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Open Access Highly Accessed Research article

Aberrant septin 9 DNA methylation in colorectal cancer is restricted to a single CpG island

Reinhold Wasserkort12*, Alexandra Kalmar3, Gabor Valcz3, Sandor Spisak3, Manuel Krispin14, Kinga Toth3, Zsolt Tulassay36, Andrew Z Sledziewski5 and Bela Molnar36

Author Affiliations

1 Epigenomics AG, Berlin, Germany

2 Current address: Delta-Vir GmbH, Leipzig, Germany

3 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary

4 Current address: Zymo Research, Irvine CA 92614, USA

5 Epigenomics Inc, Seattle, WA 98104, USA

6 Molecular Medicine Research Unit, Hungarian Academy of Science, Budapest, Hungary

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BMC Cancer 2013, 13:398  doi:10.1186/1471-2407-13-398

Published: 30 August 2013

Abstract

Background

The septin 9 gene (SEPT9) codes for a GTP-binding protein associated with filamentous structures and cytoskeleton formation. SEPT9 plays a role in multiple cancers as either an oncogene or a tumor suppressor gene. Regulation of SEPT9 expression is complex and not well understood; however, hypermethylation of the gene was recently introduced as a biomarker for early detection of colorectal cancer (CRC) and has been linked to cancer of the breast and of the head and neck. Because the DNA methylation landscape of different regions of SEPT9 is poorly understood in cancer, we analyzed the methylation patterns of this gene in distinct cell populations from normal and diseased colon mucosa.

Methods

Laser capture microdissection was performed to obtain homogeneous populations of epithelial and stromal cells from normal, adenomatous, and tumorous colon mucosa. Microdissected samples were analyzed using direct bisulfite sequencing to determine the DNA methylation status of eight regions within and near the SEPT9 gene. Septin-9 protein expression was assessed using immunohistochemistry (IHC).

Results

Regions analyzed in SEPT9 were unmethylated in normal tissue except for a methylation boundary detected downstream of the largest CpG island. In adenoma and tumor tissues, epithelial cells displayed markedly increased DNA methylation levels (>80%, p <0.0001) in only one of the CpG islands investigated. SEPT9 methylation in stromal cells increased in adenomatous and tumor tissues (≤50%, p <0.0001); however, methylation did not increase in stromal cells of normal tissue close to the tumor. IHC data indicated a significant decrease (p <0.01) in Septin-9 protein levels in epithelial cells derived from adenoma and tumor tissues; Septin-9 protein levels in stromal cells were low in all tissues.

Conclusions

Hypermethylation of SEPT9 in adenoma and CRC specimens is confined to one of several CpG islands of this gene. Tumor-associated aberrant methylation originates in epithelial cells; stromal cells appear to acquire hypermethylation subsequent to epithelial cells, possibly through field effects. The region in SEPT9 with disease-related hypermethylation also contains the CpGs targeted by a novel blood-based screening test (Epi proColon®), providing further support for the clinical relevance of this biomarker.

Keywords:
DNA methylation; Septin 9; Colorectal cancer; Adenoma; Epithelial cells; Stromal cells; Direct bisulfite sequencing; Immunohistochemistry