MOLECULAR BASIS OF THE VHL HEREDITARY CANCER SYNDROME

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1 MOLECULAR BASIS OF THE VHL HEREDITARY CANCER SYNDROME William G. Kaelin, Jr The von Hippel Lindau hereditary cancer syndrome was first described about 100 years ago. The unusual clinical features of this disorder predicted a role for the von Hippel Lindau gene (VHL) in the oxygen-sensing pathway. Indeed, recent studies of this gene have helped to decipher how cells sense changes in oxygen availability, and have revealed a previously unappreciated role of prolyl hydroxylation in intracellular signalling. These studies, in turn, are laying the foundation for the treatment of a diverse set of disorders, including cancer, myocardial infarction and stroke. PHAEOCHROMOCYTOMA A neuroendocrine tumour that typically arises in the adrenal medulla. These tumours can be benign or malignant. Symptoms often relate to the ability of these tumours to secrete catecholamines. LASER PHOTOCOAGULATION A process in which a laser is focused on a specific area of the retina. Localized coagulation is induced by the conversion of light energy to heat. RETINAL DETACHMENT A condition in which the retina the specialized lining inside the eye that is responsible for transducing light signals detaches from the underlying layers of the eye. Howard Hughes Medical Institute, Dana Farber Cancer Institute and Brigham and Women s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. william_kaelin@dfci.harvard. edu doi: /nrc885 The study of human hereditary cancer syndromes, including syndromes that are relatively rare, has often provided important insights into common, non-hereditary malignancies. Moreover, the genes that are linked to hereditary cancer syndromes are almost invariably important in fundamental cellular processes such as cell division, apoptosis and DNA repair. These principles are illustrated well by recent studies of the von Hippel Lindau (VHL) hereditary cancer syndrome. The gene (VHL) that, when mutated in the germ line, causes this syndrome is also frequently inactivated in clear-cell carcinoma of the kidney, which affects approximately 30,000 people in the United States every year. Moreover, studies of the VHL gene product, pvhl, have helped to elucidate how mammalian cells sense and respond to changes in oxygen availability. Careful clinical observations made almost a century ago, in conjunction with recent advances in molecular biology, have given rise to a more detailed understanding of the mammalian oxygen-sensing pathway and the potential role of this pathway in cancer, heart attacks and stroke. Clinical description The first reports of patients who (it is now known) had VHL disease were published in the medical literature approximately 100 years ago. Treacher Collins and, later, Eugene von Hippel described families in which individuals developed angiomas bloodvessel tumours of the retina 1,2 (FIG. 1a,b). Later, the neuropathologist Arvind Lindau reported that such patients were also at high risk of developing bloodvessel tumours of the brain (especially the cerebellum) and spinal cord, known as haemangioblastomas 3 (FIG. 1c). Retinal angiomas are histologically indistinguishable from haemangioblastomas, and so are also sometimes referred to as haemangioblastomas 4.The vascular nature of these tumours is easiest to appreciate following injection of a contrast agent such as fluorescein (FIG. 1b). A variety of other tumours have been associated with VHL disease, including clear-cell carcinoma of the kidney (FIG. 1d), PHAEOCHROMOCYTOMA, endolymphatic-sac tumours and islet-cell tumours of the pancreas 5. In addition, visceral cysts (especially of the pancreas and kidney) are also common. VHL disease affects approximately 1 in 35,000 individuals, and transmission of the disease seems to occur in an autosomal-dominant manner (see below). Symptoms typically develop in the second, third and fourth decades of life. Retinal haemangioblastomas can usually be treated with LASER PHOTOCOAGULATION if detected early, but they can cause significant problems, including RETINAL DETACHMENT and blindness, if they are undiagnosed or neglected. Lesions involving the optic nerve or MACULA are particularly problematic, as these areas are not amenable to laser or surgical intervention. Cerebellar and spinal hemangioblastomas are often treated surgically, although the presence of these tumours and the attempts to remove them frequently lead to neurological defects, including weakness or NATURE REVIEWS CANCER VOLUME 2 SEPTEMBER

2 MACULA A specific area of the retina that corresponds to the posterior pole of the eyeball in the visual axis. At its centre is the fovea centralis, which is crucial for visual acuity. DIALYSIS A process for treating patients with inadequate kidney function. Haemodialysis involves exposing blood to solute across a semipermeable membrane. Peritoneal dialysis involves instilling and removing large volumes of solute in the peritoneal cavity. Dialysis allows the removal of toxic substances from the blood and the correction of certain electrolyte imbalances. a c Summary The von Hippel Lindau (VHL) disease is caused by the germ-line mutation of the VHL tumour-suppressor gene. Kidney cancer and blood-vessel tumours (haemangioblastomas) of the central nervous system, are the two leading causes of morbidity and mortality in VHL disease. Somatic VHL mutations are also common in sporadic haemangioblastoma and kidney cancer. The VHL gene product, pvhl, is a component of an SCF (Skp1 Cdc53 F-box)-like ubiquitin-ligase complex that targets the α-subunits of the hypoxia-inducible factor (HIF) heterodimeric transcription factor for polyubiquitylation and proteasomal degradation. pvhl recognizes the HIF α-subunits only after specific proline residues within these subunits are hydroxylated by members of the EGLN family. This, and the fact that the hydroxylation is inherently oxygen dependent, is integral to how mammalian cells sense and respond to changes in oxygen. Overproduction of growth factors encoded by HIF target genes, such as vascular endothelial growth factor (VEGF), platelet-derived growth-factor B chain (PDGFβ) and transforming growth-factor-α (TGFα) probably contribute to tumour formation following pvhl inactivation. paralysis. Surgical resection of multiple and bilateral renal tumours frequently leads to renal failure and the need for DIALYSIS. Also, haemangioblastoma and renal carcinoma are the two main causes of mortality in this disorder; although the age at which patients die is highly variable, many of them do so before the age of 50. Genetics of VHL disease Linkage studies carried out in the late 1980s indicated that the VHL gene resides on chromosome 3p25, which is a region of the genome that is frequently deleted in sporadic (non-hereditary) kidney cancer 6. Using this positional information, a consortium headed by Eamon Maher (then at the University of Cambridge, United Figure 1 von Hippel Lindau-associated tumours. a Fundoscopic view of a retinal haemangioblastoma (arrow). The optic nerve is seen on the upper left. b Fluorescein angiogram showing multiple retinal haemangioblastomas (arrows). c Sagittal magnetic resonance image of a large cystic cerebellar haemangioblastoma (white arrow). Magnetic resonance imaging shows the solid component of the tumour as an enhancing (bright) lesion (yellow arrow). d Renal carcinoma (yellow arrow) arising in a large renal cyst (white arrow). Photographs courtesy of Alain Gaudric, David Goldfarb and Mika Niemela. b d Kingdom), Michael Lerman, Marston Linehan and Bert Zbar (National Cancer Institute, United States) isolated the VHL gene in 1993 (REF. 7). The gene is relatively small, consisting of three