Regulation and epigenetic modulation of EpCAM gene expression in ovarian cancer

The Epithelial Cell Adhesion Molecule (EpCAM) is a protein that is abundantly present on the surface of tumor cells of almost all carcinoma types. Inhibition of the production of this protein decreases aggressiveness of the tumor cells in some carcinoma types, for example in breast cancer. However, until now, protein production can only be reduced transiently. In this research the regulation of the gene which encodes for the EpCAM protein is studied and three methods has been developed to silence EpCAM gene expression in a permanent manner. These methods are all based on inducing DNA methylation which prevents genes to be transcribed into RNA, thereby preventing protein production. To introduce a DNA methyltransferase enzyme directly into cells an efficient protein carrier is required: the non-viral gene transfer agent SAINT was found to be able to deliver functionally active proteins into the cell. Delivery of the DNA methyltransferase into the cells indeed resulted in DNA methylation and a reduced EpCAM protein level. To prevent aspecific DNA methylation, a crippled DNA methyltransferase was coupled to a DNA binding domain designed to bind the EpCAM gene. This construct induced DNA methylation which was indeed limited to the area where the DNA binding domain was located. Alternatively, delivery of small RNAs (designed to inactivate EpCAM RNA) also showed EpCAM DNA methylation in a subpopulation of the cells which correlated with a sustained reduced EpCAM protein level. Gene-specific DNA methylation is a broadly applicable method; in principle in this way most genes can be silenced.

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