Antioxidants stimulate BACH1-dependent tumor angiogenesis.

Lung cancer progression relies on angiogenesis, which is a response to hypoxia typically coordinated by hypoxia-inducible transcription factors (HIFs), but growing evidence indicates that transcriptional programs beyond HIFs control tumor angiogenesis. Here, we show that the redox-sensitive transcription factor BTB and CNC homology 1 (BACH1) controls the transcription of a broad range of angiogenesis genes. BACH1 is stabilized by lowering ROS levels; consequently, angiogenesis gene expression in lung cancer cells, tumor organoids, and xenograft tumors increased substantially following administration of vitamins C and E and N-acetylcysteine in a BACH1-dependent fashion under normoxia. Moreover, angiogenesis gene expression increased in endogenous BACH1-overexpressing cells and decreased in BACH1-knockout cells in the absence of antioxidants. BACH1 levels also increased upon hypoxia and following administration of prolyl hydroxylase inhibitors in both HIF1A-knockout and WT cells. BACH1 was found to be a transcriptional target of HIF1α, but BACH1's ability to stimulate angiogenesis gene expression was HIF1α independent. Antioxidants increased tumor vascularity in vivo in a BACH1-dependent fashion, and overexpressing BACH1 rendered tumors sensitive to antiangiogenesis therapy. BACH1 expression in tumor sections from patients with lung cancer correlated with angiogenesis gene and protein expression. We conclude that BACH1 is an oxygen- and redox-sensitive angiogenesis transcription factor.

CD25bright NK cells display superior function and metabolic activity under regulatory T cell-mediated suppression.

Infusion of natural killer (NK) cells is an attractive therapeutic modality in patients with cancer. However, the activity of NK cells is regulated by several mechanisms operating within solid tumors. Regulatory T (Treg) cells suppress NK cell activity through various mechanisms including deprivation of IL-2 via the IL-2 receptor alpha (CD25). Here, we investigate CD25 expression on NK cells to confer persistence in Treg cells containing solid tumor models of renal cell carcinoma (RCC). Compared with IL-2, stimulation with IL-15 increases the expression of CD25 resulting in enhanced response to IL-2 as evidenced by increased phosphorylation of STAT5. Compared with CD25dim NK cells, CD25bright NK cells isolated from IL-15 primed NK cells display increased proliferative and metabolic activity as well as increased ability to persist in Treg cells containing RCC tumor spheroids. These results support strategies to enrich for or selectively expand CD25bright NK cells for adoptive cellular therapy of NK cells.

Targeting myeloid suppressive cells revives cytotoxic anti-tumor responses in pancreatic cancer.

Immunotherapy for cancer that aims to promote T cell anti-tumor activity has changed current clinical practice, where some previously lethal cancers have now become treatable. However, clinical trials with low response rates have been disappointing for pancreatic ductal adenocarcinoma (PDAC). One suggested explanation is the accumulation of dominantly immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells in the tumor microenvironment (TME). Using retrospectively collected tumor specimens and transcriptomic data from PDAC, we demonstrate that expression of the scavenger receptor MARCO correlates with poor prognosis and a lymphocyte-excluding tumor phenotype. PDAC cell lines produce IL-10 and induce high expression of MARCO in myeloid cells, and this was further enhanced during hypoxic conditions. These myeloid cells suppressed effector T and natural killer (NK) cells and blocked NK cell tumor infiltration and tumor killing in a PDAC 3D-spheroid model. Anti-human MARCO (anti-hMARCO) antibody targeting triggered the repolarization of tumor-associated macrophages and activated the inflammasome machinery, resulting in IL-18 production. This in turn enhanced T cell and NK cell functions. The targeting of MARCO thus remodels the TME and represents a rational approach to make immunotherapy more efficient in PDAC patients.

HIF-1 stabilization in T cells hampers the control of Mycobacterium tuberculosis infection.

The hypoxia-inducible factors (HIFs) regulate the main transcriptional pathway of response to hypoxia in T cells and are negatively regulated by von Hippel-Lindau factor (VHL). But the role of HIFs in the regulation of CD4 T cell responses during infection with M. tuberculosis isn't well understood. Here we show that mice lacking VHL in T cells (Vhl cKO) are highly susceptible to infection with M. tuberculosis, which is associated with a low accumulation of mycobacteria-specific T cells in the lungs that display reduced proliferation, altered differentiation and enhanced expression of inhibitory receptors. In contrast, HIF-1 deficiency in T cells is redundant for M. tuberculosis control. Vhl cKO mice also show reduced responses to vaccination. Further, VHL promotes proper MYC-activation, cell-growth responses, DNA synthesis, proliferation and survival of CD4 T cells after TCR activation. The VHL-deficient T cell responses are rescued by the loss of HIF-1α, indicating that the increased susceptibility to M. tuberculosis infection and the impaired responses of Vhl-deficient T cells are HIF-1-dependent.

