (A) Western blot analysis of EpCAM in the stable transfectants and control cell lines. Observation of the appearance of two protein species (43 and 34 kDa). Actin was used as loading control. Its molecular weight is about 45 kDa. (B) PNGase F treatment of MCF-7 cells. To analyse if the two EpCAM protein species derive from distinct glycosilations, MCF-7 lysates were incubated with the glycosidase PNGase F. As both protein bands shift in height, N-linked glycosilation is not responsible for the existence of two species (* = potential degradation products).
Figure 2
Figure 2
Microscopical analysis of EpCAM localization. Confocal microscopy of generated and endogeneously EpCAM overexpressing cell lines as well as vector controls revealed an increase of EpCAM membraneous staining with cell density. Cells with a cytosolic distribution could be observed in cultures of low confluence. Actin staining with phalloidin revealed slight changes of the actin cyctoskeleton formation upon EpCAM expression.
Figure 3
Figure 3
Migration/invasion assay. (A) Schematic presentation of an Fluoroblock BioCoat™ invasion chamber (BD) composed of a Matrigel™ coated transwell insert containing an 8 μm pore size membrane. The Matrigel matrix locks the pores of the membrane and therefore blocks non invasive cells from migrating. Invasive cells can degrade this basement membrane reconstitution and migrate through the pores. (B) Cells have been seeded at high density into the upper chamber. Migration was stimulated by a serum gradient. After 22 hours the amount of migrated cells has been measured with the Cytofluor4000 fluorescence reader (MTX Lab Systems) from the bottom after cell labelling with calcein (BD), a staining dye for vital cells only. The FluoroBlok™ membrane efficiently blocked the transmission of light into the upper chamber, therefore the measured signal derived from migrated cells only. Although all cells migrated efficiently, no difference could be detected by comparing Hs578TEpCAM and MDA-MB-231EpCAM cells with their respective empty vector control lines (set as 100%).
Figure 4
Figure 4
Cell proliferation assay. In a tetrazolium salt based assay, the proliferative behavior of cell lines with stable EpCAM expression in comparison to empty vector control and untransfected cells has been measured. EpCAM transfected Hs578T cells show enhanced doubling frequency than non-transfected cells and empty vector controls. This effect appears two days after cell seeding. No similar effect could be detected in the stable transfected MDA-MB-231EpCAM cell line. Absorbance was normalized to the 24 hours values.
Figure 5
Figure 5
Docetaxel sensitivity testing. Hs578TEpCAM and Hs578Tcontrol cells (A) as well as MDA-MB-231EpCAM and MDA-MB-231control (B) cells have been treated one day after seeding with different amounts of the chemotherapeutic Docetaxel for 24 hours. Sensitivity to the chemotherapeutic was evaluated in % proliferation (±SEM) with 100% proliferation for untreated cells. Hs578TEpCAM cells were more sensitive to Docetaxel treatment as compared to their EpCAM negative counterpart Hs578Tcontrol, whereas no difference in sensitivity could be detected in MDA-MB-231 transfectants. Docetaxel is known to inhibit mitosis by blocking the microtubule apparatus, therefore we suggest that the enhanced sensitivity of Hs578TEpCAM cells can be related to their enhanced proliferation rate.