exons, and is conserved in rodents, flies and worms VHL disease, like most human hereditary cancer syndromes, is caused by the germ-line mutation of a tumour-suppressor gene, and conforms to the KNUDSON 2-HIT MODEL. Individuals with VHL disease have typically inherited a defective VHL allele from one of their parents. Pathology, such as the development of cysts or tumours, is linked to the somatic inactivation or loss of the remaining wild-type VHL allele. Essentially all individuals who have been clinically diagnosed with VHL disease can be shown to harbour a VHL mutation if, in addition to DNA sequence analysis, one includes assays (such as semiquantitative Southern blot analysis) that can detect complete loss of the maternal or paternal VHL locus in the germ line 11. Some genotype phenotype correlations are emerging in VHL disease (TABLE 1). Most striking is the fact that VHL families with a high risk of phaeochromocytoma (type 2 VHL disease) almost invariably harbour a VHL missense mutation Mutations that lead to complete loss of the VHL gene product (pvhl), or mutations that are predicted to grossly disrupt the folding of pvhl, are associated with a markedly reduced risk of phaeochromocytoma (type 1 VHL disease). Together, these observations have led to the speculation that phaeochromocytoma development in the setting of VHL disease reflects a pvhl gain of function, or that phaeochromocytoma development requires partial, but not complete, loss of pvhl function. Type 2 VHL families might have a low risk (type 2A) or high risk (type 2B) of kidney cancer. Also noteworthy is the fact that some families with VHL disease have an increased risk of phaeochromocytoma without the other classical stigmata of VHL disease (type 2C VHL disease) (see below). In keeping with the Knudson 2-hit model, VHL inactivation is also a common feature of sporadic clear-cell carcinomas and haemangioblastomas In these settings, inactivation of the maternal and paternal VHL alleles occurs as a result of somatic 674 SEPTEMBER 2002 VOLUME 2

3 KNUDSON 2-HIT MODEL In 1971, Knudson noted that the differences between hereditary and sporadic retinoblastoma, with respect to age-specific incidence and propensity for multifocality, could be accounted for if the development of retinoblastoma required two rate-limiting mutations ( hits ) and if one of these hits had already occurred in the germ line of the hereditary patients. Later, he speculated that the two hits might be the inactivation of the maternal and paternal copies of a tumoursuppressor gene a prediction that was later proved to be correct. DOMINANT NEGATIVE A defective protein that inhibits the function of its wild-type counterpart. RAN A small GTP-binding protein that is a key component of the nuclear cytoplasmic transport machinery. CULLIN A member of a family of proteins first identified in Caenorhabditis elegans that has a similar amino-acid sequence to Cdc53 in yeast. These proteins are integral components of SCF (Skp1 Cdc53 F-Box)-like ubiquitin-ligase complexes. In this context, they serve as a bridge between the substraterecognition and ubiquitinconjugation components of the complex. F-BOX PROTEIN A protein that contains a collinear motif first identified in cyclin F. This motif allows binding to Skp1-like proteins, and hence recruitment of F-box proteins to SCF (Skp1 Cdc53 F-box) complexes. F-box proteins recognize specific substrates and present them for polyubiquitylation by other components of the SCF complex. Table 1 Characteristics of different types of VHL disease Type of VHL disease VHL mutation type Molecular defect Clinical manifestation Type 1 Loss of VHL or a mutation Upregulation of HIF-α Haemangioblastomas that affects protein folding and HIF target genes Diminshed risk of phaeochromocytoma Renal-cell carcinoma Type 2A VHL missense mutation Upregulation of HIF-α Haemangioblastomas and HIF target genes Phaeochromocytoma Low risk of renal-cell carcinoma Type 2B VHL missense mutation Upregulation of HIF-α Haemangioblastomas and HIF target genes Phaeochromocytoma High risk of renal-cell carcinoma Type 2C VHL missense mutation pvhl retains ability to Pheochromocytoma only degrade HIF-α; decreased binding to fibronectin fibronectinmatrix-assembly defect HIF, hypoxia-inducible factor; VHL, von Hippel Lindau. mutation or hypermethylation. The spectrum of VHL mutations observed in hereditary and sporadic kidney cancer might differ, although this is debated 17,27. It is possible that some VHL mutations cannot be transmitted via the germ line, because of DOMINANT-NEGATIVE or gain-of-function effects. Surprisingly, somatic VHL mutations are rare in sporadic pheochromocytomas, despite the fact that some VHL mutations predispose to pheochromocytoma 17, The VHL protein The VHL mrna encodes a protein of 213 amino-acid residues, which migrates with an apparent molecular weight of ~24 30 kda 34. A second protein isoform, with an apparent molecular weight of ~19 kda, is produced as a result of internal translation initiation from an inframe ATG at codon 54 (REFS 35 37). This smaller form of the pvhl protein seems to predominate in many tissues. At present, it is not known why cells make two forms of the pvhl protein, and the two isoforms behave fairly similarly in the biochemical and functional assays performed so far. Moreover, almost all diseaseassociated VHL mutations map carboxy-terminal to codon 54, indicating that VHL disease requires the simultaneous inactivation of both the long and short forms of pvhl. For simplicity, the term pvhl is used when referring to these two proteins generically. Biochemical fractionation and immunohistochemical studies, coupled with the analysis of pvhl green fluorescent protein fusion proteins in living cells, indicate that pvhl shuttles back and forth between the cytosol and nucleus, with most of the protein located in the cytosol 34, This shuttling is dependent on RAN, and requires ongoing transcription by RNA polymerase II; it might relate to the role of pvhl in polyubiquitylation, as described below. The primary sequence of pvhl neither closely resembles other known proteins nor reveals obvious structural motifs that might provide clues as to its function. However, a frequently mutated subdomain of pvhl binds to a protein called elongin C, which, in turn, nucleates a complex containing elongin B and cullin-2 (CUL2) (REFS 43 49; FIG. 2). As first noted by Steven Elledge and co-workers, the primary sequences of elongin C and CUL2 (a member of the CULLIN family) resemble the yeast proteins Skp1 and Cdc53, respectively 50, which bind to an F-BOX PROTEIN (so named because of a collinear motif first identified in cyclin F) to form an SCF (Skp1 Cdc53 F-box) ubiquitin-ligase (or E3) complex. This can catalyse the polyubiquitylation of a specific protein or proteins 51 (FIG. 2). In these complexes, the F-box protein serves as the substrate-recognition module, or specificity determinant. By analogy, therefore, a model emerged in which pvhl might through its association with elongin C and CUL2 target specific proteins for polyubiquitylation. Several lines of evidence have strengthened this view. First, the crystal structure of pvhl bound to elongin B and elongin C revealed that the structure of elongin C, as predicted, resembled that of Skp1, and that the pvhl elongin-c-binding domain (now called the α-domain) resembled an F-box 52. The crystal structure of pvhl also revealed a second frequently mutated subdomain, called the β-domain, that had the features of a substrate-docking site. Elongin B β CUL2 Elongin C α pvhl Target protein Skp1 F F-box CDC53 Ub Ub Ub Target destroyed Proteasome Figure 2 SCF-like ubiquitin ligases. The VHL (von Hippel Lindau) gene product, pvhl, forms complexes with elongin C, elongin B and cullin-2, which structurally resemble SCF (Skp1 Cdc53 F-Box protein) ubiquitin ligases in yeast. These polyubiquitylate target proteins that are then degraded by the proteasome. The pvhl α-domain resembles an F-box motif, which binds to Skp1. The pvhl β-domain has features of a substrate-docking site. Ub, ubiquitin. NATURE REVIEWS CANCER VOLUME 2 SEPTEMBER

