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Supplementary Figure 2 from Expression Profiling in Progressive Stages of Fumarate-Hydratase Deficiency: The Contribution of Metabolic Changes to Tumorigenesis

Supplementary Figure 1 from Expression Profiling in Progressive Stages of Fumarate-Hydratase Deficiency: The Contribution of Metabolic Changes to Tumorigenesis

Supplementary Table 1 from Expression Profiling in Progressive Stages of Fumarate-Hydratase Deficiency: The Contribution of Metabolic Changes to Tumorigenesis

Guitart et al. performed an in vivo genetic dissection of the Krebs cycle enzyme fumarate hydratase (Fh1) in the hematopoietic system. Their investigations revealed multifaceted functions of Fh1 in the regulation of hematopoietic stem cell biology and leukemic transformation., Strict regulation of stem cell metabolism is essential for tissue functions and tumor suppression. In this study, we investigated the role of fumarate hydratase (Fh1), a key component of the mitochondrial tricarboxylic acid (TCA) cycle and cytosolic fumarate metabolism, in normal and leukemic hematopoiesis. Hematopoiesis-specific Fh1 deletion (resulting in endogenous fumarate accumulation and a genetic TCA cycle block reflected by decreased maximal mitochondrial respiration) caused lethal fetal liver hematopoietic defects and hematopoietic stem cell (HSC) failure. Reexpression of extramitochondrial Fh1 (which normalized fumarate levels but not maximal mitochondrial respiration) rescued these phenotypes, indicating the causal role of cellular fumarate accumulation. However, HSCs lacking mitochondrial Fh1 (which had normal fumarate levels but defective maximal mitochondrial respiration) failed to self-renew and displayed lymphoid differentiation defects. In contrast, leukemia-initiating cells lacking mitochondrial Fh1 efficiently propagated Meis1/Hoxa9-driven leukemia. Thus, we identify novel roles for fumarate metabolism in HSC maintenance and hematopoietic differentiation and reveal a differential requirement for mitochondrial Fh1 in normal hematopoiesis and leukemia propagation.

Summary We explored the role of the Krebs cycle enzyme fumarate hydratase (FH) in glucose-stimulated insulin secretion (GSIS). Mice lacking Fh1 in pancreatic β cells (Fh1βKO 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 Fh1βKO mice led to dysregulated metabolism in β 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 Fh1βKO mice was ameliorated after culture under normoglycemic conditions. These studies highlight the role of FH and dysregulated mitochondrial metabolism in T2D., Graphical Abstract, Highlights • Fh1 loss in β cells causes progressive Hif1α-independent diabetes • Fh1 loss in β cells impairs ATP generation, electrical activity, and GSIS • Elevated fumarate is a feature of diabetic murine and human islets • “Normoglycemia” restores GSIS in Fh1βKO islets, Adam et al. have shown that progressive diabetes develops if fumarate hydratase is deleted in mouse pancreatic β cells. Such β cells exhibit elevated fumarate and protein succination and show progressively reduced ATP production and insulin secretion. The depleted insulin response to glucose recovers when diabetic islets are cultured in reduced glucose.

The contemporary oncologic pathology report conveys diagnostic, prognostic, predictive, and hereditary predisposition information. Each component may be premised on a morphologic feature or a biomarker. Clinical validity and reproducibility are paramount as is standardization of reporting and clinical response to ensure individualization of patient care. Regarding hereditary predisposition, morphology-based genetic referral systems in some instances have eclipsed genealogy-based systems, for example, cell type in ovarian cancer and BRCA screening. In other instances such as Lynch syndrome, morphology-based schemas supplement clinical schemas and there is an emerging standard of care for reflex biomarker testing. Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome predisposes patients to uterine and cutaneous leiomyomas (LMs) and renal cell carcinomas (RCCs). Several authors have emphasized the role pathologists may play in identifying this syndrome by recognizing the morphologic characteristics of syndromic uterine LMs and RCCs. Recently immunohistochemical overexpression of S-(2-succinyl) cysteine (2SC) has been demonstrated as a robust biomarker of mutation status in tumors from HLRCC patients. In this blinded control-cohort study we demonstrate that the proposed morphologic criteria used to identify uterine LMs in HLRCC syndrome are largely irreproducible among pathologists and lack sufficient robustness to serve as a trigger to triage cases for 2SC immunohistochemistry or patients for further family/personal history inquiry. Although refinement of morphologic criteria can be considered, in view of the availability of a clinically robust biomarker, consideration should be given to reflex testing of uterine LMs with an appropriate age cut off or in the setting of a suspicious family history.

Malignant pheochromocytoma (PCC) and paraganglioma (PGL) are mostly caused by germline mutations of SDHB, encoding a subunit of succinate dehydrogenase. Using whole-exome sequencing, we recently identified a mutation in the FH gene encoding fumarate hydratase, in a PCC with an 'SDH-like' molecular phenotype. Here, we investigated the role of FH in PCC/PGL predisposition, by screening for germline FH mutations in a large international cohort of patients. We screened 598 patients with PCC/PGL without mutations in known PCC/PGL susceptibility genes. We searched for FH germline mutations and large deletions, by direct sequencing and multiplex ligation-dependent probe amplification methods. Global alterations in DNA methylation and protein succination were assessed by immunohistochemical staining for 5-hydroxymethylcytosine (5-hmC) and S-(2-succinyl) cysteine (2SC), respectively. We identified five pathogenic germline FH mutations (four missense and one splice mutation) in five patients. Somatic inactivation of the second allele, resulting in a loss of fumarate hydratase activity, was demonstrated in tumors with FH mutations. Low tumor levels of 5-hmC, resembling those in SDHB-deficient tumors, and positive 2SC staining were detected in tumors with FH mutations. Clinically, metastatic phenotype (P = 0.007) and multiple tumors (P = 0.02) were significantly more frequent in patients with FH mutations than those without such mutations. This study reveals a new role for FH in susceptibility to malignant and/or multiple PCC/PGL. Remarkably, FH-deficient PCC/PGLs display the same pattern of epigenetic deregulation as SDHB-mutated malignant PCC/PGL. Therefore, we propose that mutation screening for FH should be included in PCC/PGL genetic testing, at least for tumors with malignant behavior.

