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(R)), providing further support
for the clinical relevance of this biomarker.
