AbstractThe discovery of ten-eleven translocation (TET) enzymes-mediated 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) gave rise to a new area of epigenetic study. Initially thought to be simply an intermediate in an active DNA demethylation pathway, 5hmC was since demonstrated to play critical roles in mammalian development, cellular differentiation and cancer progression. The global 5hmC content is reduced in the vast majority of studied cancers and mutations in the three TET genes are present to a varying extent in some tumour types. The reactivation of the TET proteins was proposed as a therapeutic intervention in melanoma patients. However, the preliminary data from the Murrell lab suggests that 5hmC levels increase during colorectal cancer (CRC) metastasis to the liver and few TET mutations are reported in CRC patients. Given that 90% of cancer deaths occur due to metastasis, the role of TETs and 5hmC in CRC progression warrants closer investigation.
The in-house ultrasensitive liquid chromatography-mass spectrometry method of global cytosine, 5mC and 5hmC levels detection in various DNA samples was successfully optimised and validated. It was found to robustly detect the 5hmC content in almost all DNA samples studied and offer a superior sensitivity to an external method. The primary SW480 and metastatic SW620 CRC cell lines were chosen to study the effects of 5hmC and TET levels manipulation. The SW480 cells were shown to have higher migration rate and TET2, 5mC and 5mhC content but lower TET1 expression and proliferation rate compared to the SW620 cells. The treatment of the two cell lines with vitamin C increased their 5hmC content, but its phenotypic effects were found to be 5hmC-independent. The CRISPR/Cas9-based targeting of the three TET genes in these cell lines caused an incomplete ablation of TET and 5hmC levels and resulted in lower proliferation rate in the SW480 cells and reduced migration in the SW620 cells. The changes in TET expression and 5hmC levels are also described during the differentiation of human embryonic stem cells (hESCs) to hepatocyte-like cells (HLCs). Despite the initial attempt to knockdown TET1 levels in hESC to improve differentiation efficiency failing, future experiments utilising this approach could produce HLCs closer resembling human hepatocytes, which is critical for drug testing purposes. Taken together, these results contribute to the growing body of evidence for the critical role of 5hmC and TETs in cancer progression and hESC differentiation.
|Date of Award||26 May 2021|
|Supervisor||Adele Murrell (Supervisor) & David Tosh (Supervisor)|