Summary 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., Graphical Abstract Highlights ► Fumarate inhibits Aconitase2 activity via succination of critical cysteine residues ► Endogenous Aconitase2 is succinated and inhibited in FH-deficient cells ► Succination occurs in multiple proteins with roles in diverse cellular processes ► Succination can alter metabolism in FH-deficient cells, Loss-of-function mutations in the tumor suppressor gene fumarate hydratase (FH) predispose to an aggressive type of renal cancer. FH inactivation results in accumulation of the Krebs cycle metabolite fumarate, which can irreversibly modify cysteine residues through succination. Yang, Pollard, and colleagues showed that fumarate-mediated succination occurs on three critical cysteine residues in Aconitase2, leading to its inactivation in FH-deficient cells. Succination targets multiple proteins encompassing diverse cellular pathways, potentially contributing to the oncogenesis in FH-deficient tumors.

The nuclear-encoded Krebs cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDHB, -C and -D), act as tumour suppressors. Germline mutations in FH predispose individuals to leiomyomas and renal cell cancer (HLRCC), whereas mutations in SDH cause paragangliomas and phaeochromocytomas (HPGL). In this study, we have shown that FH-deficient cells and tumours accumulate fumarate and, to a lesser extent, succinate. SDH-deficient tumours principally accumulate succinate. In situ analyses showed that these tumours also have over-expression of hypoxia-inducible factor 1alpha (HIF1alpha), activation of HIF1alphatargets (such as vascular endothelial growth factor) and high microvessel density. We found no evidence of increased reactive oxygen species in our cells. Our data provide in vivo evidence to support the hypothesis that increased succinate and/or fumarate causes stabilization of HIF1alpha a plausible mechanism, inhibition of HIF prolyl hydroxylases, has previously been suggested by in vitro studies. The basic mechanism of tumorigenesis in HPGL and HLRCC is likely to be pseudo-hypoxic drive, just as it is in von Hippel-Lindau syndrome.

Murine radiation-induced acute myeloid leukaemia (AML) is characterized by loss of one copy of chromosome 2. Previously, we positioned the critical haematopoietic-specific transcription factor PU.1 within a minimally deleted region. We now report a high frequency (>65%) of missense mutation at codon 235 in the DNA-binding Ets domain of PU.1 in murine AML. Earlier studies, outside the context of malignancy, determined that conversion of arginine 235 (R235) to any other amino-acid residue leads to ablation of DNA-binding function and loss of expression of downstream targets. We show that mutation of R235 does not lead to protein loss, and occurs specifically in those AMLs showing loss of one copy of PU.1 (P=0.001, Fisher's exact test). PU.1 mutations were not found in the coding region, UTRs or promoter of human therapy-related AMLs. Potentially regulatory elements upstream of PU.1 were located but no mutations found. In conclusion, we have identified the cause of murine radiation-induced AML and have shown that loss of one copy of PU.1, as a consequence of flanking radiation-sensitive fragile domains on chromosome 2, and subsequent R235 conversion are highly specific to this mouse model. Such a mechanism does not operate, or is extremely rare, in human AML.

Background and aims Adenomatous polyposis coli ( APC ) is a tumour suppressor gene mutated in the germline of patients with familial adenomatous polyposis (FAP) and somatically in most colorectal cancers. APC mutations impair β-catenin degradation, resulting in increased Wnt signalling. The most frequent APC mutation is a codon 1309 truncation that is associated with severe FAP. A previous study compared two mouse models of intestinal tumorigenesis, Apc R850X (Min) and Apc 1322T (1322T), the latter a model of human codon 1309 changes. 1322T mice had more severe polyposis but, surprisingly, these tumours had lower levels of nuclear β-catenin than Min tumours. The consequences of these different β-catenin levels were investigated. Methods Enterocytes were isolated from 1322T and Min tumours by microdissection and gene expression profiling was performed. Differentially expressed Wnt targets and other stem cell markers were validated using quantitative PCR, in situ hybridisation and immunohistochemistry. Results As expected, lower nuclear β-catenin levels in 1322T lesions were associated with generally lower levels of Wnt target expression. However, expression of the Wnt target and stem cell marker Lgr5 was significantly higher in 1322T tumours than in Min tumours. Other stem cell markers (Musashi1, Bmi1 and the Wnt target Cd44) were also at higher levels in 1322T tumours. In addition, expression of the Bmp antagonist Gremlin1 was higher in 1322T tumours, together with lower Bmp2 and Bmp4 expression. Conclusions The severe phenotype caused by truncation of Apc at codon 1322 is associated with an increased number of stem cells. Thus, a submaximal level of Wnt signalling favours the stem cell phenotype and this may promote tumorigenesis. A level of Wnt signalling exists that is too high for optimal tumour growth.

Germline mutations in the fumarate hydratase (FH) tumor suppressor gene predispose to leiomyomatosis, renal cysts, and renal cell cancer (HLRCC). HLRCC tumors overexpress HIF1alpha and hypoxia pathway genes. We conditionally inactivated mouse Fh1 in the kidney. Fh1 mutants developed multiple clonal renal cysts that overexpressed Hif1alpha and Hif2alpha. Hif targets, such as Glut1 and Vegf, were upregulated. We found that Fh1-deficient murine embryonic stem cells and renal carcinomas from HLRCC showed similar overexpression of HIF and hypoxia pathway components to the mouse cysts. Our data have shown in vivo that pseudohypoxic drive, resulting from HIF1alpha (and HIF2alpha) overexpression, is a direct consequence of Fh1 inactivation. Our mouse may be useful for testing therapeutic interventions that target angiogenesis and HIF-prolyl hydroxylation.

