The oncometabolite R-2-hydroxyglutarate dysregulates the differentiation of human mesenchymal stromal cells via inducing DNA hypermethylation

Limengmeng Wang,Kaimin Hu,Binsheng Wang,He Huang,Shan Fu,Xiaohong Yu,Yulin Xu,Lizhen Liu,Huafang Wang,Jingjing Feng

doi:10.1186/s12885-020-07744-x

Abstract

BackgroundIsocitrate dehydrogenase (IDH1/2) gene mutations are the most frequently observed mutations in cartilaginous tumors. The mutant IDH causes elevation in the levels of R-enantiomer of 2-hydroxylglutarate (R-2HG). Mesenchymal stromal cells (MSCs) are reasonable precursor cell candidates of cartilaginous tumors. This study aimed to investigate the effect of oncometabolite R-2HG on MSCs.MethodsHuman bone marrow MSCs treated with or without R-2HG at concentrations 0.1 to 1.5 mM were used for experiments. Cell Counting Kit-8 was used to detect the proliferation of MSCs. To determine the effects of R-2HG on MSC differentiation, cells were cultured in osteogenic, chondrogenic and adipogenic medium. Specific staining approaches were performed and differentiation-related genes were quantified. Furthermore, DNA methylation status was explored by Illumina array-based arrays. Real-time PCR was applied to examine the signaling component mRNAs involved in.ResultsR-2HG showed no influence on the proliferation of human MSCs. R-2HG blocked osteogenic differentiation, whereas promoted adipogenic differentiation of MSCs in a dose-dependent manner. R-2HG inhibited chondrogenic differentiation of MSCs, but increased the expression of genes related to chondrocyte hypertrophy in a lower concentration (1.0 mM). Moreover, R-2HG induced a pronounced DNA hypermethylation state of MSC. R-2HG also improved promotor methylation of lineage-specific genes during osteogenic and chondrogenic differentiation. In addition, R-2HG induced hypermethylation and decreased the mRNA levels of SHH, GLI1and GLI2, indicating Sonic Hedgehog (Shh) signaling inhibition.ConclusionsThe oncometabolite R-2HG dysregulated the chondrogenic and osteogenic differentiation of MSCs possibly via induction of DNA hypermethylation, improving the role of R-2HG in cartilaginous tumor development.

Highlights

Isocitrate dehydrogenase (IDH1/2) gene mutations are the most frequently observed mutations in cartilaginous tumors
R-enantiomer of 2-hydroxylglutarate (R-2HG) did not influence the proliferation and phenotype of human Mesenchymal stromal cells (MSCs) The effect of R-2HG on the proliferation of MSCs was examined by Cell Counting Kit-8 assay (CCK-8) assay
R-2HG inhibits osteogenic differentiation of MSCs Osteogenic differentiation in MSCs in the presence of R2HG (1, 1.5 mM) showed a dose dependent impaired calcification when compared to MSCs in the absence of R-2HG

Summary

Introduction

Isocitrate dehydrogenase (IDH1/2) gene mutations are the most frequently observed mutations in cartilaginous tumors. The mutant IDH causes elevation in the levels of R-enantiomer of 2-hydroxylglutarate (R-2HG). This study aimed to investigate the effect of oncometabolite R-2HG on MSCs. Mutations in isocitrate dehydrogenase (IDH) have been observed in many human malignancies [1]. The IDH1/2 mutant cells are expected to have increased levels of R-2HG, which, are at extremely low concentrations under physiological conditions [10]. The cartilaginous tumors and other neoplasms with IDH1/2 mutations have increased levels of R-2HG [4]. R-2-HG was found elevated in colon [14] and breast cancer cells [15] harboring IDH1/2 wild type, despite the R-2-HG levels are lower than IDH mutant. The role of R-2-HG in IDH wild type cells in oncogenesis still controversial

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