Hypoxia-Inducible Factor Linked to Differential Kidney Cancer Risk Seen with Type 2A and Type 2B VHL Mutations

ABSTRACT Clear cell carcinoma of the kidney is a major cause of mortality in patients with von Hippel-Lindau (VHL) disease, which is caused by germ line mutations that inactivate the VHL tumor suppressor gene. Biallelic VHL inactivation, due to mutations or hypermethylation, is also common in sporadic clear cell renal carcinomas. The VHL gene product, pVHL, is part of a ubiquitin ligase complex that targets the alpha subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF) for destruction under well-oxygenated conditions. All VHL mutations linked to classical VHL disease compromise this pVHL function although some missense mutations result in a low risk of kidney cancer (type 2A VHL disease) while others result in a high risk (type 2B VHL disease). We found that type 2A mutants were less defective than type 2B mutants when reintroduced into VHL −/− renal carcinoma cells with respect to HIF regulation. A stabilized version of HIF2α promoted tumor growth by VHL −/− cells engineered to produce type 2A mutants, while knock-down of HIF2α in cells producing type 2B mutants had the opposite effect. Therefore, quantitative differences with respect to HIF deregulation are sufficient to account for the differential risks of kidney cancer linked to VHL mutations.

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Inactivation of the Arylhydrocarbon Receptor Nuclear Translocator (Arnt) Suppresses von Hippel-Lindau Disease-Associated Vascular Tumors in Mice

Molecular and Cellular Biology ◽

10.1128/mcb.25.8.3163-3172.2005 ◽

2005 ◽

Vol 25 (8) ◽

pp. 3163-3172 ◽

Cited By ~ 112

Author(s):

Erinn B. Rankin ◽

Debra F. Higgins ◽

Jacqueline A. Walisser ◽

Randall S. Johnson ◽

Christopher A. Bradfield ◽

...

Keyword(s):

Transcription Factors ◽

Germ Line ◽

Hypoxia Inducible Factor ◽

Suppressor Gene ◽

Vascular Tumors ◽

Von Hippel Lindau ◽

Von Hippel Lindau Disease ◽

Arylhydrocarbon Receptor ◽

Vhl Disease ◽

Endothelial Growth Factor

ABSTRACT Patients with germ line mutations in the VHL tumor suppressor gene are predisposed to the development of highly vascularized tumors within multiple tissues. Loss of pVHL results in constitutive activation of the transcription factors HIF-1 and HIF-2, whose relative contributions to the pathogenesis of the VHL phenotype have yet to be defined. In order to examine the role of HIF in von Hippel-Lindau (VHL)-associated vascular tumorigenesis, we utilized Cre-loxP-mediated recombination to inactivate hypoxia-inducible factor-1α (Hif-1α) and arylhydrocarbon receptor nuclear translocator (Arnt) genes in a VHL mouse model of cavernous liver hemangiomas and polycythemia. Deletion of Hif-1α did not affect the development of vascular tumors and polycythemia, nor did it suppress the increased expression of vascular endothelial growth factor (Vegf) and erythropoietin (Epo). In contrast, phosphoglycerokinase (Pgk) expression was substantially decreased, providing evidence for target gene-dependent functional redundancy between different Hif transcription factors. Inactivation of Arnt completely suppressed the development of hemangiomas, polycythemia, and Hif-induced gene expression. Here, we demonstrate genetically that the development of VHL-associated vascular tumors in the liver depends on functional ARNT. Furthermore, we provide evidence that individual HIF transcription factors may play distinct roles in the development of specific VHL disease manifestations.

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Molecular pathology of von Hippel–Lindau disease and the VHL tumour suppressor gene

Expert Reviews in Molecular Medicine ◽

10.1017/s1462399401002654 ◽

2001 ◽

Vol 3 (8) ◽

pp. 1-27 ◽

Cited By ~ 27

Author(s):

Frances M. Richards

Keyword(s):