Human 2-oxoglutarate oxygenases catalyse a range of biological oxidations including the demethylation of histone and nucleic acid substrates and the hydroxylation of proteins and small molecules. Some of these processes are centrally involved in regulation of cellular responses to hypoxia. The ALKBH proteins are a sub-family of 2OG oxygenases that are defined by homology to the Escherichia coli DNA-methylation repair enzyme AlkB. Here we report evidence that ALKBH5 is probably unique amongst the ALKBH genes in being a direct transcriptional target of hypoxia inducible factor-1 (HIF-1) and is induced by hypoxia in a range of cell types. We show that purified recombinant ALKBH5 is a bona fide 2OG oxygenase that catalyses the decarboxylation of 2OG but appears to have different prime substrate requirements from those so far defined for other ALKBH family members. Our findings define a new class of HIF-transcriptional target gene and suggest that ALKBH5 may have a role in the regulation of cellular responses to hypoxia.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a syndrome predisposing to cutaneous and uterine leiomyomatosis as well as renal cell cancer and uterine leiomyosarcoma. Heterozygous germline mutations in the fumarate hydratase (FH, fumarase) gene are known to cause HLRCC. On occasion, no FH mutation is detected by direct sequencing, despite the evident HLRCC phenotype in a family. In the present study, to investigate whole gene or exonic deletions and amplifications in FH mutation-negative patients, we used multiplex ligation-dependent probe amplification technology. The study material comprised 7 FH mutation-negative HLRCC patients and 12 patients affected with HLRCC-associated phenotypes, including papillary RCC, early-onset RCC, uterine leiomyomas, or uterine leiomyosarcoma. A novel FH mutation, a deletion of FH exon 1 that encodes the mitochondrial signal peptide, was detected in one of the HLRCC patients (1/7). The patient with the FH mutation displayed numerous painful cutaneous leiomyomas and papillary type renal cell cancer. Our finding, together with the two patients with whole FH gene deletion who had been detected previously, suggests that exonic or whole-gene FH deletions are not a frequent cause of HLRCC syndrome.

Autosomal dominant polycystic kidney disease (PKD) is an inherited disease that results from mutations in either polycystin (PKD1) or polycystin 2 (PKD2), both of which are large, complex, and multifunctional proteins whose loss results in the development of numerous fluid-filled cysts and fibrosis that compromise renal function. A number of spontaneous and engineered mouse models of PKD have provided some understanding of many aspects of cyst development, modifier genes, and mechanistic pathways, but fall short of reproducing the human disease accurately. Two recent papers in The Journal of Pathology set out new models using miRNA, or inducible and targeted recombination, that achieve partial or timed suppression of Pkd1. Instead of knocking out Pkd1 immediately, or completely, these more subtle approaches may help deliver more faithful models of this significant human renal disease.

The SDHA, SDHB, SDHC, SDHD genes encode the four subunits of succinate dehydrogenase (SDH; mitochondrial complex II), a mitochondrial enzyme involved in two essential energy-producing metabolic processes of the cell, the Krebs cycle and the electron transport chain. Germline loss-of-function mutations in any of the SDH genes or assembly factor (SDHAF2) cause hereditary paraganglioma/phaeochromocytoma syndrome (HPGL/PCC) through a mechanism which is largely unknown. Owing to the central function of SDH in cellular energy metabolism it is important to understand its role in tumor suppression. Here is reported an overview of genetics, clinical and molecular progress recently performed in understanding the basis of HPGL/PCC tumorigenesis. © 2011 Elsevier B.V. All rights reserved.

The heterogeneity of renal cell carcinoma (RCC) poses a challenge for designing clinically applicable diagnostic and screening investigations, predictive and prognostic biomarkers, and targeted molecular therapies. Hereditary RCC syndromes harbor specific driver gene mutations, and their discoveries have provided unequivocal insight into the pathogenomic landscape of RCCs. These observed genetic aberrations correspond to a diverse range of dysplastic metabolic processes, including mutations in genes encoding tricarboxylic acid (TCA) cycle enzymes, defects in hypoxic and antioxidant signaling, and abnormalities in nutrient-sensing phosphorylation cascades. Medical management of RCC focused on understanding and correcting these metabolic abnormalities may refine current RCC screening, diagnosis, and treatment. This review describes RCC subtypes associated with TCA and intermediary metabolic defects, outlining salient clinical features, genetic and molecular pathologies, medical management, and dynamic research areas that may affect future practice. © 2013 International Society of Nephrology.

Objective To examine differences between sporadic and familial uterine leiomyomata related to expression of apoptosis-related proteins and tumor ultrastructure. Design Expression of apoptosis-related proteins was measured by immunohistochemistry. Tumor ultrastructure was evaluated by transmission electron microscopy. Setting Human genetics laboratory. Patient(s) Patients confirmed for hereditary leiomyomatosis and renal cell carcinoma (HLRCC), and anonymous archival sporadic leiomyoma patients. Intervention(s) Samples for electron microscopy were collected from myomectomy and hysterectomy with informed consent. Other samples were archival. Main Outcome Measure(s) Intensity of immunohistochemistry staining and evaluation of electron micrographs. Result(s) Immunohistochemistry revealed increases in expression of antiapoptotic Bcl-2 and the proliferation factor proliferating cell nuclear antigen (PCNA) in both sporadic and HLRCC uterine leiomyomata. Furthermore, we observed an increase in antiapoptotic Bcl-x and a concurrent decrease in proapoptotic Bak solely in HLRCC leiomyomas. We also observed ultrastructural alterations in HLRCC and sporadic leiomyomas, particularly pertaining to extracellular matrix and intermediate filament aggregation. Conclusion(s) The observed alterations in expression of apoptosis-related proteins indicate a shift in both HLRCC and sporadic leiomyomas to increased resistance to apoptosis compared with myometrium, which appears to be stronger in HLRCC leiomyomas. The changes observed in HLRCC leiomyomas appear to be related to activation of the hypoxia pathways. The results suggest not only a partial overlap in the pathogenic mechanism of the two tumor types, but also intriguing differences.

