Independence of HIF1a and androgen signaling pathways in prostate cancer.

BACKGROUND: Therapeutic targeting of the androgen signaling pathway is a mainstay treatment for prostate cancer. Although initially effective, resistance to androgen targeted therapies develops followed by disease progression to castrate-resistant prostate cancer (CRPC). Hypoxia and HIF1a have been implicated in the development of resistance to androgen targeted therapies and progression to CRCP. The interplay between the androgen and hypoxia/HIF1a signaling axes was investigated. METHODS: In vitro stable expression of HIF1a was established in the LNCaP cell line by physiological induction or retroviral transduction. Tumor xenografts with stable expression of HIF1a were established in castrated and non-castrated mouse models. Gene expression analysis identified transcriptional changes in response to androgen treatment, hypoxia and HIF1a. The binding sites of the AR and HIF transcription factors were identified using ChIP-seq. RESULTS: Androgen and HIF1a signaling promoted proliferation in vitro and enhanced tumor growth in vivo. The stable expression of HIF1a in vivo restored tumor growth in the absence of endogenous androgens. Hypoxia reduced AR binding sites whereas HIF binding sites were increased with androgen treatment under hypoxia. Gene expression analysis identified seven genes that were upregulated both by AR and HIF1a, of which six were prognostic. CONCLUSIONS: The oncogenic AR, hypoxia and HIF1a pathways support prostate cancer development through independent signaling pathways and transcriptomic profiles. AR and hypoxia/HIF1a signaling pathways independently promote prostate cancer progression and therapeutic targeting of both pathways simultaneously is warranted.

Targeted inactivation of fh1 causes proliferative renal cyst development and activation of the hypoxia pathway.

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.

Deletion of the von Hippel-Lindau gene in pancreatic beta cells impairs glucose homeostasis in mice.

Defective insulin secretion in response to glucose is an important component of the beta cell dysfunction seen in type 2 diabetes. As mitochondrial oxidative phosphorylation plays a key role in glucose-stimulated insulin secretion (GSIS), oxygen-sensing pathways may modulate insulin release. The von Hippel-Lindau (VHL) protein controls the degradation of hypoxia-inducible factor (HIF) to coordinate cellular and organismal responses to altered oxygenation. To determine the role of this pathway in controlling glucose-stimulated insulin release from pancreatic beta cells, we generated mice lacking Vhl in pancreatic beta cells (betaVhlKO mice) and mice lacking Vhl in the pancreas (PVhlKO mice). Both mouse strains developed glucose intolerance with impaired insulin secretion. Furthermore, deletion of Vhl in beta cells or the pancreas altered expression of genes involved in beta cell function, including those involved in glucose transport and glycolysis, and isolated betaVhlKO and PVhlKO islets displayed impaired glucose uptake and defective glucose metabolism. The abnormal glucose homeostasis was dependent on upregulation of Hif-1alpha expression, and deletion of Hif1a in Vhl-deficient beta cells restored GSIS. Consistent with this, expression of activated Hif-1alpha in a mouse beta cell line impaired GSIS. These data suggest that VHL/HIF oxygen-sensing mechanisms play a critical role in glucose homeostasis and that activation of this pathway in response to decreased islet oxygenation may contribute to beta cell dysfunction.

Hypoxia-inducible transcription factors stabilization in the thick ascending limb protects against ischemic acute kidney injury.

Hypoxia-inducible transcription factors (HIF) protect cells against oxygen deprivation, and HIF stabilization before ischemia mitigates tissue injury. Because ischemic acute kidney injury (AKI) often involves the thick ascending limb (TAL), modulation of HIF in this segment may be protective. Here, we generated mice with targeted TAL deletion of the von Hippel-Lindau protein (Vhl), which mediates HIF degradation under normoxia, using Tamm-Horsfall protein (Thp)-driven Cre expression. These mice showed strong expression of HIF-1α in TALs but no changes in kidney morphology or function under control conditions. Deficiency of Vhl in the TAL markedly attenuated proximal tubular injury and preserved TAL function following ischemia-reperfusion, which may be partially a result of enhanced expression of glycolytic enzymes and lactate metabolism. These results highlight the importance of the thick ascending limb in the pathogenesis of AKI and suggest that pharmacologically targeting the HIF system may have potential to prevent and mitigate AKI.

Inactivation of the von Hippel-Lindau tumour suppressor gene induces Neuromedin U expression in renal cancer cells.

