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.     

Antitumor effect of parathyroid hormone-related protein neutralizing antibody in human renal cell carcinoma in vitro and in vivo

Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene occurs in 40-80% of human conventional renal cell carcinomas (RCCs). We showed recently that VHL-deficient RCCs expressed large amounts of parathyroid hormone-related protein (PTHrP), and that PTHrP, acting through the PTH1 receptor (PTH1R), plays an essential role in tumor growth. We also showed that PTHrP expression is negatively regulated by the VHL gene products (pVHL). Our goal was to determine whether blocking the PTHrP/PTH1R system might be of therapeutic value against RCC, independent of VHL status and PTHrP expression levels. The antitumor activity of PTHrP neutralizing antibody and of PTH1R antagonist were evaluated in vitro and in vivo in a panel of human RCC lines expressing or not pVHL. PTHrP is upregulated compared with normal tubular cells. In vitro, tumor cell growth and viability was decreased by up to 80% by the antibody in all cell lines. These effects resulted from apoptosis. Exogenously added PTHrP had no effect on cell growth and viability, but reversed the inhibitory effects of the antibody. The growth inhibition was reproduced by a specific PTH1R antagonist in all cell lines. In vivo, the treatment of nude mice bearing the Caki-1 RCC tumor with the PTHrP antibody inhibited tumor growth by 80%, by inducing apoptosis. Proliferation and neovascularization were not affected by the antiserum. Anti-PTHrP treatment induced no side effects as assessed by animal weight and blood chemistries. Current therapeutic strategies are only marginally effective against metastatic RCC, and adverse effects are common. This study provides a rationale for evaluating the blockade of PTHrP signaling as therapy for human RCC in a clinical setting.     

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.     

The 104-123 amino acid sequence of the beta-domain of von Hippel-Lindau gene product is sufficient to inhibit renal tumor growth and invasion

The von Hippel-Lindau (VHL) tumor suppressor gene is mutated in patients with VHL disease and in the majority of patients with sporadic renal cell carcinomas (RCCs). RCCs are dependent on insulin-like growth factor-1 receptor-mediated signaling for tumor growth and invasion in vivo. Reintroduction of the VHL gene product (pVHL) can inhibit on insulin-like growth factor-I receptor-mediated signaling in RCC cells in vitro through interaction with protein kinase C delta and is mediated by a specific amino acid sequence (104-123) in the beta-domain of the pVHL. In the present study, the amino acid sequence (104-123) of the pVHL was conjugated to the protein transduction domain of HIV-TAT protein (TATFLAGVHL-peptide) to facilitate entry into cells, and we demonstrate that this amino acid region of VHL is sufficient to block proliferation and invasion of 786-O renal cancer cells in vitro. Furthermore, daily i.p. injections with the TATFLAGVHL peptide retarded and, in some cases, caused partial regression of renal tumors that were implanted in the dorsal flank of nude mice. Treatment with this peptide also inhibits the invasiveness of renal tumors. A 56% decrease in the proliferative index in tumors treated with the TATFLAGVHL-peptide versus control-peptide-treated mice was observed. Taken together, these results show the novel importance of a 20-amino acid sequence of the beta-domain of the VHL gene product capable of inhibiting tumor growth and invasion. These results lay the foundation for a unique approach toward treating RCCs using this small-molecular-weight peptide fused to the TAT-sequence, which may, in the future, be used alone or in conjunction with other therapies.     

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.