title: Persistent downregulation of the pancarcinoma-associated epithelial cell adhesion molecule via active intranuclear methylation.
abstract: The epithelial cell adhesion molecule (EpCAM) is expressed at high levels on the surface of most carcinoma cells. SiRNA silencing of EpCAM expression leads to reduced metastatic potential of tumor cells demonstrating its importance in oncogenesis and tumor progression. However, siRNA therapy requires either sequential delivery or integration into the host cell genome. Hence we set out to explore a more definite form to influence EpCAM gene expression. The mechanisms underlying the transcriptional activation of the EpCAM gene, both in normal epithelial tissue as well as in carcinogenesis, are poorly understood. We show that DNA methylation plays a crucial role in EpCAM expression, and moreover, active silencing of endogenous EpCAM via methylation of the EpCAM promoter results in a persistent downregulation of EpCAM expression. In a panel of carcinoma derived cell lines, bisulfite analyses showed a correlation between the methylation status of the EpCAM promoter and EpCAM expression. Treatment of EpCAM-negative cell lines with a demethylating agent induced EpCAM expression, both on mRNA and protein level, and caused upregulation of EpCAM expression in an EpCAM-positive cell line. After delivery of the DNA methyltransferase M.SssI into EpCAM-positive ovarian carcinoma cells, methylation of the EpCAM promoter resulted in silencing of EpCAM expression. SiRNA-mediated silencing remained for 4 days, after which EpCAM re-expression increased in time, while M.SssI-mediated downregulation of EpCAM maintained through successive cell divisions as the repression persisted for at least 17 days. This is the first study showing that active DNA methylation leads to sustained silencing of endogenous EpCAM expression.
authors.affiliation: Department of Pathology and Laboratory Medicine, Section Medical Biology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands.
authors.forename: Bernardina T F
authors.initials: BTF
authors.lastname: van der Gun
authors.affiliation: Epigenomics AG, Kleine Praesidentenstrasse 1, D-10178, Berlin, Germany.
authors.forename: Reinhold
authors.initials: R
authors.lastname: Wasserkort
authors.affiliation: Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, D-52056 Aachen, Germany.
authors.forename: Amélie
authors.initials: A
authors.lastname: Monami
authors.affiliation: School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, D-28725 Bremen, Germany.
authors.forename: Albert
authors.initials: A
authors.lastname: Jeltsch
authors.affiliation: Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, 6726 Szeged, Temesvári krt. 62, Hungary.
authors.forename: Tamás
authors.initials: T
authors.lastname: Raskó
authors.affiliation: Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, 6726 Szeged, Temesvári krt. 62, Hungary.
authors.forename: Krystyna
authors.initials: K
authors.lastname: Ślaska-Kiss
authors.affiliation: Epigenomics AG, Kleine Praesidentenstrasse 1, D-10178, Berlin, Germany.
authors.forename: Rene
authors.initials: R
authors.lastname: Cortese
authors.affiliation: Department of Pathology and Laboratory Medicine, Section Medical Biology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands.
authors.forename: Marianne G
authors.initials: MG
authors.lastname: Rots
authors.affiliation: Department of Pathology and Laboratory Medicine, Section Medical Biology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands.
authors.forename: Lou F M H
authors.initials: LFMH
authors.lastname: de Leij
authors.affiliation: Department of Pathology and Laboratory Medicine, Section Medical Biology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands.
authors.forename: Marcel H J
authors.initials: MHJ
authors.lastname: Ruiters
authors.affiliation: Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, 6726 Szeged, Temesvári krt. 62, Hungary.
authors.forename: Antal
authors.initials: A
authors.lastname: Kiss
authors.affiliation: Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, D-52056 Aachen, Germany.
authors.forename: Elmar
authors.initials: E
authors.lastname: Weinhold
authors.affiliation: Department of Pathology and Laboratory Medicine, Section Medical Biology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands.
authors.forename: Pamela M J
authors.initials: PMJ
authors.lastname: McLaughlin
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D001374:Azacitidine / Q000494:pharmacology; D002277:Carcinoma / Q000188:drug therapy / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D019175:DNA Methylation / Q000187:drug effects; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D015536:Down-Regulation; D000071858:Epithelial Cell Adhesion Molecule; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D010051:Ovarian Neoplasms / Q000378:metabolism; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D034741:RNA, Small Interfering; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases; D001374:Azacitidine