Expression and activation of nuclear hormone receptors result in neuronal differentiation and favorable prognosis in neuroblastoma.

BACKGROUND: Neuroblastoma (NB), a childhood tumor derived from the sympathetic nervous system, presents with heterogeneous clinical behavior. While some tumors regress spontaneously without medical intervention, others are resistant to therapy, associated with an aggressive phenotype. MYCN-amplification, frequently occurring in high-risk NB, is correlated with an undifferentiated phenotype and poor prognosis. Differentiation induction has been proposed as a therapeutic approach for high-risk NB. We have previously shown that MYCN maintains an undifferentiated state via regulation of the miR-17 ~ 92 microRNA cluster, repressing the nuclear hormone receptors (NHRs) estrogen receptor alpha (ERα) and the glucocorticoid receptor (GR). METHODS: Cell viability was determined by WST-1. Expression of differentiation markers was analyzed by Western blot, RT-qPCR, and immunofluorescence analysis. Metabolic phenotypes were studied using Agilent Extracellular Flux Analyzer, and accumulation of lipid droplets by Nile Red staining. Expression of angiogenesis, proliferation, and neuronal differentiation markers, and tumor sections were assessed by immunohistochemistry. Gene expression from NB patient as well as adrenal gland cohorts were analyzed using GraphPad Prism software (v.8) and GSEA (v4.0.3), while pseudo-time progression on post-natal adrenal gland cells from single-nuclei transcriptome data was computed using scVelo. RESULTS: Here, we show that simultaneous activation of GR and ERα potentiated induction of neuronal differentiation, reduced NB cell viability in vitro, and decreased tumor burden in vivo. This was accompanied by a metabolic reprogramming manifested by changes in the glycolytic and mitochondrial functions and in lipid droplet accumulation. Activation of the retinoic acid receptor alpha (RARα) with all-trans retinoic acid (ATRA) further enhanced the differentiated phenotype as well as the metabolic switch. Single-cell nuclei transcriptome analysis of human adrenal glands indicated a sequential expression of ERα, GR, and RARα during development from progenitor to differentiated chromaffin cells. Further, in silico analysis revealed that patients with higher combined expression of GR, ERα, and RARα mRNA levels had elevated expression of neuronal differentiation markers and a favorable outcome. CONCLUSION: Together, our findings suggest that combination therapy involving activation of several NHRs could be a promising pharmacological approach for differentiation treatment of NB patients.

Impaired oxygen-sensitive regulation of mitochondrial biogenesis within the von Hippel-Lindau syndrome.

Mitochondria are the main consumers of oxygen within the cell. How mitochondria sense oxygen levels remains unknown. Here we show an oxygen-sensitive regulation of TFAM, an activator of mitochondrial transcription and replication, whose alteration is linked to tumours arising in the von Hippel-Lindau syndrome. TFAM is hydroxylated by EGLN3 and subsequently bound by the von Hippel-Lindau tumour-suppressor protein, which stabilizes TFAM by preventing mitochondrial proteolysis. Cells lacking wild-type VHL or in which EGLN3 is inactivated have reduced mitochondrial mass. Tumorigenic VHL variants leading to different clinical manifestations fail to bind hydroxylated TFAM. In contrast, cells harbouring the Chuvash polycythaemia VHLR200W mutation, involved in hypoxia-sensing disorders without tumour development, are capable of binding hydroxylated TFAM. Accordingly, VHL-related tumours, such as pheochromocytoma and renal cell carcinoma cells, display low mitochondrial content, suggesting that impaired mitochondrial biogenesis is linked to VHL tumorigenesis. Finally, inhibiting proteolysis by targeting LONP1 increases mitochondrial content in VHL-deficient cells and sensitizes therapy-resistant tumours to sorafenib treatment. Our results offer pharmacological avenues to sensitize therapy-resistant VHL tumours by focusing on the mitochondria.

VEGFR2 inhibition hampers breast cancer cell proliferation via enhanced mitochondrial biogenesis.

