Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type-Specific and Time-Resolved Responses to Vhl Inactivation in the Kidney.

Defining the initial events in oncogenesis and the cellular responses they entrain, even in advance of morphologic abnormality, is a fundamental challenge in understanding cancer initiation. As a paradigm to address this, we longitudinally studied the changes induced by loss of the tumor suppressor gene von Hippel Lindau (VHL), which ultimately drives clear cell renal cell carcinoma. Vhl inactivation was directly coupled to expression of a tdTomato reporter within a single allele, allowing accurate visualization of affected cells in their native context and retrieval from the kidney for single-cell RNA sequencing. This strategy uncovered cell type-specific responses to Vhl inactivation, defined a proximal tubular cell class with oncogenic potential, and revealed longer term adaptive changes in the renal epithelium and the interstitium. Oncogenic cell tagging also revealed markedly heterogeneous cellular effects including time-limited proliferation and elimination of specific cell types. Overall, this study reports an experimental strategy for understanding oncogenic processes in which cells bearing genetic alterations can be generated in their native context, marked, and analyzed over time. The observed effects of loss of Vhl in kidney cells provide insights into VHL tumor suppressor action and development of renal cell carcinoma. SIGNIFICANCE: Single-cell analysis of heterogeneous and dynamic responses to Vhl inactivation in the kidney suggests that early events shape the cell type specificity of oncogenesis, providing a focus for mechanistic understanding and therapeutic targeting.

Recent advances in the biology of tumour hypoxia with relevance to diagnostic practice and tissue-based research.

In this review article, we examine the importance of low levels of oxygen (hypoxia) in cancer biology. We provide a brief description of how mammalian cells sense oxygen. The hypoxia-inducible factor (HIF) pathway is currently the best characterised oxygen-sensing system, but recent work has revealed that mammals also use an oxygen-sensing system found in plants to regulate the abundance of some proteins and peptides with an amino-terminal cysteine residue. We discuss how the HIF pathway is affected during the growth of solid tumours, which develop in microenvironments with gradients of oxygen availability. We then introduce the concept of 'pseudohypoxia', a state of constitutive, oxygen-independent HIF system activation that occurs due to oncogenic stimulation in a number of specific tumour types that are of immediate relevance to diagnostic histopathologists. We provide an overview of the different methods of quantifying tumour hypoxia, emphasising the importance of pre-analytic factors in interpreting the results of tissue-based studies. Finally, we review recent approaches to targeting hypoxia/HIF system activation for therapeutic benefit, the application of which may require knowledge of which hypoxia signalling components are being utilised by a given tumour. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Somatostatin secretion by Na+-dependent Ca2+-induced Ca2+ release in pancreatic delta-cells.

Pancreatic islets are complex micro-organs consisting of at least three different cell types: glucagon-secreting α-, insulin-producing ß- and somatostatin-releasing δ-cells1. Somatostatin is a powerful paracrine inhibitor of insulin and glucagon secretion2. In diabetes, increased somatostatinergic signalling leads to defective counter-regulatory glucagon secretion3. This increases the risk of severe hypoglycaemia, a dangerous complication of insulin therapy4. The regulation of somatostatin secretion involves both intrinsic and paracrine mechanisms5 but their relative contributions and whether they interact remains unclear. Here we show that dapagliflozin-sensitive glucose- and insulin-dependent sodium uptake stimulates somatostatin secretion by elevating the cytoplasmic Na+ concentration ([Na+]i) and promoting intracellular Ca2+-induced Ca2+ release (CICR). This mechanism also becomes activated when [Na+]i is elevated following the inhibition of the plasmalemmal Na+-K+ pump by reductions of the extracellular K+ concentration emulating those produced by exogenous insulin in vivo 6. Islets from some donors with type-2 diabetes hypersecrete somatostatin, leading to suppression of glucagon secretion that can be alleviated by a somatostatin receptor antagonist. Our data highlight the role of Na+ as an intracellular second messenger, illustrate the significance of the intraislet paracrine network and provide a mechanistic framework for pharmacological correction of the hormone secretion defects associated with diabetes that selectively target the δ-cells.

Inosine pranobex enhances human NK cell cytotoxicity by inducing metabolic activation and NKG2D ligand expression.