4 Hypoxia Normoxia Hydroxyproline Hydroxyasparagine HIF-α HYPOXIA-INDUCIBLE GENES A gene for which mrna abundance is markedly enhanced under conditions of low oxygen. In the laboratory, low-oxygen conditions are usually achieved with environments that contain 1% oxygen or less. Air contains 21% oxygen. P300 AND CBP Two large nuclear proteins that serve as co-activators when bound to DNA through other transcription factors. p300/cbp EGLN, FIH1 O 2 NTAD Elongin B Oxygen-dependent degradation domain pvhl p300/cbp CTAD Elongin C CUL2 Activate transcription Elongin B Elongin C pvhl CUL2 Second, anti-pvhl immunoprecipitates were found to contain ubiquitin-ligase activity if supplemented with a ubiquitin-conjugating enzyme (or E2) 53,54. Finally, a protein implicated in SCF ubiquitin ligase function named Rbx1 (also known as ROC1 or Hrt1) was also found to associate with pvhl elongin CUL2 complexes 55. Collectively, these findings strongly indicated that pvhl regulated the polyubiquitylation of specific proteins. Control of HIF and gene expression by pvhl Haemangioblastomas and renal-cell carcinomas share two characteristics, the second of which (see below) is also shared by phaeochromocytomas. First, both tumours are highly vascular and are known to overproduce the angiogenic peptide vascular endothelial growth factor (VEGF). Second, the three tumour types can produce erythropoietin (EPO), which leads to increases in red-blood-cell count (polycythaemia). VEGF and EPO are both encoded by HYPOXIA-INDUCIBLE GENES. On the basis of these clues, several groups went on to show that pvhl-defective cells produce high levels of hypoxia-inducible mrnas (such as the VEGF mrna), irrespective of changes in ambient oxygen, and that this defect could be corrected by the restoration of pvhl function So, a molecular signature of pvhl inactivation is the finding that hypoxia-inducible gene expression has been uncoupled from changes in oxygen availability. β HIF-α polyubiquitylated and destroyed Figure 3 Regulation of HIF-α by pvhl. In hypoxic conditions, the transcriptional co-activators p300 and camp-response-element-binding protein (CREB)-binding protein (CBP) bind to the carboxy-terminal transactivation domain (CTAD) of hypoxia-inducible factor-α (HIF-α) to activate transcription of HIF-regulated genes. However, in normoxic conditions, an asparagine in the CTAD is hydroxylated by FIH1 (factor inhibiting HIF1), which prevents this binding. Proline residues in the oxygen-dependent degradation domain, which is found within the amino-terminal transactivation domain (NTAD), are also hydroxylated, this time by EGLN proteins. This facilitates the binding of the pvhl elongin cullin-2 complex, which polyubiquitylates HIF-α, targeting it for destruction by the proteasome. For simplicity, only one of two prolyl hydroxylation sites in the oxygen-dependent degradation domain is shown. pvhl, von Hippel Lindau gene product. Many hypoxia-inducible genes, including those that encode the above-mentioned VEGF and EPO, are under the control of a heterodimeric transcription factor, hypoxia-inducible factor (HIF), which consists of an α-subunit (usually HIF-1α; other family members are HIF-2α and HIF-3α) and HIF-1β also called aryl hydrocarbon receptor nuclear translocator (ARNT) 60. In the presence of oxygen, the HIF-α subunits are normally polyubiquitylated rapidly, which targets them for proteasomal degradation. However, when cells are hypoxic, the HIF-α subunits accumulate and following dimerization with HIF-1β bind to specific DNA sequences in the cis-regulatory regions of hypoxiainducible genes to activate transcription. Importantly, Maxwell and co-workers showed that cells lacking pvhl are unable to degrade HIF-α subunits in the presence of oxygen 61. On the basis of this lead, several groups went on to show that the pvhl β-domain binds directly to the HIF-α subunits and that pvhl is indeed capable of directing the polyubiquitylation, and hence destruction, of the HIF-α subunits The interaction of pvhl with HIF-α is governed by the enzymatic hydroxylation of conserved proline residues that are located within highly conserved, peptidic, pvhl-binding domains within the HIF-α subunits (FIG. 3). The hydroxyl group mediates the pvhl HIF-α interaction by participating in two essential hydrogen bonds with hydrophilic side chains that are located in the pvhl β-domain 70,71. Importantly, hydroxylation is inherently oxygen dependent, as the oxygen of the hydroxyl group is derived from molecular oxygen 72,73 (BOX 1). In Caenorhabditis elegans and Drosophila, this modification is clearly carried out by the product of the egl-9 gene 9,10. Human cells contain three members of the EGL-9 family (EGLN1, EGLN2 and EGLN3) 74. In vitro studies indicate that all three human EGL-9 orthologues encode proteins that can hydroxylate the HIF-α subunits, although genetic experiments will be required to determine their relative contribution to HIF regulation in vivo 9,10,75. The HIF-α subunits contain two transactivation domains the amino-terminal transactivation domain (NTAD) and the carboxy-terminal transactivation domain (CTAD) 76 (FIG. 3). The NTAD overlaps with a HIF-1α region the oxygen-dependent degradation domain (ODD) which is sufficient to render heterologous proteins unstable in the presence of oxygen and contains the pvhl-binding site. By contrast, the CTAD, as an isolated polypeptide, remains stable in the presence of oxygen, but only activates transcription under hypoxic conditions. This behaviour is due to hydroxylation by FIH1, of a specific asparagine residue in the CTAD in the presence of oxygen which, in turn, prevents recruitment of the co-activator proteins p300 AND CBP (cyclic-amp-response-element-binding protein(creb)-binding protein) (FIG. 3).So, HIF is regulated at the level of protein turnover by prolyl hydroxylation, which serves as a signal for pvhl binding, and at the level of co-activator recruitment by asparaginyl hydroxylation. 676 SEPTEMBER 2002 VOLUME 2

5 Box 1 Prolyl hydroxylases Prolyl hydroxylases modify peptide or protein-bound prolyl residues. The best-studied of these enzymes are the collagen prolyl hydroxylases, which reside in the endoplasmic reticulum and are necessary for collagen maturation. The hydroxylation of hypoxia-inducible factor, however, is carried out by members of the EGLN family. Prolyl hydroxylases require several cofactors, including 2-oxoglutarate, iron and ascorbate (vitamin C), in addition to oxygen (see figure). The hydroxylase reaction is coupled to the decarboxylation of 2-oxoglutarate to succinate. Ascorbate is thought to prevent the auto-oxidation of the enzyme. Note that the oxygen atom that is incorporated into the hydroxyl group is derived from molecular oxygen. Smallmolecule 2-oxoglutarate antagonists and iron antagonists can prevent prolyl hydroxylation. 