Methylation status of the Ep-CAM promoter region in human breast cancer cell lines and breast cancer tissue
Introduction
Ep-CAM (also known as 17-1A, GA733-2, KSA, KS1/4, 323/A3) is a calcium-independent homophilic cell adhesion molecule of 39–42 kDa [1], [2] which is expressed by the majority of epithelial tissues. The Ep-CAM antigen does not structurally resemble any of the major families of the adhesion molecules (cadherins, selectins, integrins, or cell adhesion molecules of the Ig superfamily). It is a type I transmembrane glycoprotein, consisting of an extracellular domain with two EGF-like repeats, and a short cytoplasmic domain of 26 amino acids (for review see Balzar et al. [3]). The cDNA for Ep-CAM was cloned in 1989 from the lung adenocarcinoma cell line UCLA-P3 and designated KSA [4]. The gene was mapped to human chromosome 2 by analysis of human–mouse somatic cell hybrids, by fluorescence in situ hybridisation and by PCR analysis [5], [6], [7].
Ep-CAM is also abundantly and homogeneously expressed on human carcinomas of different origins. Recent immunohistochemical studies of prostate cancer and cervical intraepithelial neoplasia have shown that Ep-CAM expression can increase with disease progression and proliferation [8], [9]. This apparent overexpression has also been described in 35–42% of patients with invasive breast cancer and was a strong predictor of poor disease-free and overall survival [10], [11], [12]. In line with these results, silencing Ep-CAM gene expression with Ep-CAM short interfering RNA (siRNA) resulted in decreased cell proliferation of human breast cancer cell lines [13]. Furthermore, Ep-CAM signalling seems to impact cell proliferation through up-regulation of the proto-oncogene c-myc [14]. Intriguingly, beside its function as adhesion molecule, Ep-CAM can inhibit CD4+T-cell dependent immune responses and thus enable tumour cells to evade T-cell mediated antitumour immunity [15].
The Ep-CAM antigen has attracted major interest as a target for passive and active immunotherapy. Anti-tumour responses have been observed in metastatic colorectal cancer after treatment with the Ep-CAM specific monoclonal antibody edrecolomab [16]. Adjuvant treatment of radically resected Dukes' C colorectal cancer patients using the same antibody induced a 32% relative reduction in mortality as compared to the results of surgery alone [17]. However, as compared to chemotherapy, treatment with the edrecolomab antibody in a phase III study was shown to be less effective [18]. Interestingly, in vitro and in vivo analysis have shown that Ep-CAM expression can be up-regulated by chemotherapeutic agents increasing the effectiveness of Ep-CAM directed antibodies in terms of antibody-dependent cellular cytotoxicity [19]. Thus, the Ep-CAM antigen has become an attractive target for antitumour immune interventions, especially in breast cancer patients, for whose clinical trials are currently under way.
To date, information about molecular mechanisms underlying the regulation of Ep-CAM expression is scarce. Gires and colleagues have described down-regulation of Ep-CAM expression by TNF-α [20]. Other authors have described a modulating effect of cytokines such as IFN-α on Ep-CAM expression [21]. However, the molecular events responsible for overexpression or silencing of the Ep-CAM expression in breast cancer have yet to be elucidated. DNA methylation is an important mechanism for inactivating various genes during tumourigenesis and progression of breast carcinoma, such as the genes encoding the estrogen receptor [22], [23], [24], retinoic acid receptor [25], E-cadherin [26], [27] or BRCA1 [28], [29]. The presence of a CpG island in the Ep-CAM promoter and first exon led us to investigate in this study whether Ep-CAM expression can be influenced by DNA methylation.
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Cell lines, breast cancer tissue, normal breast tissue and colon mucosa samples
Searching a large panel of human breast cancer cell lines of the American Type Culture Collection, we chose the MCF-7 and Hs 578T because of their opposing Ep-CAM protein expression. The cell lines were cultured in RPMI 1640 containing 10% heat-inactivated fetal calf serum (FCS), 1% glutamine, and 1% penicillin/streptomycin. Ep-CAM protein expression was evaluated using flow cytometry; Ep-CAM RNA expression was assessed by real-time PCR and DNA methylation by MethyLight technology and bisulfite
Ep-CAM expression, promoter methylation status and sequence analysis in human breast cancer cell lines
Ep-CAM expression of two established breast cancer cell lines Hs 578T and MCF-7 was assessed by FACS and real-time PCR. MCF-7 cell line stained Ep-CAM positive, whereas the Hs 578T cell line was negative (Fig. 2). Real-time PCR revealed high amounts of Ep-CAM RNA in the cell line MCF-7 but no Ep-CAM RNA in the cell line Hs 578T. As calculated with the ΔΔCt method, MCF-7 cell lines presented over 100,000-fold higher Ep-CAM RNA expression as compared to Hs 578T cell line. Among these two cell
Discussion
In previous publications [10], [11], [12], we have demonstrated that Ep-CAM overexpression in breast cancer correlates with early tumour recurrence and decreased overall survival. Thus, understanding of the molecular mechanisms responsible for the up- or down-regulation of Ep-CAM expression is of utmost interest. To approach this issue we put forward the hypothesis that gene methylation may at least in part impact Ep-CAM expression.
The Ep-CAM promoter region extending into the first exon from
Acknowledgements
This work was supported by the ‘Österreichische Krebshilfe-Krebsgesellschaft/Tirol’ and the ‘MFF Tirol’.
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