Alterations of the epigenome in brain cancer: loss of 5-hydroxymethylcytosine

Abstract : 5-Methylcytosine is an epigenetic mark, which can be oxidized to 5-hydroxymethylcytosine (5hmC) in DNA by ten-eleven translocation (TET) oxygenases. It is an initial step in the demethylation of 5mC. Levels of 5hmC is relatively high in the brain compared to other organs, but these levels are known to be significantly reduced during the development of a brain tumor, especially in glioblastoma multiforme (GBM). However, no known mechanisms may fully explain this abnormality. The objectives of my project were to (1) understand the implications of the demethylation pathway mediated by TET, and (2) gain a deeper insight in the epigenetic make-up of brain tumors. (1) U87 cells were incubated with 5mC, 5hmC, 5-formylcytosine (5fC) or co-incubated of 5hmC with 3,4,5,6-tetrahydro-2’-deoxyuridine (dTHU) over a timeline of 0, 24, 48 and 96 hours. (2) 130 brain tumors (GBM= 79; grade II/III= 51) were obtained directly from surgery and immediately suspended in DNA extraction buffer. Both cell samples and tumor tissues underwent DNA extraction and DNA digestion protocols. The percent per cytosine (%/C) was obtained by quantification of 5mC, 5hmC, 5fC, 5-hydroxymethyluracil (5hmU) and 5formyluracil (5fU) using LC-MS/MS. (1) Cellular incubations showed that it is possible to increase levels of 5hmC in DNA, but also a slight increase in 5mC levels throughout the experiment. 5HmC levels dramatically increased by 1.9-fold after 96h. On the other hand, no increase was observed in 5fC levels. Both 5hmC and 5fC incubations were accompanied by high increases in 5hmU and 5fU levels respectively. The addition of dTHU to the 5hmC incubation decreased 5hmU incorporation by 65%. (2) The average levels of 5mC, 5hmC and 5fC, in brain tumors, were 4.0, 0.15 and 0.021 %/C respectively. 5HmU and 5fU levels were present at comparable levels of 5hmC and 5fC. Levels of 5hmC, 5hmU and 5fU were significantly lower in the DNA of GBM specimens. There was a strong correlation between 5mC with 5hmC and 5fC in GBM, but this was absent in low grade tumors. The presence of 5hmU and 5fU in brain tumor and the increase in their levels during cell incubations indicate a deamination activity in these cancerous cells, which may impinge on the cellular levels of 5hmC, in particular. Furthermore, upon the incubations with 5hmC, downstream levels of 5fC did not increase suggesting a TET malfunction. TET activity is maintained in GBMs, but impaired in low grade tumors due to isocitrate dehydrogenase-1 (IDH1) mutations. Therefore, in brain tumors, a strong deamination activity and TET impairment may lead to epigenetic reduction of 5hmC.