BACKGROUND: 209 000 new cases of renal carcinoma are diagnosed each year worldwide and new therapeutic targets are urgently required. The great majority of clear cell renal cancer involves inactivation of VHL, which acts as a gatekeeper tumour suppressor gene in renal epithelial cells. However how VHL exerts its tumour suppressor function remains unclear. A gene expression microarray comparing RCC10 renal cancer cells expressing either VHL or an empty vector was used to identify novel VHL regulated genes. FINDINGS: NMU (Neuromedin U) is a neuropeptide that has been implicated in energy homeostasis and tumour progression. Here we show for the first time that VHL loss-of-function results in dramatic upregulation of NMU expression in renal cancer cells. The effect of VHL inactivation was found to be mediated via activation of Hypoxia Inducible Factor (HIF). Exposure of VHL expressing RCC cells to either hypoxia or dimethyloxalylglycine resulted in HIF activation and increased NMU expression. Conversely, suppression of HIF in VHL defective RCC cells via siRNA of HIF-α subunits or expression of Type 2C mutant VHLs reduced NMU expression levels. We also show that renal cancer cells express a functional NMU receptor (NMUR1), and that NMU stimulates migration of renal cancer cells. CONCLUSIONS: These findings suggest that NMU may act in an autocrine fashion, promoting progression of kidney cancer. Hypoxia and HIF expression are frequently observed in many non-renal cancers and are associated with a poor prognosis. Our study raises the possibility that HIF may also drive NMU expression in non-renal tumours.

Regulation of myeloperoxidase-specific T cell responses during disease remission in antineutrophil cytoplasmic antibody-associated vasculitis: the role of Treg cells and tryptophan degradation.

OBJECTIVE: T lymphocytes have been implicated in the pathogenesis of antineutrophil cytoplasmic antibody-associated vasculitis (AAV). Patients with myeloperoxidase (MPO) antineutrophil cytoplasmic antibody (ANCA) experience relapses less frequently than those with proteinase 3 ANCA, suggesting greater immune regulation. This study was undertaken to investigate MPO-specific T cell reactivity during disease remission and the factors regulating their responsiveness. METHODS: MPO-specific T cells were quantified by enzyme-linked immunospot assay with additional Treg cell depletion or exogenous interleukin-2. Serum tryptophan and its metabolites were measured. In vivo blockade of indoleamine 2,3-dioxygenase (IDO) was performed, and its effect on MPO reactivity was assessed. RESULTS: During disease remission, MPO-specific interferon-gamma-producing T cell frequencies were comparable with those found in healthy controls and significantly lower than those found in patients with acute disease. CD4+CD25+ regulatory cells did not play a role in maintaining these low MPO-specific T cell frequencies, since depletion of Treg cells did not augment MPO-specific responses, and FoxP3 levels were diminished in patients compared with controls. Treg cell function, however, was comparable in patients and controls, suggesting numerical rather than functional deficiency. We found diminished serum tryptophan levels and elevated levels of its metabolite kynurenine in patients with MPO AAV as compared with controls. To confirm the effect of tryptophan degradation on MPO responses in vivo, we inhibited degradation in MPO-immunized WKY rats and found greater immune responsiveness to MPO and a tendency to more severe glomerulonephritis. CONCLUSION: Our findings indicate that MPO-specific T cell frequencies are regulated during disease remission in association with tryptophan degradation. The tryptophan regulatory pathway is induced during active disease and persists during disease remission.

VHL inactivation induces HEF1 and Aurora kinase A.

The ciliary hypothesis for cystic renal diseases postulates that most of these conditions result from abnormalities in the primary cilium, a microtubule-based structure that acts as a sensor for extracellular cues. Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes to renal cysts and clear cell renal cell carcinoma. VHL plays a critical role in the formation of primary cilia in kidney epithelium, but the underlying mechanisms are poorly understood. Here, we demonstrate that VHL inactivation induces HEF1/Cas-L/NEDD9 and Aurora kinase A via the stabilization of hypoxia-inducible factors 1 and 2. Aurora kinase A is a mitotic kinase commonly upregulated in cancer that causes regression of the primary cilium by promoting histone deacetylase-dependent tubulin depolymerization of the ciliary axoneme. HEF1/Cas-L/NEDD9 is a component of focal adhesions that has a prominent role in inducing metastasis and that colocalizes with Aurora kinase A at the centrosome, thereby enhancing the harmful effect of Aurora kinase A on the cilium. Suppression of this pathway improved the formation of primary cilia and reduced cell motility in VHL-defective renal cancer cells. Our results highlight the gatekeeper role of VHL in the kidney epithelium.

Low-Temperature As-Grown Crystalline ß-Ga2O3 Films via Plasma-Enhanced Atomic Layer Deposition.