Inosine pranobex (IP) is a synthetic immunomodulating compound, indicated for use in the treatment of human papillomavirus-associated warts and subacute sclerosing panencephalitis. Previous studies demonstrate that the immunomodulatory activity of IP is characterized by enhanced lymphocyte proliferation, cytokine production, and NK cell cytotoxicity. The activation of NKG2D signaling on NK cells, CD8+ T cells, and γδ T cells also produces these outcomes. We hypothesized that IP alters cellular immunity through the induction of NKG2D ligand expression on target cells, thereby enhancing immune cell activation through the NKG2D receptor. We tested this hypothesis and show that exposure of target cells to IP leads to increased expression of multiple NKG2D ligands. Using both targeted metabolic interventions and unbiased metabolomic studies, we found that IP causes an increase in intracellular concentration of purine nucleotides and tricarboxylic acid (TCA) cycle intermediates and NKG2D ligand induction. The degree of NKG2D ligand induction was functionally significant, leading to increased NKG2D-dependent target cell immunogenicity. These findings demonstrate that the immunomodulatory properties of IP are due to metabolic activation with NKG2D ligand induction.

Insulin inhibits glucagon release by SGLT2-induced stimulation of somatostatin secretion.

Hypoglycaemia (low plasma glucose) is a serious and potentially fatal complication of insulin-treated diabetes. In healthy individuals, hypoglycaemia triggers glucagon secretion, which restores normal plasma glucose levels by stimulation of hepatic glucose production. This counterregulatory mechanism is impaired in diabetes. Here we show in mice that therapeutic concentrations of insulin inhibit glucagon secretion by an indirect (paracrine) mechanism mediated by stimulation of intra-islet somatostatin release. Insulin's capacity to inhibit glucagon secretion is lost following genetic ablation of insulin receptors in the somatostatin-secreting δ-cells, when insulin-induced somatostatin secretion is suppressed by dapagliflozin (an inhibitor of sodium-glucose co-tranporter-2; SGLT2) or when the action of secreted somatostatin is prevented by somatostatin receptor (SSTR) antagonists. Administration of these compounds in vivo antagonises insulin's hypoglycaemic effect. We extend these data to isolated human islets. We propose that SSTR or SGLT2 antagonists should be considered as adjuncts to insulin in diabetes therapy.

Dysregulation of Glucagon Secretion by Hyperglycemia-Induced Sodium-Dependent Reduction of ATP Production.

Diabetes is a bihormonal disorder resulting from combined insulin and glucagon secretion defects. Mice lacking fumarase (Fh1) in their ß cells (Fh1ßKO mice) develop progressive hyperglycemia and dysregulated glucagon secretion similar to that seen in diabetic patients (too much at high glucose and too little at low glucose). The glucagon secretion defects are corrected by low concentrations of tolbutamide and prevented by the sodium-glucose transport (SGLT) inhibitor phlorizin. These data link hyperglycemia, intracellular Na+ accumulation, and acidification to impaired mitochondrial metabolism, reduced ATP production, and dysregulated glucagon secretion. Protein succination, reflecting reduced activity of fumarase, is observed in α cells from hyperglycemic Fh1ßKO and ß-V59M gain-of-function KATP channel mice, diabetic Goto-Kakizaki rats, and patients with type 2 diabetes. Succination is also observed in renal tubular cells and cardiomyocytes from hyperglycemic Fh1ßKO mice, suggesting that the model can be extended to other SGLT-expressing cells and may explain part of the spectrum of diabetic complications.

PHD2 inactivation in Type I cells drives HIF-2α-dependent multilineage hyperplasia and the formation of paraganglioma-like carotid bodies.

