Xenon treatment protects against cold ischemia associated delayed graft function and prolongs graft survival in rats.

Prolonged hypothermic storage causes ischemia-reperfusion injury (IRI) in the renal graft, which is considered to contribute to the occurrence of the delayed graft function (DGF) and chronic graft failure. Strategies are required to protect the graft and to prolong renal graft survival. We demonstrated that xenon exposure to human proximal tubular cells (HK-2) led to activation of range of protective proteins. Xenon treatment prior to or after hypothermia-hypoxia challenge stabilized the HK-2 cellular structure, diminished cytoplasmic translocation of high-mobility group box (HMGB) 1 and suppressed NF-κB activation. In the syngeneic Lewis-to-Lewis rat model of kidney transplantation, xenon exposure to donors before graft retrieval or to recipients after engraftment decreased caspase-3 expression, localized HMGB-1 within nuclei and prevented TLR-4/NF-κB activation in tubular cells; serum pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α were reduced and renal function was preserved. Xenon treatment of graft donors or of recipients prolonged renal graft survival following IRI in both Lewis-to-Lewis isografts and Fischer-to-Lewis allografts. Xenon induced cell survival or graft functional recovery was abolished by HIF-1α siRNA. Our data suggest that xenon treatment attenuates DGF and enhances graft survival. This approach could be translated into clinical practice leading to a considerable improvement in long-term graft survival.

Epigenetic regulation of HIF-1α in renal cancer cells involves HIF-1α/2α binding to a reverse hypoxia-response element.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene underlies the majority of sporadic clear cell renal cell carcinomas (CCRCCs) and is also responsible for the hereditary VHL cancer syndrome. VHL loss of function results in constitutive stabilization of hypoxia-inducible factors (HIF-1α and HIF-2α) due to insufficient proteolysis in the presence of oxygen. This activates multiple genes relevant to tumorigenesis, allowing cells to acquire further mutations and undergo malignant transformation. However, the specific role of each HIF-α subunit in CCRCC tumorigenesis is not yet well understood. The current paradigm supports that in the first stages of CCRCC formation the stabilization of HIF-1α is dominant and this limits proliferation, but later on HIF-2α increases and this induces a more aggressive cell behavior. Understanding how this transition happens is highly relevant, as it may provide novel ways to treat these cancers. Here, we show that VHL inactivation in CCRCC cells results in HIF-1α/2α-dependent downregulation of HIF-1α mRNA through direct binding of either subunit to a reverse hypoxia-response element in the HIF-1α proximal promoter. This binding activates a series of repressive histone modification marks including histone 3 lysine 27 trimethylation (H3K27me3) to make the changes stable, and if overturned reduces CCRCC cell proliferation due to excessive HIF-1α expression level. Our findings thus help understand how HIF-α subunits influence each other and also reinforce the idea that epigenetic mechanisms are a key step of CCRCC progression.

Factor inhibiting HIF (FIH-1) promotes renal cancer cell survival by protecting cells from HIF-1α-mediated apoptosis.

BACKGROUND: Clear cell renal cell carcinoma (CCRCC) is the commonest form of kidney cancer. Up to 91% have biallelic inactivation of VHL, resulting in stabilisation of HIF-α subunits. Factor inhibiting HIF-1 is an enzyme that hydroxylates HIF-α subunits and prevents recruitment of the co-activator CBP/P300. An important question is whether FIH-1 controls HIF activity in CCRCC. METHODS: Human VHL defective CCRCC lines RCC10, RCC4 and 786-O were used to determine the role of FIH-1 in modulating HIF activity, using small interfering RNA knockdown, retroviral gene expression, quantitative RT-PCR, western blot analysis, Annexin V and propidium iodide labelling. RESULTS: Although it was previously suggested that FIH-1 is suppressed in CCRCC, we found that FIH-1 mRNA and protein are actually present at similar levels in CCRCC and normal kidney. The FIH-1 inhibition or knockdown in the VHL defective CCRCC lines RCC10 and RCC4 (which express both HIF-1α and HIF-2α) resulted in increased expression of HIF target genes. In the 786-O CCRCC cell line, which expresses only HIF-2α, FIH-1 attenuation showed no significant effect on expression of these genes; introduction of HIF-1α resulted in sensitivity of HIF targets to FIH-1 knockdown. In RCC4 and RCC10, knockdown of FIH-1 increased apoptosis. Suppressing HIF-1α expression in RCC10 prevented FIH-1 knockdown from increasing apoptosis. CONCLUSION: Our results support a unifying model in which HIF-1α has a tumour suppressor action in CCRCC, held in check by FIH-1. Inhibiting FIH-1 in CCRCC could be used to bias the HIF response towards HIF-1α and decrease tumour cell viability.

