von Hippel-Lindau Syndrome: Target for Anti-Vascular
`Endothelial Growth Factor (VEGF) Receptor Therapy
`ADRIAN L. HARRIS
`
`Imperial Cancer Research Fund, Medical Oncology Laboratories, University of Oxford, Institute of Molecular
`Medicine, John Radcliffe Hospital, Oxford, England
`
`Key Words. von Hippel-Lindau Syndrome · Vascular endothelial growth factor · Angiogenesis · Hypoxia-inducible factors
`
`ABSTRACT
`von Hippel-Lindau (VHL) syndrome is a familial
`cancer syndrome caused by germline mutations in the
`VHL tumor suppressor gene. Mutations in the VHL
`gene result in the constitutive stabilization of transcrip-
`tion factors hypoxia-inducible factors 1a and 2a
`, which
`bind to specific enhancer elements in the vascular
`endothelial growth factor (VEGF) gene and stimulate
`angiogenesis. This increase in angiogenesis under nor-
`moxic conditions in key target organs such as the brain,
`kidney, and eye leads to high morbidity and reduced life
`expectancy. Drugs designed to block the VEGF signaling
`
`INTRODUCTION
`von Hippel-Lindau (VHL) syndrome is an autosomal
`dominant familial cancer syndrome caused by germline
`mutations in the VHL tumor suppressor gene. This disease
`is characterized by abnormalities of vascular proliferation
`and an increased risk of certain cancers including clear cell
`renal carcinomas, pheochromocytomas, endolymphatic sac
`tumors, central nervous system hemangioblastomas, cysts
`of the kidney, liver, and pancreas, epididymal cystadeno-
`mas, neuroendocrine tumors of the pancreas, and retinal
`angiomas (Table 1) [1-4]. The age of onset for VHL varies
`depending on the manifestation, for example, retinal
`angiomas can develop from age 5 on, whereas renal cancers
`are not common until the mid-20s [5]. The cloning of the
`VHL gene in 1993 was a major step in understanding the
`disease [6]. Identification of the VHL gene on chromosome
`3p25 revealed that it possesses a unique sequence and has
`characteristics of a tumor suppressor gene.
`Families with VHL syndrome are classified as having
`either type 1 (without pheochromocytomas) or type 2 (with
`pheochromocytomas) disease. Type 2 disease is further
`classified into three subgroups: group A, patients with renal
`
`pathway may prevent the long-term complications of the
`disease. To test this hypothesis, a clinical study was initi-
`ated to evaluate the effect of the VEGF tyrosine kinase
`receptor inhibitor SU5416 in patients with VHL syn-
`drome. Preliminary data on SU5416 indicate that it is well
`tolerated when administered chronically in such patients.
`However, since little is known about the long-term use of
`such inhibitors, patients will need careful monitoring. Data
`obtained from monitoring these patients will provide valu-
`able information for adjuvant treatment trials in cancer
`patients. The Oncologist 2000;5(suppl 1):32-36
`
`cancer; group B, patients without renal cancer and group C,
`patients with only pheochromocytoma. Genetic analyses of
`VHL kindreds have shown that patterns of mutation in the
`VHL gene correlate with the distinct clinical phenotypes
`[7-9]. For example, mutations in codon 167 and missense
`mutations correlate with type 2 disease [10-13]. Mutations
`leading to truncations of the gene are associated with a
`higher risk of renal cancer and are more common in spo-
`radic renal cancer.