Objective: Vascular endothelial growth factor (VEGF), apart from its predominant roles in angiogenesis, can enhance cancer cell proliferation, but its mechanisms remain elusive. The purpose of the present study was therefore to identify how VEGF regulates cancer cell proliferation. Methods: VEGF effects on cancer cell proliferation were investigated with the VEGF receptor 2 inhibitor, Ki8751, and the breast cancer cell lines, MCF-7 and MDA-MB-231, using flow cytometry, mass spectrometry, immunoblotting, and confocal microscopy. Data were analyzed using one-way analysis of variance followed by Tukey's multiple comparison test. Results: VEGF blockade by Ki8751 significantly reduced cancer cell proliferation, and enhanced breast cancer cell apoptosis. Mass spectrometric analyses revealed that Ki8751 treatment significantly upregulated the expression of mitochondrial proteins, suggesting the involvement of mitochondrial biogenesis. Confocal microscopy and flow cytometric analyses showed that Ki8751 treatment robustly increased the mitochondrial masses of both cancer cells, induced endomitosis, and arrested cancer cells in the high aneuploid phase. VEGFR2 knockdown by shRNAs showed similar effects to those of Ki8751, confirming the specificity of Ki8751 treatment. Enhanced mitochondrial biogenesis increased mitochondrial oxidative phosphorylation and stimulated reactive oxygen species (ROS) production, which induced cancer cell apoptosis. Furthermore, Ki8751 treatment downregulated the phosphorylation of Akt and PGC1α, and translocated PGC1α into the nucleus. The PGC1α alterations increased mitochondrial transcription factor A (TFAM) expression and subsequently increased mitochondrial biogenesis. Conclusions: VEGF enhances cancer cell proliferation by decreasing Akt-PGC1α-TFAM signaling-mediated mitochondrial biogenesis, ROS production, and cell apoptosis. These findings suggested the anticancer potential of Ki8751 via increased mitochondrial biogenesis and ROS production.

Aberrant splicing in neuroblastoma generates RNA-fusion transcripts and provides vulnerability to spliceosome inhibitors.

The paucity of recurrent mutations has hampered efforts to understand and treat neuroblastoma. Alternative splicing and splicing-dependent RNA-fusions represent mechanisms able to increase the gene product repertoire but their role in neuroblastoma remains largely unexplored. Here we investigate the presence and possible roles of aberrant splicing and splicing-dependent RNA-fusion transcripts in neuroblastoma. In addition, we attend to establish whether the spliceosome can be targeted to treat neuroblastoma. Through analysis of RNA-sequenced neuroblastoma we show that elevated expression of splicing factors is a strong predictor of poor clinical outcome. Furthermore, we identified >900 primarily intrachromosomal fusions containing canonical splicing sites. Fusions included transcripts from well-known oncogenes, were enriched for proximal genes and in chromosomal regions commonly gained or lost in neuroblastoma. As a proof-of-principle that these fusions can generate altered gene products, we characterized a ZNF451-BAG2 fusion, producing a truncated BAG2-protein which inhibited retinoic acid induced differentiation. Spliceosome inhibition impeded neuroblastoma fusion expression, induced apoptosis and inhibited xenograft tumor growth. Our findings elucidate a splicing-dependent mechanism generating altered gene products in neuroblastoma and show that the spliceosome is a potential target for clinical intervention.

Platelet factor 4 enhances CD4+ T effector memory cell responses via Akt-PGC1α-TFAM signaling-mediated mitochondrial biogenesis.

BACKGROUND: Cell metabolism drives T cell functions, while platelets regulate overall CD4+ T cell immune responses. OBJECTIVE: To investigate if platelets influence cell metabolism and thus regulate CD4+ T effector memory cell (Tem) responses. METHODS: Human CD4+ Tem cells were activated with αCD3/αCD28 and cultured without or with platelets or platelet-derived mediators. RESULTS: Polyclonal stimulation induced rapid and marked Th1 and Treg cell activation of CD4+ Tem cells. Platelet co-culture enhanced Th1 response transiently, while it persistently enhanced Treg cell activation of Tem cells, with an enhancement that plateaued by day 3. Platelet factor 4 (PF4) was the key platelet-derived mediator regulating CD4+ Tem cell responses, which involved cellular metabolisms as indicated by mass spectrometric analyses. PF4 exerted its effects via its receptor CXCR3, attenuated Akt activity, and reduced PGC1α phosphorylation, and resulted in elevations of PGC1α function and mitochondrial transcription factor A (TFAM) synthesis. The latter increased mitochondrial biogenesis, and subsequently enhanced Th1 and Treg responses. Consistent with these observations, inhibition of mitochondrial function by rotenone counteracted the enhancements by recombinant PF4, and TFAM overexpression by TFAM-adenovirus infection mimicked PF4 effects. Furthermore, increased mitochondrial mass elevated oxygen consumption, and enhanced adenosine triphosphate and reactive oxygen species production, which, in turn, stimulated Th1 (T-bet) and Treg (FoxP3) transcription factor expression and corresponding CD4+ T effector cell responses. CONCLUSIONS: Platelets enhance CD4+ T cell responses of Tem cells through PF4-dependent and Akt-PGC1α-TFAM signaling-mediated mitochondrial biogenesis. Hence, PF4 may be a promising intervention target of platelet-regulated immune responses.