2-oxoglutarate, O 2 CO 2, succinate Fe 2+, ascorbate In view of the above considerations, it is perhaps surprising that hypoxia-inducible genes are activated in the presence of oxygen following inactivation of pvhl 59,61,82. One possibility, suggested by the work of Greg Semenza and colleagues, is that pvhl is required for HIF asparaginyl hydroxylation 61,82. However, as an isolated polypeptide, the CTAD remains oxygen dependent in cells that lack pvhl 78. A second possibility is that the activation of HIF target genes in pvhl-defective cells reflects largely the activity of the NTAD, which can function in the absence of the CTAD 83. It is possible that hypoxia-inducible genes are differentially controlled by the CTAD and NTAD, in which case, pvhl loss would not completely mimic bona fide hypoxia with respect to changes in gene expression. Preliminary studies using gene-expression arrays are consistent with this view 82. A second paradox relates to two earlier reports indicating that regulation of hypoxia-inducible gene expression by pvhl was due primarily to changes in mrna stability, rather than to changes in transcription 57,59. One possibility, among several, is that HIF regulates (at least indirectly) both transcription and mrna turnover, although this question has yet to be resolved. Other pvhl targets An important question is whether pvhl has targets or functions that are unrelated to HIF. The existence of multiple functions would offer an explanation for the genotype phenotype correlations that are observed in VHL disease. The degree to which these functions were quantitatively or qualitatively altered by various disease-associated VHL mutations would determine the corresponding phenotype with respect to site-specific HO N N tumour risk. In this regard, VHL alleles associated with type 2C VHL disease (phaeochromocytoma only) encode proteins that retain the ability to polyubiquitylate the HIF-α subunits 62,84. This, presumably, accounts for the absence of haemangioblastomas and renal-cell carcinomas in type 2C families, as dysregulation of HIF seems to have a causal role in these tumours (see below). Although it remains possible that type 2C pvhl mutants are quantitatively defective with respect to HIF control in the adrenal gland in vivo, the simplest model would indicate that phaeochromocytoma development is linked to a pvhl loss of function or gain of function that is unrelated to HIF. Gene-expression analysis also indicates that pvhl has targets other than HIF. Specifically, it is becoming clear that the set of genes regulated by pvhl and that are regulated by hypoxia (or HIF) are overlapping, but not congruent 82. Moreover, forced activation of HIF target genes in vivo by using HIF-1α variants that escape pvhl control does not lead to cyst or tumour formation 85,86. A caveat to these studies, however, is that HIF-1α was targeted to muscle and skin, which are not affected in VHL disease; moreover, it is possible that HIF-2α is more oncogenic than HIF-1α, at least in the human kidney. In keeping with these ideas, further functions have been ascribed to pvhl. pvhl has been reported to bind to specific isoforms of protein kinase C (PKC) and might serve as an E3 for PKC-λ In addition, pvhl can bind to, and suppress, the SP1 transcription factor, although there is no evidence that pvhl targets SP1 for degradation 91,92. pvhl also interacts with a cytosolic protein-folding complex called CCT 93,94. Elongin C is required for the proper folding of pvhl, and this folding is facilitated by CCT 93,94. pvhl mutants that cannot bind to elongin C are retained by CCT and might therefore influence the rate at which CCT processes other cellular proteins 94. pvhl is clearly important for extracellular-matrix formation and epithelial differentiation The extent to which these activities ultimately relate to the control of HIF is still uncertain. pvhl can bind to fibronectin and this activity, rather than HIF binding, seems to be impaired in type 2C pvhl mutants 84,95.How intracellular pvhl interacts with the secreted protein fibronectin is uncertain. Some pvhl associates with the endoplasmic reticulum (ER), but there is no evidence that pvhl is normally secreted into the ER lumen 95,98. One possibility is that some of the fibronectin molecules (perhaps those that are malfolded or malprocessed) undergo retrograde transport to the cytosolic surface of the ER membrane, and are available to interact with pvhl. A potential role of pvhl in ER surveillance is indicated by the sensitivity of VHL / cells to agents that induce ER stress 99. Alternatively, it is possible that binding of pvhl to fibronectin is a post-lysis artefact, although the mixing experiments carried out to date argue against this 95. What is clear, however, is that cells lacking pvhl secrete fibronectin, but are defective with respect to fibronectin-matrix assembly 95. This phenotype is likely to be complex, and the degree to which this might relate NATURE REVIEWS CANCER VOLUME 2 SEPTEMBER

6 Mast cell * Stromal cell TGF-α PDGF-β * Pericyte VEGF * EPO Blood capillary Endothelial cell * Lack VHL Figure 4 Histopathology of haemangioblastomas. Haemangioblastomas consist of poorly understood stromal cells that are mixed with blood vessels. In von Hippel Lindau (VHL) disease, blood-vessel cells endothelial cells and pericytes are VHL +/ ; in sporadic haemangioblastoma, they are VHL +/+. In VHL disease and often in sporadic haemangioblastomas stromal cells are VHL / and so accumulate high levels of hypoxiainducible factor-α (HIF-α), which leads to overproduction of growth factors such as transforming growth factor-α (TGF-α), platelet-derived growth factor B chain (PDGF-β), vascular endothelial growth factor (VEGF) and erythropoietin (EPO). TGF-α probably acts in an autocrine loop, whereas PDGF-β and VEGF drive the proliferation of pericytes and endothelial cells, respectively. Modified with permission from REF. 4 (1998) Blackwell Science. communication; FIG. 4). Moreover, these cells almost invariably overproduce the epidermal growth-factor receptor (EGFR) 101,105, which is the receptor for TGF-α, thereby establishing a potential autocrine loop. VEGF and PDGF-β are likely to support the proliferation of the endothelial cells and pericytes, respectively. An earlier study showed that forced production of VEGF in the mouse brain was sufficient to cause the development of haemangioblastoma-like lesions 110. This study, however, used xenografts of poorly tumorigenic C6 glioma cells that were engineered to produce VEGF as a delivery vehicle. So, the contribution of other C6- derived factors in this model is unknown. In other models, however, it is clear that VEGF is sufficient to induce the formation of immature blood vessels 111,112. Studies of VHL inactivation in the mouse indicate that loss of pvhl function is sufficient to cause the emergence of blood-vessel tumours that loosely resemble haemangioblastomas 113. Collectively, these studies indicate that VHL inactivation might be sufficient to account for the development of retinal and central nervous system (CNS) haemangioblastomas, with TGF-α acting as an autocrine growth factor, and factors such as VEGF and PDGF-β driving blood-vessel proliferation. TIMPS Tissue inhibitors of metalloproteinases; a family of proteins that inhibit the functions of the matrix metalloproteinases. MMPS Matrix metalloproteinases. This protein family degrades specific proteins that are found in the extracellular matrix. LASER-CAPTURE MICRODISSECTION A method for selectively removing and analysing specific cells from a histological tissue section. A laser light is focused on the cells of interest. The heat energy results in the cells adhering to a specialized solid support. to the above-mentioned ability of pvhl to bind to fibronectin is still being unravelled. It is possible that pvhl-defective cells secrete a form of fibronectin that interferes with the orderly assembly of macroscopic fibronectin arrays. A recent study showed that VHL / renal carcinoma cells could not assemble exogenous fibronectin into a matrix, and linked this to abnormalities of integrin assembly on their cell surface 97. pvhl also regulates the transcription, possibly via HIF, of several genes that are