Identification of novel VHL target genes and relationship to hypoxic response pathways

Upregulation of hypoxia-inducible factors HIF-1 and HIF-2 is frequent in human cancers and may result from tissue hypoxia or genetic mechanisms, in particular the inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene (TSG). Tumours with VHL inactivation are highly vascular, but it is unclear to what extent HIF-dependent and HIF-independent mechanisms account for pVHL tumour suppressor activity. As the identification of novel pVHL targets might provide insights into pVHL tumour suppressor activity, we performed gene expression microarray analysis in VHL-wild-type and VHL-null renal cell carcinoma (RCC) cell lines. We identified 30 differentially regulated pVHL targets (26 of which were 'novel') and the results of microarray analysis were confirmed in all 11 novel targets further analysed by real-time RT-PCR or Western blotting. Furthermore, nine of 11 targets were dysregulated in the majority of a series of primary clear cell RCC with VHL inactivation. Three of the nine targets had been identified previously as candidate TSGs (DOC-2/DAB2, CDKN1C and SPARC) and all were upregulated by wild-type pVHL. The significance for pVHL function of two further genes upregulated by wild-type pVHL was initially unclear, but re-expression of GNG4 (G protein gamma-4 subunit/guanine nucleotide-binding protein-4) and MLC2 (myosin light chain) in a RCC cell line suppressed tumour cell growth. pVHL regulation of CDKN1C, SPARC and GNG4 was not mimicked by hypoxia, whereas for six of 11 novel targets analysed (including DOC-2/DAB2 and MLC2) the effects of pVHL inactivation and hypoxia were similar. For GPR56 there was evidence of a tissue-specific hypoxia response. Such a phenomenon might, in part, explain organ-specific tumorigenesis in VHL disease. These provide insights into mechanisms of pVHL tumour suppressor function and identify novel hypoxia-responsive targets that might be implicated in tumorigenesis in both VHL disease and in other cancers with HIF upregulation.     

von Hippel-Lindau protein promotes Skp2 destabilization on DNA damage

Germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene cause VHL disease, a rare and autosomal-dominant genetic syndrome. Because VHL protein (pVHL) is the master regulator of hypoxia-inducible factor alpha (HIFα), the most prominent feature of VHL disease is the deregulation of HIFα proteins. However, the precise mechanism by which the loss of pVHL function contributes to tumorigenesis is not fully understood. Here, we show that pVHL destabilizes the F-box protein Skp2, a chief component of Skp, Cullin, F-box-containing complex that promotes DNA synthesis in the S phase. The β-domain of pVHL interacts with Skp2, stimulating proteasome-dependent Skp2 degradation, but the destabilization of Skp2 does not depend on the E3 ubiquitin ligase activity of pVHL. Notably, the generation of DNA damage induces Skp2 degradation, which is attenuated by the suppression of endogenous pVHL expression. One possible mechanism of pVHL-dependent Skp2 degradation entails the antagonizing of Akt-mediated Skp2 phosphorylation, which maintains Skp2 stability. Reintroduction of VHL into VHL-null renal cell carcinoma (RCC) cells decreased Skp2 levels and restored DNA damage-dependent Skp2 degradation. These results identify the tumor suppressor function of pVHL in delaying the S-phase progression to inhibit cell proliferation on DNA damage. Clinically, this report explains as to why Skp2 accumulates abnormally in RCC tissues.     

von Hippel-Lindau tumor suppressor protein regulates the assembly of intercellular junctions in renal cancer cells through hypoxia-inducible factor-independent mechanisms