`Screening programs aimed at detecting and treating com-
`plications at an early stage have improved both detection and
`life expectancy of patients with VHL [14, 15]. Early detec-
`tion makes it possible to treat retinal angiomas with laser
`ablation, to remove renal cysts 3 cm in size (risk of malig-
`nancy at this size is much greater, but metastasis is of very
`low frequency), and to treat cerebellar or spinal cord heman-
`gioblastomas neurosurgically. However, many patients still
`develop severe disease complications, including blindness
`from untreatable damage due to retinal neovascularization
`and the occurrence or recurrence of inoperable central ner-
`vous system (CNS) hemangioblastomas that require bilat-
`eral nephrectomy and transplantation. Thus, a well-tolerated
`
`Correspondence: Adrian L. Harris, M.D., Imperial Cancer Research Fund, Medical Oncology Laboratories, University of
`Oxford, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, England. Telephone: +44-1865-222-
`457; Fax: +44-1865-222-431; e-mail: aharris.lab@icrf.icnet.uk Accepted for publication February 14, 2000. ©AlphaMed
`Press 1083-7159/2000/$5.00/0
`
`The Oncologist 2000;5(suppl 1):32-36 www.TheOncologist.com
`
`Roxane Labs., Inc.
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`Harris
`
`33
`
`Table 1. Incidence of tumors associated with VHL syndrome
`
`Lesion
`Retinal
`angioma
`Cerebellar
`hemangioblastoma
`Spinal cord
`hemangioblastoma
`Renal cell
`carcinoma
`Pheochromocytoma
`
`%
`58
`
`57
`
`14
`
`25
`
`13
`
`Median age
`of onset (yr)
`25
`
`Age
`range (yr)
`4-68
`
`29
`
`34
`
`44
`
`20
`
`13-61
`
`11-60
`
`23-68
`
`12-58
`
`chronic drug therapy that could effectively block the effects
`of the VHL mutation would be of great value to patients with
`VHL syndrome. Recent biochemical studies of the VHL pro-
`tein (pVHL) support a model that it plays a critical role in
`modulating transcription factors that regulate vascular
`endothelial growth factor (VEGF) expression. This finding
`has helped highlight the potential of antiangiogenic agents as
`rational targets for the treatment of VHL.
`
`ROLE OF VHL IN UBIQUITINATION
`Ubiquitination is well defined and involves activation
`of ubiquitin by a ubiquitin-activating enzyme, transfer of
`ubiquitin to protein substrate by a ubiquitin-conjugating
`enzyme, and ligation of ubiquitin to the substrate by the E3
`ligase, followed by capture and degradation of the
`ubiquinated protein by the 26S proteasome [16]. E3 ligases
`are arranged in a complex modular pattern as seen in the
`stem cell factor (SCF) (Skp1-Cdc53/CUL-1/F-box protein)
`ligase. In the SCF E3 ligase, the F-box protein serves as an
`adaptor that recognizes and recruits target substrates to the
`ubiquitin ligase. Although SCF ligase complex was origi-
`nally described in regulating the degradation of cyclins in
`yeast, the human homolog (hSKP1-human cullin-1-SKP2)
`is also involved in regulating the cell cycle [17].
`The VHL gene encodes a protein with two major
`domains, which have recently been elucidated by crystal-
`lography [18, 19]. One of these domains, the a -domain,
`interacts with ElonginB-ElonginC-cullin-2 (hCUL-2) com-
`plex to form a multiprotein complex, CBCVHL. The struc-
`ture and function of the CBCVHL complex resemble that of
`the SCF E3 ubiquitin ligase family (Fig. 1) [16, 18, 20-22].
`In the CBCVHL complex, VHL functions as the recruitment
`protein for the substrates to the ligase. A further component
`of the complex is the ubiquitin-like protein NEDD8, and
`conjugation of NEDD8 to hCUL-2 is linked to pVHL activ-
`ity [20]. The overall complex requires functional pVHL and
`does not form or function with mutant pVHL. This provides
`
`key biochemical evidence for the role of VHL and also
`links VHL to mechanisms involved in cell cycle regulation.
`Indeed, normal functioning of VHL is needed for cells to
`exit the cell cycle after serum starvation [23, 24].