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.

This study investigated the function of each of the hypoxia inducible factor (HIF) prolyl-4-hydroxylase enzymes (PHD1–3) in the first 24 h following transient focal cerebral ischaemia by using mice with each isoform genetically suppressed. Male, 8- to 12-week old PHD1−/−, PHD2+/− and PHD3−/− mice and their wild-type (WT) littermate were subjected to 45 min of middle cerebral artery occlusion (MCAO). During the experiments, regional cerebral blood flow (rCBF) was recorded by laser Doppler flowmetry. Behaviour was assessed at both 2 h and 24 h after reperfusion with a common neuroscore. Infarct volumes, blood–brain barrier (BBB) disruption, cerebral vascular density, apoptosis, reactive oxygen species (ROS), HIF1α, and glycogen levels were then determined using histological and immunohistochemical techniques. When compared to their WT littermates, PHD2+/− mice had significantly increased cerebral microvascular density and more effective restoration of CBF upon reperfusion. PHD2+/− mice showed significantly better functional outcomes and higher activity rates at both 2 h and 24 h after MCAO, associated with significant fewer apoptotic cells in the penumbra and less BBB disruption; PHD3−/− mice had impaired rCBF upon early reperfusion but comparable functional outcomes; PHD1−/− mice did not show any significant changes following the MCAO. Production of ROS, HIF1α staining and glycogen content in the brain were not different in any comparison. Life-long genetic inhibition of PHD enzymes produces different effects on outcome in the first 24 h after transient cerebral ischaemia. These need to be considered in optimizing therapeutic effects of PHD inhibitors, particularly when isoform specific inhibitors become available.

Summary 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., Graphical Abstract, Highlights • Idh1R132H knockin in the mouse brain SVZ recapitulates features of gliomagenesis • Self-renewal and proliferation of neural stem cells and progenitors increase • SVZ cells proliferate ectopically, infiltrate the brain parenchyma, and form nodules • Increases occur in 2HG levels, Wnt and telomere pathway activity, and DNA methylation, Bardella et al. report that expression of IDH1R132H in the subventricular zone of the adult mouse brain causes features of gliomagenesis, including increased numbers of neural stem cells, cellular infiltration into surrounding brain regions, and a gene expression profile overlapping that of human gliomas.

Adenomatous polyposis coli (APC) mutations are found in most colorectal tumours. These mutations are almost always protein-truncating, deleting both central domains that regulate Wnt signalling and C-terminal domains that interact with the cytoskeleton. The importance of Wnt dysregulation for colorectal tumourigenesis is well characterized. It is, however, unclear whether loss of C-terminal functions contributes to tumourigenesis, although this protein region has been implicated in cellular processes—including polarity, migration, mitosis, and chromosomal instability (CIN)—that have been postulated as critical for the development and progression of intestinal tumours. Since almost all APC mutations in human patients disrupt both central and C-terminal regions, we created a mouse model to test the role of the C-terminus of APC in intestinal tumourigenesis. This mouse (Apc) carries an internal deletion within Apc that dysregulates Wnt by removing the beta-catenin-binding and SAMP repeats, but leaves the C-terminus intact. We compared Apc mice with Apc animals. The latter allele represented the most commonly found human APC mutation and was identical to Apc except for absence of the entire C-terminus. Apc mice developed numerous intestinal adenomas indistinguishable in number, location, and dysplasia from those seen in Apc mice. No carcinomas were found in Apc or Apc animals. While similar disruption of the Wnt signalling pathway was observed in tumours from both mice, no evidence of differential C-terminus functions (such as cell migration, CIN, or localization of APC and EB1) was seen. We conclude that the C-terminus of APC does not influence intestinal adenoma development or progression. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Germline mutations in the FH gene encoding the Krebs cycle enzyme fumarate hydratase predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. FH-deficient cells and tissues accumulate high levels of fumarate, which may act as an oncometabolite and contribute to tumourigenesis. A recently proposed role for fumarate in the covalent modification of cysteine residues to S-(2-succinyl) cysteine (2SC) (termed protein succination) prompted us to assess 2SC levels in our existing models of HLRCC. Herein, using a previously characterized antibody against 2SC, we show that genetic ablation of FH causes high levels of protein succination. We next hypothesized that immunohistochemistry for 2SC would serve as a metabolic biomarker for the in situ detection of FH-deficient tissues. Robust detection of 2SC was observed in Fh1 (murine FH)-deficient renal cysts and in a retrospective series of HLRCC tumours (n = 16) with established FH mutations. Importantly, 2SC was undetectable in normal tissues (n = 200) and tumour types not associated with HLRCC (n = 1342). In a prospective evaluation of cases referred for genetic testing for HLRCC, the presence of 2SC-modified proteins (2SCP) correctly predicted genetic alterations in FH in every case. In two series of unselected type II papillary renal cancer (PRCC), prospectively analysed by 2SCP staining followed by genetic analysis, the biomarker accurately identified previously unsuspected FH mutations (2/33 and 1/36). The investigation of whether metabolites in other tumour types produce protein modification signature(s) that can be assayed using similar strategies will be of interest in future studies of cancer.