Recurrence of complement factor H-related protein 5 nephropathy in a renal transplant.

Complement factor H-related protein 5 (CFHR5) nephropathy is a familial renal disease endemic in Cyprus. It is characterized by persistent microscopic hematuria, synpharyngitic macroscopic hematuria and progressive renal impairment. Isolated glomerular accumulation of complement component 3 (C3) is typical with variable degrees of glomerular inflammation. Affected individuals have a heterozygous internal duplication in the CFHR5 gene, although the mechanism through which this mutation results in renal disease is not understood. Notably, the risk of progressive renal failure in this condition is higher in males than females. We report the first documented case of recurrence of CFHR5 nephropathy in a renal transplant in a 53-year-old Cypriot male. Strikingly, histological changes of CFHR5 nephropathy were evident in the donor kidney 46 days post-transplantation. This unique case demonstrates that renal-derived CFHR5 protein cannot prevent the development of CFHR5 nephropathy.

The hypoxia response pathway and ß-cell function.

ß-cells sense glucose and secrete appropriate amounts of insulin by coupling glucose uptake and glycolysis with quantitative ATP production via mitochondrial oxidative pathways. Therefore, oxidative phosphorylation is essential for normal ß-cell function. Multiple cell types adapt to hypoxia by inducing a transcriptional programme coordinated by the transcription factor hypoxia-inducible factor (HIF). HIF activity is regulated by the von Hippel-Lindau (Vhl) protein, which targets the HIFα subunit for proteasomal degradation in the presence of oxygen. Several recent studies have shown that Vhl deletion in ß-cells results in Hif1α activation, impaired glucose-stimulated insulin secretion (GSIS) and glucose intolerance. This was found to be because of alterations in ß-cell gene expression inducing a switch from aerobic glucose metabolism to anaerobic glycolysis, thus disrupting the GSIS triggering pathway. Situations in which islets may become hypoxic are discussed, in particular islet transplantation which has been reported to cause islet hypoxia because of an inadequate blood supply post-transplant. Aside from this principal role for HIF in negatively regulating ß-cell glucose sensing, other aspects of hypoxia signalling are discussed including ß-cell differentiation, development and vascularization. In conclusion, recent studies clearly show that hypoxia response mechanisms can negatively impact on glucose sensing mechanisms in the ß-cell and this has the potential to impair ß-cell function in a number of physiological and clinical situations.

Mutation of the von Hippel-Lindau gene alters human cardiopulmonary physiology.

Intracellular responses to hypoxia are coordinated by the von Hippel-Lindau--hypoxia-inducible factor (VHL-HIF) transcriptional system. This study investigated the potential role of the VHL-HIF pathway in human systems-level physiology. Patients diagnosed with Chuvash polycythaemia, a rare disorder in which VHL signalling is specifically impaired, were studied during acute hypoxia and hypercapnia. Subjects breathed through a mouthpiece and ventilation was measured while pulmonary vascular tone was assessed echocardiographically. The patients were found to have elevated basal ventilation and pulmonary vascular tone, and ventilatory, pulmonary vasoconstrictive and heart rate responses to acute hypoxia were greatly increased, as were heart rate responses to hypercapnia. The patients also had abnormal pulmonary function on spirometry. This study's findings demonstrate that the VHL-HIF signalling pathway, which is so central to intracellular oxygen sensing, also regulates the organ systems upon which cellular oxygen delivery ultimately depends.

From critters to cancers: bridging comparative and clinical research on oxygen sensing, HIF signaling, and adaptations towards hypoxia.