`Recent reports indicate that other proteins also interact
`with VHL, including VHL-binding protein 1 [25]; Rbx-1
`[26] (an evolutionary conserved RING-H2 finger protein
`that interacts with Cullins and activates ubiquitin ligase);
`and atypical protein kinase C isoforms pKCs
`and l
`(that
`interact with the b domain of VHL) [27]. There are three
`exons in the VHL gene and two splice variants generating
`proteins of 24 and 18 kDa, both of which can interact in
`these complexes [28-30]. ElonginB-ElonginC complex was
`initially identified as a positive regulator of RNA poly-
`merase II and then as a component of the VHL complex. In
`addition to binding to VHL, it also interacts with other mul-
`tiprotein complexes involved in regulation of cytokine sig-
`naling (suppressor of cytokine signaling-1, SOCS-1) [31].
`
`HYPOXIA-INDUCIBLE FACTORS 1a AND 2a
`The key to clarifying the role of VHL in the ubiqui-
`tin activation complex is to determine the target sub-
`strates for CBCVHL complex and where they bind to the com-
`plex. At least two substrates are now known—the hypoxia-
`inducible factors 1a
`and 2a
`(HIF-1a
`and HIF-2a ) [32, 33].
`These transcription factors are unstable in intact cells under
`normoxia but are stabilized under hypoxic conditions. Under
`hypoxic conditions, HIF-1a
`and HIF-2a
`translocate to the
`nucleus where they form a heterodimer with the aryl hydro-
`carbon nuclear translocator. As shown in Figure 2, these het-
`erodimers bind to specific hypoxia-regulated response
`elements and activate genes such as VEGF and isozymes of
`the glycolytic pathway, as well as the transferrin and uroki-
`nase receptors and inducible nitric oxide synthase [34]. Thus
`in hypoxia, several genes of potential importance in tumor
`growth, invasion, or metastasis are regulated by this path-
`way. In normoxia, the a -subunits of HIF are rapidly degraded
`by ubiquitin-mediated proteolysis that is mediated by pVHL
`[32]. Recent studies indicate that mutations in the VHL gene
`result in the constitutive stabilization of HIF-a
`subunits and
`the activation of HIF-1 in normoxia [32]. Consequently,
`VEGF as well as other HIF-responsive pathways are consti-
`tutively upregulated to levels that normally only occur under
`hypoxic conditions [33]. The association of mutations in VHL
`with familial cancer demonstrates that this is a key pathway in
`tumorigenesis.
`The role of HIF-1 in nonfamilial cancer has been evalu-
`ated using the mouse xenograft of hepatoma cell line Hepa-1,
`which was deficient in hypoxia signaling pathway [35]. The
`tumor cells with a normal hypoxia signaling pathway entered
`a rapid exponential growth phase after an initial lag, whereas
`
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`34
`
`VHL Syndrome and VEGFR Therapy
`
`Figure 1. Structure of von
`Hippel-Lindau (VHL)-Elon-
`ginB-ElonginC complex and
`the stem cell factor (SCF)
`ligase complex. The similar
`structure of the VHL-Elon-
`ginB-ElonginC and SCF
`complexes support the hypo-
`thesis that the VHL-Elon-
`ginB-ElonginC complex may
`function to regulate the ubi-
`quitin-mediated degredation
`of target proteins in a manner
`analogous to that of the SCF
`complex. Reproduced with
`permission [18].
`
`Cul2
`
`ElonginB
`
`ElonginC
`
`Cdc34
`
`Ubiquitin
`
`Cul1
`
`Skp 1
`
`Target binding
`
`ElonginC binding
`
`Skp 1 binding
`
`Target binding
`
`b –domain
`
`SH2
`
`WD40
`
`Ank
`
`a –domain
`
`VHL
`
`Mutational patch
`
`SOCS-box
`
`SOCS
`
`Cdc4
`
`F-box
`
`Met30
`
`F-box
`
`SOCS-box WSB
`
`Grr1
`
`F-box
`
`WD40
`
`WD40
`
`LRR
`
`SOCS-box
`
`ASB
`
`cells deficient for hypoxia signaling did not. Hypoxia deficient
`cells also showed much slower growth in vivo with less angio-
`genesis and failed to induce VEGF [35]. These results demon-
`strated the important role of HIF pathway in tumor
`angiogenesis and growth, even in a fully transformed cell line.
