Persistent downregulation of the pancarcinoma-associated epithelial cell adhesion molecule via active intranuclear methylation.
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.
van der Gun
Bernardina T F
BTF
Department of Pathology and Laboratory Medicine, Section Medical Biology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands.
Wasserkort
Reinhold
R
Epigenomics AG, Kleine Praesidentenstrasse 1, D-10178, Berlin, Germany.
Monami
Amélie
A
Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, D-52056 Aachen, Germany.
Jeltsch
Albert
A
School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, D-28725 Bremen, Germany.
Raskó
Tamás
T
Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, 6726 Szeged, Temesvári krt. 62, Hungary.
Ślaska-Kiss
Krystyna
K
Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, 6726 Szeged, Temesvári krt. 62, Hungary.
Cortese
Rene
R
Epigenomics AG, Kleine Praesidentenstrasse 1, D-10178, Berlin, Germany.
Rots
Marianne G
MG
Department of Pathology and Laboratory Medicine, Section Medical Biology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands.
de Leij
Lou F M H
LFMH
Department of Pathology and Laboratory Medicine, Section Medical Biology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands.
Ruiters
Marcel H J
MHJ
Department of Pathology and Laboratory Medicine, Section Medical Biology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands.
Kiss
Antal
A
Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, 6726 Szeged, Temesvári krt. 62, Hungary.
Weinhold
Elmar
E
Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, D-52056 Aachen, Germany.
McLaughlin
Pamela M J
PMJ
Department of Pathology and Laboratory Medicine, Section Medical Biology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands.
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
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