The objective of this symposium at the First International Congress of Respiratory Biology (ICRB) was to enhance communication between comparative biologists and cancer researchers working on O(2) sensing via the HIF pathway. Representatives from both camps came together on August 13-16, 2006, in Bonn, Germany, to discuss molecular adaptations that occur after cells have been challenged by a reduced (hypoxia) or completely absent (anoxia) supply of oxygen. This brief "critters-to-cancer" survey discusses current projects and new directions aimed at improving understanding of hypoxic signaling and developing therapeutic interventions.

Evolution of VHL tumourigenesis in nerve root tissue.

Haemangioblastomas are the key central nervous system manifestation of von Hippel-Lindau (VHL) disease, which is caused by germline mutation of the VHL gene. We have recently shown that 'tumour-free' spinal cord from patients with VHL disease contains microscopic, poorly differentiated cellular aggregates in nerve root tissue, which we descriptively designated 'mesenchymal tumourlets'. Here we have investigated spinal cord tissue affected by multiple tumours. We show that a small subset of mesenchymal tumourlets extends beyond the nerve root to form proliferative VHL-deficient mesenchyme and frank haemangioblastoma. We thus demonstrate that tumourlets present potential, but true precursor material for haemangioblastoma. We further show that intraradicular tumourlets consist of scattered VHL-deficient cells with activation of HIF-2alpha and HIF-dependent target proteins including CAIX and VEGF, and are associated with an extensive angiogenic response. In contrast, activation of HIF-1alpha was only observed in the later stages of tumour progression. In addition, ultrastructural examination reveals gradual transition from poorly differentiated VHL-deficient cells into vacuolated cells with a 'stromal' cell phenotype. The evolution of frank haemangioblastoma seems to involve multiple steps from a large pool of precursor lesions.

Epididymal cystadenomas and epithelial tumourlets: effects of VHL deficiency on the human epididymis.

Although epididymal cystadenomas (ECAs) are among the most frequent VHL disease-associated tumours, fundamental questions about their pathogenesis have remained unanswered. Classification of ECAs is controversial, and the cell of origin is unknown. It is also unknown whether ECAs-like other VHL disease-associated tumours-arise as a result of VHL gene inactivation, and whether ECAs exhibit subsequent activation of hypoxia-inducible factor HIF. Moreover, the morphological spectrum of earliest ECA formation is unknown. In a detailed molecular pathological analysis of a series of epididymides collected from VHL patients at autopsy, we found that ECAs are true neoplasms that arise secondary to inactivation of the wild-type copy of the VHL gene, followed by early and simultaneous activation of HIF1 and HIF2 associated with up-regulation of downstream targets, including CAIX and GLUT-1. The observations also indicate that ECA formation evolves from a variety of microscopic epithelial tumourlets, and that these tumourlets are confined to the efferent ductular system. Although genetic and immunohistochemical analysis of precursor structures consistently revealed VHL gene inactivation and activation of HIF in the precursor lesions, only a small subset appears to progress into frank cystadenoma. Thus, ECA tumorigenesis in VHL disease shares fundamental principles with tumorigenesis in other affected organ systems.

The HIF pathway: implications for patterns of gene expression in cancer.

Regulation of the growth and metabolism of large organisms is tightly constrained by the need for precise oxygen homeostasis. Work on control of the haematopoietic growth factor erythropoietin has led to the recognition of a widespread transcriptional response to hypoxia which provides insights into how this is achieved. The central mediator of this response is a DNA binding complex termed hypoxia inducible factor 1 (HIF-1), which plays a key role in the regulation by oxygen of a large and rapidly growing panel of genes. In cancer, activity of the HIF system is up-regulated both by microenvironmental hypoxia and by genetic changes. The clearest example of genetic activation is seen in the hereditary cancer syndrome von Hippel-Lindau (VHL) disease. In normal cells the product of the VHL tumour suppressor gene targets the regulatory HIF subunits (HIF-1alpha and HIF-2alpha) for oxygen-dependent proteolysis, acting as the substrate recognition component of an E3 ubiquitin ligase. In pVHL defective cells this process is blocked leading to constitutive up-regulation of HIF-1alpha subunits, activation of the HIF complex and overexpression of HIF target genes. Using gene array screens we have defined a large number of VHL-regulated genes. The majority of these show hypoxia-inducible responses, supporting the central involvement of pVHL in gene regulation by oxygen. In addition to known HIF target genes involved in angiogenesis, glucose metabolism and vasomotor control, these new targets include examples with functions in matrix metabolism, apoptosis, carbon dioxide metabolism and secondary cascades of transcriptional control. Thus activation of HIF provides insights into the classical metabolic alterations in cancer cells, and into the mechanisms by which microenvironmental hypoxia might influence tumour behaviour. In the case of VHL disease, this activation can be linked to mutations in a defined tumour suppressor gene. Equally regulation of the HIF-1alpha/pVHL interaction in normal cells should provide insights into the physiological mechanisms operating in cellular oxygen sensing.

