Introduction. Glycosylation is a fundamental post-translational modification (PTM) which when altered in cancer, plays an integral role in tumour progression and cancer cell survival. There is a clear link between chemoresistance and hypoxia, hypoxia and epigenetics and more recently glycosylation and epigenetics . Our remit is to interrogate these paradigms together, to establish new approaches for the detection, diagnosis and treatment of ovarian and breast cancer.
Materials and Methods. Ovarian and breast cancer cells were treated with the DNA methyltransferase inhibitor, 5-AZA-2-deoxycytidine (5-AZA-dC). Cells were exposed to normoxia and differential hypoxic conditions. The methylation status of hypoxia-exposed cells and the normoxia-controls as well as demethylation status following 5-AZA-dC treatment, was confirmed by flow cytometry. N-glycans from cell secreted glycoproteins were analysed using hydrophilic interaction liquid chromatography (HILIC) and mass spectrometry. Western blot analyses assessed apoptosis, senescence, autophagy and markers of epithelial to mesenchymal transition (EMT). The Oris Migration Assay monitored the cell migration, while qRT-PCR measured candidate glycosyltransferases and transcription factor (TFs) gene expression in these samples. Subsequent knock down of GATA transcription factors, transient siRNA approaches were confirmed by qRT-PCR and Western blot analyses.
Results and Discussion. Branching and sialylation, known to aid in tumour survival, were increased on secreted N-glycans from chemosensitive cells compared to chemoresistant cells following treatment with 5-AZA-dC and in all cells under hypoxic conditions. These changes correlated with increases in MGAT5 and ST3GAL4 expression in demethylated ovarian cancer cells. GATA2/3 were identified in-silico, as possible TFs for these genes. Results showed that there is a correlation between, ST3GAL4 and GATA2 and MGAT5 and GATA3, respectively. 5-AZA-dC-treated and hypoxia-exposed cells displayed increased migration, with a greater effect in chemosensitive demethylated- and 0.5% hypoxia-exposed cells compared to chemoresistant cells. Apoptotic and senescence markers were increased in 5-AZA-dC treated cells. Knocking down GATA2 and 3 showed a decrease in MGAT5 and ST3GAL4 expression.
Conclusion. These results give insight into the effects epigenetic alterations have on cancer cell glycosylation and how these may impact on the overall fate of those cells. The GATA2/3 TFs are linked to cancer stage, increased invasiveness and are possible therapeutic targets. Our data demonstrate a potential correlation between GATA2/3 and the levels of glycosyltransferases involved in branching and sialylation which are involved in cancer cell survival and metastases.
- Greville G, McCann A, Rudd PM, Saldova R. Epigenetic regulation of glycosylation and the impact on chemo-resistance in breast and ovarian cancer. Epigenetics, 2016, 11(12): 845–857.