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is responsible for the development of renal cell cancers (RCC), pheochromocytomas, and tumors in other organs. The best known function of VHL protein (VHL) is to target the hypoxia-inducible factor (HIF) for proteasome degradation. VHL is also required for the establishment of an epithelial-like cell shape in otherwise fibroblastic-like RCC cell lines. However, the underlying mechanisms and whether this is linked to HIF remain undetermined. Because the breakage of intercellular junctions induces a fibroblastic-like phenotype in multiple cancer cell models, we hypothesized that VHL may be required for the assembly of intercellular junctions in RCC cells. Our experiments showed that VHL in RCC cell lines is necessary for the normal organization of adherens and tight intercellular junctions, the maintenance of cell polarity, and control of paracellular permeability. Additionally, 786-O cells reconstituted with wild-type VHL and with a constitutively active form of HIF-2alpha did not reproduce any of the phenotypic alterations of VHL-negative cells. In summary, we show that VHL inactivation in RCC cells disrupts intercellular junctions and cell shape through HIF-independent events, supporting the concept that VHL has additional functions beside its role in the regulation of HIF.     

Role of VHL gene mutation in human cancer.

Germline inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene causes the von Hippel-Lindau hereditary cancer syndrome, and somatic mutations of this gene have been linked to the development of sporadic hemangioblastomas and clear-cell renal carcinomas. The VHL tumor suppressor protein (pVHL), through its oxygen-dependent polyubiquitylation of hypoxia-inducible factor (HIF), plays a central role in the mammalian oxygen-sensing pathway. This interaction between pVHL and HIF is governed by post-translational prolyl hydroxylation of HIF in the presence of oxygen by a conserved family of Egl-nine (EGLN) enzymes. In the absence of pVHL, HIF becomes stabilized and is free to induce the expression of its target genes, many of which are important in regulating angiogenesis, cell growth, or cell survival. Moreover, preliminary data indicate that HIF plays a critical role in pVHL-defective tumor formation, raising the possibility that drugs directed against HIF or its downstream targets (such as vascular endothelial growth factor) might one day play a role in the treatment of hemangioblastoma and renal cell carcinoma. On the other hand, clear genotype-phenotype correlations are emerging in VHL disease and can be rationalized if pVHL has functions separate from its control of HIF.     

Cyclin D1 Genotype and Expression in Sporadic Hemangioblastomas

Summary Central nervous system (CNS) hemangioblastomas are highly-vascularized tumors occurring in sporadic form or as a manifestation of von Hippel–Lindau disease (VHL). The VHL protein (pVHL) regulates various target genes, one of which is the CCND1 gene, encoding cyclin D1, a protein that plays a critical role in the control of the cell cycle. Overexpression of cyclin D1 is found in many cancers. The CCND1 gene contains a common G → A polymorphism (870G > A) that enhances alternative splicing of the gene. CCND1 genotype is associated with clinical outcome in a number of cancers although prognostic significance varies with tumor type. In VHL disease, CCND1 genotype has been suggested as a genetic modifier that influences susceptibility to hemangioblastomas. In order to analyze whether CCND1 genotype plays a role in sporadic CNS hemangioblastomas, we investigated CCND1 genotype in tumor tissue of 17 sporadic and also in five VHL-related CNS hemangioblastomas. In addition, in these tumors the extent and localization of cyclin D1 expression was investigated by immunohistochemistry. We found no deviation in CCND1 genotype distribution and allele frequencies from expected values. Also, there was no correlation between age at onset and CCND1 genotype. The expression of cyclin D1 as detected by immunohistochemistry was highly variable within and between tumors, without a clear correlation with CCND1 genotype. We conclude that, whereas variable but sometimes high cyclin D1 expression is a feature of sporadic hemangioblastomas, CCND1 genotype is unlikely to be an important genetic modifier in the oncogenesis of these tumors.     

Loss of von Hippel-Lindau protein causes cell density dependent deregulation of CyclinD1 expression through hypoxia-inducible factor

