Investigation of hallmark epigenetic changes in a cancer stem cell model.

Epigenetic control of gene expression is vital for normal development and differentiation of cells, and is also important in the development of disease. In particular, there is a strong association between hallmark epigenetic changes and cancer – namely, genome wide hypomethylation, gene specific hypermethylation and characteristic histone modification. Almost all studies of cancer epigenetics to date have been conducted in malignant tissues or already transformed cell lines, and therefore do not take into account epigenetic changes occurring during the process of transformation. Our lab has developed a line of primary mesenchymal stem cells (MSC; thought to be the origin of various types of sarcoma) in which five oncogenic steps towards a fully transformed state are sequentially introduced including: human telomerase, necessary to extend the life span of MSC in culture, genes to inactivate the p53 and pRb tumour suppressor genes and genes to activate the oncogenes c-Myc and Ras. I hypothesized that hallmark epigenetic changes take place in this step-wise model of transformation, and aimed to investigate genome wide hypomethylation and the activity of the polycomb repressive 2 (PRC2) complex in this system. Utilizing the PCR based technique MethyLight, I show that transformed MSC are hypomethylated compared to parental MSC, with this decrease in methylation occurring on the introduction of oncogenic H-Ras in the final step. I also show that this hypomethylation is a gradual event following H-Ras expression, and transformation can take place in the absence of hypomethylation. I demonstrate that the three core components of the PRC2 complex are up-regulated during step-wise transformation and that PRC2 target genes are down-regulated. Finally, I show that MSC are able to be transformed when the PRC2 components EZH2 and SUZ12 are knocked down before the final oncogenic hit. These studies show that hallmark epigenetic changes occur during step-wise transformation and suggest that tumour-associated epigenetic changes occur following genetic aberrations. This model is valuable and relevant to further explore the mechanisms behind epigenetic alterations in cancer.