UpToDate: Molecular biology and pathogenesis of von Hippel-Lindau disease
von Hippel-Lindau (VHL) disease is an inherited, autosomal dominant syndrome manifested by a variety of benign and malignant tumors. VHL is diagnosed in about 1 in 36,000 people [1-3].
The initial manifestations of disease can occur in childhood or adolescence or later (mean age about 26 years). The spectrum of VHL-associated tumors includes:
- Hemangioblastomas of the central nervous system
- Retinal hemangioblastomas
- Clear cell renal cell carcinomas (RCCs)
- Endolymphatic sac tumors of the middle ear
- Serous cystadenomas and neuroendocrine tumors of the pancreas
- Papillary cystadenomas of the epididymis and broad ligament
Molecular biology and pathogenesis
The VHL gene has been mapped to chromosome 3p25 and cloned . Its gene product, pVHL, functions as a tumor suppressor protein . As with mutations in certain other tumor suppressor genes (eg, the RB1 gene), a «two-hit» model has been validated for VHL disease, in which a germline mutation inactivates one copy of the VHL gene in all cells. For VHL-associated tumors to develop, there must be loss of expression of the second, normal allele, through either somatic mutation or deletion of the second allele, or through hypermethylation of its promoter. In sporadic renal cell cancers, inactivation of VHL through somatic mutation of both alleles is very common.
Major advances have been made over the last two decades in understanding the biology that underlies the formation of VHL-associated tumors [5-9]. The VHL protein (pVHC) forms a stable complex with several other proteins including elongin B, elongin C, and cullin 2. This VBC complex targets several proteins for proteasomal degradation, thereby regulating their levels within the cell [7-9]. The pVHL component of this complex functions as an E3 ubiquitin ligase for the target molecules. Once bound to the pVHL complex, the target molecules are covalently bound to ubiquitin, facilitating degradation by the proteasome.
In addition to its function as an E3 ubiquitin ligase, pVHL performs several other important cellular functions, including maintenance of the primary cilium, regulation of cytokinesis, control of microtubule function, extracellular matrix integrity, and regulation of the cell cycle.
Hypoxia-inducible factor-1 and 2 — Hypoxia-inducible factor-1 alpha and 2 alpha (HIF-1 and HIF2) are two of the major proteins regulated by pVHL. HIF-1a is involved in erythropoiesis, through its ability to induce transcription of mRNA coding for erythropoietin. The role of HIF-1 in erythropoiesis is discussed in detail elsewhere, but will be briefly reviewed here to permit understanding of the events that occur in VHL disease.
Transcriptional activation by HIF requires the heterodimerization and nuclear translocation of alpha and beta subunits. The beta subunit is not influenced by the oxygen tension and is not bound by the pVHL protein complex. In contrast, the alpha subunits are sensitive to oxygen levels and are a substrate for the pVHL protein complex. In the presence of normal oxygen tension, HIF-1a and 2a are enzymatically hydroxylated. The hydroxylated HIF- subunits are bound by the VHL protein complex and covalently linked to ubiquitin. Once this occurs, HIFa subunits are rapidly degraded by proteasomes (figure 1).
Under conditions of hypoxia, however, hydroxylation does not occur, and HIFa and HIF2a are not bound to the VHL protein complex and cannot undergo ubiquitination. The levels of HIF1a and HIF2a rise, resulting in increased mRNA transcription of a variety of proteins, thus inducing a physiologic angiogenic response.
In patients with VHL disease, loss of the sole functioning VHL allele in somatic tissues causes a situation analogous to hypoxia, despite the presence of normal oxygen tension [5,6,9,10]. Mutations in the VHL gene can result either in pVHL failing to form the necessary protein complex to bind HIF1a and HIF2a or in failure of the binding site on the complex to recognize HIFa proteins . In either case, HIF1a and HIF2a are not linked to ubiquitin and are not degraded in proteasomes. Elevated levels of HIF1a and HIF2a can then induce abnormal production of the same factors that would be produced in conditions of physiologic hypoxia.