KEY POINTS: The carotid body is a peripheral arterial chemoreceptor that regulates ventilation in response to both acute and sustained hypoxia. Type I cells in this organ respond to low oxygen both acutely by depolarization and dense core vesicle secretion and, over the longer term, via cellular proliferation and enhanced ventilatory responses. Using lineage analysis, the present study shows that the Type I cell lineage itself proliferates and expands in response to sustained hypoxia. Inactivation of HIF-2α in Type I cells impairs the ventilatory, proliferative and cell intrinsic (dense core vesicle) responses to hypoxia. Inactivation of PHD2 in Type I cells induces multilineage hyperplasia and ultrastructural changes in dense core vesicles to form paraganglioma-like carotid bodies. These changes, similar to those observed in hypoxia, are dependent on HIF-2α. Taken together, these findings demonstrate a key role for the PHD2-HIF-2α couple in Type I cells with respect to the oxygen sensing functions of the carotid body. ABSTRACT: The carotid body is a peripheral chemoreceptor that plays a central role in mammalian oxygen homeostasis. In response to sustained hypoxia, it manifests a rapid cellular proliferation and an associated increase in responsiveness to hypoxia. Understanding the cellular and molecular mechanisms underlying these processes is of interest both to specialized chemoreceptive functions of that organ and, potentially, to the general physiology and pathophysiology of cellular hypoxia. We have combined cell lineage tracing technology and conditionally inactivated alleles in recombinant mice to examine the role of components of the HIF hydroxylase pathway in specific cell types within the carotid body. We show that exposure to sustained hypoxia (10% oxygen) drives rapid expansion of the Type I, tyrosine hydroxylase expressing cell lineage, with little transdifferentiation to (or from) that lineage. Inactivation of a specific HIF isoform, HIF-2α, in the Type I cells was associated with a greatly reduced proliferation of Type I cells and hypoxic ventilatory responses, with ultrastructural evidence of an abnormality in the action of hypoxia on dense core secretory vesicles. We also show that inactivation of the principal HIF prolyl hydroxylase PHD2 within the Type I cell lineage is sufficient to cause multilineage expansion of the carotid body, with characteristics resembling paragangliomas. These morphological changes were dependent on the integrity of HIF-2α. These findings implicate specific components of the HIF hydroxylase pathway (PHD2 and HIF-2α) within Type I cells of the carotid body with respect to the oxygen sensing and adaptive functions of that organ.

Purine nucleotide metabolism regulates expression of the human immune ligand MICA.

Expression of the cell-surface glycoprotein MHC class I polypeptide-related sequence A (MICA) is induced in dangerous, abnormal, or "stressed" cells, including cancer cells, virus-infected cells, and rapidly proliferating cells. MICA is recognized by the activating immune cell receptor natural killer group 2D (NKG2D), providing a mechanism by which immune cells can identify and potentially eliminate pathological cells. Immune recognition through NKG2D is implicated in cancer, atherosclerosis, transplant rejection, and inflammatory diseases, such as rheumatoid arthritis. Despite the wide range of potential therapeutic applications of MICA manipulation, the factors that control MICA expression are unclear. Here we use metabolic interventions and metabolomic analyses to show that the transition from quiescent cellular metabolism to a "Warburg" or biosynthetic metabolic state induces MICA expression. Specifically, we show that glucose transport into the cell and active glycolytic metabolism are necessary to up-regulate MICA expression. Active purine synthesis is necessary to support this effect of glucose, and increases in purine nucleotide levels are sufficient to induce MICA expression. Metabolic induction of MICA expression directly influences NKG2D-dependent cytotoxicity by immune cells. These findings support a model of MICA regulation whereby the purine metabolic activity of individual cells is reflected by cell-surface MICA expression and is the subject of surveillance by NKG2D receptor-expressing immune cells.

Fumarate Hydratase Deletion in Pancreatic ß Cells Leads to Progressive Diabetes.

We explored the role of the Krebs cycle enzyme fumarate hydratase (FH) in glucose-stimulated insulin secretion (GSIS). Mice lacking Fh1 in pancreatic ß cells (Fh1ßKO mice) appear normal for 6-8 weeks but then develop progressive glucose intolerance and diabetes. Glucose tolerance is rescued by expression of mitochondrial or cytosolic FH but not by deletion of Hif1α or Nrf2. Progressive hyperglycemia in Fh1ßKO mice led to dysregulated metabolism in ß cells, a decrease in glucose-induced ATP production, electrical activity, cytoplasmic [Ca2+]i elevation, and GSIS. Fh1 loss resulted in elevated intracellular fumarate, promoting succination of critical cysteines in GAPDH, GMPR, and PARK 7/DJ-1 and cytoplasmic acidification. Intracellular fumarate levels were increased in islets exposed to high glucose and in islets from human donors with type 2 diabetes (T2D). The impaired GSIS in islets from diabetic Fh1ßKO mice was ameliorated after culture under normoglycemic conditions. These studies highlight the role of FH and dysregulated mitochondrial metabolism in T2D.