Hypoxia Inducible Factor ◽

Suppressor Gene ◽

Tumour Suppressor Gene ◽

Tumour Suppressor ◽

Missense Mutations ◽

Cancer Syndrome ◽

Inherited Cancer ◽

Von Hippel Lindau ◽

Adrenal Chromaffin ◽

Vhl Disease

von Hippel–Lindau (VHL) disease is a dominantly inherited cancer syndrome characterised by predisposition to multiple tumours of the eyes and central nervous system (haemangioblastomas), kidneys (renal cell carcinoma; RCC), adrenal chromaffin cells (phaeochromocytoma), and other organs. The VHL gene was isolated in 1993 and mutations or deletions in the VHL gene have been identified in the germline of nearly all tested individuals with VHL disease. Genotype–phenotype correlations have been observed: individuals with missense mutations are more likely to develop phaeochromocytoma than those with deletions or protein-truncating mutations are, and specific missense mutations at certain codons might not predispose to RCC. In accordance with its role as a tumour suppressor gene, the normal allele of the VHL gene is deleted, mutated or silenced by promoter methylation in the tumours from VHL patients, and in a large proportion of sporadic tumours of the same histological types as observed in VHL disease. Thus, the VHL gene is of major importance in the development of RCC in the general population. Recent advances in understanding the structure and function of the VHL protein (pVHL) have revealed insights into the different phenotypes, with indications that some retention of function might be required for predisposition to phaeochromocytoma. pVHL interacts with many cellular proteins, mainly via one of two protein-binding domains (α and β). The best-characterised interaction is that of pVHL with elongin C, which forms a complex with elongin B and Cullin 2 proteins. This complex has E3 ubiquitin ligase activity and promotes ubiquitin-mediated proteasomal degradation of the hypoxia-inducible factor 1α (HIF-1α) transcription factor under normal oxygen (normoxic) conditions. Loss of pVHL function leads to stabilisation of HIF-1 and expression under normoxic conditions of hypoxia-inducible genes including vascular endothelial growth factor (VEGF), which might explain the hypervascular phenotype of VHL tumours. Several other genes implicated in intra- and intercellular signalling and control of tumour growth are overexpressed in the absence of pVHL, but it is not yet clear which features of pVHL function are most significant for tumour suppression in different tissues. Further advances in understanding pVHL function might eventually enable development of specific therapies for prevention or treatment of VHL tumours and RCC.

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Downregulation of integrins by von Hippel-Lindau (VHL) tumor suppressor protein is independent of VHL-directed hypoxia-inducible factor alpha degradation

Biochemistry and Cell Biology ◽

10.1139/o08-035 ◽

2008 ◽

Vol 86 (3) ◽

pp. 227-234 ◽

Cited By ~ 12

Author(s):

Qingzhou Ji ◽

Robert D. Burk

Keyword(s):

Tumor Suppressor ◽

Clear Cell ◽

Hypoxia Inducible Factor ◽

Intercellular Junctions ◽

Suppressor Gene ◽

Wild Type ◽

Von Hippel Lindau ◽

Degradation Activity ◽

Factor Alpha ◽

Vhl Tumor Suppressor Protein

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene occurs in the majority of clear-cell renal cell carcinomas (RCCs). It was previously shown that VHL decreased the abundance of integrins α2, α5, and β1, which is consistent with VHL-associated changes in cell–cell and cell – extracellular matrix adhesions. We investigated the mechanism by which VHL downregulates integrins. Although VHL can target hypoxia-inducible factor alpha (HIFα) subunits for degradation, VHL-dependent reduction of integrins was independent of O2 concentration and HIFα levels. VHL reduced the half-lives of integrins, and this activity was blocked by proteasomal inhibition. Although ectopically expressed FLAG-VHL retained HIFα degradation activity, it neither downregulated integrins nor promoted adherens and tight intercellular junctions, in contrast to expressed wild-type VHL. Moreover, integrins co-immunoprecipitated with wild-type VHL, but not FLAG-VHL. These data indicate that the downregulation of integrins by VHL is distinct from the regulation of HIFα subunits by VHL, and suggests that the loss of this activity contributes to VHL-associated RCC development through disruption of adherens and tight junctions.

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363: Somatic Mutations of the Von Hippel-Lindau (VHL) Tumor Suppressor Gene, Expression of Hypoxia Inducible Factor-α and Vascula Endothelial Growth Factor in Chinese Sporadic Clear Cell Renal Cell Carcinomas and Their Relationships to Angiogenesis

The Journal of Urology ◽

10.1016/s0022-5347(18)34616-0 ◽

2005 ◽

Vol 173 (4S) ◽

pp. 99-99

Author(s):

Kan Gong ◽

Ning Zhang ◽

Yanqun Na

Keyword(s):

Gene Expression ◽

Growth Factor ◽

Somatic Mutations ◽

Clear Cell ◽

Hypoxia Inducible Factor ◽

Suppressor Gene ◽

Renal Cell Carcinomas ◽

Von Hippel Lindau ◽

Factor Α ◽

Endothelial Growth Factor

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pVHL Modification by NEDD8 Is Required for Fibronectin Matrix Assembly and Suppression of Tumor Development

Molecular and Cellular Biology ◽

10.1128/mcb.24.8.3251-3261.2004 ◽

2004 ◽

Vol 24 (8) ◽

pp. 3251-3261 ◽

Cited By ~ 128

Author(s):

Natalie H. Stickle ◽

Jacky Chung ◽

Jeffery M. Klco ◽

Richard P. Hill ◽

William G. Kaelin ◽

...