C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation.

HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. Recent studies have defined posttranslational modification by prolyl hydroxylation as a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Here, we define a conserved HIF-VHL-prolyl hydroxylase pathway in C. elegans, and use a genetic approach to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, we show that the HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF.

Hypoxia and oxidative stress in breast cancer. Hypoxia signalling pathways.

Hypoxia-inducible factor-1 (HIF), which is centrally involved in physiological oxygen homeostasis, is also activated in the majority of tumours. Activation of HIF can occur through genetic mechanisms or as a result of hypoxia within the tumour microenvironment. In some cases HIF activation appears to be intimately linked to the proliferative stimulus itself. HIF affects patterns of gene expression and tumour growth, although precise effects vary between tumour types. Modulation of HIF activity, if correctly applied, may be therapeutically beneficial in tumour therapy.

Selection of mutant CHO cells with constitutive activation of the HIF system and inactivation of the von Hippel-Lindau tumor suppressor.

Hypoxia-inducible factor (HIF) mediates a widespread transcriptional response to hypoxia through binding to cis-acting DNA sequences termed hypoxia response elements (HREs). Activity of the transcriptional complex is suppressed in the presence of oxygen by processes that include the targeting of HIF-alpha subunits for ubiquitin-mediated proteolysis. To provide further insights into these processes we constructed Chinese hamster ovary (CHO) cells bearing stably integrated plasmids that expressed HRE-linked surface antigens and used these cells in genetic screens for mutants that demonstrated constitutive up-regulation of HRE activity. From mutagenized cultures, clones were isolated that demonstrated up-regulation of HRE activity and increased HIF-1alpha protein levels in normoxic culture. Transfection and cell fusion studies suggested that these cells possess recessive defects that affect one or more pathways involved in HIF-alpha proteolysis. Two lines were demonstrated to harbor truncating mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. In these cells, defects in ubiquitylation of exogenous human HIF-1alpha in vitro could be complemented by wild type pVHL, and re-expression of a wild type VHL gene restored a normal pattern of HIF/HRE activity, demonstrating the critical dependence of HIF regulation on pVHL in CHO cells. In contrast, other mutant cells had no demonstrable mutation in the VHL gene, and ubiquitylated exogenous HIF-1alpha normally, suggesting that they contain defects at other points in the oxygen-regulated processing of HIF-alpha subunits.

Independent function of two destruction domains in hypoxia-inducible factor-alpha chains activated by prolyl hydroxylation.

Oxygen-dependent proteolytic destruction of hypoxia-inducible factor-alpha (HIF-alpha) subunits plays a central role in regulating transcriptional responses to hypoxia. Recent studies have defined a key function for the von Hippel-Lindau tumour suppressor E3 ubiquitin ligase (VHLE3) in this process, and have defined an interaction with HIF-1 alpha that is regulated by prolyl hydroxylation. Here we show that two independent regions within the HIF-alpha oxygen-dependent degradation domain (ODDD) are targeted for ubiquitylation by VHLE3 in a manner dependent upon prolyl hydroxylation. In a series of in vitro and in vivo assays, we demonstrate the independent and non-redundant operation of each site in regulation of the HIF system. Both sites contain a common core motif, but differ both in overall sequence and in the conditions under which they bind to the VHLE3 ligase complex. The definition of two independent destruction domains implicates a more complex system of pVHL-HIF-alpha interactions, but reinforces the role of prolyl hydroxylation as an oxygen-dependent destruction signal.