Succination of Protein Disulfide Isomerase Links Mitochondrial Stress and Endoplasmic Reticulum Stress in the Adipocyte During Diabetes.

AIMS: Protein succination by fumarate increases in the adipose tissue of diabetic mice and in adipocytes matured in high glucose as a result of glucotoxicity-driven mitochondrial stress. The endoplasmic reticulum (ER) oxidoreductase protein disulfide isomerase (PDI) is succinated in adipocytes that are matured in high glucose, and in this study we investigated whether succination would alter PDI oxidoreductase activity, directly linking mitochondrial stress and ER stress. RESULTS: Protein succination and the ER stress marker C/EBP homologous protein (CHOP) were diminished after pharmaceutical targeting of mitochondrial stress with the chemical uncoupler niclosamide in adipocytes matured in high-glucose concentrations. PDI was succinated by fumarate on both CXXC-containing active sites, contributing to reduced enzymatic activity. Succinated PDI decreased reductase activity in adipocytes matured in high glucose, and in db/db epididymal adipose tissue, in association with increased levels of CHOP. PDI succination was increased in fumarase knockdown adipocytes, leading to reduced PDI oxidoreductase activity, increased CHOP levels, and pro-inflammatory cytokine secretion, confirming the specific role of elevated fumarate levels in contributing to ER stress. In addition, PDI succination and ER stress were decreased, and PDI reductase activity was restored when exposure to chronic high glucose was limited, highlighting the importance of calorie restriction in the improvement of adipocyte metabolic function. INNOVATION: These experiments identify PDI succination as a novel biochemical mechanism linking altered mitochondrial metabolism to ER stress in the adipocyte during diabetes. CONCLUSION: The current study demonstrates that early biochemical changes in mitochondrial metabolism have important implications for the development of adipocyte stress. Antioxid. Redox Signal. 27, 1281-1296.

Remodelling of microRNAs in colorectal cancer by hypoxia alters metabolism profiles and 5-fluorouracil resistance.

Solid tumours have oxygen gradients and areas of near and almost total anoxia. Hypoxia reduces sensitivity to 5-fluorouracil (5-FU)-chemotherapy for colorectal cancer (CRC). MicroRNAs (miRNAs) are hypoxia sensors and were altered consistently in six CRC cell lines (colon cancer: DLD-1, HCT116 and HT29; rectal cancer: HT55, SW837 and VACO4S) maintained in hypoxia (1 and 0.2% oxygen) compared with normoxia (20.9%). CRC cell lines also showed altered amino acid metabolism in hypoxia and hypoxia-responsive miRNAs were predicted to target genes in four metabolism pathways: beta-alanine; valine, leucine, iso-leucine; aminoacyl-tRNA; and alanine, aspartate, glutamate. MiR-210 was increased in hypoxic areas of CRC tissues and hypoxia-responsive miR-21 and miR-30d, but not miR-210, were significantly increased in 5-FU resistant CRCs. Treatment with miR-21 and miR-30d antagonists sensitized hypoxic CRC cells to 5-FU. Our data highlight the complexity and tumour heterogeneity caused by hypoxia. MiR-210 as a hypoxic biomarker, and the targeting of miR-21 and miR-30d and/or the amino acid metabolism pathways may offer translational opportunities.

Expression of Idh1R132H in the Murine Subventricular Zone Stem Cell Niche Recapitulates Features of Early Gliomagenesis.

Isocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed Idh1R132H in the subventricular zone (SVZ) of the adult mouse brain. The mice developed hydrocephalus and grossly dilated lateral ventricles, with accumulation of 2-hydroxyglutarate and reduced α-ketoglutarate. Stem and transit amplifying/progenitor cell populations were expanded, and proliferation increased. Cells expressing SVZ markers infiltrated surrounding brain regions. SVZ cells also gave rise to proliferative subventricular nodules. DNA methylation was globally increased, while hydroxymethylation was decreased. Mutant SVZ cells overexpressed Wnt, cell-cycle and stem cell genes, and shared an expression signature with human gliomas. Idh1R132H mutation in the major adult neurogenic stem cell niche causes a phenotype resembling gliomagenesis.

Loss of Fumarate Hydratase and Aberrant Protein Succination Detected With S-(2-Succino)-Cysteine Staining to Identify Patients With Multiple Cutaneous and Uterine Leiomyomatosis and Hereditary Leiomyomatosis and Renal Cell Cancer Syndrome.