Contemporary research indicates that HIF1a and HIF2a may possess distinct functional characteristics. Skewing of the ratio of HIF1a and HIF2a towards HIF2a may alter cell signaling, and upregulate Myc activity in cells . The ratio switching is likely due to several factors. Hypoxia activated factor (HAF) was shown to increase HIF2a transactivation  and HIF1a instability , resulting in a more aggressive cellular phenotype. In addition, in RCC tissue, preferential loss of chromosome 14q, the locus for the HIF1a gene, results in decreased levels of HIF1a .
In addition to erythropoietin, other factors known to be regulated through the HIF1a and HIF2a include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-beta and transforming growth factor (TGF)-alpha [5,8,9]. In addition, pVHL affects several other factors potentially involved in tumorigenesis that are not regulated through the HIF-1alpha system. These targets include matrix metalloproteinases (MMP) such as MMP1, MMP inhibitors, and atypical protein kinase C [5,8].
Although the mechanism of tumorigenesis remains unproven, the combined effect of various angiogenic factors and other growth factors may be to create an autocrine loop that provides an uncontrolled growth stimulus consistent with the highly vascular CNS tumors found in VHL patients . Additionally, pVHL regulates several key cellular processes whose disruption may result in a malignant phenotype. These processes include extracellular matrix control, microtubule regulation, cilia centrosome cycle control, and cell cycle control.
Extracellular matrix control
Presence of functional pVHL is required to maintain proper assembly of an extracellular fibronectin matrix. pVHL binds to and regulates fibronectin in a phosphorylation dependent fashion . Mutations that preserve HIF regulation but are collagen IV binding incompetent, for example the L188V mutation, demonstrate malignant behavior .
Cilia centrosome regulation and microtubule control
The primary cilium is a nonmotile organelle involved in mechanosensing, cell signaling, and regulation of cellular entry into mitosis . Loss of ciliary proteins or of signaling via canonical or noncanonical Wnt signaling pathways disrupts regulation of planar cell polarity, and results in cyst formation . Coordinate inactivation of pVHL and GSK3B is sufficient to induce loss of primary cilium , and in animal models of Vhlh loss and Pten loss, increases cyst formation . pVHL was found to bind to and stabilize microtubules . The binding of pVHL to microtubules is regulated by glycogen synthase 3, which phosphorylates pVHL at serine 68 and requires a priming phosphorylation event on serine 72 by casein kinase 1 . Loss of pVHL or expression of mutated pVHL in cells results in unstable astral microtubules, dysregulation of the spindle assembly checkpoint, and an increase in aneuploidy .
Cell cycle control
Adding back pVHL to the VHL deficient 786-0 cell line results in acquisition of cell cycle arrest upon serum withdrawal with concomitant upregulation of p27 . Nuclear localization and intensity of p27 is inversely associated with tumor grade . pVHL is responsible for Skp2 destabilization, and concomitant upregulation of p27 after DNA damage .
There are emerging data that suggest pVHL may also regulate p53. It has been shown that p53 is an important regulator of mitotic checkpoints, and loss of p53 permits aneuploid cells to survive . pVHL has been shown to bind to, stabilize and transactivate p53 , and this binding may be regulated by phosphorylation [27,30]. Further work is required to dissect out the significance of these findings and their role in driving tumorigenesis.
Animal models of VHL disease
To date no satisfactory animal models of VHL disease have been generated. Knockout of the homologous Vhlh gene in mice does not cause renal cell carcinoma formation or the development of hemangioblastomas . Attempts to generate murine homologues of human phenotypes have resulted in a replication of Chuvash polycythemia  but an R167Q homologue did not generate renal cell carcinoma . The combination of Vhlh and Pten inactivation resulted in accelerated cyst formation . Reasons for the failure of Vhlh mutations in mice to generate a phenocopy may be due to species-specificity, or to the lack of hitherto unspecified additional obligate hits that occur in human tissue.