The pVHL-associated SCF ubiquitin ligase complex: molecular genetic analysis of elongin B and C, Rbx1 and HIF-1alpha in renal cell carcinoma.

The VHL gene product (pVHL) forms a multimeric complex with the elongin B and C, Cul2 and Rbx1 proteins (VCBCR complex), which is homologous to the SCF family of ubiquitin ligase complexes. The VCBCR complex binds HIF-1alpha and HIF-2alpha, transcription factors critically involved in cellular responses to hypoxia, and targets them for ubiquitin-mediated proteolysis. Germline mutations in the VHL gene cause susceptibility to haemangioblastomas, renal cell carcinoma (RCC), phaeochromocytoma and other tumours. In addition somatic inactivation of the VHL gene occurs in most sporadic clear cell RCC (CC-RCC). However, the absence of somatic VHL inactivation in 30-40% of CC-RCC implies the involvement of other gatekeeper genes in CC-RCC development. We reasoned that in CC-RCC without VHL inactivation, other pVHL-interacting proteins might be defective. To assess the role of elongin B/C, Rbx1 and HIF-1alpha in RCC tumorigenesis we (a) mapped the genes to chromosomes 8q(cen) (elongin C), 16p13.3 (elongin B) and 22q11.2 (Rbx1) by FISH, monochromosomal somatic cell hybrid panel screening and in silico GenBank homology searching; (b) determined the genomic organisation of elongin C (by direct sequencing of PAC clones), Rbx1 and elongin B (by GenBank homology searching); and (c) performed mutation analysis of exons comprising the coding regions of elongins B, C and Rbx1 and the oxygen-dependent degradation domain of HIF-1alpha by SSCP screening and direct sequencing in 35 sporadic clear cell RCC samples without VHL gene inactivation and in 13 individuals with familial non-VHL clear cell RCC. No coding region sequence variations were detected for the elongin B, elongin C or Rbx1 genes. Two amino acid substitutions (Pro582Ser and Ala588Thr) were identified in the oxygen-dependent degradation/pVHL binding domain of HIF-1alpha, however neither substitution was observed exclusively in tumour samples. Association analysis in panels of CC-RCC and non-neoplastic samples using the RFLPs generated by each variant did not reveal allelic frequency differences between RCC patients and controls (P>0.32 by chi-squared analysis). Nevertheless, the significance of these variations and their potential for modulation of HIF-1alpha function merits further investigation in both other tumour types and in non-neoplastic disease. Taken together with our previous Cul2 mutation analysis these data suggest that development of sporadic and familial RCC is not commonly contributed to by genetic events altering the destruction domain of HIF-1alpha, or components of the HIF-alpha destruction complex other than VHL itself. Although (a) activation of HIF could occur through mutation of another region of HIF-a, and (b) epigenetic silencing of elongin B/C, Cul2 or Rbx1 cannot be excluded, these findings suggest that pVHL may represent the sole mutational target through which the VCBR complex is disrupted in CC-RCC. HIF response is activated in CC-RCC tumorigenesis.

Constitutive activation of hypoxia-inducible genes related to overexpression of hypoxia-inducible factor-1alpha in clear cell renal carcinomas.

