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Cindy Weidmann, Colm F. Quirke, Christine Yao, Carolyne Mary Lowry, Jade Pomerleau, J. Richard Wagner, Solange Landreville; The effect of oncometabolite 2-hydroxyglutarate on the biological behavior of differentiated melanocytes and uveal melanoma cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4117.
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© ARVO (1962-2015); The Authors (2016-present)
Liver metastasis is a dreaded complication of uveal melanoma (UM). Ocular tumors with a propensity to metastasize are less differentiated than their non-metastatic counterparts. Cell differentiation and metastatic potential are controlled, among others, by the precise regulation of DNA methylation. Hydroxylation of 5-methylcytosine (5-mC) in 5-hydroxymethylcytosine (5-hmC) is a new demethylation mechanism mediated by Ten-eleven translocation 5-methylcytosine oxygenases (TETs) and isocitrate dehydrogenases (IDHs). The overall level of DNA hydroxymethylation in the genome of several cancers is significantly lower than that observed in the corresponding differentiated tissues and is correlated with metastatic stage. Our preliminary data suggest a similar 5-hmC loss in UM. About 10% of cutaneous melanomas harbor a mutation of IDH genes that leads to the production of the oncometabolite 2-hydroxyglutarate (2-HG), which inhibits TET enzymes. Our research project aims to understand the mechanisms that deregulate DNA hydroxymethylation in UM.
mRNA expression of DNA methylases and demethylases was determined in UM, choroid, and melanocytes by gene expression profiling. Mutations in these enzymes were screened by Sanger sequencing. Percentage of 5-hmC of total cytosines was quantified in DNA from UM, choroid, and melanocytes by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Low grade UMs and melanocytes were treated with the TET inhibitor 2-HG before studying their state of differentiation using morphologic analyses, cell proliferation assays, as well as quantification of 5-hmC using ELISA.
IDH1 and IDH2 transcripts were repressed in UM (melanocyte/UM ratio: 9.1 and 12.6, respectively). IDH1 or IDH2 mutations were not detected in UM cell lines screened so far. We measured a significant loss of DNA hydroxymethylation in UM genome compared to melanocytes and choroid (mean % on total cytosines: UM, 0.0035%; choroid, 0.185%; melanocytes, 0.0345%). 2-HG exposure increased proliferation and decreased melanocytic dendrite length in melanocytes.
Our study will yield novel information about the UM epigenome, and how it contributes to metastasis. Alterations of DNA methylation are reversible, and can thus be targeted with drugs.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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