Gene mutations and clinical manifestations of disease
Specific germline mutations or deletions of the VHL gene can influence the clinical manifestations of VHL disease [5,34]. Deletions in VHL and nonsense and frameshift mutations appear to be more common in type I disease, while missense mutations may be more common in type II disease .
These relationships can be illustrated by the following observations:
- In a study of 138 families with VHL disease, large deletions and mutations in the VHL gene resulting in a truncated protein were associated with a much lower risk of pheochromocytoma than missense mutations (6 versus 40 percent and 9 versus 59 percent at 30 and 50 years, respectively) . In particular, missense mutations at codon 167 were associated with a particularly high risk of pheochromocytoma (over 80 percent by age 50). The cumulative probability of RCC and the age-related risk for hemangioblastoma were similar in the two mutation groups. It is important to note that although the risk for pheochromocytomas is diminished with deletions or truncating mutations, the residual risk is high enough to warrant continued screening for pheochromocytomas in this patient population.
- By contrast, in a report of 274 individuals in 126 unrelated families, gene abnormalities resulting in a truncated protein or large rearrangements led to an increased incidence of RCC compared with missense mutations (81 versus 63 percent) . However, missense mutations within two narrow cluster regions of the VHL gene were associated with higher incidence of RCC than missense mutations elsewhere in the VHL gene.
- Correlations between the VHL gene abnormality and the frequency of retinal capillary hemangioblastomas (RCH) have also been observed . In a series of 196 patients, RCH were twice as common among patients who had a substitution mutation rather than a gene abnormality resulting in a truncated protein.
- von Hippel-Lindau (VHL) disease is an inherited, autosomal dominant syndrome manifested by a variety of benign and malignant neoplasms, including clear cell renal cell carcinoma, hemangioblastomas, pheochromocytomas, and other rare tumors. (See «Clinical features, diagnosis, and management of von Hippel-Lindau disease».)
- The pathogenesis of VHL disease has been linked to mutations in the VHL gene. pVHL, the product of the VHL gene, is a tumor suppressor protein, which performs a number of important cellular functions, including targeting proteins for proteasomal degradation, maintaining an intact primary cilium, and regulating the extracellular matrix (See ‘Molecular biology and pathogenesis’ above.)
- The type of mutation in the VHL gene impacts the disease phenotype. In particular, patients with missense mutations have a significantly greater risk of developing pheochromocytomas compared with patients carrying truncating mutations or deletions.
- The alpha subunits of hypoxia-inducible factor-1 and 2 (HIF1 and HIF2) are substrates for the product of the VHL gene. In the absence of VHL-induced degradation, HIF1 and HIF2 may contribute to increased levels of erythropoietin, VEGF, and other growth factors, providing a stimulus for tumor growth. (See ‘Hypoxia-inducible factor-1 and 2’ above.)
- Satisfactory animal models for VHL disease have not yet been developed. Efforts are underway to define the necessary molecular steps required to replicate the human VHL disease phenotype in mice.
Oxygen sensor model
Schematic showing a proposed model for the oxygen sensor mechanism of erythropoiesis. Under hypoxic conditions (upper left), both the alpha and beta subunits of HIF are stabilized, leading to subsequent erythropoietin (Epo) gene transcription via the aggregate effects of hepatic nuclear factor (HNF)-4, p300, and HIF-alpha and -beta. The Epo, once formed, binds to Epo receptors on committed erythroid precursor cells. Under normoxic conditions (upper middle), only the beta unit of HIF is fully expressed. The alpha unit undergoes proline hydroxylation (PH) in the presence of iron and oxygen. When hydroxylated HIF-alpha binds to the Von Hippel-Lindau protein (VHL), a ubiquitin ligase complex (UL) is activated, leading to ubiquitination and subsequent degradation of HIF-alpha via the proteasome, preventing the subsequent transcription of Epo. Mutations affecting proteins along this pathway have been associated with congenital erythrocytosis.
EpoR: erythropoietin receptor; SF: steel factor; IL-3R: interleukin-3 receptor; BFU-E: erythroid blast-forming units; CFU-E: erythroid colony-forming units.
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