Abstract
Familial paraganglioma (PGL) is a rare neuroendocrine cancer associated with defects in the genes encoding the subunits of succinate dehydrogenase (SDH), a tricarboxylic acid (TCA) cycle enzyme. For unknown reasons, a higher prevalence of PGL has been reported for humans living at higher altitude, with increased disease aggressiveness and morbidity. In this study, we evaluate the effects of oxygen on epigenetic changes due to succinate accumulation in three SDH loss cell culture models. We test the hypothesis that the mechanism of α-ketoglutarate (α-KG)-dependent dioxygenase enzymes explains the inhibitory synergy of hypoxia and succinate accumulation. We confirm that SDH loss leads to profound succinate accumulation. We further show that hypoxia and succinate accumulation synergistically inhibit α-KG-dependent dioxygenases leading to increased stabilization of transcription factor HIF1α, HIF2α, and hypermethylation of histones and DNA. Increasing oxygen suppresses succinate inhibition of α-KG-dependent dioxygenases. This result provides a possible explanation for the association between hypoxia and PGL, and suggests hyperoxia as a potential novel therapy.
Highlights
The succinate dehydrogenase (SDH) complex is a tricarboxylic acid (TCA) cycle enzyme composed of four highly conserved nuclear-encoded subunits (SDHA-D) localized to the inner mitochondrial membrane
We report that cells with SDHB knockdown or Sdhc knockout dramatically accumulate succinate, but inhibition of prolyl hydroxylases (PHD), Jumonji domain histone demethylases (JMHD), and TET dioxygenases is only observed as oxygen is reduced from ambient conditions to the lower levels expected in tissues
Western blot analysis showed that HEK293 cells transduced with shRNA lentiviral vectors targeting SDHB mRNA resulted in nearly complete depletion of SDHB protein within 12 h (Fig 1A)
Summary
The SDH complex is a TCA cycle enzyme composed of four highly conserved nuclear-encoded subunits (SDHA-D) localized to the inner mitochondrial membrane. The SDHA and SDHB subunits protrude into the mitochondrial matrix, anchored to the inner mitochondrial membrane by the SDHC and SDHD subunits. SDHA catalyzes the oxidation of succinate to fumarate, and the SDHB subunit contains iron-sulfur clusters that guide the flow of electrons from succinate to ubiquinone in the electron transport chain. Mutations in the genes encoding SDH subunits and SDH assembly factor 2, required for flavination of SDH, predispose carriers to develop PGL in an autosomal dominant fashion [1,2,3,4,5,6]. Cells in carriers heterozygous for germline SDH defects presumably suffer loss of heterozygosity through a second SDH mutation, leading to tumorigenesis through an unknown mechanism. The succinate accumulation hypothesis proposes that this mechanism involves some combination of pseudohypoxia [7,8,9], PLOS ONE | DOI:10.1371/journal.pone.0127471 May 18, 2015
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