Figure 6
Figure 6
Schematic representation of TCF7L2, SFRP1 and ITF-2. (A) TCF7L2 can be divided in 3 major domains: the β-catenin binding domain (CTNNB1), the DNA-binding HMG box and a C-terminal domain which is variable in many isoforms. Only the long form is able to bind to the COOH-terminal binding protein (CtPB), the C-terminus has an important regulatory function. The presence of the CtBP motif is required for the possible repressor function of TCF7L2. Many splicing variants (>16) have been described and each cell expresses more than one variant [32,37]. (B) SFRP1 is a secreted protein and contains a frizzled typical cysteine rich domain (Fz-CRD) which displays the putative Wnt-ligand binding site. The C-terminal part of the protein shows homology to netrin (NTR), an extracelluar protein involved in axonal guidance. NTR domains are involved in protein binding and contain segments rich of positively charged residues. This region is reported to bind to heparin [34]. (C) ITF-2 binds to DNA via its basic-helix-loop-helix (bHLH) domain and can be regulated by β-catenin. The Pitt-Hopkins syndrome (PTHS), a rare syndromic encephalopathy, is caused by ITF-2 haploinsufficency. At least 3 isoformal sequences have been described. The two major isoforms, ITF-2A and ITF-2B, have different N-termini resulting from alternative promoter usage [30]. (⋆ = glycosilation, p = phosphorylation. Swissprot accession numbers Q9NQB0, Q8N474, P15884)
Figure 7
Figure 7
TCF7L2 protein expression. Western Blot showing lower amounts of TCF7L2 protein expression in MDA-MB-231EpCAM cells compared to their empty vector counterparts.
Figure 8
Figure 8
Subcellular fractionation analysis. In Hs578TEpCAM and MDA-MB-231EpCAM cells and their respective control cell lines, β-catenin was highly expressed in all cell lines, but only in the MDA-MB-231EpCAM cell line a significant and reproducible accumulation of β-catenin could be observed in the nuclear compartment, which resulted in an increased Wnt signaling activity. The mainly cytosolic protein GAPDH was used to control the purity of the cytosolic fractionation. To show integrity of the nuclear fraction lamin a/c antibody was used. Pan-actin was used as loading control. As cells were cultivated to 70-80% density, a majority of the EpCAM protein was present in the cytosol. Additionally to the N-terminal directed anti-EpCAM antibody C-10, EpCAM was detected with a C-terminal directed antibody (E144). Interestingly, EpCAM could be detected in the nuclear fraction with both antibodies.
Figure 9
Figure 9
Wnt signaling activity assay. Upon activation of Wnt signaling a luciferase emitted bioluminescence signal is measured. A reporter plasmid containing a constitutive active TCF/LEF motif determined the 100% activation value while a mutated motif lacking any consensus site for β-catenin binding represented the background luminescence level. Hs578TEpCAM cells showed only a modest activation, whereas EpCAM overexpression in MDA-MB-231EpCAM could activate Wnt signaling about 20% in comparison to their empty vector controls.
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References

    1. Tandon AK, Clark GM, Chamness GC, McGuire WL. Association of the 323/A3 surface glycoprotein with tumor characteristics and behavior in human breast cancer. Cancer Res. 1990;50:3317–3321. - PubMed
    1. Gastl G, Spizzo G, Obrist P, Dunser M, Mikuz G. Ep-CAM overexpression in breast cancer as a predictor of survival. Lancet. 2000;356:1981–1982. doi: 10.1016/S0140-6736(00)03312-2. - DOI - PubMed
    1. Spizzo G, Went P, Dirnhofer S, Obrist P, Simon R, Spichtin H, Maurer R, Metzger U, Von Castelberg B, Bart R. et al.High Ep-CAM Expression is Associated with Poor Prognosis in Node-positive Breast Cancer. Breast Cancer Res Treat. 2004;86:207–213. doi: 10.1023/B:BREA.0000036787.59816.01. - DOI - PubMed
    1. Schmidt M, Hasenclever D, Schaeffer M, Boehm D, Cotarelo C, Steiner E, Lebrecht A, Siggelkow W, Weikel W, Schiffer-Petry I. et al.Prognostic effect of epithelial cell adhesion molecule overexpression in untreated node-negative breast cancer. Clin Cancer Res. 2008;14:5849–5855. doi: 10.1158/1078-0432.CCR-08-0669. - DOI - PubMed
    1. Spizzo G, Obrist P, Ensinger C, Theurl I, Dunser M, Ramoni A, Gunsilius E, Eibl G, Mikuz G, Gastl G. Prognostic significance of Ep-CAM AND Her-2/neu overexpression in invasive breast cancer. Int J Cancer. 2002;98:883–888. doi: 10.1002/ijc.10270. - DOI - PubMed

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