EglN3 hydroxylase stabilizes BIM-EL linking VHL type 2C mutations to pheochromocytoma pathogenesis and chemotherapy resistance.

Despite the discovery of the oxygen-sensitive regulation of HIFα by the von Hippel-Lindau (VHL) protein, the mechanisms underlying the complex genotype/phenotype correlations in VHL disease remain unknown. Some germline VHL mutations cause familial pheochromocytoma and encode proteins that preserve their ability to down-regulate HIFα. While type 1, 2A, and 2B VHL mutants are defective in regulating HIFα, type 2C mutants encode proteins that preserve their ability to down-regulate HIFα. Here, we identified an oxygen-sensitive function of VHL that is abolished by VHL type 2C mutations. We found that BIM-EL, a proapoptotic BH3-only protein, is hydroxylated by EglN3 and subsequently bound by VHL. VHL mutants fail to bind hydroxylated BIM-EL, regardless of whether they have the ability to bind hydroxylated HIFα or not. VHL binding inhibits BIM-EL phosphorylation by extracellular signal-related kinase (ERK) on serine 69. This causes BIM-EL to escape from proteasomal degradation, allowing it to enhance EglN3-induced apoptosis. BIM-EL was rapidly degraded in cells lacking wild-type VHL or in which EglN3 was inactivated genetically or by lack of oxygen, leading to enhanced cell survival and chemotherapy resistance. Combination therapy using ERK inhibitors, however, resensitizes VHL- and EglN3-deficient cells that are otherwise cisplatin-resistant.

PHD3 Regulates p53 Protein Stability by Hydroxylating Proline 359.

Cellular p53 protein levels are regulated by a ubiquitination/de-ubiquitination cycle that can target the protein for proteasomal destruction. The ubiquitination reaction is catalyzed by a multitude of ligases, whereas the removal of ubiquitin chains is mediated by two deubiquitinating enzymes (DUBs), USP7 (HAUSP) and USP10. Here, we show that PHD3 hydroxylates p53 at proline 359, a residue that is in the p53-DUB binding domain. Hydroxylation of p53 upon proline 359 regulates its interaction with USP7 and USP10, and its inhibition decreases the association of p53 with USP7/USP10, increases p53 ubiquitination, and rapidly reduces p53 protein levels independently of mRNA expression. Our results show that p53 is a PHD3 substrate and that hydroxylation by PHD3 regulates p53 protein stability through modulation of ubiquitination.

Combined epigenetic and differentiation-based treatment inhibits neuroblastoma tumor growth and links HIF2α to tumor suppression.

Neuroblastoma is a pediatric cancer characterized by variable outcomes ranging from spontaneous regression to life-threatening progression. High-risk neuroblastoma patients receive myeloablative chemotherapy with hematopoietic stem-cell transplant followed by adjuvant retinoid differentiation treatment. However, the overall survival remains low; hence, there is an urgent need for alternative therapeutic approaches. One feature of high-risk neuroblastoma is the high level of DNA methylation of putative tumor suppressors. Combining the reversibility of DNA methylation with the differentiation-promoting activity of retinoic acid (RA) could provide an alternative strategy to treat high-risk neuroblastoma. Here we show that treatment with the DNA-demethylating drug 5-Aza-deoxycytidine (AZA) restores high-risk neuroblastoma sensitivity to RA. Combined systemic distribution of AZA and RA impedes tumor growth and prolongs survival. Genome-wide analysis of treated tumors reveals that this combined treatment rapidly induces a HIF2α-associated hypoxia-like transcriptional response followed by an increase in neuronal gene expression and a decrease in cell-cycle gene expression. A small-molecule inhibitor of HIF2α activity diminishes the tumor response to AZA+RA treatment, indicating that the increase in HIF2α levels is a key component in tumor response to AZA+RA. The link between increased HIF2α levels and inhibited tumor growth is reflected in large neuroblastoma patient datasets. Therein, high levels of HIF2α, but not HIF1α, significantly correlate with expression of neuronal differentiation genes and better prognosis but negatively correlate with key features of high-risk tumors, such as MYCN amplification. Thus, contrary to previous studies, our findings indicate an unanticipated tumor-suppressive role for HIF2α in neuroblastoma.

