Abstract
The Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in many cancers including ovarian cancer and EpCAM overexpression correlates with decreased survival of patients. It was the aim of this study to achieve a targeted methylation of the EpCAM promoter and silence EpCAM gene expression using an engineered zinc finger protein that specifically binds the EpCAM promoter fused to the catalytic domain of the Dnmt3a DNA methyltransferase. We show that transient transfection of this construct increased the methylation of the EpCAM promoter in SKOV3 cells from 4–8% in untreated cells to 30%. Up to 48% methylation was observed in stable cell lines which express the chimeric methyltransferase. Control experiments confirmed that the methylation was dependent on the fusion of the Zinc finger and the methyltransferase domains and specific for the target region. The stable cell lines with methylated EpCAM promoter showed a 60–80% reduction of EpCAM expression as determined at mRNA and protein level and exhibited a significantly reduced cell proliferation. Our data indicate that targeted methylation of the EpCAM promoter could be an approach in the therapy of EpCAM overexpressing cancers.
Highlights
Cancer occurring in the peritoneal cavity of the ovaries is the seventh most common cancer in women and second leading cause of death worldwide among gynecological cancers [1,2,3]
The Epithelial Cell Adhesion Molecule (EpCAM) promoter showed basal level of methylation in SKOV3 cells that were transfected with control vectors, either vector control alone, zinc finger without Dnmt3aCD or Dnmt3aCD without zinc finger, respectively (Fig. 3)
The finding that the expression of untargeted DNA methyltransferase 3a (Dnmt3a) catalytic domain did not lead to a large increase in DNA methylation at the EpCAM promoter is in agreement with previous observations at another target locus [62]
Summary
Cancer occurring in the peritoneal cavity of the ovaries is the seventh most common cancer in women and second leading cause of death worldwide among gynecological cancers [1,2,3]. Ovarian cancer is difficult to treat with a five year survival rate of around 20% in cancers diagnosed in advanced stage [4,5,6]. Platinum-based analogues such as Cisplatin or Carboplatin are the major standard chemotherapy agents to treat ovarian cancer in initial stages [7]. Their use is hindered by the acquired or intrinsic resistance of the cancer cells to the drug [8]. In spite of an increased understanding in the etiology of ovarian cancer there has been little change in the survival of patients over the past 30 years, because in the early stages ovarian cancer is asymptomatic and there are no efficient tumor specific and sensitive markers to monitor epithelial ovarian cancer [9]. There is an immediate need for new strategies for the treatment of ovarian cancer
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