implicated in extracellular-matrix turnover, such as tissue inhibitors of metalloproteinases (TIMPS ) and matrix metalloproteinases (MMPS ) 100. Why does pvhl loss lead to tumour formation? Both VHL and its principal target, HIF-1α, are ubiquitously expressed, and yet VHL inactivation is linked to only a small subset of human tumours. This, coupled with the genotype phenotype correlations described above, indicates that the role of VHL in tumorigenesis depends on tissue context. Haemangioblastomas. Microscopic examination of haemangioblastomas shows a mixture of stromal cells and blood vessels (pericytes and endothelial cells). The embryological origin of the stromal cells is still debated, but LASER-CAPTURE MICRODISSECTION and immunohistochemical studies clearly indicate that these cells are the tumour cells: they lack pvhl function and, consequently, overproduce the products of HIF target genes, such as the growth factors VEGF and platelet-derived growth-factor B chain (PDGF-β). Moreover, loss of pvhl leads to overproduction of transforming growth factor-α (TGF-α), which is suspected of being a HIF target because it is induced by hypoxia and downregulated in the presence of a dominant-negative HIF-1α mutant(refs 18, 58, , and S. Lee, personal Renal carcinoma. Patients with VHL disease develop preneoplastic renal cysts that, over time, can degenerate into renal-cell carcinomas of the clear-cell type (FIG. 1d). Lasercapture microdissection studies indicate that the epithelial cells that line these renal cysts are VHL / (REFS 19,25). So, it is presumed that additional genetic alterations are required for conversion of the cysts to frank renal carcinomas. Several recurrent genetic alterations have been described in kidney cancer, including abnormalities involving 3p14, 3p21, 5q, 6q and 10q Restoration of pvhl function in VHL / renal carcinoma cells is sufficient to suppress their ability to form tumours in nude mice, indicating an ongoing requirement for VHL inactivation in such tumours, despite the existence of additional mutations 34,57.In cell-culture models, reintroduction of pvhl into VHL / renal carcinoma cells can promote cell-cycle exit and differentiation, both of which might translate into impaired tumour growth in vivo 96, Restoration of pvhl function also corrects the overproduction of HIF target genes in renal carcinoma cells 56 59,61,108. The importance of HIF with respect to renal carcinogenesis is highlighted by two recent studies showing that HIF-α variants that were engineered to escape recognition by pvhl could override pvhl s tumour-suppressor activities in vitro and in vivo 122,123.HIF-2α, but not HIF-1α, was shown in assays to restore tumour formation in nude mice, whereas HIF-1α was sufficient to override certain pvhl-dependent phenotypes in cell-culture experiments. This suggests that HIF-1α and HIF-2α might conspire to induce renal tumorigenesis. A broader panel of pvhl-defective renal carcinoma cells will need to be studied to ascertain the relative contributions of HIF-1α and HIF-2α to renal tumorigenesis in different genetic backgrounds. 678 SEPTEMBER 2002 VOLUME 2

7 FIELD DEFECT An alteration (such as a mutation) that involves most or all of the cells in a contiguous region (field). HOMOLOGOUS RECOMBINATION The process by which segments of homologous DNA are exchanged between two DNA duplexes that share high sequence similarity. Often used to inactivate a gene of interest. a b β Drug α pvhl VEGF HIF-α HIF-α HIF-α PDGF-β TGF-α KDR PDGFR EGFR Drug A O 2 EGLN Stable Drug B Drug C Protection against low O 2 Unstable Figure 5 Potential therapeutics based on the pvhl HIF pathway. a pvhl-defective haemangioblastomas and renal carcinomas are characterized by high levels of hypoxia inducible factor-α (HIF-α), and consequently overproduce growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor-β (PDGF-β) and transforming growth factor-α (TGF-α), which activate membrane-bound receptor tyrosine kinases VEGF receptor (KDR), PDGF receptor (PDGFR) and epidermal growth-factor receptor (EGFR), respectively. Control of TGF-α by HIF-α might be direct or indirect, and is therefore indicated with a dashed arrow. Drugs that inhibit these receptors are in various stages of development, and might be used to treat such tumours. b HIF controls a variety of genes that facilitate acute and chronic adaptation to oxygen insufficiency. Transient inhibition of EGLN, and hence of HIF prolyl hydroxylation, leads to HIF stabilization, which might therefore confer protection against hypoxic insults. pvhl, von Hippel Lindau gene product. Among the HIF target genes, TGF-α is notable for two reasons. First, renal epithelial cells seem to be particularly sensitive to the mitogenic effects of TGF-α compared with the other epithelial cells tested, and overproduction of both TGF-α and its receptor, EGFR, is common in renal carcinoma Second, transgenic production of TGF-α in the kidney has been linked to renal-cyst formation 127. So, overproduction of TGF-α, perhaps in conjunction with abnormalities in extracellular-matrix formation and vascular permeability (due to the vascular-permeability effects of VEGF), might provide a platform for renal-cyst formation following VHL inactivation. After malignant conversion of a renal cyst, angiogenic peptides such as VEGF and PDGF-β would presumably promote the development of a tumour blood supply. Phaeochromocytoma. As described above, phaeochromocytoma presents a paradox as certain germ-line VHL mutations predispose individuals to develop this tumour, and yet VHL mutations are rare in sporadic phaeochromocytomas. Indeed, many cases of seemingly sporadic phaeochromocytomas have been linked, on further analysis, to unsuspected germ-line VHL mutations 28 30,128. Among several possibilities, this might suggest that phaeochromocytoma development in VHL disease requires a FIELD DEFECT related to VHL hemizygosity and/or requires that some aspect of pvhl function is lost (or, as described above, perhaps gained) during an early developmental window. The exact role of HIF in this disorder is also uncertain. Some phaeochromocytomas overproduce HIF target genes, including EPO, in keeping with impaired pvhl function. It is also intriguing that HIF-2α is highly expressed in the adrenal medulla and the organ of Zuckerkandl, which are the precursor tissues for pheochromocytoma 129. In addition, the tyrosine hydoxylase gene, which plays an important role in adrenal catecholamine production, has the features of a HIF target gene 130,131. As described above, however, the products of type 2C VHL alleles (phaeochromocytoma only) seem to retain the ability to downregulate HIF in the assays performed so far. At face value, this would link phaeochromocytoma development to loss or gain of a non-hif function. Implications and future directions Treatments for pvhl-defective tumours. Given the above considerations, it would seem reasonable to treat haemangioblastomas and renal carcinomas with agents that block the function of HIF or its downstream targets (FIG. 5a), although it has not yet been formally proven that downregulation of HIF is sufficient to impair the growth of these tumours. However, disruption of HIF function in other tumour cell types, using HOMOLOGOUS RECOMBINATION or dominant-negative approaches, has been shown to impair tumour growth in vivo Agents that inhibit signalling by VEGF, PDGF-β, and/or TGF-α have been developed and are at various stages of clinical development 136,137. Small-molecule VEGF antagonists have shown promise in a variety of preclinical solid-tumour models, including renal-cellcarcinoma models 138. A recent Phase II trial of a neutralizing anti-vegf antibody showed a modest, but statistically significant, improvement in time to progression. Likewise, EGFR inhibitors have activity in preclinical models of renal cysts and renal carcinomas Such agents can now be tested in humans, alone or in combination with agents that interfere with other HIF targets. One recent case report described a marked and sustained visual improvement in a VHL patient with an optic-nerve haemangioblastoma following treatment with SU5416, which inhibits the VEGF receptor KDR 142. Interestingly, this marked improvement in visual function was not associated with tumour shrinkage, but instead seemed to reflect a decrease in pressure on the optic nerve. Conceivably, this pressure decrease was due to the inhibition of the vascular permeability effects of VEGF. Although anecdotal, the failure of this lesion to regress might relate to the fact that newly sprouting blood vessels require VEGF as a survival factor, whereas more mature vessels, which have become stabilized by pericytes and surrounding stroma, do not 111,136. So, it is possible that the use of VEGF antagonists will cause disease stabilization, but NATURE REVIEWS CANCER VOLUME 2 SEPTEMBER

