The von Hippel Lindau Hereditary Cancer Syndrome

VHL disease is inherited as an autosomal dominant trait affecting 1 in 36,000 of the population. Affected individuals bear a germ-line mutation in the VHL tumor suppressor gene. However, it is the predisposition to cancer rather than the cancer itself that is inherited, and the presence of a single mutation at the susceptibility locus is insufficient for tumor formation. Tumors are associated with somatic loss or inactivation of the remaining wild-type allele, in accordance with the Knudson "two-hit" hypothesis. The high likelihood of somatic mutation affecting the single remaining wildtype allele over the lifetime of an individual with the inherited syndrome explains the dominant inheritance pattern of tumors, the greater than 90 percent penetrance by 65 of years age, and the multifocal nature of associated tumors. Sporadic tumors can arise in nonaffected individuals, by the occurrence of somatic loss of both alleles within the same cell. However, on average, it takes longer to accrue two "hits" within the same cell, accounting for the lower prevalence (within the much larger at-risk population) and older age distribution of sporadic tumors, compared to those associated with the hereditary VHL syndrome.

The first report of patients with what was probably VHL syndrome was by Treacher Collins in 1894, when he reported two siblings with retinal angiomas. In 1904 Eugene von Hippel, who gives his name to the syndrome, reported families with blood-vessel tumors (angiomas) of the retina.

The correlation with microscopically indistinguishable tumors (hemangioblastomas) of the central nervous system (especially cerebellum and spinal cord) was first described, in 1926, by Arvind Lindau. This description also included cysts in the kidney, pancreas, and epididymis. However, the first diagnostic criteria to include renal cell carcinoma as part of the syndrome was that of Melmon and Rosen in 1964. Other tumors now known also to be associated with the syndrome include pheochromocytoma (a hormone-secreting tumor of the adrenal medulla), endolymphatic-sac tumors of the inner ear, and islet-cell tumors of the pancreas.

Hemangioblastomas and renal-cell carcinomas are both highly vascular tumors and demonstrate disordered growth of the microvasculature. Hemangioblastomas are the commonest tumors in the VHL syndrome, affecting 60 to 80 percent of individuals, and have a predilection for the posterior fossa and spinal cord. They are well-defined thinly encapsulated benign tumors formed from a mixture of "stromal" cells and a rich plexus of sometimes telangiectatic capillary blood vessels, and are frequently associated with edema and cysts. It is the stromal cells that are the tumor cells. These cells present a finely vacuolated or foamy cytoplasm, and sometimes evoke an epitheloid appearance of the tumor mimicking that of metastatic renal cell carcinoma. They lack functional pVHL and overproduce angiogenic growth factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor B chain (PDGF-b). These in turn drive proliferation of endothelial cells and pericytes, respectively, leading to the angiogenic phenotype. Furthermore, VEGF, also known as vascular permeability factor (VPF), increases capillary leakiness, leading to edema.

Renal cell carcinomas, also known as clear-cell carcinomas, arise from the renal tubular epithelium and again are highly vascular, giving them their characteristic red color at operation. The tumors develop from preneoplastic cysts lined with VHL-/- epithelial cells. Importantly, the very large number of associated benign lesions compared to malignant lesions suggests a complex oncogenic process, most probably involving several additional genetic alterations following VHL inactivation. However, restoration of pVHL function in fully transformed VHL-/- renal carcinoma cells suppresses their ability to form tumors in nude mice, indicating an ongoing requirement for VHL inactiva-tion. Interestingly, restoration of pVHL does not affect the ability of tumor cells to grow in adherent tissue culture, suggesting that the tumor suppressor action is in some way environmentally specific. Like the stromal cells in hemangioblastomas, renal-cell carcinoma cells also overexpress VEGF and PDGF-b, leading to their highly vascular appearance.

Although the angiogenic tumor phenotype is most apparent in VHL-associated tumors, this increased angiogenesis is not unique to them. A considerable body of evidence spanning more than three decades has documented that tumor growth and metastasis of many cancers requires persistent new blood-vessel growth. When tumor cells were transplanted into avascular sites, such as the cornea, the implants attracted new capillary growth. In the absence of access to an adequate vasculature, tumor cells became necrotic and/or apoptotic. Furthermore, ingress of new blood vessels in these tumor transplantation models suggested that tumors released diffusible activators of angio-genesis that could stimulate a quiescent vasculature to begin capillary sprouting. In a variety of transgenic mouse tumor models, an "angiogenic switch" could be detected during the early stages of tumorigenesis, preceding the development of solid tumors. Recent analyses of von Hippel-Lindau disease have provided the first direct link between a tumor suppressor gene function and the molecular mechanisms of angiogenesis.

The VHL Protein (pVHL)

The VHL gene was first cloned from chromosome 3p25-26 by a large consortium in 1993, and comprises just three exons coding for a protein of 213 amino acids. A second isoform of 150 residues is produced from an in frame ATG at codon 54. Notably all disease-causing mutations affect sequences C-terminus to this second start site, so that tumorigenesis is associated with inactivation of both forms.

The majority of familial point mutations lie within two regions of the protein, suggesting two major functional domains. Although the primary VHL sequence did not immediately suggest a function, protein association experiments defined a series of pVHL interacting proteins, including elongins B and C, CUL2, and Rbx1, which bound to the frequently mutated a-domain. Cul2, a member of the cullin family, resembles yeast Cdc53, and elongin C resembles yeast Skp1. In yeast, Cdc53 and Skp1 bind to one another to form ubiquitin ligases referred to as SCF complexes (Skp1/Cdc53/F-box protein), which covalently attach a ubiquitin polymer to cell-cycle control proteins targeting them for destruction by the proteasome. In such complexes, the target protein destined for polyubiquitination, and hence destruction by the proteasome, is recognized or bound by the F-box protein (so named because of a collinear Skp1-binding motif present in cyclin F), suggesting an analogous role for pVHL. This putative role was strengthened by recognition of a second frequently mutated subdomain of pVHL, the b-domain, which has features of a substrate-docking site. Experimental evidence confirmed that anti-pVHL immunoprecipitates exhibit ubiquitin ligase activity in vitro.

The discovery that pVHL was acting as a ubiquitin ligase substrate recognition module raised the question of what was its target protein, and how was this related to the angio-genic phenotype of the tumors? Repetition of the protein association studies in the presence of proteasomal blockade demonstrated two novel pVHL binding partners, the a-subunits of the transcription factors hypoxia inducible factor 1 and 2. Furthermore, in vitro ubiquitylation studies showed that these two proteins were targeted for ubiquitylation by pVHL (see Figure 1).

Figure 1 Regulation of HIF-a by pVHL. Under oxygen-replete conditions HIF-a is enzymatically modified by prolyl hydroxylase activity to a form that is recognized by pVHL. The pVHL ubiquitin ligase complex covalently attaches a polyubiquitin tag, which then targets HIF-a for rapid destruction in by the proteasome, so that it is no longer available to effect target gene transcription. (see color insert)

Figure 1 Regulation of HIF-a by pVHL. Under oxygen-replete conditions HIF-a is enzymatically modified by prolyl hydroxylase activity to a form that is recognized by pVHL. The pVHL ubiquitin ligase complex covalently attaches a polyubiquitin tag, which then targets HIF-a for rapid destruction in by the proteasome, so that it is no longer available to effect target gene transcription. (see color insert)

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