In a model of autosomally recessive Alport syndrome, mice that lack the alpha3 chain of collagen IV (Col4alpha3(-/-)) develop progressive glomerular damage leading to renal failure. The proposed mechanism is that podocytes fail to synthesize normal glomerular basement membrane, so the collagen IV network is unstable and easily degraded. We used this model to study whether bone marrow (BM) transplantation can rectify this podocyte defect by correcting the deficiency in Col4alpha3. Female C57BL/6 Col4alpha3(-/-) (-/-) mice were transplanted with whole BM from male wild-type (+/+) mice. Control female -/- mice received BM from male -/- littermates. Serum urea and creatinine levels were significantly lower in recipients of +/+ BM compared with those of -/- BM 20 weeks post-transplant. Glomerular scarring and interstitial fibrosis were also significantly decreased. Donor-derived cells were detected by in situ hybridization (ISH) for the Y chromosome, and fluorescence and confocal microscopy indicated that some showed an apparent podocyte phenotype in mice transplanted with +/+ BM. Glomeruli of these mice showed small foci of staining for alpha3(IV) protein by immunofluorescence. alpha3(IV) mRNA was detectable by reverse transcription-polymerase chain reaction and ISH in some mice transplanted with +/+ BM but not -/- BM. However, a single injection of mesenchymal stem cells from +/+ mice to irradiated -/- recipients did not improve renal disease. Our data show that improved renal function in Col4alpha3(-/-) mice results from BM transplantation from wild-type donors, and the mechanism by which this occurs may in part involve generation of podocytes without the gene defect.

The Mendelian tumour syndromes hereditary leiomyomatosis and renal cell cancer (HLRCC) and hereditary paragangliomatosis with phaeochromocytomas (HPGL) result from mutations in nuclear genes (FH and SDHB/C/D, respectively) that encode Krebs cycle enzymes. HPGL tumours are highly vascular and there is evidence that inactivation of SDH leads to activation of the hypoxia/angiogenesis pathway. In contrast, uterine leiomyomas are not generally regarded as particularly vascular lesions. In order to test the possibility that activation of the hypoxia/angiogenesis pathway contributes to tumourigenesis in HLRCC, increased vascularity and hypoxia pathway activation were searched for in HLRCC tumours. Microvessel density was markedly higher in uterine leiomyomas from HLRCC than in the surrounding myometrium; it was notable that sporadic uterine leiomyomas were actually less vascular than normal myometrium. In HLRCC tumours, there was increased expression of transcripts from the hypoxia-responsive genes vascular endothelial growth factor (VEGF) and BNIP3; sporadic uterine leiomyomas did not show these changes. All uterine leiomyomas showed decreased expression of thrombospondin 1. Although sporadic and HLRCC uterine leiomyomas appear to have identical morphology, their pathways of tumourigenesis may be fundamentally different. As is the case in HPGL, it is probable that failure of the Krebs cycle in HLRCC tumours causes inappropriate signalling that the cell is in a hypoxic state, leading to angiogenesis and perhaps directly to clonal expansion and tumour growth through some uncharacterized, cell-autonomous effect.

Germline mutations of the fumarate hydratase (FH, fumarase) gene are found in the recessive FH deficiency syndrome and in dominantly inherited susceptibility to multiple cutaneous and uterine leiomyomatosis (MCUL). We have previously reported a number of germline FH mutations from MCUL patients. In this study, we report additional FH mutations in MCUL and FH deficiency patients. Mutations can readily be found in about 75% of MCUL cases and most cases of FH deficiency. Some of the more common FH mutations are probably derived from founding individuals. Protein-truncating FH mutations are functionally null alleles. Disease-associated missense FH changes map to highly conserved residues, mostly in or around the enzyme's active site or activation site; we predict that these mutations severely compromise enzyme function. The mutation spectra in FH deficiency and MCUL are similar, although in the latter mutations tend to occur earlier in the gene and, perhaps, are more likely to result in a truncated or absent protein. We have found that not all mutation-carrier parents of FH deficiency children have a strong predisposition to leiomyomata. We have confirmed that renal carcinoma is sometimes part of MCUL, as part of the variant hereditary leiomyomatosis and renal cancer (HLRCC) syndrome, and have shown that these cancers may have either type II papillary or collecting duct morphology. We have found no association between the type or site of FH mutation and any aspect of the MCUL phenotype. Biochemical assay for reduced FH functional activity in the germline of MCUL patients can indicate carriers of FH mutations with high sensitivity and specificity, and can detect reduced FH activity in some patients without detectable FH mutations. We conclude that MCUL is probably a genetically homogeneous tumour predisposition syndrome, primarily resulting from absent or severely reduced fumarase activity, with currently unknown functional consequences for the smooth muscle or kidney cell.

Cancer-associated mutations have been identified in the metabolic genes succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH), advancing and challenging our understanding of cellular function and disease mechanisms and providing direct links between dysregulated metabolism and cancer. Some striking parallels exist in the cellular consequences of the genetic mutations within this triad of cancer syndromes, including accumulation of oncometabolites and competitive inhibition of 2-oxoglutarate-dependent dioxygenases, particularly, hypoxia-inducible factor (HIF) prolyl hydroxylases, JmjC domain-containing histone demethylases (part of the JMJD family) and the ten-eleven translocation (TET) family of 5methyl cytosine (5mC) DNA hydroxylases. These lead to activation of HIF-dependent oncogenic pathways and inhibition of histone and DNA demethylation. Mutations in FH, resulting in loss of enzyme activity, predispose affected individuals to a rare cancer, hereditary leiomyomatosis and renal cell cancer (HLRCC), characterised by benign smooth muscle cutaneous and uterine tumours (leiomyomata) and an aggressive form of collecting duct and type 2 papillary renal cancer. Interestingly, loss of FH activity results in the accumulation of high levels of fumarate that can lead to the non-enzymatic modification of cysteine residues in multiple proteins (succination) and in some cases to their disrupted function. Here we consider that the study of rare diseases such as HLRCC, combining analyses of human tumours and cell lines with in vitro and in vivo murine models has provided novel insights into cancer biology associated with dysregulated metabolism and represents a useful paradigm for cancer research.