Loss of the von Hippel-Lindau gene (VHL) expression ca-uses deregulation of contact inhibition of cell growth, which might be one of the bases of the tumor suppressor function of VHL. Here we show that this function of the VHL gene product (pVHL) depends on cell autonomous events. To identify the target gene of pVHL, which is directly involved in the contact inhibition, we compared the gene expression profile between VHL-deficient renal carcinoma 786-O cells and those infected with an adenovirus vector encoding VHL. In addition to known pVHL-regulated genes, such as vascular endothelial growth factor and carbonic anhydrase, we found cyclinD1 as a new target of pVHL at a high cell density. In VHL-expressing cells (VHL (+) cells), the cyclinD1 mRNA expression level diminishes at a high cell density, while it remains at a relatively high level in VHL-deficient cells (VHL (-) cells). The cyclinD1 expression level was also abnormally high in VHL (-) cells at a high cell density. Consequently, the phosporylation level of the retinoblastoma (Rb) protein remained high in these cells, whereas there was no phosporylated Rb in VHL (+) cells under the contact inhibition. The abnormal expression of cyclinD1 at a high cell density was observed even in VHL (+) cells under the hypoxic state. Moreover, ectopic expression of a HIF mutant resistant to pVHL-mediated proteolysis causes the abnormal cyclinD1 expression in VHL (+) cells. Taken together, these observations indicate that VHL is required for the downregulation of cyclinD1 at a high cell density through HIF.     

In vitro and in vivo models analyzing von Hippel-Lindau disease-specific mutations

Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene cause tissue-specific tumors, with a striking genotype-phenotype correlation. Loss of VHL expression predisposes to hemangioblastoma and clear cell renal cell carcinoma, whereas specific point mutations predispose to pheochromocytoma, polycythemia, or combinations of hemangioblastoma, renal cell carcinoma, and/or pheochromocytoma. The VHL protein (pVHL) has been implicated in many cellular activities including the hypoxia response, cell cycle arrest, apoptosis, and extracellular matrix remodeling. We have expressed missense pVHL mutations in Vhl(-/-) murine embryonic stem cells to test genotype-phenotype correlations in euploid cells. We first examined the ability of mutant pVHL to direct degradation of the hypoxia inducible factor (HIF) subunits HIF1alpha and HIF2alpha. All mutant pVHL proteins restored proper hypoxic regulation of HIF1alpha, although one VHL mutation (VHL(R167Q)) displayed impaired binding to Elongin C. This mutation also failed to restore HIF2alpha regulation. In separate assays, these embryonic stem cells were used to generate teratomas in immunocompromised mice, allowing independent assessment of the effects of specific VHL mutations on tumor growth. Surprisingly, teratomas expressing the VHL(Y112H) mutant protein displayed a growth disadvantage, despite restoring HIFalpha regulation. Finally, we observed increased microvessel density in teratomas derived from Vhl(-/-) as well as VHL(Y112H), VHL(R167Q), and VHL(R200W) embryonic stem cells. Together, these observations support the hypothesis that pVHL plays multiple roles in the cell, and that these activities can be separated via discrete VHL point mutations. The ability to dissect specific VHL functions with missense mutations in a euploid model offers a novel opportunity to elucidate the activities of VHL as a tumor suppressor.     

Mutations of the von Hippel-Lindau gene confer increased susceptibility to natural killer cells of clear-cell renal cell carcinoma

The tumor suppressor gene von Hippel-Lindau (VHL) is involved in the development of sporadic clear-cell renal cell carcinoma (RCC). VHL interferes with angiogenesis and also controls cell adhesion and invasion. Therapies that target VHL-controlled genes are currently being evaluated in RCC patients. RCC is a immunogenic tumor and treatment with interleukin-2 (IL2) or interferon (IFN)-α results in regression in some patients. We used two renal tumor cell lines (RCC6 and RCC4) carrying VHL loss-of-function mutations to investigate the role of mutant VHL in susceptibility to natural killer (NK) cell-mediated lysis. The RCC6 and RCC4 cell lines were transfected with the wild-type gene to restore the function of VHL. The presence of the gene in RCC cells downregulated hypoxia-inducible factor (HIF)-1α and subsequently decreased vascular endothelial growth factor (VEGF) production. Relative to control transfectants and parental cells, pVHL-transfected cell lines activated resting and IL2-activated NK cells less strongly, as assessed by IFNγ secretion, NK degranulation and cell lysis. NKG2A, a human leukocyte antigen (HLA)-I-specific inhibitory NK receptor, controls the lysis of tumor targets. We show that HLA-I expression in RCC-pVHL cells is stronger than that in parental and controls cells, although the expression of activating receptor NK ligands remains unchanged. Blocking NKG2A/HLA-I interactions substantially increased lysis of RCC-pVHL, but had little effect on the lysis of VHL-mutated RCC cell lines. In addition, in response to IFNα, the exponential growth of RCC-pVHL was inhibited more than that of RCC-pE cells, indicating that VHL mutations may be involved in IFNα resistance. These results indicate that a decreased expression of HLA-I molecules in mutated VHL renal tumor cells sensitizes them to NK-mediated lysis. These results suggest that combined immunotherapy with anti-angiogenic drugs may be beneficial for patients with mutated VHL.     