We report on the low-temperature growth of crystalline Ga2O3 films on Si, sapphire, and glass substrates using plasma-enhanced atomic layer deposition (PEALD) featuring a hollow-cathode plasma source. Films were deposited by using triethylgallium (TEG) and Ar/O2 plasma as metal precursor and oxygen co-reactant, respectively. Growth experiments have been performed within 150-240 °C substrate temperature and 30-300 W radio-frequency (rf) plasma power ranges. Additionally, each unit AB-type ALD cycle was followed by an in situ Ar plasma annealing treatment, which consisted of an extra (50-300 W) Ar plasma exposure for 20 s ending just before the next TEG pulse. The growth per cycle (GPC) of the films without Ar plasma annealing step ranged between 0.69 and 1.31 Å/cycle, and as-grown refractive indices were between 1.67 and 1.75 within the scanned plasma power range. X-ray diffraction (XRD) measurements showed that Ga2O3 films grown without in situ Ar plasma annealing exhibited amorphous character irrespective of substrate temperature and rf power values. With the incorporation of the in situ Ar plasma annealing process, the GPC of Ga2O3 films ranged between 0.76 and 1.03 Å/cycle along with higher refractive index values of 1.75-1.79. The increased refractive index (1.79) and slightly reduced GPC (1.03 Å/cycle) at 250 W plasma annealing indicated possible densification and crystallization of the films. Indeed, X-ray measurements confirmed that in situ plasma annealed films grow in a monoclinic ß-Ga2O3 crystal phase. The film crystallinity and density further enhance (from 5.11 to 5.60 g/cm3) by increasing the rf power value used during in situ Ar plasma annealing process. X-ray photoelectron spectroscopy (XPS) measurement of the ß-Ga2O3 sample grown under optimal in situ plasma annealing power (250 W) revealed near-ideal film stoichiometry (O/Ga of ∼1.44) with relatively low carbon content (∼5 at. %), whereas 50 W rf power treated film was highly non-stoichiometric (O/Ga of ∼2.31) with considerably elevated carbon content. Our results demonstrate the effectiveness of in situ Ar plasma annealing process to transform amorphous wide bandgap oxide semiconductors into crystalline films without needing high-temperature post-deposition annealing treatment.

Atomic layer deposition of metal oxides for efficient perovskite single-junction and perovskite/silicon tandem solar cells.

Aluminum-doped and undoped zinc oxide films were investigated as potential front and rear contacts of perovskite single and perovskite/silicon tandem solar cells. The films were prepared by atomic layer deposition (ALD) at low (<200 °C) substrate temperatures. The deposited films were crystalline with a single-phase wurtzite structure and exhibit excellent uniformity and low surface roughness which was confirmed by XRD and SEM measurements. Necessary material characterizations allow for realizing high-quality films with low resistivity and high optical transparency at the standard growth rate. Spectroscopic ellipsometry measurements were carried out to extract the complex refractive index of the deposited films, which were used to study the optics of perovskite single junction and perovskite/silicon tandem solar cells. The optics was investigated by three-dimensional finite-difference time-domain simulations. Guidelines are provided on how to realize perovskite solar cells exhibiting high short-circuit current densities. Furthermore, detailed guidelines are given for realizing perovskite/silicon tandem solar cells with short-circuit current densities exceeding 20 mA cm-2 and potential energy conversion efficiencies beyond 31%.

Elucidating the role of nitrogen plasma composition in the low-temperature self-limiting growth of indium nitride thin films.

In this work, we have studied the role varying nitrogen plasma compositions play in the low-temperature plasma-assisted growth of indium nitride (InN) thin films. Films were deposited on Si (100) substrates using a plasma-enhanced atomic layer deposition (PE-ALD) reactor featuring a capacitively-coupled hollow-cathode plasma source. Trimethylindium (TMI) and variants of nitrogen plasma (N2-only, Ar/N2, and Ar/N2/H2) were used as the metal precursor and nitrogen co-reactant, respectively. In situ ellipsometry was employed to observe individual ligand exchange and plasma-assisted ligand removal events in real-time during the growth process. Only the samples grown under hydrogen-free nitrogen plasmas (Ar/N2 or N2-only) resulted in nearly stoichiometric single-phase crystalline hexagonal InN (h-InN) films at substrate temperatures higher than 200 °C under 100 W rf-plasma power. Varying the plasma gas composition by adding H2 led to rather drastic microstructural changes resulting in a cubic phase oxide (c-In2O3) film. Combining the in situ measured growth evolution with ex situ materials characterization, we propose a simplified model describing the possible surface reactions/groups during a unit PE-ALD cycle, which depicts the highly efficient oxygen incorporation in the presence of hydrogen radicals. Further structural, chemical, and optical characterization have been carried out on the optimal InN films grown with Ar/N2 plasma to extract film properties. Samples grown at lower substrate temperature (160 °C) and reduced/elevated rf-plasma power levels (50/150 W) displayed similar amorphous character, which is attributed to either insufficient surface energy or plasma-induced crystal damage. InN samples grown at 240 °C under 100 W rf-plasma showed clear polycrystalline h-InN layers with ∼20 nm average-sized single crystal domains exhibiting hexagonal symmetry.