AIMS: Hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome is an autosomal dominant disorder caused by heterozygotic germline mutations in fumarate hydratase (FH) with incomplete penetrance, and clinically challenging to diagnose. Immunohistochemical stainings may favor an earlier diagnosis. METHODS AND RESULTS: The authors have tested 31 smooth muscle neoplasms. Ten of the 13 lesions from patients with HLRCC syndrome showed negative FH staining. Most sporadic piloleiomyomas presented strongly positive FH staining although 5 cases were negative. Sensitivity of FH staining in our series is 83.3% but specificity is 75%. Anti-S-(2-succino)-cysteine (2SC) showed the opposite intensity staining pattern and showed great correlation with anti-FH (rho spearman = -0.797). Anti-2SC staining increased the diagnostic accuracy in 19% of the cases. LIMITATIONS: The main limitation of this study is the lack additional clinical data to further classify the cases as the case inclusion was histopathological. CONCLUSIONS: Negative FH staining could indicate a high risk of HLRCC but it could also suggest the presence of a syndrome in up to 25% of sporadic cases. Thus, when there is a doubtful case, anti-2SC may be added to exclude the syndrome if a negative staining is found.

Increased Expression of the Diabetes Gene SOX4 Reduces Insulin Secretion by Impaired Fusion Pore Expansion.

The transcription factor Sox4 has been proposed to underlie the increased type 2 diabetes risk linked to an intronic single nucleotide polymorphism in CDKAL1 In a mouse model expressing a mutant form of Sox4, glucose-induced insulin secretion is reduced by 40% despite normal intracellular Ca(2+) signaling and depolarization-evoked exocytosis. This paradox is explained by a fourfold increase in kiss-and-run exocytosis (as determined by single-granule exocytosis measurements) in which the fusion pore connecting the granule lumen to the exterior expands to a diameter of only 2 nm, which does not allow the exit of insulin. Microarray analysis indicated that this correlated with an increased expression of the exocytosis-regulating protein Stxbp6. In a large collection of human islet preparations (n = 63), STXBP6 expression and glucose-induced insulin secretion correlated positively and negatively with SOX4 expression, respectively. Overexpression of SOX4 in the human insulin-secreting cell EndoC-ßH2 interfered with granule emptying and inhibited hormone release, the latter effect reversed by silencing STXBP6 These data suggest that increased SOX4 expression inhibits insulin secretion and increased diabetes risk by the upregulation of STXBP6 and an increase in kiss-and-run exocytosis at the expense of full fusion. We propose that pharmacological interventions promoting fusion pore expansion may be effective in diabetes therapy.

The Succinated Proteome of FH-Mutant Tumours.

Inherited mutations in the Krebs cycle enzyme fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity in HLRCC tumours causes accumulation of the Krebs cycle intermediate fumarate to high levels, which may act as an oncometabolite through various, but not necessarily mutually exclusive, mechanisms. One such mechanism, succination, is an irreversible non-enzymatic modification of cysteine residues by fumarate, to form S-(2-succino)cysteine (2SC). Previous studies have demonstrated that succination of proteins including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), kelch-like ECH-associated protein 1 (KEAP1) and mitochondrial aconitase (ACO2) can have profound effects on cellular metabolism. Furthermore, immunostaining for 2SC is a sensitive and specific biomarker for HLRCC tumours. Here, we performed a proteomic screen on an FH-mutant tumour and two HLRCC-derived cancer cell lines and identified 60 proteins where one or more cysteine residues were succinated; 10 of which were succinated at cysteine residues either predicted, or experimentally proven, to be functionally significant. Bioinformatic enrichment analyses identified most succinated targets to be involved in redox signaling. To our knowledge, this is the first proteomic-based succination screen performed in human tumours and cancer-derived cells and has identified novel 2SC targets that may be relevant to the pathogenesis of HLRCC.

OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation.