Neuroblast differentiation during development and in neuroblastoma requires KIF1Bß-mediated transport of TRKA.

We recently identified pathogenic KIF1Bß mutations in sympathetic nervous system malignancies that are defective in developmental apoptosis. Here we deleted KIF1Bß in the mouse sympathetic nervous system and observed impaired sympathetic nervous function and misexpression of genes required for sympathoadrenal lineage differentiation. We discovered that KIF1Bß is required for nerve growth factor (NGF)-dependent neuronal differentiation through anterograde transport of the NGF receptor TRKA. Moreover, pathogenic KIF1Bß mutations identified in neuroblastoma impair TRKA transport. Expression of neuronal differentiation markers is ablated in both KIF1Bß-deficient mouse neuroblasts and human neuroblastomas that lack KIF1Bß. Transcriptomic analyses show that unfavorable neuroblastomas resemble mouse sympathetic neuroblasts lacking KIF1Bß independent of MYCN amplification and the loss of genes neighboring KIF1B on chromosome 1p36. Thus, defective precursor cell differentiation, a common trait of aggressive childhood malignancies, is a pathogenic effect of KIF1Bß loss in neuroblastomas. Furthermore, neuropathy-associated KIF1Bß mutations impede cargo transport, providing a direct link between neuroblastomas and neurodegeneration.

The 2-oxoglutarate analog 3-oxoglutarate decreases normoxic hypoxia-inducible factor-1α in cancer cells, induces cell death, and reduces tumor xenograft growth.

The cellular response to hypoxia is primarily regulated by the hypoxia-inducible factors (HIFs). HIF-1α is also a major mediator of tumor physiology, and its abundance is correlated with therapeutic resistance in a broad range of cancers. Accumulation of HIF-1α under hypoxia is mainly controlled by the oxygen-sensing HIF prolyl 4-hydroxylases (EGLNs, also known as PHDs). Here, we identified a high level of normoxic HIF-1α protein in various cancer cell lines. EGLNs require oxygen and 2-oxoglutarate for enzymatic activity. We tested the ability of several cell-permeable 2-oxoglutarate analogs to regulate the abundance of HIF-1α protein. We identified 3-oxoglutarate as a potent regulator of HIF-1α in normoxic conditions. In contrast to 2-oxoglutarate, 3-oxoglutarate decreased the abundance of HIF-1α protein in several cancer cell lines in normoxia and diminished HIF-1α levels independent of EGLN enzymatic activity. Furthermore, we observed that 3-oxoglutarate was detrimental to cancer cell survival. We show that esterified 3-oxoglutarate, in combination with the cancer chemotherapeutic drug vincristine, induces apoptosis and inhibits tumor growth in vitro and in vivo. Our data imply that a novel treatment strategy targeting HIF-1α in combination with the use of existing cytotoxic agents could serve as potent, future antitumor chemotherapies.

XAF1 promotes neuroblastoma tumor suppression and is required for KIF1Bß-mediated apoptosis.

Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system. Current treatment modalities do not fully exploit the genetic basis between the different molecular subtypes and little is known about the targets discovered in recent mutational and genetic studies. Neuroblastomas with poor prognosis are often characterized by 1p36 deletion, containing the kinesin gene KIF1B. Its beta isoform, KIF1Bß, is required for NGF withdrawal-dependent apoptosis, mediated by the induction of XIAP-associated Factor 1 (XAF1). Here, we showed that XAF1 low expression correlates with poor survival and disease status. KIF1Bß deletion results in loss of XAF1 expression, suggesting that XAF1 is indeed a downstream target of KIF1Bß. XAF1 silencing protects from NGF withdrawal and from KIF1Bß-mediated apoptosis. Overexpression of XAF1 impairs tumor progression whereas knockdown of XAF1 promotes tumor growth, suggesting that XAF1 may be a candidate tumor suppressor in neuroblastoma and its associated pathway may be important for developing future interventions.