Hotspot mutations in the promoter of the telomerase reverse transcriptase (TERT) gene have been recently reported in human cancers and proposed as a novel mechanism of telomerase activation. To exploreTERTpromoter mutations in tumors originating from the adrenal gland and extra-adrenal paraganglia, a set of 253 tumors (38 adrenocortical carcinomas (ACCs), 127 pheochromocytomas (PCCs), 18 extra-adrenal paragangliomas (ea PGLs), 37 head and neck PGLs (HN PGLs), and 33 peripheral neuroblastic tumors) was selected along with 16 human neuroblastoma (NBL) and two ACC cell lines to assessTERTpromoter mutations by the Sanger sequencing method. All mutations detected were confirmed by a SNaPshot assay. Additionally, 36 gastrointestinal stromal tumors (GISTs) were added to explore an association betweenTERTpromoter mutations and SDH deficiency.TERTpromoter mutations were found in seven out of 289 tumors and in three out of 18 human cell lines; fourC228Tmutations in 38 ACCs (10.5%), twoC228Tmutations in 18 ea PGLs (11.1%), oneC250Tmutation in 36 GISTs (2.8%), and threeC228Tmutations in 16 human NBL cell lines (18.75%). No mutation was detected in PCCs, HN PGLs, neuroblastic tumors as well as ACC cell lines.TERTpromoter mutations preferentially occurred in a SDH-deficient setting (P=0.01) being present in three out of 47 (6.4%) SDH-deficient tumors vs zero out of 171 (0%) SDH-intact tumors. We conclude thatTERTpromoter mutations occur in ACCs and ea PGLs. In addition, preliminary evidence indicates a potential association with the acquisition ofTERTpromoter mutations in SDH-deficient tumors.

The class V myosins are actin-based motors that move a variety of cellular cargoes [1]. In budding yeast, their activity includes the relocation of a portion of the vacuole from the mother cell to the bud [2, 3]. Fission yeast cells contain numerous (approximately 80) small vacuoles. When S. pombe cells are placed in water, vacuoles fuse in response to osmotic stress [4]. Fission yeast possess two type V myosin genes, myo51(+) and myo52(+) [5]. In a myo51Delta strain, vacuoles were distributed throughout the cell, and mean vacuole diameter was identical to that seen in wild-type cells. When myo51Delta and wild-type cells were placed in water, vacuoles enlarged by fusion. In myo52Delta cells, by contrast, vacuoles were smaller and mostly clustered around the nucleus, and fusion in water was largely inhibited. When cells containing GFP-Myo52 were placed in water, Myo52 was seen to redistribute from the cell poles to the surface of the fusing vacuoles. Vacuole fusion in fission yeast was inhibited by the microtubule drug thiabendazole (TBZ) but not by the actin inhibitor latrunculin B. This is the first demonstration of the involvement of a type V myosin, possibly via an interaction with microtubules, in homotypic membrane fusion.

Mutations in a gene that encodes a metabolic enzyme have been linked to certain brain tumors, but is the gene a tumor suppressor or an oncogene?

The discovery of cancer-associated mutations in genes encoding key metabolic enzymes has provided a direct link between altered metabolism and cancer. Advances in mass spectrometry and nuclear magnetic resonance technologies have facilitated high-resolution metabolite profiling of cells and tumors and identified the accumulation of metabolites associated with specific gene defects. Here we review the potential roles of such "oncometabolites" in tumor evolution and as clinical biomarkers for the detection of cancers characterized by metabolic dysregulation.

Epigenetic reprogramming is a feature of many human cancers. In this issue of Cancer Cell, Letouze and colleagues describe DNA hypermethylation in paragangliomas harboring mutations in succinate dehydrogenase genes. These tumors accumulate succinate, which inhibits 2-oxoglutarate-dependent histone and DNA demethylase enzymes, resulting in epigenetic silencing that affects neuroendocrine differentiation.

Summary 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., Graphical Abstract, Highlights • FH-deficient cells accumulate fumarate and urea cycle metabolites • Cytosolic FH is critical for renal cyst development in FH1-deficient mice • Restoration of cytosolic FH activity restores urea cycle defects • Arginine depletion reduces viability of FH1-deficient cells, In this study, Pollard and colleagues demonstrate the importance of cytosolic fumarate hydratase (FH) in renal cyst development in mice. Metabolite analysis of FH-deficient cysts and tumors identified alterations in urea cycle metabolism. re-expression of extramitochondrial FH in mice with renal-specific FH1 deletion was sufficient to ameliorate cyst development and metabolic alterations in the urea cycle. Significantly, FH-deficient cells exhibit increased sensitivity to arginine depletion. Exploitation of this vulnerability may be a potential route for therapy for FH-mutant tumors.

Summary The citric acid cycle (CAC) metabolite fumarate has been proposed to be cardioprotective; however, its mechanisms of action remain to be determined. To augment cardiac fumarate levels and to assess fumarate's cardioprotective properties, we generated fumarate hydratase (Fh1) cardiac knockout (KO) mice. These fumarate-replete hearts were robustly protected from ischemia-reperfusion injury (I/R). To compensate for the loss of Fh1 activity, KO hearts maintain ATP levels in part by channeling amino acids into the CAC. In addition, by stabilizing the transcriptional regulator Nrf2, Fh1 KO hearts upregulate protective antioxidant response element genes. Supporting the importance of the latter mechanism, clinically relevant doses of dimethylfumarate upregulated Nrf2 and its target genes, hence protecting control hearts, but failed to similarly protect Nrf2-KO hearts in an in vivo model of myocardial infarction. We propose that clinically established fumarate derivatives activate the Nrf2 pathway and are readily testable cytoprotective agents., Highlights ► Cardiac fumarase deletion (cFH1-KO) results in mice with elevated cardiac fumarate ► cFH1-KO is compensated for by amino acid influx into the citric acid cycle ► Nrf2 and its target genes are activated in the hearts of cFH1-KO mice ► Fumarate-related Nrf2 activation is cytoprotective and may be of therapeutic use

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 (IDH1), 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 tumour suppressor, whose mutation is associated with the development of leiomyomata, renal cysts, and tumours. 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 signalling. Here we discuss the evidence for proposing fumarate as an oncometabolite.