8 will not cause frank regressions of established lesions. In another study, three VHL patients with CNS haemangioblastomas were treated with SU5416. These patients also had stable disease without evidence of tumour shrinkage. Curiously, all three patients developed mild polycythaemia, although the mechanism underlying this phenomenon was not elucidated 143. Development of SU5416 has now been halted due to lack of efficacy in a colon cancer trial. Nonetheless, a number of other VEGF/KDR antagonists are currently being tested 144. Treatment of ischaemic disorders. Many of the major diseases of the developed world, including myocardial ischaemia, stroke and peripheral vascular disease, are characterized by tissue hypoxia. HIF plays a principal role in adaptation to acute and chronic hypoxia. Acute changes induced by HIF include changes in glucose uptake and metabolism, which allow continued energy generation in a hypoxic environment, and changes in angiogenesis and red-blood-cell production, which increase oxygen delivery. In theory, transient stabilization of HIF with small molecules might augment the cellular and tissue response to hypoxia. Such stabilization might be achieved through pharmacological inhibition of EGLN, which would prevent hydroxylation of HIF (FIG. 5b). Earlier studies of the collagen prolyl hydroxylases established the feasibility of inhibiting this class of enzymes with drugs that antagonize iron or 2-oxoglutarate, which are essential cofactors for the hydroxylation reaction (BOX 1). One of these compounds, FG0041, was shown earlier to preserve myocardial function when given to rats following experimental myocardial infarction 145. Although the rationale for these studies related to the potential inhibition of myocardial fibrosis, it now seems more likely that the beneficial effects were related to the stabilization of HIF 75. Questions still outstanding. The study of pvhl has led to the identification of a previously unappreciated role of hydroxylation in intracellular signalling. Clearly, it will be important to determine whether this modification takes place on other intracellular proteins and, if so, how it affects their function. Why does inactivation of VHL lead to tumour formation in such a restricted set of tissues and cell types? It is to be hoped that this question, as well as an understanding of the genotype phenotype correlations in VHL disease, will be answered with a more complete biochemical description of pvhl function. A related question is whether dysregulation of HIF is sufficient to cause tumour growth in specific contexts, and whether inhibition of HIF is sufficient to account for pvhl tumour-suppressor activity in vivo. The answer will clearly have an impact on the success or failure of treating pvhl-defective tumours with small molecules that antagonize HIF-dependent growth factors. 1. Collins, E. T. Intra-ocular growths (two cases, brother and sister, with peculiar vascular new growth, probably retinal, affecting both eyes). Trans. Ophthalmol. Soc. UK 14, (1894). 2. von Hippel, E. Ueber eine sehr seltene Erkrankung der Nethaut. Graefe Arch. Ophthalmol. 59, (1904). 3. Lindau, A. Zur Frage der Angiomatosis Retinae und Ihrer Hirncomplikation. Acta Ophthalmol. 4, (1927). 4. Richard, S., Campello, C., Taillandier, L., Parker, F. & Resche, F. Haemangioblastoma of the central nervous system in von Hippel Lindau disease. J. Int. Med. 243, (1998). 5. Maher, E. & Kaelin, W. G. von Hippel Lindau disease. Medicine 76, (1997). 6. Seizinger, B. R. et al. Von Hippel Lindau disease maps to the region of chromosome 3 associated with renal cell carcinoma. Nature 332, (1988). 7. Latif, F. et al. Identification of the von Hippel Lindau disease tumor suppressor gene. Science 260, (1993). Describes the identification and cloning of the VHL tumour-suppressor gene. The gene was authenticated by the demonstration of intragenic mutations in affected members of VHL kindreds. 8. Woodward, E. et al. Comparative sequence analysis of the VHL tumor suppressor gene. Genomics 65, (2000). 9. Epstein, A. et al. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107, (2001). This paper, along with reference 10, shows that the C. elegans and Drosophila egl-9 genes, hydroxylate the HIF-α proteins that are identified in these two species, respectively, and that the three human EGL-9 orthologues can hydroxylate human HIF-1α in vitro. 10. Bruick, R. & McKnight, S. A conserved family of prolyl-4- hydroxylases that modify HIF. Science 294, (2001). 11. Stolle, C. et al. Improved detection of germline mutations in the von Hippel Lindau disease tumor suppressor gene. Hum. Mutat. 12, (1998). 12. Chen, F. et al. Germline mutations in the von Hippel Lindau disease tumor suppressor gene: correlations with phenotype. Hum. Mutat. 5, (1995). 13. Zbar, B. et al. Germline mutations in the von Hippel Lindau (VHL) gene in families from North America, Europe, and Japan. Hum. Mutat. 8, (1996). 14. Neumann, H. & Bender, B. Genotype phenotype correlations in von Hippel Lindau disease. J. Intern. Med. 243, (1998). 15. Kanno, H. et al. Somatic mutations of the von Hippel Lindau tumor supressor gene in sporadic central nervous systems hemangioblastomas. Cancer Res. 54, (1994). 16. Shuin, T. et al. Germline and somatic mutations in von Hippel Lindau disease gene and its significance in the development of kidney cancer. Contrib. Nephrol. 128, 1 10 (1999). 17. Gnarra, J. R. et al. Mutations of the VHL tumour suppressor gene in renal carcinoma. Nature Genet. 7, (1994). 18. Vortmeyer, A. et al. von Hippel Lindau gene deletion detected in the stromal cell component of a cerebellar hemangioblastoma associated with von Hippel Lindau disease. Hum. Pathol. 28, (1997). 19. Zhuang, Z. et al. A microscopic dissection technique for archival DNA analysis of specific cell populations in lesions <1mm in size. Am. J. Path. 146, (1995). 20. Zhuang, Z. et al. Detection of von Hippel Lindau disease gene mutations in paraffin-embedded sporadic renal cell carcinoma specimens. Modern Pathol. 9, (1996). 21. Clifford, S., Prowse, A., Affara, N., Buys, C. & Maher, E. Inactivation of the von Hippel Lindau (VHL) tumour suppressor gene and allelic losses at chromosome arm 3p in primary renal cell carcinoma: evidence for a VHLindependent pathway in clear cell renal tumourigenesis. Genes Chromosom. Cancer 22, (1998). 22. Foster, K. et al. Somatic mutations of the von Hippel Lindau disease tumor suppressor gene in non-familial clear cell renal carcinoma. Hum. Mol. Genet. 3, (1994). 23. Gallou, C. et al. Mutations of the VHL gene in sporadic renal cell carcinoma: definition of a risk factor for VHL patients to develop an RCC. Hum. Mutat. 