The 1p36 Tumor Suppressor KIF 1Bß Is Required for Calcineurin Activation, Controlling Mitochondrial Fission and Apoptosis.

KIF1Bß is a candidate 1p36 tumor suppressor that regulates apoptosis in the developing sympathetic nervous system. We found that KIF1Bß activates the Ca(2+)-dependent phosphatase calcineurin (CN) by stabilizing the CN-calmodulin complex, relieving enzymatic autoinhibition and enabling CN substrate recognition. CN is the key mediator of cellular responses to Ca(2+) signals and its deregulation is implicated in cancer, cardiac, neurodegenerative, and immune disease. We show that KIF1Bß affects mitochondrial dynamics through CN-dependent dephosphorylation of Dynamin-related protein 1 (DRP1), causing mitochondrial fission and apoptosis. Furthermore, KIF1Bß actuates recognition of all known CN substrates, implying a general mechanism for KIF1Bß in Ca(2+) signaling and how Ca(2+)-dependent signaling is executed by CN. Pathogenic KIF1Bß mutations previously identified in neuroblastomas and pheochromocytomas all fail to activate CN or stimulate DRP1 dephosphorylation. Importantly, KIF1Bß and DRP1 are silenced in 1p36 hemizygous-deleted neuroblastomas, indicating that deregulation of calcineurin and mitochondrial dynamics contributes to high-risk and poor-prognosis neuroblastoma.

RNA helicase A is a downstream mediator of KIF1Bß tumor-suppressor function in neuroblastoma.

UNLABELLED: Inherited KIF1B loss-of-function mutations in neuroblastomas and pheochromocytomas implicate the kinesin KIF1B as a 1p36.2 tumor suppressor. However, the mechanism of tumor suppression is unknown. We found that KIF1B isoform ß (KIF1Bß) interacts with RNA helicase A (DHX9), causing nuclear accumulation of DHX9, followed by subsequent induction of the proapoptotic XIAP-associated factor 1 (XAF1) and, consequently, apoptosis. Pheochromocytoma and neuroblastoma arise from neural crest progenitors that compete for growth factors such as nerve growth factor (NGF) during development. KIF1Bß is required for developmental apoptosis induced by competition for NGF. We show that DHX9 is induced by and required for apoptosis stimulated by NGF deprivation. Moreover, neuroblastomas with chromosomal deletion of 1p36 exhibit loss of KIF1Bß expression and impaired DHX9 nuclear localization, implicating the loss of DHX9 nuclear activity in neuroblastoma pathogenesis. SIGNIFICANCE: KIF1Bß has neuroblastoma tumor-suppressor properties and promotes and requires nuclear-localized DHX9 for its apoptotic function by activating XAF1 expression. Loss of KIF1Bß alters subcellular localization of DHX9 and diminishes NGF dependence of sympathetic neurons, leading to reduced culling of neural progenitors, and, therefore, might predispose to tumor formation.

Mutation analysis of HIF prolyl hydroxylases (PHD/EGLN) in individuals with features of phaeochromocytoma and renal cell carcinoma susceptibility.

Germline mutations in the von Hippel-Lindau disease (VHL) and succinate dehydrogenase subunit B (SDHB) genes can cause inherited phaeochromocytoma and/or renal cell carcinoma (RCC). Dysregulation of the hypoxia-inducible factor (HIF) transcription factors has been linked to VHL and SDHB-related RCC; both HIF dysregulation and disordered function of a prolyl hydroxylase domain isoform 3 (PHD3/EGLN3)-related pathway of neuronal apoptosis have been linked to the development of phaeochromocytoma. The 2-oxoglutarate-dependent prolyl hydroxylase enzymes PHD1 (EGLN2), PHD2 (EGLN1) and PHD3 (EGLN3) have a key role in regulating the stability of HIF-α subunits (and hence expression of the HIF-α transcription factors). A germline PHD2 mutation has been reported in association with congenital erythrocytosis and recurrent extra-adrenal phaeochromocytoma. We undertook mutation analysis of PHD1, PHD2 and PHD3 in two cohorts of patients with features of inherited phaeochromocytoma (n=82) and inherited RCC (n=64) and no evidence of germline mutations in known susceptibility genes. No confirmed pathogenic mutations were detected suggesting that mutations in these genes are not a frequent cause of inherited phaeochromocytoma or RCC.