Characteristic profiles of DNA epigenetic modifications in colon cancer and its predisposing conditions-benign adenomas and inflammatory bowel disease.

Background: Active demethylation of 5-methyl-2'-deoxycytidine (5-mdC) in DNA occurs by oxidation to 5-(hydroxymethyl)-2'-deoxycytidine (5-hmdC) and further oxidation to 5-formyl-2'-deoxycytidine (5-fdC) and 5-carboxy-2'-deoxycytidine (5-cadC), and is carried out by enzymes of the ten-eleven translocation family (TETs 1, 2, 3). Decreased level of epigenetic DNA modifications in cancer tissue may be a consequence of reduced activity/expression of TET proteins. To determine the role of epigenetic DNA modifications in colon cancer development, we analyzed their levels in normal colon and various colonic pathologies. Moreover, we determined the expressions of TETs at mRNA and protein level.The study included material from patients with inflammatory bowel disease (IBD), benign polyps (AD), and colorectal cancer (CRC). The levels of epigenetic DNA modifications and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in examined tissues were determined by means of isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS). The expressions of TET mRNA were measured with RT-qPCR, and the expressions of TET proteins were determined immunohistochemically.

Results: IBD was characterized by the highest level of 8-oxodG among all analyzed tissues, as well as by a decrease in 5-hmdC and 5-mdC levels (at a midrange between normal colon and CRC). AD had the lowest levels of 5-hmdC and 5-mdC of all examined tissues and showed an increase in 8-oxodG and 5-(hydroxymethyl)-2'-deoxyuridine (5-hmdU) levels. CRC was characterized by lower levels of 5-hmdC and 5-mdC, the lowest level of 5-fdC among all analyzed tissues, and relatively high content of 5-cadC. The expression of TET1 mRNA in CRC and AD was significantly weaker than in IBD and normal colon. Furthermore, CRC and AD showed significantly lower levels of TET2 and AID mRNA than normal colonic tissue.

Conclusions: Our findings suggest that a complex relationship between aberrant pattern of DNA epigenetic modification and cancer development does not depend solely on the transcriptional status of TET proteins, but also on the characteristics of premalignant/malignant cells. This study showed for the first time that the examined colonic pathologies had their unique epigenetic marks, distinguishing them from each other, as well as from normal colonic tissue. A decrease in 5-fdC level may be a characteristic feature of largely undifferentiated cancer cells.