13, (1999). 24. Herman, J. G. et al. Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc. Natl Acad. Sci. USA 91, (1994). 25. Lubensky, I. A. et al. Allelic deletions of the VHL gene detected in multiple microscopic clear cell renal lesions in von Hippel Lindau disease patients. Am. J. Pathol. 149, (1996). 26. Shuin, T. et al. Frequent somatic mutations and loss of heterozygosity of the von Hippel Lindau tumor suppressor gene in primary human renal cell carcinomas. Cancer Res. 54, (1994). 27. Whaley, J. M. et al. Germ-line mutations in the von Hippel Lindau tumor suppressor gene are similar to somatic von Hippel Lindau abberations in sporadic renal cell carcinoma. Am. J. Hum. Genet. 55, (1994). 28. Neumann, H. et al. Germ-line mutations in nonsyndromic pheochromocytoma. N. Engl. J. Med. 343, (2002). 29. Neumann, H. P. H. et al. Pheochromocytomas, multiple endocrine neoplasia type 2, and von Hippel Lindau disease. N. Engl. J. Med. 329, (1993). 30. Bender, B. et al. Differential genetic alterations in von Hippel Lindau syndrome-associated and sporadic pheochromocytomas. J. Clin. Endocrinol. Metab. 85, (2000). 31. Woodward, E. et al. Genetic predisposition to phaeochromocytoma: analysis of candidate genes GDNF, RET and VHL. Hum. Mol. Genet. 6, (1997). 32. Bar, M. et al. Sporadic phaeochromocytomas are rarely associated with germline mutations in the von Hippel Lindau and RET genes. Clin. Endocrinol. 47, (1997). 33. Brauch, H. et al. Sporadic pheochromocytomas are rarely associated with germline mutations in the VHL tumor suppressor gene or the RET protooncogene. J. Clin. Endocrinol. Metab. 82, (1997). 34. Iliopoulos, O., Kibel, A., Gray, S. & Kaelin, W. G. Tumor suppression by the human von Hippel Lindau gene product. Nature Med. 1, (1995). This paper was the first to identify the VHL gene product, pvhl, in cells, and the first to show that restoring pvhl function in VHL / renal carcinoma cells suppressed their ability to form tumours in nude mice. 35. Iliopoulos, O., Ohh, M. & Kaelin, W. pvhl19 is a biologically active product of the von Hippel Lindau gene arising from internal translation initiation. Proc. Natl Acad. Sci. USA 95, (1998). 36. Blankenship, C., Naglich, J., Whaley, J., Seizinger, B. & Kley, N. Alternate choice of initiation codon produces a biologically 680 SEPTEMBER 2002 VOLUME 2

9 active product of the von Hippel Lindau gene with tumor suppressor activity. Oncogene 18, (1999). 37. Schoenfeld, A., Davidowitz, E. & Burk, R. A second major native von Hippel Lindau gene product, initiated from an internal translation start site, functions as a tumor suppressor. Proc. Natl Acad. Sci. USA 95, (1998). 38. Los, M. et al. Expression pattern of the von Hippel Lindau protein in human tissues. Lab. Invest. 75, (1996). 39. Corless, C. L., Kibel, A., Iliopoulos, O. & Kaelin, W. G. J. Immunostaining of the von Hippel Lindau gene product (pvhl) in normal and neoplastic human tissues. Hum. Pathol. 28, (1997). 40. Lee, S. et al. Transcription-dependent nuclear-cytoplasmic trafficking is required for the function of the von Hippel Lindau tumor suppressor protein. Mol. Cell. Biol. 19, (1999). 41. Groulx, I., Bonicalzi, M. & Lee, S. Ran-mediated nuclear export of the von Hippel Lindau tumor suppressor protein occurs independently of its assembly with cullin-2. J. Biol. Chem. 275, (2000). 42. Bonicalzi, M., Groulx, I., de Paulsen, N. & Lee, S. Role of exon 2-encoded β-domain of the von Hippel Lindau tumor suppressor protein. J. Biol. Chem. 12, (2001). 43. Duan, D. R. et al. Characterization of the VHL tumor suppressor gene product: localization, complex formation, and the effect of natural inactivating mutations. Proc. Natl Acad. Sci. USA 92, (1995). 44. Duan, D. R. et al. Inhibition of transcriptional elongation by the VHL tumor suppressor protein. Science 269, (1995). This paper and reference 47 were the first to show that pvhl binds to elongins B and C. 45. Pause, A. et al. The von Hippel Lindau tumor-suppressor gene product forms a stable complex with human CUL-2, a member of the Cdc53 family of proteins. Proc. Natl Acad. Sci. USA 94, (1997). This paper and reference 48 were the first to show that the pvhl complex also contains CUL2, and therefore provided an indication that pvhl might be involved in polyubiquitylation. 46. Pause, A., Peterson, B., Schaffar, G., Stearman, R. & Klausner, R. Studying interactions of four proteins in the yeast two-hybrid system: structural resemblance of the pvhl/elongin BC/hCUL-2 complex with the ubiquitin ligase complex SKP1/cullin/F-box protein. Proc. Natl Acad. Sci. USA 96, (1999). 47. Kibel, A., Iliopoulos, O., DeCaprio, J. D. & Kaelin, W. G. Binding of the von Hippel Lindau tumor suppressor protein to elongin B and C. Science 269, (1995). 48. Lonergan, K. M. et al. Regulation of hypoxia-inducible mrnas by the von Hippel Lindau protein requires binding to complexes containing elongins B/C and Cul2. Mol. Cell. Biol. 18, (1998). 49. Kishida, T., Stackhouse, T. M., Chen, F., Lerman, M. I. & Zbar, B. Cellular proteins that bind the von Hippel Lindau disease gene product: mapping of binding domains and the effect of missense mutations. Cancer Res. 55, (1995). 50. Bai, C. et al. SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell 86, (1996). 51. Deshaies, R. SCF and Cullin/Ring H2-based ubiquitin ligases. Annu. Rev. Cell Dev. Biol. 15, (1999). 52. Stebbins, C. E., Kaelin, W. G. & Pavletich, N. P. Structure of the VHL elongin-c elongin-b complex: implications for VHL tumor suppressor function. Science 284, (1999). This paper described the three-dimensional structure of the pvhl elongin-b elongin-c complex, which revealed the presence of two frequently mutated subdomains, α and β. 53. Lisztwan, J., Imbert, G., Wirbelauer, C., Gstaiger, M. & Krek, W. The von Hippel Lindau tumor suppressor protein is a component of an E3 ubiquitin protein ligase activity. Genes Dev. 13, (1999). 54. Iwai, K. et al. Identification of the von Hippel Lindau tumorsuppressor protein as part of an active E3 ubiquitin ligase complex. Proc. Natl Acad. Sci. USA 96, (1999). 55. Kamura, T. et al. Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase. Science 284, (1999). 56. Siemeister, G. et al. Reversion of deregulated expression of vascular endothelial growth factor in human renal carcinoma cells by von Hippel Lindau tumor suppressor protein. Cancer Res. 56, (1996). 57. Gnarra, J. R. et al. Post-transcriptional regulation of vascular endothelial growth factor mrna by the VHL tumor suppressor gene product. Proc. Natl Acad. Sci. USA 93, (1996). 58. Stratmann, R., Krieg, M., Haas, R. & Plate, K. Putative control of angiogenesis in hemangioblastomas by the von Hippel Lindau tumor suppressor gene. J. Neuropathol. Exp. Neurol. 56, (1997). 59. Iliopoulos, O., Jiang, C., Levy, A. P., Kaelin, W. G. & Goldberg, M. A. Negative regulation of hypoxia-inducible genes by the von Hippel Lindau protein. Proc. Natl Acad. Sci. USA 93, (1996). This paper showed that cells lacking pvhl produced high levels of hypoxia-inducible mrnas irrespective of changes in oxygen. In keeping with this, references 56 and 57 reported that cells lacking pvhl overproduce VEGF mrna. 60. Semenza, G. Regulation of mammalian O 2 homeostasis by hypoxia-inducible factor 1. Annu. Rev. Cell Dev. Biol. 15, (1999). 61. Maxwell, P. et al. The von Hippel Lindau gene product is necessary for oxgyen-dependent proteolysis of hypoxiainducible factor-α subunits. Nature 399, (1999). A landmark paper showing that cells lacking pvhl do not degrade HIF-α subunits. 62. Cockman, M. et al. Hypoxia inducible factor-α binding and ubiquitylation by the von Hippel Lindau tumor suppressor protein. J. Biol. Chem. 275, (2000). This paper and references were the first to show directly that pvhl polyubiquitylates HIF-α subunits. 