Epigenetic study of histone demethylases enzymes Fe (II) and ±-ketoglutarate dependent of the jumonji family in the context of tumoral metabolism and glutaminase activity

Abstract

Enzymes that add or remove methyl groups on lysine tails of histones are sensitive to changes in cellular metabolism, because they use metabolic as co-substrates. The histone demethylases (KDMs) containing the JmjC domain (JKDMs) uses ±- ketoglutarate (±-KG) derived from the glycolytic or glutaminolitic pathways in a hydroxylation reaction that removes methyl-lysine groups from tails of histones H3 and H4. The conversion of glutamine from ±-KG is particularly important for some types of tumor cells and confers the metabolic flux required to ensure the growth and viability of these cells. It was showed that the intracellular concentration of ±-KG varies between normal and tumor cells, which may be associated with regulation of the activity of JHDMs, the tumorigenic process and maintenance of cancer stem cells (CSCs). Triple negative breast cancer (TNBC) tumors with expression profile of mesenchymal stem-like (MSL) and claudin low shows unregulated JKDM activity, are more aggressive, resistant to therapy and less differentiated, a phenotype associated with the maintenance of CSCs populations and epithelial to mesenchymal transition. In addition, these tumors consume more glutamine and have increased expression of glutaminase C (GAC). Thus, the aim of this work is to study the importance of glutaminolysis in the production of ±-KC cofactor in breast cancer model (focusing on TNBC), as well as its relationship to the maintenance of CSCs populations, their importance for EMT and cellular behaviors associated with these processes. For this, the knockdown against glutaminase 1 (GLS1) gene and/or GAC isoform, and overexpression of wild-type GAC and K320A mutation (with enhanced catalytic activity) will be conducted in three breast cell lines, MCF-10A, a non-tumoral breast lineage, MCF-7, a luminal well differentiated non-TNBC and MDA-MB-231, a lineage TNBC with MSL expression profile similar to undifferentiated claudin low tumors. Overexpression and knockdown will be confirmed by Western blot (WB) and the level of intracellular ±-KG will be measured fluorometrically. The overall methylation status of histones will be monitored passage by passage with flow cytometry, while CSCs populations will be quantified by flow cytometry using the ALDEFLUOR system. The overall methylation profile in H3 and H4 will be determined by mass spectrometry LS-MS-MS and methylation status of gene promoters associated with cell differentiation, stem-like phenotype and EMT, including TGF-², NOTCH and WNT pathways and cluster HOX genes, previously described as regulated by JHDMs, will be determined using commercial qPCR and ChIP-qPCR arrays. The occurrence of EMT will be determined by WB and immunofluorescence with antibodies against E-cadherin, N-cadherin, Vimentin and BMI-1 proteins. Finally, if determined that the EMT and increase of CSCs populations are dependent on the availability of ±-KG produced by glutaminolysis, we will perform a sphere-formation assay (functional in vitro assay to detect stem cells), cell migration assay using scratch method and invasion assay in Boyden chamber coated with Matrigel. The enzyme glutaminase has been shown to be essential for cell proliferation, but is still unknown its relationship with cellular epigenetic status. This project proposes unravel this relationship, which will help understanding this enzyme as a target in cancer research. (AU)