Abstract:
Epigenetic modification plays an important role in regulating gene expression as well as maintaining the integrity of the genome. One such epigenetic modifier is histone lysine demethylase 6A (KDM6A) which removes the repressive histone 3 lysine 27 trimethylated (H3K27me3) mark along the promoter of genes to allow for their activation and expression. KDM6A undergoes frequent somatic mutation in cancer and has been shown to have a tumour suppressor role in oncogenesis. Previous studies conducted at the ACSRC demonstrated that KDM6A RNA interference (RNAi) reduced the viability of cancer cell lines that were reported to express low levels of KDM6A while cell lines with reported high KDM6A expression were unaffected, suggesting a possible haploinsufficient tumour suppressor role. To investigate this observation, a panel of 14 human cancer cell lines was generated and KDM6A expression was characterised by both RT-qPCR and western immunoblotting. Treatment of KDM6A RNAi to the low-expressing KDM6A cell line MIA PaCa-2 showed anti-proliferative effect however, characterisation of this cell line showed no KDM6A expression suggesting a possible off-target effect from RNAi. To better understand the pathobiology of KDM6A, KDM6A expression was reintroduced into three KDM6A-deficient cell lines (MIA PaCa-2, THP-1 and HeLa) to generate isogenic cell line pairs. MIA PaCa-2 cells were effectively transfected with KDM6A expression construct, however, expression was not maintained. THP-1 cells expressed low levels of KDM6A, yet displayed clear phenotypic changes including reduced proliferation rate and cell cycle changes. HeLa cells displayed sustained expression of KDM6A therefore, HeLa cells were then treated with 5 μM of GSK343, an inhibitor of the H3K27 methyltransferase, EZH2. Gene expression analysis showed that GSK343 treatment upregulated the expression of EZH2-mediated repressed gene, IGFBP3, in wild-type, KDM6A-deficient HeLa cells. In contrast, no increase in IGFBP3 expression was seen in KDM6A-expressing HeLa cells. These findings suggest that in KDM6A-deficient cells, dysregulated EZH2 activity leads to aberrant accumulation of H3K27me3 and repression of certain genes. Our results show that inhibition of EZH2 in KDM6A-deficient cells can re-express these repressed genes. This work uncovers a new strategy for targeting cancers with this genetic feature.