63. Ohh, M. et al. Ubiquitination of HIF requires direct binding to the von Hippel Lindau protein β-domain. Nature Cell Biol. 2, (2000). 64. Kamura, T. et al. Activation of HIF1α ubiquitination by a reconstituted von Hippel Lindau tumor suppressor complex. Proc. Natl Acad. Sci. USA 97, (2000). 65. Tanimoto, K., Makino, Y., Pereira, T. & Poellinger, L. Mechanism of regulation of the hypoxia-inducible factor-1α by the von Hippel Lindau tumor suppressor protein. EMBO J. 19, (2000). 66. Ivan, M. et al. HIFα targeted for VHL-mediated destruction by proline hydroxylation: implications for O 2 sensing. Science 292, (2001). This paper and reference 67 were the first to show that the interaction of pvhl with HIF-α is regulated by oxygen-dependent prolyl hydroxylation. 67. Jaakkola, P. et al. Targeting of HIF-α to the von Hippel Lindau ubiquitylation complex by O 2 -regulated prolyl hydroxylation. Science 292, (2001). 68. Yu, F., White, S., Zhao, Q. & Lee, F. HIF1α binding to VHL is regulated by stimulus-sensitive proline hydroxylation. Proc. Natl Acad. Sci. USA 98, (2001). 69. Yu, F., White, S., Zhao, Q. & Lee, F. Dynamic, site-specific interaction of hypoxia-inducible factor-1α with the von Hippel Lindau tumor suppressor protein. Cancer Res. 61, (2001). 70. Hon, W. C. et al. Structural basis for the recognition of hydroxyproline in HIF1α by pvhl. Nature 417, (2002). This paper and reference 71 report the structure of pvhl bound to HIF-1α, and explain the requirement for prolyl hydroxylation. 71. Min, J.-H. et al. Structure of a pvhl HIF1α complex: hydroxyproline recognition in intracellular signaling. Science 296, (2002). 72. Schofield, C. J. & Zhang, Z. Structural and mechanistic studies on 2-oxoglutarate-dependent oxygenases and related enzymes. Curr. Opin. Struct. Biol. 9, (1999). 73. Kivirikko, K. I. & Myllyharju, J. Prolyl 4-hydroxylases and their protein disulfide isomerase subunit. Matrix Biol. 16, (1998). 74. Taylor, M. S. Characterization and comparative analysis of the EGLN gene family. Gene 275, (2001). 75. Ivan, M. et al. Biochemical purification and pharmacological inhibition of a mammalian HIF prolyl hydroxylase. Proc. Natl Acad. Sci. USA (in the press). 76. Bruick, R. & McKnight, S. Transcription. Oxygen sensing gets a second wind. Science 295, (2002). 77. Lando, D. et al. Asparagine hydroxylation of the HIF transactivation domain: a hypoxic switch. Science 295, (2002). 78. Sang, N., Fang, J., Srinivas, V., Leshchinsky, I. & Caro, J. Carboxyl-terminal transactivation activity of hypoxiainducible factor-1α is governed by a von Hippel Lindau protein-independent, hydroxylation-regulated association with p300/cbp. Mol. Cell. Biol. 22, (2002). 79. Freedman, S. et al. Structural basis for recruitment of CBP/p300 by hypoxia-inducible factor-1α. Proc. Natl Acad. Sci. USA 99, (2002). 80. Dames, S., Martinez-Yamout, M., De Guzman, R., Dyson, H. & Wright, P. From the cover: structural basis for Hif-1α/CBP recognition in the cellular hypoxic response. Proc. Natl Acad. Sci. USA 99, (2002). 81. Hewitson, K. S. et al. Hypoxia-inducible Factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural family. J. Biol. Chem. 277, (2002). 82. Wykoff, C., Pugh, C., Maxwell, P., Harris, A. & Ratcliffe, P. Identification of novel hypoxia dependent and independent target genes of the von Hippel Lindau (VHL) tumour suppressor by mrna differential expression profiling. Oncogene 19, (2000). 83. Gothie, E., Richard, D., Berra, E., Pages, G. & Pouyssegur, J. Identification of alternative spliced variants of human hypoxia-inducible factor-1α. J. Biol. Chem. 275, (2000). 84. Hoffman, M. et al. von Hippel Lindau protein mutants linked to type 2C VHL disease preserve the ability to downregulate HIF. Hum. Mol. Genet. 10, (2001). 85. Elson, D. et al. Induction of hypervascularity without leakage or inflammation in transgenic mice overexpressing hypoxiainducible factor-1α. Genes Dev. 15, (2001). 86. Vincent, K. et al. Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1α/VP16 hybrid transcription factor. Circulation 102, (2000). 87. Okuda, H. et al. Direct interaction of the β-domain of VHL tumor suppressor protein with the regulatory domain of atypical PKC isotypes. Biochem. Biophys. Res. Commun. 263, (1999). 88. Okuda, H. et al. The von Hippel Lindau (VHL) tumor suppressor protein mediates ubiquitination of activated atypical protein kinase C. J. Biol. Chem. 276, (2001). 89. Pal, S., Claffey, K., Dvorak, H. & Mukhopadhyay, D. The von Hippel Lindau gene product inhibits vascular permeability factor/vascular endothelial growth factor expression in renal cell carcinoma by blocking protein kinase C pathways. J. Biol. Chem. 272, (1997). 90. Pal, S., Claffey, K., Cohen, H. & Mukhopadhyay, D. Activation of Sp1-mediated vascular permeability factor/vascular endothelial growth factor transcription requires specific interaction with protein kinase C. J. Biol. Chem. 273, (1998). 91. Cohen, H. et al. An important von Hippel Lindau tumor suppressor domain mediates Sp1-binding and selfassociation. Biochem. Biophys. Res. Commun. 266, (1999). 92. Mukhopadhyay, D., Knebelmann, B., Cohen, H., Ananth, S. & Sukhatme, V. The von Hippel Lindau tumor suppressor gene product interacts with Sp1 to repress vascular endothelial growth factor promoter activity. Mol. Cell. Biol. 17, (1997). 93. Feldman, D., Thulasiraman, V., Ferreyra, R. & Frydman, J. Formation of the VHL elongin-b/c tumor suppressor complex is mediated by the chaperonin TRiC. Mol. Cell 4, (1999). 94. Hansen, W. et al. Diverse effects of mutations in exon II of the von Hippel Lindau (VHL) tumor suppressor gene on the interaction of pvhl with the cytosolic chaperonin and pvhl-dependent ubiquitin ligase activity. Mol. Cell. Biol. 22, (2002). 95. Ohh, M. et al. The von Hippel Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix. Mol. Cell 1, (1998). 96. Lieubeau-Teillet, B. et al. von Hippel Lindau gene-mediated growth suppression and induction of differentiation in renal cell carcinoma cells grown as multicellular tumor spheroids. Cancer Res. 58, (1998). 97. Esteban-Barragan, M. et al. Role of the von Hippel Lindau tumor suppressor gene in the formation of β1-integrin fibrillar adhesions. Cancer Res, 62, (2002). 98. Schoenfeld, A., Davidowitz, E. & Burk, R. Endoplasmic reticulum/cytosolic localization of von Hippel Lindau gene products is mediated by a 64-amino acid region. Int. J. Cancer 91, (2001). 99. Gorospe, M. et al. Protective function of von Hippel Lindau protein against impaired protein processing in renal carcinoma cells. Mol. Cell. Biol. 19, (1999) Koochekpour, S. et al. The von Hippel Lindau tumor suppressor gene inhibits hepatocyte growth factor/scatter factor-induced invasion and branching morphogenesis in renal carcinoma cells. Mol. Cell. Biol. 19, (1999) Bohling, T. et al. Expression of growth factors and growth factor receptors in capillary hemangioblastoma. J. Neuropathol. Exp. Neurol. 55, (1996) Flamme, I., Krieg, M. & Plate, K. Up-regulation of vascular endothelial growth factor in stromal cells of hemangioblastomas is correlated with up-regulation of the transcription factor HRF/HIF-2α. Am. J. Pathol. 153, (1998) Grossniklaus, H., Thomas, J., Vigneswaran, N. & Jarrett, W. H. Retinal hemangioblastoma. A histologic, immunohistochemical, and ultrastructural evaluation. Ophthalmology 99, (1992) Morii, K., Tanaka, R., Washiyama, K., Kumanishi, T. & Kuwano, R. Expression of vascular endothelial growth factor in capillary hemangioblastoma. Biochem. Biophys. Res. Commun. 194, (1993) Reifenberger, G., Reifenberger, J., Bilzer, T., Wechsler, W. & Collins, V. Coexpression of transforming growth factor-α and NATURE REVIEWS CANCER VOLUME 2 SEPTEMBER