Carcinogenesis Advance Access originally published online on March 27, 2009
Carcinogenesis 2009 30(6):1041-1048; doi:10.1093/carcin/bgp073
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Promoter CpG island hypermethylation- and H3K9me3 and H3K27me3-mediated epigenetic silencing targets the deleted in colon cancer (DCC) gene in colorectal carcinogenesis without affecting neighboring genes on chromosomal region 18q21
Department of Pathology, Maastricht University Medical Center, PO Box 616, 6200 MD Maastricht, The Netherlands
1 Department of Pathology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands
2 Department of Molecular Genetics
3 The European Fine Art Foundation (TEFAF) Oncology Chair, GROW––School for Oncology and Developmental Biology, Maastricht University Medical Center, PO Box 616, 6200 MD Maastricht, The Netherlands
4 Department of Oncology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231, USA
* To whom correspondence should be addressed. Department of Pathology, GROW––School for Oncology and Developmental Biology, Maastricht University Medical Center, PO Box 616, 6200 MD Maastricht, The Netherlands. Tel: +31 43 3874622; Fax: +31 43 3876613; Email: manon.van.engeland{at}mumc.nl
Chromosomal loss of 18q21 is a frequent event in colorectal cancer (CRC) development, suggesting that this region harbors tumor suppressor genes (TSGs). Several candidate TSGs, among which methyl-CpG-binding domain protein 1 (MBD1), CpG-binding protein CXXC1, Sma- and Mad-related protein 4 (SMAD4), deleted in colon cancer (DCC) and methyl-CpG-binding domain protein 2 (MBD2) are closely linked on a 4-Mb DNA region on chromosome18q21. As TSGs can be epigenetically silenced, this study investigates whether MBD1, CXXC1, SMAD4, DCC and MBD2 are subject to epigenetic silencing in CRC. Methylation-specific polymerase chain reaction and sodium bisulfite sequencing of these genes show that DCC, but not MBD1, CXXC1, SMAD4 and MBD2, has promoter CpG island methylation in CRC cell lines and tissues {normal mucosa [29.5% (18/61)], adenomas [81.0% (47/58)] and carcinomas [82.7% (62/75)] (P = 8.6 x 10–9)} that is associated with reduced DCC expression, independent of 18q21 loss analyzed by multiplex ligation-dependent probe amplification. Reduced gene expression of CXXC1, SMAD4 and MBD2 correlates with 18q21 loss in CRC cell lines (P = 0.04, 0.02 and 0.02, respectively). Treatment with the demethylating agent 5-aza-2'-deoxycytidine, but not with the histone deacetylase inhibitor trichostatin A exclusively restored DCC expression in CRC cell lines. Chromatin immunoprecipitation studies reveal that the DCC promoter is marked with repressive histone-tail marks H3K9me3 and H3K27me3, whereas activity related H3K4me3 was absent. Only active epigenetic marks were detected for MBD1, CXXC1, SMAD4 and MBD2. This study demonstrates specific epigenetic silencing of DCC in CRC as a focal process not affecting neighboring genes on chromosomal region 18q21.
Abbreviations: Aza, 5-aza-2'-deoxycytidine; ChIP, chromatin immunoprecipitation; CRC, colorectal cancer; DCC, deleted in colon cancer; MBD1, methyl-CpG-binding domain protein 1; MBD2, methyl-CpG-binding domain protein 2; MLPA, multiplex ligation-dependent probe amplification; MSP, methylation-specific polymerase chain reaction; PCR, polymerase chain reaction; SMAD4, Sma- and Mad-related protein 4; TSA, trichostatin A; TSG, tumor suppressor gene; TSS, transcription start site
Received February 3, 2009; revised March 19, 2009; accepted March 22, 2009.