Inhibition of hypoxia inducible factor hydroxylases protects against renal ischemia-reperfusion injury.

Acute renal failure resulting from hypoperfusion and hypoxia is a significant clinical problem. Hypoxia activates the heterodimeric transcription factor hypoxia inducible factor (HIF), leading to changes in gene expression that promote tissue adaptation and survival. To determine whether HIF may protect the kidney from ischemia-reperfusion injury, we subjected hif1a(+/-) and hif2a(+/-) mice to renal ischemia-reperfusion injury. Injury was substantially more severe in hif(+/-) than in littermate controls, consistent with a protective role for HIF. Because wild-type mice exhibited submaximal HIF accumulation in response to no-flow ischemia, we tested compounds that might augment the protective HIF response following ischemia-reperfusion in these animals. We found that l-mimosine and dimethyloxalylglycine, two small molecules that activate HIF by inhibiting HIF hydroxylases, protected mouse kidneys from ischemia-reperfusion injury. Therefore, pharmacological activation of HIF may offer an effective strategy to protect the kidney from ischemic injury.

Development of a new low cost high sensitivity system for behavioural ecotoxicity testing.

The amphipod Gammarus pulex has been extensively used for ecotoxicological studies. However, the tests used are either labourious to perform and/or require relatively expensive equipment. We report the development of a new low cost infra red actograph system that measures relative activity, and can detect the behavioural effects of very low concentrations of heavy metals. Trials demonstrated that the home built system can distinguish significantly different behaviour between G. pulex exposed to clean water and that contaminated with as low as 10 microg L(-1) copper. This highly sensitive low cost automated system has the potential to become an important tool for ecotoxicity testing and water quality monitoring.

HIF-1 reduces ischaemia-reperfusion injury in the heart by targeting the mitochondrial permeability transition pore.

AIMS: Hypoxia-inducible factor-1 (HIF-1) has been reported to promote tolerance against acute myocardial ischaemia-reperfusion injury (IRI). However, the mechanism through which HIF-1 stabilization actually confers this cardioprotection is not clear. We investigated whether HIF-1α stabilization protects the heart against acute IRI by preventing the opening of the mitochondrial permeability transition pore (MPTP) and the potential mechanisms involved. METHODS AND RESULTS: Stabilization of myocardial HIF-1 was achieved by pharmacological inhibition of prolyl hydroxylase (PHD) domain-containing enzyme using GSK360A or using cardiac-specific ablation of von Hippel-Lindau protein (VHL(fl/fl)) in mice. Treatment of HL-1 cardiac cells with GSK360A stabilized HIF-1, increased the expression of HIF-1 target genes pyruvate dehydrogenase kinase-1 (PDK1) and hexokinase II (HKII), and reprogrammed cell metabolism to aerobic glycolysis, thereby resulting in the production of less mitochondrial oxidative stress during IRI, and less MPTP opening, effects which were shown to be dependent on HKII. These findings were further confirmed when HIF-1 stabilization in the rat and murine heart resulted in smaller myocardial infarct sizes (both in vivo and ex vivo), decreased mitochondrial oxidative stress, and inhibited MPTP opening following IRI, effects which were also found to be dependent on HKII. CONCLUSIONS: We have demonstrated that acute HIF-1α stabilization using either a pharmacological or genetic approach protected the heart against acute IRI by promoting aerobic glycolysis, decreasing mitochondrial oxidative stress, activating HKII, and inhibiting MPTP opening.

Human CHCHD4 mitochondrial proteins regulate cellular oxygen consumption rate and metabolism and provide a critical role in hypoxia signaling and tumor progression.