`Further evidence for the role of HIF-1 in tumorigenesis is
`derived from studies using immunohistochemical techniques
`to assess HIF expression in tumor specimens. Using mono-
`clonal antibodies specific for HIF-1, we were able to show that
`all common tumors switch on this pathway, whereas it is
`hardly detectable in normal tissues [36]. Therefore, HIF-1a
`and HIF-2a are key targets for developing new therapy against
`cancer, and genes downstream in the HIF pathway are also
`potential targets.
`
`OTHER GENES REGULATED BY PVHL
`Several genes important in cancer growth are regulated
`by mutant and wild-type pVHL. Transforming growth fac-
`tor b
`[37], hepatocyte growth factor receptor [38], PAI-1
`[39], and carbonic anhydrases [40] are downregulated by
`wild-type pVHL and upregulated by mutant pVHL. The
`pVHL also binds fibronectin intracellularly and is neces-
`sary for proper assembly of fibronectin in the extracellular
`matrix [41]. Thus, there is a pleiotropic response to VHL
`mutations, and whether this is common to all cell lines with
`VHL mutations is not known. In normal tissues, erythro-
`poietin is regulated by the HIF pathway and is aberrantly
`upregulated in hemangioblastomas [42].
`
`VEGF: TARGET FOR THERAPY IN VHL SYNDROME
`Although many genes seem to be regulated by the VHL
`gene, the clearest role is in regulation of the hypoxia
`response pathway. Among these genes, VEGF is particu-
`larly important because it is responsible for many of the
`complications associated with VHL syndrome [43], includ-
`ing angiomas and hemangioblastomas [42-45].
`The clinical evaluation of current antiangiogenic ther-
`apy in cancer is problematic due to the short treatment time
`and difficulty in determining the optimal dose. In phase I
`trials, patients are treated only for a few weeks with antian-
`giogenic agents at what may be less than the optimal dose.
`Additionally, many patients have bulky tumors that
`progress rapidly while on treatment. Since antiangiogenic
`agents tend to stabilize disease rather than produce regres-
`sion and may induce less normal tissue toxicity than con-
`ventional agents, their optimal dose is harder to evaluate.
`While antiangiogenic therapy will probably be most suc-
`cessful as long-term adjuvant treatment, protocols are diffi-
`cult to develop without knowing long-term toxicity.
`Patients with the VHL syndrome provide an opportu-
`nity to study the long-term effects of antiangiogenic therapy
`while preventing the progression of their disease; these
`patients can be treated for many years unlike patients with
`advanced cancers who may only have a few months to live.
`Optimal drug doses will be easier to determine because the
`main pathway for renal proliferation seems to be VEGF,
`and therefore, specific inhibitors may be more likely to
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`35
`
`Harris
`
`Figure 2. Pathways of regulation of HIF-1a
`and von Hippel-Lindau (VHL). The labile
`transcription factor HIF-1a
`is stabilized in the
`cytoplasm by hypoxic stress via a sensor and
`translocates to the nucleus where it het-
`erodimerizes with aryl hydrocarbon nuclear
`translocator (ARNT) to bind to specific
`hypoxia response elements (see text for
`details). There are three members of the HIF-
`1a
`family and three of the ARNT family. The
`VHL gene product is involved in targeting
`HIF-1a to the proteasome where it is degraded
`in normoxia.
`
`show an effect in VHL compared with
`cancers that may have multiple angio-
`genic pathways. Patients could be treated for years with
`long-term therapy to prevent lesion progression as well as
`to help define long-term toxicity of adjuvant treatment in
`cancer trials.
`To test this hypothesis, we have initiated a multicenter
`trial to evaluate the effect of chronic administration of a
`VEGF-Flk-1/KDR tyrosine kinase inhibitor (SU5416) in
`patients with VHL who had complications or risks of com-
`plications, but in whom existing conventional modalities
`could no longer be used. These complications included
`
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