Renal cyst development in mice with conditional inactivation of the von Hippel-Lindau tumor suppressor

Inactivation of the von Hippel-Lindau tumor suppressor, pVHL, is associated with both hereditary and sporadic renal cysts and renal cell carcinoma, which are commonly thought to arise from the renal proximal tubule. pVHL regulates the protein stability of hypoxia-inducible factor (HIF)-alpha subunits and loss of pVHL function leads to HIF stabilization. The role of HIF in the development of VHL-associated renal lesions remains to be determined. To investigate the functional consequences of pVHL inactivation and the role of HIF signaling in renal epithelial cells, we used the phosphoenolpyruvate carboxykinase (PEPCK) promoter to generate transgenic mice in which Cre-recombinase is expressed in the renal proximal tubule and in hepatocytes. We found that conditional inactivation of VHL in PEPCK-Cre mutants resulted in renal cyst development that was associated with increased erythropoietin levels and polycythemia. Increased expression of the HIF target gene erythropoietin was limited to the liver, whereas expression of carbonic anhydrase 9 and multidrug resistance gene 1 was up-regulated in the renal cortex of mutant mice. Inactivation of the HIF-alpha binding partner, arylhydrocarbon receptor nuclear translocator (Arnt), but not Hif-1alpha, suppressed the development of renal cysts. Here, we present the first mouse model of VHL-associated renal disease that will provide a basis for further genetic studies to define the molecular events that are required for the progression of VHL-associated renal cysts to clear cell renal cell carcinoma.     

Overexpression of von Hippel-Lindau tumor suppressor protein and antisense HIF-1alpha eradicates gliomas

The von Hippel-Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the alpha subunits of hypoxia-inducible-factors responsible for stimulating tumor angiogenesis and glycolysis, and targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to tumorigenesis and development of sporadic renal cell carcinomas and central nervous system hemangioblastomas. In the present study, we investigated whether engineered overexpression of pVHL in C6 glioma cells, which already express endogenous pVHL, would suppress the tumorigenicity of this particular tumor cell type. C6 cells overexpressing VHL displayed a reduced growth rate (70% inhibition) compared to the parental cell line when subcutaneously implanted in athymic (nu/nu) mice. Growth inhibition was associated with a 50% reduction in the number of tumor vessels and a 60% increase in tumor cell apoptosis, due in part to downregulation of HIF-1, VEGF, and the antiapoptotic factor Bcl-2, respectively. Gene transfer of VHL suppressed the growth of established C6 gliomas, and synergized with antisense HIF-1 to completely eradicate tumors. The data suggest that VHL gene therapy and/or agents that increase VHL expression could have utility in the treatment of gliomas, particularly when combined with agents that inhibit the expression or function of HIF-1.     

Von Hippel-Lindau syndrome: molecular mechanisms of the disease

Inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene is responsible for the development of renal carcinomas, pheochromocytomas and tumours in other organs. The gene product (pVHL) is a central component in the oxygen-sensing pathway through its role in the regulation of the hypoxia-inducible factor (HIF). Loss of pVHL leads to activation of the HIF pathway in normoxia with the concomitant increase in tumour vascularisation due to the up-regulation of pro-angiogenic genes. However, although the role of pVHL in the regulation of HIF has proved to be important for tumour growth, other pVHL functions independent of HIF have been reported and help to explain why loss of VHL leads to renal cancer. Studies aimed to characterise other molecular pathways that shed light on its physiological roles as a gatekeeper gene in kidney and other organs will be very helpful for the development of novel anticancer therapies.     