Keyword(s):

Hypoxia Inducible Factor ◽

Tumor Development ◽

Three Dimensional ◽

Suppressor Gene ◽

Matrix Assembly ◽

Von Hippel Lindau ◽

Fibronectin Matrix ◽

Matrix Expression ◽

Vhl Disease ◽

Hif Pathway

ABSTRACT Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is the cause of the familial VHL disease and most sporadic renal clear-cell carcinomas (RCC). pVHL has been shown to play a role in the destruction of hypoxia-inducible factor α (HIF-α) subunits via ubiquitin-mediated proteolysis and in the regulation of fibronectin matrix assembly. Although most disease-causing pVHL mutations hinder the regulation of the HIF pathway, every disease-causing pVHL mutant tested to date has failed to promote the assembly of the fibronectin matrix, underscoring its potential importance in VHL disease. Here, we report that a ubiquitin-like molecule called NEDD8 covalently modifies pVHL. A nonneddylateable pVHL mutant, while retaining its ability to ubiquitylate HIF, failed to bind to and promote the assembly of the fibronectin matrix. Expression of the neddylation-defective pVHL in RCC cells, while restoring the regulation of HIF, failed to promote the differentiated morphology in a three-dimensional growth assay and was insufficient to suppress the formation of tumors in SCID mice. These results suggest that NEDD8 modification of pVHL plays an important role in fibronectin matrix assembly and that in the absence of such regulation, an intact HIF pathway is insufficient to prevent VHL-associated tumorigenesis.

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The role of von Hippel-Lindau tumor suppressor protein and hypoxia in renal clear cell carcinoma

AJP Renal Physiology ◽

10.1152/ajprenal.00424.2003 ◽

2004 ◽

Vol 287 (1) ◽

pp. F1-F6 ◽

Cited By ~ 38

Author(s):

Roxana I. Sufan ◽

Michael A. S. Jewett ◽

Michael Ohh

Keyword(s):

Tumor Suppressor ◽

Molecular Mechanisms ◽

Clear Cell ◽

Hypoxia Inducible Factor ◽

Suppressor Gene ◽

Renal Clear Cell Carcinoma ◽

Von Hippel Lindau ◽

Ubiquitin Ligase Complex ◽

Vhl Protein

The majority of kidney cancers are caused by the mutation of the von Hippel-Lindau ( VHL) tumor suppressor gene. VHL protein (pVHL) is part of an E3 ubiquitin ligase complex called VEC that is composed of elongin B, elongin C, cullin 2, NEDD8, and Rbx1. VEC targets a hypoxia-inducible factor (HIF) transcription factor for ubiquitin-mediated destruction selectively in the presence of oxygen. In the absence of wild-type pVHL, as in VHL patients or in the majority of sporadic clear cell renal cell carcinomas, HIF-responsive genes are inappropriately activated even under normoxia. Recent insights into the molecular mechanisms regulating the function of pVHL, and thereby HIF, in the context of kidney cancer are the focus of this review.

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Parallel Regulation of von Hippel-Lindau Disease by pVHL-Mediated Degradation of B-Myb and Hypoxia-Inducible Factor α

Molecular and Cellular Biology ◽

10.1128/mcb.00067-16 ◽

2016 ◽

Vol 36 (12) ◽

pp. 1803-1817 ◽

Cited By ~ 10

Author(s):

Fumihiko Okumura ◽

Keiji Uematsu ◽

Stuart D. Byrne ◽

Mie Hirano ◽

Akiko Joo-Okumura ◽

...

Keyword(s):

Growth Factor ◽

Critical Role ◽

Control Cell ◽

Hypoxia Inducible Factor ◽

Suppressor Gene ◽

Protein Product ◽

Von Hippel Lindau ◽

Microarray Screening ◽

Factor Α ◽

Vhl Disease

pVHL, the protein product of the von Hippel-Lindau (VHL) tumor suppressor gene, is a ubiquitin ligase that targets hypoxia-inducible factor α (HIF-α) for proteasomal degradation. Although HIF-α activation is necessary for VHL disease pathogenesis, constitutive activation of HIF-α alone did not induce renal clear cell carcinomas and pheochromocytomas in mice, suggesting the involvement of an HIF-α-independent pathway in VHL pathogenesis. Here, we show that the transcription factor B-Myb is a pVHL substrate that is degraded via the ubiquitin-proteasome pathway and that vascular endothelial growth factor (VEGF)- and/or platelet-derived growth factor (PDGF)-dependent tyrosine 15 phosphorylation of B-Myb prevents its degradation. Mice injected with B-Myb knockdown 786-O cells developed dramatically larger tumors than those bearing control cell tumors. Microarray screening of B-Myb-regulated genes showed that the expression of HIF-α-dependent genes was not affected by B-Myb knockdown, indicating that B-Myb prevents HIF-α-dependent tumorigenesis through an HIF-α-independent pathway. These data indicate that the regulation of B-Myb by pVHL plays a critical role in VHL disease.