2-Oxoglutarate (2OG) and Fe(II)-dependent oxygenase domain-containing protein 1 (OGFOD1) is predicted to be a conserved 2OG oxygenase, the catalytic domain of which is related to hypoxia-inducible factor prolyl hydroxylases. OGFOD1 homologs in yeast are implicated in diverse cellular functions ranging from oxygen-dependent regulation of sterol response genes (Ofd1, Schizosaccharomyces pombe) to translation termination/mRNA polyadenylation (Tpa1p, Saccharomyces cerevisiae). However, neither the biochemical activity of OGFOD1 nor the identity of its substrate has been defined. Here we show that OGFOD1 is a prolyl hydroxylase that catalyzes the posttranslational hydroxylation of a highly conserved residue (Pro-62) in the small ribosomal protein S23 (RPS23). Unusually OGFOD1 retained a high affinity for, and forms a stable complex with, the hydroxylated RPS23 substrate. Knockdown or inactivation of OGFOD1 caused a cell type-dependent induction of stress granules, translational arrest, and growth impairment in a manner complemented by wild-type but not inactive OGFOD1. The work identifies a human prolyl hydroxylase with a role in translational regulation.

A role for cytosolic fumarate hydratase in urea cycle metabolism and renal neoplasia.

The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

Inhibition of mitochondrial aconitase by succination in fumarate hydratase deficiency.

The gene encoding the Krebs cycle enzyme fumarate hydratase (FH) is mutated in hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity causes accumulation of intracellular fumarate, which can directly modify cysteine residues to form 2-succinocysteine through succination. We undertook a proteomic-based screen in cells and renal cysts from Fh1 (murine FH)-deficient mice and identified 94 protein succination targets. Notably, we identified the succination of three cysteine residues in mitochondrial Aconitase2 (ACO2) crucial for iron-sulfur cluster binding. We show that fumarate exerts a dose-dependent inhibition of ACO2 activity, which correlates with increased succination as determined by mass spectrometry, possibly by interfering with iron chelation. Importantly, we show that aconitase activity is impaired in FH-deficient cells. Our data provide evidence that succination, resulting from FH deficiency, targets and potentially alters the function of multiple proteins and may contribute to the dysregulated metabolism observed in HLRCC.

The emerging role of fumarate as an oncometabolite.

The drive to understand how altered cellular metabolism and cancer are linked has caused a paradigm shift in the focus of cancer research. The discovery of a mutated metabolic enzyme, isocitrate dehydrogenase 1, that leads to accumulation of the oncometabolite 2-hydroxyglutarate, provided significant direct evidence that dysfunctional metabolism plays an important role in oncogenesis. Striking parallels exist with the Krebs cycle enzyme fumarate hydratase (FH), a tumor suppressor, whose mutation is associated with the development of leiomyomata, renal cysts, and tumors. Loss of FH enzymatic activity results in accumulation of intracellular fumarate which has been proposed to act as a competitive inhibitor of 2-oxoglutarate-dependent oxygenases including the hypoxia-inducible factor (HIF) hydroxylases, thus activating oncogenic HIF pathways. Interestingly, our studies have questioned the role of HIF and have highlighted other candidate mechanisms, in particular the non-enzymatic modification of cysteine residues (succination) that could lead to disruption or loss of protein functions, dysfunctional cell metabolism and cell signaling. Here, we discuss the evidence for proposing fumarate as an onco-metabolite.

Cells lacking the fumarase tumor suppressor are protected from apoptosis through a hypoxia-inducible factor-independent, AMPK-dependent mechanism.

Loss-of-function mutations of the tumor suppressor gene encoding fumarase (FH) occur in individuals with hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC). We found that loss of FH activity conferred protection from apoptosis in normal human renal cells and fibroblasts. In FH-defective cells, both hypoxia-inducible factor 1α (HIF-1α) and HIF-2α accumulated, but they were not required for apoptosis protection. Conversely, AMP-activated protein kinase (AMPK) was activated and required, as evidenced by the finding that FH inactivation failed to protect AMPK-null mouse embryo fibroblasts (MEFs) and AMPK-depleted human renal cells. Activated AMPK was detected in renal cysts, which occur in mice with kidney-targeted deletion of Fh1 and in kidney cancers of HLRCC patients. In Fh1-null MEFs, AMPK activation was sustained by fumarate accumulation and not by defective energy metabolism. Addition of fumarate and succinate to kidney cells led to extracellular signal-regulated kinase 1/2 (ERK1/2) and AMPK activation, probably through a receptor-mediated mechanism. These findings reveal a new mechanism of tumorigenesis due to FH loss and an unexpected pro-oncogenic role for AMPK that is important in considering AMPK reactivation as a therapeutic strategy against cancer.