Renal oncometabolite L-2-hydroxyglutarate imposes a block in kidney tubulogenesis: Evidence for an epigenetic basis for the L-2HG-induced impairment of differentiation

Abstract

2-Hydroxyglutarate (2HG) overproducing tumors arise in a number of tissues, including the kidney. The tumorigenesis resulting from overproduced 2HG has been attributed to the ability of 2HG alter gene expression by inhibiting α-ketoglutarate (αKG)-dependent dioxygenases, including Ten-eleven-Translocation (TET) enzymes. Genes that regulate cellular differentiation are reportedly repressed, blocking differentiation of mesenchymal cells into myocytes, and adipocytes. In this report, the expression of the enzyme responsible for L2HG degradation, L-2HG dehydrogenase (L2HGDH), is knocked down, using lentiviral shRNA, as well as siRNA, in primary cultures of normal Renal Proximal Tubule (RPT) cells. The knockdown (KD) results in increased L-2HG levels, decreased demethylation of 5mC in genomic DNA, and increased methylation of H3 Histones. Consequences include reduced tubulogenesis by RPT cells in matrigel, and reduced expression of molecular markers of differentiation, including membrane transporters as well as HNF1α and HNF1β, which regulate their transcription. These results are consistent with the hypothesis that oncometabolite 2HG blocks RPT differentiation by altering the methylation status of chromatin in a manner that impedes the transcriptional events required for normal differentiation. Presumably, similar alterations are responsible for promoting the expansion of renal cancer stem-cells, increasing their propensity for malignant transformation.

Oncometabolite L-2-Hydroxyglutarate Promotes Oncogenesis of Renal Cell Carcinomas by Down-Regulating Differentiation

“…Consistent with this hypothesis, 5hmC blots of genomic DNA derived from primary RPT cells with an L2HGDH KD indicated that the level of 5hmC was reduced in primary RPT cells with an L2HGDH KD (vs. controls) [43]. In addition, Western blots indicated increases in the levels of a number of different classes of methylated histones in primary RPT cells with an L2HGD KD [43]. Not only was there an increase in the level of the activating histone, H3K4me3, but also an increase in the level of the repressive histone H3K27me3.…”

“…Included amongst these αKG dependent dioxygenases are Jmj-KDM histone demethylases, as well as TET 5mC demethylases, which are inhibited not only in tumor cells with IDH1 and IDH2 mutations [45], but also in ccRCCs with reduced L2HGDH levels [11]. Consistent with this hypothesis, 5hmC blots of genomic DNA derived from primary RPT cells with an L2HGDH KD indicated that the level of 5hmC was reduced in primary RPT cells with an L2HGDH KD (vs. controls) [43]. In addition, Western blots indicated increases in the levels of a number of different classes of methylated histones in primary RPT cells with an L2HGD KD [43].…”

“…The results (illustrated in Figure 7) indicated that CB-839 relieved the inhibition of tubulogenesis caused by an L2HGDH KD, presumably by reducing L2HG levels (a consequence of inhibiting the metabolism of Gln to Glu, and ultimately to L2HG). The involvement of the elevated L2HG in mediating the inhibition of tubulogenesis in cultures with an L2HGDH KD was further substantiated by the observed inhibitory effect of cell permeable L2HG octyl ester on tubulogenesis [43].…”

“…In order to examine this hypothesis initially, primary RPT cell cultures were transduced with lentiviral particles containing a vector (pLKO-TRC) encoding either L2HGDH shRNA or control shRNA [43]. The effect on tubulogenesis in matrigel was examined.…”

“…Tubules were quantitated and L2HGDH mRNA level were determined. From Taub et al [43] with permission of Frontiers.…”