Increased expression of the regulatory subunit of HIFs (HIF-1α or HIF-2α) is associated with metabolic adaptation, angiogenesis, and tumor progression. Understanding how HIFs are regulated is of intense interest. Intriguingly, the molecular mechanisms that link mitochondrial function with the HIF-regulated response to hypoxia remain to be unraveled. Here we describe what we believe to be novel functions of the human gene CHCHD4 in this context. We found that CHCHD4 encodes 2 alternatively spliced, differentially expressed isoforms (CHCHD4.1 and CHCHD4.2). CHCHD4.1 is identical to MIA40, the homolog of yeast Mia40, a key component of the mitochondrial disulfide relay system that regulates electron transfer to cytochrome c. Further analysis revealed that CHCHD4 proteins contain an evolutionarily conserved coiled-coil-helix-coiled-coil-helix (CHCH) domain important for mitochondrial localization. Modulation of CHCHD4 protein expression in tumor cells regulated cellular oxygen consumption rate and metabolism. Targeting CHCHD4 expression blocked HIF-1α induction and function in hypoxia and resulted in inhibition of tumor growth and angiogenesis in vivo. Overexpression of CHCHD4 proteins in tumor cells enhanced HIF-1α protein stabilization in hypoxic conditions, an effect insensitive to antioxidant treatment. In human cancers, increased CHCHD4 expression was found to correlate with the hypoxia gene expression signature, increasing tumor grade, and reduced patient survival. Thus, our study identifies a mitochondrial mechanism that is critical for regulating the hypoxic response in tumors.

Renal tubular HIF-2α expression requires VHL inactivation and causes fibrosis and cysts.

The Hypoxia-inducible transcription Factor (HIF) represents an important adaptive mechanism under hypoxia, whereas sustained activation may also have deleterious effects. HIF activity is determined by the oxygen regulated α-subunits HIF-1α or HIF-2α. Both are regulated by oxygen dependent degradation, which is controlled by the tumor suppressor "von Hippel-Lindau" (VHL), the gatekeeper of renal tubular growth control. HIF appears to play a particular role for the kidney, where renal EPO production, organ preservation from ischemia-reperfusion injury and renal tumorigenesis are prominent examples. Whereas HIF-1α is inducible in physiological renal mouse, rat and human tubular epithelia, HIF-2α is never detected in these cells, in any species. In contrast, distinct early lesions of biallelic VHL inactivation in kidneys of the hereditary VHL syndrome show strong HIF-2α expression. Furthermore, knockout of VHL in the mouse tubular apparatus enables HIF-2α expression. Continuous transgenic expression of HIF-2α by the Ksp-Cadherin promotor leads to renal fibrosis and insufficiency, next to multiple renal cysts. In conclusion, VHL appears to specifically repress HIF-2α in renal epithelia. Unphysiological expression of HIF-2α in tubular epithelia has deleterious effects. Our data are compatible with dedifferentiation of renal epithelial cells by sustained HIF-2α expression. However, HIF-2α overexpression alone is insufficient to induce tumors. Thus, our data bear implications for renal tumorigenesis, epithelial differentiation and renal repair mechanisms.

HIF-1alpha and HIF-2alpha are differentially activated in distinct cell populations in retinal ischaemia.

BACKGROUND: Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs) that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR) in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF) and erythropoietin (Epo) by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO. CONCLUSIONS/SIGNIFICANCE: Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.

Expression profiling in progressive stages of fumarate-hydratase deficiency: the contribution of metabolic changes to tumorigenesis.

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.

Regulation of renal epithelial tight junctions by the von Hippel-Lindau tumor suppressor gene involves occludin and claudin 1 and is independent of E-cadherin.

Epithelial-to-mesenchymal transitions (EMT) are important in renal development, fibrosis, and cancer. Loss of function of the tumor suppressor VHL leads to many features of EMT, and it has been hypothesized that the pivotal mediator is down-regulation of the adherens junction (AJ) protein E-cadherin. Here we show that VHL loss-of-function also has striking effects on the expression of the tight junction (TJ) components occludin and claudin 1 in vitro in VHL-defective clear cell renal cell carcinoma (CCRCC) cells and in vivo in VHL-defective sporadic CCRCCs (compared with normal kidney). Occludin is also down-regulated in premalignant foci in kidneys from patients with germline VHL mutations, consistent with a contribution to CCRCC initiation. Reexpression of E-cadherin was sufficient to restore AJ but not TJ assembly, indicating that the TJ defect is independent of E-cadherin down-regulation. Additional experiments show that activation of hypoxia inducible factor (HIF) contributes to both TJ and AJ abnormalities, thus the VHL/HIF pathway contributes to multiple aspects of the EMT phenotype that are not interdependent. Despite the independent nature of the defects, we show that treatment with the histone deacetylase inhibitor sodium butyrate, which suppresses HIF activation, provides a method for reversing EMT in the context of VHL inactivation.