The aberrant expression and functional activity of proteins involved in ATP production pathways may cause a crisis in energy generation for cells and compromise their survival under stressful conditions such as excitation, starvation, pharmacological treatment or disease states. Under resting conditions such defects are often compensated for, and therefore masked by, alternative pathways which have significant spare capacity. Here we present a multiplexed 'cell energy budget' platform which facilitates metabolic assessment and cross-comparison of different cells and the identification of genes directly or indirectly involved in ATP production. Long-decay emitting O(2) and pH sensitive probes and time-resolved fluorometry are used to measure changes in cellular O(2) consumption, glycolytic and total extracellular acidification (ECA), along with the measurement of total ATP and protein content in multiple samples. To assess the extent of spare capacity in the main energy pathways, the cells are also analysed following double-treatment with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and oligomycin. The four-parametric platform operating in a high throughput format has been validated with two panels of transformed cells: mouse embryonic fibroblasts (MEFs) lacking the Krebs cycle enzyme fumarate hydratase (Fh1) and HeLa cells with reduced expression of pyrimidine nucleotide carrier 1. In both cases, a marked reduction in both respiration and spare respiratory capacity was observed, accompanied by a compensatory activation of glycolysis and consequent maintenance of total ATP levels. At the same time, in Fh1-deficient MEFs the contribution of non-glycolytic pathways to the ECA did not change.

SummaryThe Krebs cycle enzyme fumarate hydratase (FH) is a human tumor suppressor whose inactivation is associated with the development of leiomyomata, renal cysts, and tumors. It has been proposed that activation of hypoxia inducible factor (HIF) by fumarate-mediated inhibition of HIF prolyl hydroxylases drives oncogenesis. Using a mouse model, we provide genetic evidence that Fh1-associated cyst formation is Hif independent, as is striking upregulation of antioxidant signaling pathways revealed by gene expression profiling. Mechanistic analysis revealed that fumarate modifies cysteine residues within the Kelch-like ECH-associated protein 1 (KEAP1), abrogating its ability to repress the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant response pathway, suggesting a role for Nrf2 dysregulation in FH-associated cysts and tumors.

Germline mutations in the FH gene encoding the Krebs cycle enzyme fumarate hydratase predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. FH-deficient cells and tissues accumulate high levels of fumarate, which may act as an oncometabolite and contribute to tumourigenesis. A recently proposed role for fumarate in the covalent modification of cysteine residues to S-(2-succinyl) cysteine (2SC) (termed protein succination) prompted us to assess 2SC levels in our existing models of HLRCC. Herein, using a previously characterized antibody against 2SC, we show that genetic ablation of FH causes high levels of protein succination. We next hypothesized that immunohistochemistry for 2SC would serve as a metabolic biomarker for the in situ detection of FH-deficient tissues. Robust detection of 2SC was observed in Fh1 (murine FH)-deficient renal cysts and in a retrospective series of HLRCC tumours (n = 16) with established FH mutations. Importantly, 2SC was undetectable in normal tissues (n = 200) and tumour types not associated with HLRCC (n = 1342). In a prospective evaluation of cases referred for genetic testing for HLRCC, the presence of 2SC-modified proteins (2SCP) correctly predicted genetic alterations in FH in every case. In two series of unselected type II papillary renal cancer (PRCC), prospectively analysed by 2SCP staining followed by genetic analysis, the biomarker accurately identified previously unsuspected FH mutations (2/33 and 1/36). The investigation of whether metabolites in other tumour types produce protein modification signature(s) that can be assayed using similar strategies will be of interest in future studies of cancer.

The observation that altered metabolism is the fundamental cause of cancer was made by Otto Warburg nearly a century ago. However, the subsequent identification of oncogenes and tumor suppressor genes has displaced Warburg's theory pointing towards genetic aberrations as the underlining cause of cancer. Nevertheless, in the last decade, cancer-associated mutations have been identified in genes coding for tricarboxylic acid cycle (TCA cycle, also known as Krebs cycle) and closely related enzymes that have essential roles in cellular metabolism. These observations have revived interest in Warburg's hypothesis and prompted a flurry of functional studies in the hope of gaining mechanistic insight into the links between mitochondrial dysfunction, metabolic alterations, and cancer. In this review, we discuss the potential pro-oncogenic signaling role of some TCA cycle metabolites and their derivatives (oncometabolites). In particular, we focus on their effects on dioxygenases, a family of oxygen and α-ketoglutarate-dependent enzymes that control, among other things, the levels and activity of the hypoxia-inducible transcription factors and the activity of DNA and histone demethylases.

Metabolic reprogramming is a characteristic of cancer cells. Three studies published in this month's Molecular Cell provide novel insights into the role of mitochondrial pyruvate in tumor metabolism and describe how targeting pyruvate transport and metabolism may afford therapeutic benefit.

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.

