Regulation of the urokinase-type plasminogen activator system by the von Hippel-Lindau tumor suppressor gene.

The urokinase-type plasminogen activator (uPA) system plays an important role in tumor cell invasion, metastases, and angiogenesis. uPA, uPA receptor, and plasminogen activator inhibitor 1 (PAI-1) are prognostic factors in different solid tumors, e.g., renal cell carcinomas (RCCs). von Hippel-Lindau (VHL) disease is an inherited cancer syndrome that is characterized by extensively vascularized tumors, including hemangioblastomas and RCCs. In 75% of sporadic RCCs, the VHL gene is also inactivated. It has been recognized in sporadic RCC that PAI-1 mRNA levels are up-regulated and uPA mRNA levels are down-regulated. We determined the role of the VHL tumor suppressor gene in the regulation of the uPA system in RCC. In 786-O RCC cells expressing the wild-type (wt) VHL gene, we measured a 3-fold higher overall urokinase activity than in 786-O cells expressing a mutant VHL gene or lacking VHL. uPA mRNA and protein levels were higher in cells with wt VHL compared with cells with mutant VHL or lacking VHL. In addition, PAI-1 mRNA and protein levels were dramatically increased in 786-O cells with mutant VHL or lacking VHL, compared with cells expressing wt VHL. Our results provide further evidence that the VHL gene plays an important role in the process of angiogenesis by regulation of plasmin-mediated proteolysis of the extracellular matrix and may explain why VHL-induced RCCs grow slowly and metastasize relatively late.     

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 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.     

Expression of heparanase in renal cell carcinomas: implications for tumor invasion and prognosis

PURPOSE: Heparanase activity has been detected in many malignant tumors, showing a correlation with the metastatic potential. The present study was undertaken to investigate the expression of heparanase and its prognostic significance in renal cell carcinomas (RCC). EXPERIMENTAL DESIGN: Nineteen RCCs and 6 nonneoplastic renal tissues were analyzed for heparanase mRNA expression by real-time PCR. Heparanase protein expression was semiquantitatively investigated by immunohistochemistry in 70 RCCs. Involvement of heparanase in the invasiveness of RCC cell lines, 786-O and Caki-2 cells, was examined by down-regulating the gene expression with small interfering RNA (siRNA) using the Matrigel invasion assay. RESULTS: The expression level of heparanase mRNA was significantly higher in clear cell RCCs than in papillary RCCs, chromophobe RCCs, and nonneoplastic renal tissues. Heparanase was predominantly immunolocalized to cell surface and cytoplasm of clear cell RCCs and mean expression levels of heparanase were significantly higher in clear cell RCCs than in papillary and chromophobe RCCs. The protein expression levels were positively correlated with primary tumor stage, distant metastasis, and histologic grade. Targeting of heparanase mRNA expression in 786-O and Caki-2 cells with siRNA down-regulated the mRNA expression and inhibited the Matrigel invasion by these cells, whereas nonsilencing siRNA showed no effect. Multivariate Cox analysis revealed that elevated heparanase expression was a significant and an independent predictor of disease-specific survival (odds ratio, 8.814; P = 0.019). CONCLUSIONS: These data suggest that heparanase plays an important role in invasion and metastasis and silencing of the gene might be a potential therapeutic target in clear cell RCCs.     

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.     

A molecule targeting VHL-deficient renal cell carcinoma that induces autophagy

Renal cell carcinomas (RCCs) are refractory to standard therapies. The von Hippel-Lindau (VHL) tumor suppressor gene is inactivated in 75% of RCCs. By screening for small molecules selectively targeting VHL-deficient RCC cells, we identified STF-62247. STF-62247 induces cytotoxicity and reduces tumor growth of VHL-deficient RCC cells compared to genetically matched cells with wild-type VHL. STF-62247-stimulated toxicity occurs in a HIF-independent manner through autophagy. Reduction of protein levels of essential autophagy pathway components reduces sensitivity of VHL-deficient cells to STF-62247. Using a yeast deletion pool, we show that loss of proteins involved in Golgi trafficking increases killing by STF-62247. Thus, we have found a small molecule that selectively induces cell death in VHL-deficient cells, representing a paradigm shift for targeted therapy.     

