Alterations in the DNA methylation profile of cells are among the earliest molecular changes in cancer . Both locus-specific hypermethylation and genome-wide hypomethylation commonly occur in different types of tumors . 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 .
Septin 9 (SEPT9)a involvement in cancer was first discovered as a fusion product with the MLL gene in leukemia . Subsequent studies showed that SEPT9 was frequently deleted in sporadic ovarian tumors  or amplified in breast cancer ; it was suggested that the gene might be a candidate ovarian tumor suppressor gene that may also act like an oncogene. A comprehensive screen of a wide variety of tissue samples and cell lines revealed that SEPT9 was ubiquitously expressed, although, its isoform expression appeared to be tissue specific . Moreover, SEPT9 mRNA and protein were overexpressed in diverse human tumors further suggesting an important role of the gene in tumorigenesis.
SEPT9 belongs to a highly conserved family of septin genes coding for GTP-binding proteins. These multidomain proteins assemble into complexes and form filamentous structures which comprise part of the cytoskeleton [8, 9]. The septin proteins play important roles in many cellular processes by providing rigidity to the cell membrane, serving as scaffolds to recruit proteins to specific subcellular locales, and creating membrane diffusion barriers to establish discrete cellular domains . SEPT9 is characterized by its complex genomic organization that spans 219 kb and has 18 distinct transcripts encoding 15 isoforms [10, 11]. Alternative splicing at both 5′ and 3′ ends generates this transcript diversity . The gene also harbors several CpG islands that when methylated may impact the expression of different transcripts.
Several analyses have been conducted on the expression of the SEPT9 transcripts. The transcript SEPT9_v1 was overexpressed in breast, ovarian, and prostate cancer while no SEPT9_v1 expression was observed in the normal tissue of these organs [12, 13]. Two distinct transcripts, SEPT_v4 and SEPT_v4*, code for the same polypeptide, but are translated with different efficiencies and respond differently to cellular stresses; the SEPT9_v4* polypeptide is thought to play a role in neoplasia [14, 15]. The transcript SEPT9_v3 expression was up-regulated in some cancer cell lines and repressed in others in which there was also a reduction in protein levels . This transcript was also associated with promoter hypermethylation.
Recently, SEPT9 was shown to be epigenetically modified in colorectal cancer (CRC) . Subsequent studies using refined and more sensitive assays confirmed SEPT9 as a biomarker for CRC [18, 19]. A complete workflow was developed on the basis of these findings and permits a minimally invasive, blood-based screening test (Epi proColon®) for colorectal cancer , which is currently available for clinical application in Europe . The test detects methylated SEPT9 in circulating DNA in plasma which is believed to be derived from apoptotic cells shed from the tumor . However, it is unknown whether SEPT9 hypermethylation is present in all types of tumor cells of the colon and to what extent the hypermethylation affects the promoter region of SEPT9. To advance our understanding of cancer-associated epigenetic changes in SEPT9, we investigated the methylation profile of SEPT9 in epithelial and stromal cells microdissected from normal and diseased fresh-frozen biopsies and tissue colon samples. Since earlier studies analyzed DNA methylation changes only in a single SEPT9 CpG island, and only in heterogeneous 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.
Our results provide new insights into the cellular origin of aberrant DNA methylation in the SEPT9 gene for CRC and the clinical relevance of DNA methylation for early CRC detection.