Third International Meeting on von Hippel-Lindau disease

Five years after the identification of the von Hippel-Lindau (VHL) gene, physicians, scientists and concerned VHL family members met to review the current state of knowledge on the diagnosis and treatment of VHL and to summarize the latest information on the biochemistry of the VHL protein (pVHL). The NIH and University of Pennsylvania groups reported the detection of germ-line mutations in 100% (93 of 93) of VHL families studied. Several studies determined the frequency of VHL germ-line mutations in individuals with a single manifestation of VHL without a family history of VHL. National groups to improve the diagnosis and treatment of individuals with VHL disease have been established in Great Britain, Denmark, France, Holland, Italy, Japan, Poland, and the United States. Evidence for the existence of genes that modify the expression of VHL was presented. The VHL protein appears to have several distinct functions: (a) down-regulation of hypoxia-inducible mRNAs; (b) proper assembly of the extracellular fibronectin matrix; (c) regulation of exit from the cell cycle; and (d) regulation of expression of carbonic anhydrases 9 and 12.     

von Hippel-Lindau disease: recent advances and therapeutic perspectives

von Hippel-Lindau disease is a hereditary cancer syndrome predisposing carriers to the development of a panel of highly vascularized tumors such as central nervous system and retinal hemangioblastomas, endolymphatic sac tumors, clear-cell renal cell carcinomas, pheochromocytomas and pancreatic neuroendocrine tumors. The disease is the foremost cause of inherited renal cell carcinomas, which is induced by germline mutations of the VHL tumor-suppressor gene also inactivated in most sporadic renal cell carcinomas. VHL appears to be a pivotal gene in the oxygen-sensing pathway, mainly involved in targeting the hypoxia-inducible factors for ubiquitination. This discovery is opening the way for the development of new specific drugs inhibiting hypoxia-inducible factors and/or their downstream targets, possibly representing an attractive treatment not only for von Hippel-Lindau disease but also for sporadic renal cell carcinomas and others cancers.     

Renal cell carcinoma- and pheochromocytoma-specific altered gene expression profiles in VHL mutant clones

Von Hippel-Lindau (VHL) disease is associated with various missense germline mutations in the VHL tumor suppressor gene. Some are associated with type 1 VHL disease, renal cell carcinoma (RCC) without pheochromocytoma, while others are associated with type 2A or 2B VHL disease, pheochromocytoma without and with RCC, respectively. These mutations may cause substitutions of specific amino acid residue and functional change of VHL protein (pVHL), which leads to the oncogenesis of the particular tumor types that characterize the different VHL disease types. To investigate, we transfected a pVHL-null RCC cell line with plasmids expressing wild-type pVHL (WT) or pVHL bearing 1 of 3 point mutations. These occur in the pVHL regions that bind hypoxia-inducible factor alpha (HIF-alpha ) or Elongin C. Microarray analysis showed that the clones bearing a mutation in the elongin-binding region (mutant 167) were unique, as many more genes were suppressed than up-regulated. The other two mutant groups, which bear a mutation in the HIF-alpha -binding region (mutants 98 and 111), showed the opposite pattern. The 167 mutation is associated with type 2B VHL disease. Real-time PCR analysis confirmed the altered expression of selected genes in the clones. Relative to WT, stratifin (14-3-3 sigma) and lysyl oxidase-like 1 were down-regulated in the 167 mutants, while the transforming growth factor beta-induced protein (beta ig-h3) was up-regulated in the 111 mutants. Thus, the location of pVHL mutations results in distinct gene expression patterns. Moreover, a mutation in the elongin-binding domain may induce type 2B tumors through different molecular pathways compared to those induced by type 1- or 2A-associated mutations in the HIF-alpha -binding region.