The transcription factor hypoxia-inducible factor (HIF)-1 is an important mediator of hypoxic adaptation of tumor cells and controls several genes that have been implicated in tumor growth. Oxygen-dependent degradation of HIF-1alpha, the regulatory subunit, requires binding to the von Hippel Lindau (VHL) protein. Because functional inactivation of the VHL tumor suppressor gene occurs in up to 70% of clear cell renal carcinomas, we investigated whether this results in overexpression of HIF-1alpha and its target genes. Immunoblotting revealed increased expression of HIF-1alpha in 24 of 32 (75%) clear cell renal carcinomas but only 3 of 8 non-clear cell renal tumors. Somatic mutations of the VHL gene were detected only in clear cell renal carcinomas that overexpressed HIF-1alpha. None of the HIF-1alpha-negative tumors displayed a VHL mutation. The level of HIF-1alpha mRNA was not different between tumors and adjacent kidney tissue. Immunohistochemistry revealed distinct patterns of nuclear staining for HIF-1alpha, depending on histological type and overall abundance of HIF-1alpha. In those clear cell renal carcinomas that showed increased expression on immunoblots, HIF-1alpha was expressed in almost all cells. In the remaining clear cell and in non-clear cell tumors, staining was focal; these different patterns thus were compatible with genetic stabilization in contrast to microenvironmental stimulation of HIF-1alpha as the primary mechanism. The mRNA expression of two known target genes of HIF-1alpha, vascular endothelial growth factor and glucose transporter 1, increased progressively with increasing amounts of HIF-1alpha in tumor extracts. In addition, glucose transporter 1 protein levels correlated with HIF-1alpha abundance. In conclusion, the data provide in vivo evidence for a constitutive up-regulation of HIF-1alpha in the majority of clear cell renal carcinomas, which leads to more widespread accumulation of this transcription factor than hypoxic stimulation. These observations are most likely linked to functional inactivation of the VHL gene product. Increased expression of HIF-1alpha is associated with alterations in gene expression patterns that are likely to contribute to tumor phenotype and progression.

Insights into the role of the von Hippel-Lindau gene product. A key player in hypoxic regulation.

Many adaptive responses to hypoxia involve changes in gene transcription mediated by the hypoxia-inducible factor 1 complex. Central to this is oxygen-dependent proteolysis of the alpha subunit, which has recently been shown to require the von Hippel-Lindau tumour-suppressor protein. This observation provides one mechanism by which inherited defects in the von Hippel-Lindau gene could cause features of the clinical syndrome, and offers insight into the events leading to sporadic clear cell renal cancer. Furthermore, it clearly implicates the von Hippel-Lindau tumour-suppressor protein in the biochemistry of oxygen sensing.

Activation of the HIF pathway in cancer.

The maintenance of oxygen homeostasis is required both in physiological development and tumour growth. Hypoxia inducible factor (HIF) plays a central role in both processes. Reliable methods for visualising HIF alpha subunits have established that HIF activation occurs in the majority of common cancers. This occurs both by genetic mechanisms and through microenvironmental hypoxia. Activation of the HIF pathway has important effects on patterns of gene expression in tumours.

Contrasting effects on HIF-1alpha regulation by disease-causing pVHL mutations correlate with patterns of tumourigenesis in von Hippel-Lindau disease.

The von Hippel-Lindau tumour suppressor gene product (pVHL) associates with the elongin B and C and Cul2 proteins to form a ubiquitin-ligase complex (VCBC). To date, the only VCBC substrates identified are the hypoxia-inducible factor alpha subunits (HIF-1alpha and HIF-2alpha). However, pVHL is thought to have multiple functions and the significance of HIF-1alpha and HIF-2alpha regulation for tumour suppressor activity has not been defined. VHL disease is characterized by distinct clinical subtypes. Thus haemangioblastomas (HABs) and renal cell carcinoma (RCC) but not phaeochromocytoma (PHE) occur in type 1 VHL disease. Type 2 subtypes are characterized by PHE susceptibility but differ with respect to additional tumours (type 2A, PHE+HAB but not RCC; type 2B, PHE+ HAB+RCC; type 2C, PHE only). We investigated in detail the effect of 13 naturally occurring VHL mutations (11 missense), representing each phenotypic subclass, on HIF-alpha subunit regulation. Consistent effects on pVHL function were observed for all mutations within each subclass. Mutations associated with the PHE-only phenotype (type 2C) promoted HIF-alpha ubiquitylation in vitro and demonstrated wild-type binding patterns with pVHL interacting proteins, suggesting that loss of other pVHL functions are necessary for PHE susceptibility. Mutations causing HAB susceptibility (types 1, 2A and 2B) demonstrated variable effects on HIF-alpha subunit and elongin binding, but all resulted in defective HIF-alpha regulation and loss of p220 (fibronectin) binding. All RCC-associated mutations caused complete HIF-alpha dysregulation and loss of p220 (fibronectin) binding. Our findings are consistent with impaired ability to degrade HIF-alpha subunit being required for HAB development and RCC susceptibility.