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Germline VHL gene mutations in Hungarian families with von Hippel–Lindau disease and patients with apparently sporadic unilateral pheochromocytomas

Acta Endocrinologica ◽

10.1530/eje-09-0399 ◽

2009 ◽

Vol 161 (3) ◽

pp. 495-502 ◽

Cited By ~ 13

Author(s):

Peter Gergics ◽

Attila Patocs ◽

Miklos Toth ◽

Peter Igaz ◽

Nikolette Szucs ◽

...

Keyword(s):

Direct Sequencing ◽

Gene Mutations ◽

Suppressor Gene ◽

Genetic Alterations ◽

Missense Mutations ◽

Gene Deletions ◽

Von Hippel Lindau ◽

Hereditary Tumor Syndrome ◽

Large Gene ◽

Vhl Disease

ObjectiveVon Hippel–Lindau (VHL) disease is a hereditary tumor syndrome caused by mutations or deletions of theVHLtumor-suppressor gene. GermlineVHLgene alterations may be also present in patients with apparently sporadic pheochromocytoma (ASP), although a wide variation in mutation frequencies has been reported in different patient cohorts.DesignHerein, we report the analysis of theVHLgene in Hungarian families with VHL disease and in those with ASP.MethodsSeven families (35 members) with VHL disease and 37 unrelated patients with unilateral ASP were analyzed. Patients were clinically evaluated and theVHLgene was analyzed using direct sequencing, multiplex ligation-dependent probe amplification, and real-time PCR with SYBR Green chemistry.ResultsDisease-causing genetic abnormalities were identified in each of the seven VHL families and in 3 out of the 37 patients with ASP (one nonsense and six missense mutations, two large gene deletions and one novel 2 bp deletion). Large gene deletions and other genetic alterations resulting in truncated VHL protein were found only in families with VHL type 1, whereas missense mutations were associated mainly, although not exclusively, with VHL type 2B and type 2C.ConclusionsThe spectrum ofVHLgene abnormalities in the Hungarian population is similar to that observed in Western, Japanese, or Chinese VHL kindreds. The presence ofVHLgene mutations in 3 out of the 37 patients with ASP suggests that genetic testing is useful not only in patients with VHL disease but also in those with ASP.

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Key Role for Activin B in Cellular Transformation after Loss of the von Hippel-Lindau Tumor Suppressor

Molecular and Cellular Biology ◽

10.1128/mcb.01184-07 ◽

2009 ◽

Vol 29 (7) ◽

pp. 1707-1718 ◽

Cited By ~ 17

Author(s):

Ingrid Wacker ◽

Martin Sachs ◽

Karl Knaup ◽

Michael Wiesener ◽

Jörg Weiske ◽

...

Keyword(s):

Tumor Suppressor ◽

Transforming Growth Factor ◽

Clear Cell ◽

Cell Transformation ◽

Hypoxia Inducible Factor ◽

Cellular Transformation ◽

Transforming Growth Factor Β ◽

Suppressor Gene ◽

Normal Kidney ◽

Von Hippel Lindau

ABSTRACT The von Hippel-Lindau tumor suppressor gene (VHL) is mutated in clear cell renal cell carcinomas (RCC), leading to the activation of hypoxia-inducible factor (HIF)-mediated gene transcription. Several VHL/HIF targets, such as glycolysis, angiogenesis, cell growth, and chemotaxis of tumor cells, have been implicated in the transformed phenotype of RCC-regulating properties. Here, we show that VHL suppresses key features of cell transformation through downregulation of the HIF-dependent expression of activin B, a member of the transforming growth factor β superfamily. Activin B expression is repressed by restoration of VHL in VHL-deficient RCC cells and upregulated by hypoxia. RCC tumor samples show increased expression of activin B compared to that in the normal kidney. VHL increases cell adhesion to the extracellular matrix, promotes cell flattening, and reduces invasiveness. These effects are completely phenocopied by RNA interference-mediated knockdown of activin B and reverted by treatment with recombinant activin B. Finally, knockdown of activin B reduces tumor growth of RCC cells in nude mice. Our data indicate that activin B is a key mediator of VHL/HIF-induced transformation in RCC.

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