Fumarate hydratase (FH) is an enzyme of the tricarboxylic acid cycle (TCA cycle) that catalyses the hydration of fumarate into malate. Germline mutations of FH are responsible for hereditary leiomyomatosis and renal-cell cancer (HLRCC)1. It has previously been demonstrated that the absence of FH leads to the accumulation of fumarate, which activates hypoxia-inducible factors (HIFs) at normal oxygen tensions2-4. However, so far no mechanism that explains the ability of cells to survive without a functional TCA cycle has been provided. Here we use newly characterized genetically modified kidney mouse cells in which Fh1 has been deleted, and apply a newly developed computer model of the metabolism of these cells to predict and experimentally validate a linear metabolic pathway beginning with glutamine uptake and ending with bilirubin excretion from Fh1-deficient cells. This pathway, which involves the biosynthesis and degradation of haem, enables Fh1-deficient cells to use the accumulated TCA cycle metabolites and permits partial mitochondrial NADH production. We predicted and confirmed that targeting this pathway would render Fh1-deficient cells non-viable, while sparing wild-type Fh1-containing cells. This work goes beyond identifying a metabolic pathway that is induced in Fh1-deficient cells to demonstrate that inhibition of haem oxygenation is synthetically lethal when combined with Fh1 deficiency, providing a new potential target for treating HLRCC patients. © 2011 Macmillan Publishers Limited. All rights reserved.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is caused by mutations in the Krebs cycle enzyme fumarate hydratase (FH). It has been proposed that “pseudohypoxic” stabilization of hypoxia-inducible factor-α (HIF-α) by fumarate accumulation contributes to tumorigenesis in HLRCC. We hypothesized that an additional direct consequence of FH deficiency is the establishment of a biosynthetic milieu. To investigate this hypothesis, we isolated primary mouse embryonic fibroblast (MEF) lines from Fh1-deficient mice. As predicted, these MEFs upregulated Hif-1α and HIF target genes directly as a result of FH deficiency. In addition, detailed metabolic assessment of these MEFs confirmed their dependence on glycolysis, and an elevated rate of lactate efflux, associated with the upregulation of glycolytic enzymes known to be associated with tumorigenesis. Correspondingly, Fh1-deficient benign murine renal cysts and an advanced human HLRCC-related renal cell carcinoma manifested a prominent and progressive increase in the expression of HIF-α target genes and in genes known to be relevant to tumorigenesis and metastasis. In accord with our hypothesis, in a variety of different FH-deficient tissues, including a novel murine model of Fh1-deficient smooth muscle, we show a striking and progressive upregulation of a tumorigenic metabolic profile, as manifested by increased PKM2 and LDHA protein. Based on the models assessed herein, we infer that that FH deficiency compels cells to adopt an early, reversible, and progressive protumorigenic metabolic milieu that is reminiscent of that driving the Warburg effect. Targets identified in these novel and diverse FH-deficient models represent excellent potential candidates for further mechanistic investigation and therapeutic metabolic manipulation in tumors. Cancer Res; 70(22); 9153–65. ©2010 AACR.

Contains fulltext : 80017.pdf (Publisher’s version ) (Closed access) BACKGROUND: Phaeochromocytomas and paragangliomas are neuro-endocrine tumours that occur sporadically and in several hereditary tumour syndromes, including the phaeochromocytoma-paraganglioma syndrome. This syndrome is caused by germline mutations in succinate dehydrogenase B (SDHB), C (SDHC), or D (SDHD) genes. Clinically, the phaeochromocytoma-paraganglioma syndrome is often unrecognised, although 10-30% of apparently sporadic phaeochromocytomas and paragangliomas harbour germline SDH-gene mutations. Despite these figures, the screening of phaeochromocytomas and paragangliomas for mutations in the SDH genes to detect phaeochromocytoma-paraganglioma syndrome is rarely done because of time and financial constraints. We investigated whether SDHB immunohistochemistry could effectively discriminate between SDH-related and non-SDH-related phaeochromocytomas and paragangliomas in large retrospective and prospective tumour series. METHODS: Immunohistochemistry for SDHB was done on 220 tumours. Two retrospective series of 175 phaeochromocytomas and paragangliomas with known germline mutation status for phaeochromocytoma-susceptibility or paraganglioma-susceptibility genes were investigated. Additionally, a prospective series of 45 phaeochromocytomas and paragangliomas was investigated for SDHB immunostaining followed by SDHB, SDHC, and SDHD mutation testing. FINDINGS: SDHB protein expression was absent in all 102 phaeochromocytomas and paragangliomas with an SDHB, SDHC, or SDHD mutation, but was present in all 65 paraganglionic tumours related to multiple endocrine neoplasia type 2, von Hippel-Lindau disease, and neurofibromatosis type 1. 47 (89%) of the 53 phaeochromocytomas and paragangliomas with no syndromic germline mutation showed SDHB expression. The sensitivity and specificity of the SDHB immunohistochemistry to detect the presence of an SDH mutation in the prospective series were 100% (95% CI 87-100) and 84% (60-97), respectively. INTERPRETATION: Phaeochromocytoma-paraganglioma syndrome can be diagnosed reliably by an immunohistochemical procedure. SDHB, SDHC, and SDHD germline mutation testing is indicated only in patients with SDHB-negative tumours. SDHB immunohistochemistry on phaeochromocytomas and paragangliomas could improve the diagnosis of phaeochromocytoma-paraganglioma syndrome. FUNDING: The Netherlands Organisation for Scientific Research, Dutch Cancer Society, Vanderes Foundation, Association pour la Recherche contre le Cancer, Institut National de la Sante et de la Recherche Medicale, and a PHRC grant COMETE 3 for the COMETE network.

This article is open access and is available at the linked URL, Hypoxia-inducible factor (HIF) controls an extensive range of adaptive responses to hypoxia. To better understand this transcriptional cascade we performed genome-wide chromatin immunoprecipitation using antibodies to two major HIF-alpha subunits, and correlated the results with genome-wide transcript profiling. Within a tiled promoter array we identified 546 and 143 sequences that bound, respectively, to HIF-1alpha or HIF-2alpha at high stringency. Analysis of these sequences confirmed an identical core binding motif for HIF-1alpha and HIF-2alpha (RCGTG) but demonstrated that binding to this motif was highly selective, with binding enriched at distinct regions both upstream and downstream of the transcriptional start. Comparison of HIF-promoter binding data with bidirectional HIF-dependent changes in transcript expression indicated that whereas a substantial proportion of positive responses (>20% across all significantly regulated genes) are direct, HIF-dependent gene suppression is almost entirely indirect. Comparison of HIF-1alpha- versus HIF-2alpha-binding sites revealed that whereas some loci bound HIF-1alpha in isolation, many bound both isoforms with similar affinity. Despite high-affinity binding to multiple promoters, HIF-2alpha contributed to few, if any, of the transcriptional responses to acute hypoxia at these loci. Given emerging evidence for biologically distinct functions of HIF-1alpha versus HIF-2alpha understanding the mechanisms restricting HIF-2alpha activity will be of interest.