Patrick J. Pollard

Heterozygous Deficiency of PHD2 Restores Tumor Oxygenation and Inhibits Metastasis via Endothelial Normalization

A key function of blood vessels, to supply oxygen, is impaired in tumors because of abnormalities in their endothelial lining. PHD proteins serve as oxygen sensors and may regulate oxygen delivery. We therefore studied the role of endothelial PHD2 in vessel shaping by implanting tumors in PHD2(+/-) mice. Haplodeficiency of PHD2 did not affect tumor vessel density or lumen size, but normalized the endothelial lining and vessel maturation. This resulted in improved tumor perfusion and oxygenation and inhibited tumor cell invasion, intravasation, and metastasis. Haplodeficiency of PHD2 redirected the specification of endothelial tip cells to a more quiescent cell type, lacking filopodia and arrayed in a phalanx formation. This transition relied on HIF-driven upregulation of (soluble) VEGFR-1 and VE-cadherin. Thus, decreased activity of an oxygen sensor in hypoxic conditions prompts endothelial cells to readjust their shape and phenotype to restore oxygen supply. Inhibition of PHD2 may offer alternative therapeutic opportunities for anticancer therapy.

An immunohistochemical procedure to detect patients with paraganglioma and phaeochromocytoma with germline SDHB, SDHC, or SDHD gene mutations: a retrospective and prospective analysis.

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 Santé et de la Recherche Médicale, and a PHRC grant COMETE 3 for the COMETE network.

Genome-wide Association of Hypoxia-inducible Factor (HIF)-1α and HIF-2α DNA Binding with Expression Profiling of Hypoxia-inducible Transcripts

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-α 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-1α or HIF-2α at high stringency. Analysis of these sequences confirmed an identical core binding motif for HIF-1α and HIF-2α (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-1α- versus HIF-2α-binding sites revealed that whereas some loci bound HIF-1α in isolation, many bound both isoforms with similar affinity. Despite high-affinity binding to multiple promoters, HIF-2α contributed to few, if any, of the transcriptional responses to acute hypoxia at these loci. Given emerging evidence for biologically distinct functions of HIF-1α versus HIF-2α understanding the mechanisms restricting HIF-2α activity will be of interest.

Renal Cyst Formation in Fh1-Deficient Mice Is Independent of the Hif/Phd Pathway: Roles for Fumarate in KEAP1 Succination and Nrf2 Signaling

Summary The 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.

Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase

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). 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 tensions. 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.

Regulation of Jumonji-domain-containing histone demethylases by hypoxia-inducible factor (HIF)-1alpha.

The transcription factor HIF (hypoxia-inducible factor) mediates a highly pleiotrophic response to hypoxia. Many recent studies have focused on defining the extent of this transcriptional response. In the present study we have analysed regulation by hypoxia among transcripts encoding human Fe(II)- and 2-oxoglutarate-dependent oxygenases. Our results show that many of these genes are regulated by hypoxia and define two groups of histone demethylases as new classes of hypoxia-regulated genes. Patterns of induction were consistent across a range of cell lines with JMJD1A (where JMJD is Jumonji-domain containing) and JMJD2B demonstrating robust, and JMJD2C more modest, up-regulation by hypoxia. Functional genetic and chromatin immunoprecipitation studies demonstrated the importance of HIF-1alpha in mediating these responses. Given the importance of histone methylation status in defining patterns of gene expression under different physiological and pathophysiological conditions, these findings predict a role for the HIF system in epigenetic regulation.

Bone Marrow‐Derived Cells Contribute to Podocyte Regeneration and Amelioration of Renal Disease in a Mouse Model of Alport Syndrome

In a model of autosomally recessive Alport syndrome, mice that lack the α3 chain of collagen IV (Col4α3−/−) 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 Col4α3. Female C57BL/6 Col4α3−/− (−/−) 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 α3(IV) protein by immunofluorescence. α3(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 Col4α3−/− 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.

SDH mutations in cancer.

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.

Fumarate Is Cardioprotective via Activation of the Nrf2 Antioxidant Pathway

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 fumarates 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.

Aberrant succination of proteins in fumarate hydratase-deficient mice and HLRCC patients is a robust biomarker of mutation status

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. Copyright