Abstract
BackgroundThe 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.MethodsLaser 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).ResultsRegions 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.ConclusionsHypermethylation 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.
Highlights
The septin 9 gene (SEPT9) codes for a Guanosine triphosphate (GTP)-binding protein associated with filamentous structures and cytoskeleton formation
Since earlier studies analyzed DNA methylation changes only in a single SEPT9 Cytosine guanine dinucleotide (CpG) island, and only in heterogeneous colorectal cancer (CRC) tissue specimens, this study aims at determining whether the aberrant methylation changes are unique to only this CpG island or whether hypermethylation affects multiple CpG dense regions associated with this gene and how SEPT9 methylation is affected in homogeneous populations of epithelial and stromal cells
The multiplex PCR (mPCR) assay established for this study included primer pairs for 13 different loci (Additional file 3: Table S3; only the results for SEPT9-related loci are presented in this paper)
Summary
The septin 9 gene (SEPT9) codes for a GTP-binding protein associated with filamentous structures and cytoskeleton formation. Alterations in the DNA methylation profile of cells are among the earliest molecular changes in cancer [1]. Both locus-specific hypermethylation and genome-wide hypomethylation commonly occur in different types of tumors [2]. Hypermethylation of tumor suppressor genes has been identified as a critical step in tumor initiation as the silenced expression of such genes affects whether cells maintain normal growth. Such epigenetic events, along with mutations, provide cells with a selective advantage that may lead to their clonal expansion [3]. SEPT9 mRNA and protein were overexpressed in diverse human tumors further suggesting an important role of the gene in tumorigenesis
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