Dysregulation of hypoxia pathways in fumarate hydratase-deficient cells is independent of defective mitochondrial metabolism

Abstract

Mutations in the gene encoding the Krebs cycle enzyme fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer in affected individuals. FH-associated neoplasia is characterized by defective mitochondrial function and by upregulation of transcriptional pathways mediated by hypoxia-inducible factor (HIF), although whether and by what means these processes are linked has been disputed. We analysed the HIF pathway in Fh1-/- mouse embryonic fibroblasts (MEFs), in FH-defective neoplastic tissues and in Fh1-/- MEFs re-expressing either wild-type or an extra-mitochondrial restricted form of FH. These experiments demonstrated that upregulation of HIF-1alpha occurs as a direct consequence of FH inactivation. Fh1-/- cells accumulated intracellular fumarate and manifested severe impairment of HIF prolyl but not asparaginyl hydroxylation which was corrected by provision of exogenous 2-oxoglutarate (2-OG). Re-expression of the extra-mitochondrial form of FH in Fh1-/- cells was sufficient to reduce intracellular fumarate and to correct dysregulation of the HIF pathway completely, even in cells that remained profoundly defective in mitochondrial energy metabolism. The findings indicate that upregulation of HIF-1alpha arises from competitive inhibition of the 2-OG-dependent HIF hydroxylases by fumarate and not from disruption of mitochondrial energy metabolism.

Oncometabolites: linking altered metabolism with cancer

“…In SDH-and FH-deficient cells and tumors, the activation of a HIF-orchestrated "pseudohypoxic" response has been reported (41)(42)(43)(44), which could at least in part be attributed to the allosteric inhibition of HIF prolyl hydroxylases (PHDs) by elevated levels of succinate or fumarate ( Figure 2). Although generally regarded as a major player in human cancer by transcriptionally regulating pathways that facilitate tumor growth and progression, the exact role of HIF in tumorigenesis is unclear (45).…”

Expression Profiling in Progressive Stages of Fumarate-Hydratase Deficiency: The Contribution of Metabolic Changes to Tumorigenesis

“…MEFs were isolated by standard techniques from littermate embryos as previously described (18). Fh1/ Ksp-Cre conditional knockout mice have been previously described (19).…”

“…We hypothesized that as well as activating a pseudohypoxic response (18), FH deficiency activates a biosynthetic milieu, i.e., an environment that is both permissive of and contributory to tumorigenesis. To define the progressive consequences of FH deficiency in detail, we developed a novel Fh1 (murine orthologue of FH)-deficient mouse embryonic fibroblast (MEF) line and characterized changes in the expression profile of these cells.…”

Cells Lacking the Fumarase Tumor Suppressor Are Protected from Apoptosis through a Hypoxia-Inducible Factor-Independent, AMPK-Dependent Mechanism

“…In leiomyomas and renal tumors of HLRCC patients, high levels of intracellular fumarate accumulate, HIF-␣ subunits are stabilized, and HIF target genes are transcribed in ambient normoxia, mimicking hypoxic conditions (pseudohypoxia) (23,42). There is strong evidence to suggest that fumaratemediated inhibition of PHD activity and subsequent HIF accumulation and activation are direct consequences of FH loss and the cause of the observed pseudohypoxia (19,39). Further evidence has come from the conditional inactivation of mouse Fh1 in the kidney, which leads to the development of multiple clonal renal cysts that overexpress Hif-1␣, Hif-2␣, and Hif target genes such as Glut1 and Vegf (44).…”