The expression of hypoxia-inducible factor 1alpha is a favorable independent prognostic factor in renal cell carcinoma.

PURPOSE: Renal cell carcinoma (RCC) is the most common malignancy of the kidney composed of specific tumor types. The sporadic conventional RCCs are, in contrast to the other RCC types, characterized by a high rate of von Hippel-Lindau (VHL) mutations and hypermethylation. The majority of these tumors lack functional VHL protein (pVHL) that leads to increased hypoxia-inducible factor 1alpha (HIF-1alpha) expression. The pVHL is the physiologic regulator of the activity of HIF-1alpha by targeting it to the proteasome for degradation under normoxia. Both pVHL and HIF-1alpha target other genes that are important for cancer survival and proliferation. Expression of HIF-1alpha has been linked to poor prognosis in different malignancies, although few studies have been done on the relation between HIF-1alpha and clinical variables in RCC. EXPERIMENTAL DESIGN: HIF-1alpha protein expression was analyzed in tumor tissue from 92 patients with RCC. HIF-1alpha was quantified by Western blot relative to a positive control. RESULTS: The HIF-1alpha protein was expressed as two bands which strongly correlated (r = 0.906, P < 0.001); therefore, they were added and the sum evaluated against clinicopathologic variables. There was no association between HIF-1alpha and gender, stage, grade, tumor size, or vein invasion. Conventional RCCs had significantly higher HIF-1alpha expression compared with papillary and chromophobe RCCs and kidney cortex. In conventional RCC, HIF-1alpha was an independent prognostic factor. CONCLUSION: HIF-1alpha levels varied significantly between the different RCC types. In conventional RCC, HIF-1alpha was an independent prognostic factor. These data indicate that HIF-1alpha is involved in tumorogenesis and progression of RCC. Evaluation of other HIF target gene products and correlation to angiogenesis seems warranted.     

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.     

JunB promotes cell invasion and angiogenesis in VHL-defective renal cell carcinoma

Inactivation of the von Hippel-Lindau (VHL) tumor-suppressor gene causes both hereditary and sporadic clear-cell renal-cell carcinoma (ccRCC). Although the best-characterized function of the VHL protein (pVHL) is regulation of hypoxia-inducible factor-α (HIFα), pVHL also controls the development of pheochromocytoma through HIF-independent pathways by regulating JunB. However, it is largely unknown how these pathways contribute to the development and progression of ccRCC. In the present study, we confirmed that JunB was upregulated in VHL-defective ccRCC specimens by immunostaining. Short-hairpin RNA (shRNA)-mediated knockdown of JunB in 786-O and A498 VHL null ccRCC cells suppressed their invasiveness. In addition, JunB knockdown significantly repressed tumor growth and microvessel density in xenograft tumor assays. Conversely, forced expression of wild-type, but not dimerization-defective, JunB in a VHL-restored 786-O subclone promoted invasion in vitro and tumor growth and vessel formation in vivo. Quantitative PCR array analysis revealed that JunB regulated multiple genes relating to tumor invasion and angiogenesis such as matrix metalloproteinase-2 (MMP-2), MMP-9 and chemokine (C-C motif) ligand-2 (CCL2) in 786-O cells. JunB knockdown in these cells reduced the proteolytic activity of both MMPs in gelatin zymography and the amount of CCL2 in the culture supernatant. Moreover, shRNA-mediated knockdown of MMP-2 or inhibition of CCL2 activity with a neutralizing antibody repressed xenograft tumor growth and angiogenesis. Collectively, these results suggest that JunB promotes tumor invasiveness and enhances angiogenesis in VHL-defective ccRCCs.     

Blockade of the vascular endothelial growth factor-receptor 2 pathway inhibits the growth of human renal cell carcinoma, RBM1-IT4, in the kidney but not in the bone of nude mice

Primary and metastatic RCCs are consistently resistant to radiotherapy, chemotherapy, or immunotherapy. As recurrent or metastatic RCC after surgery is related with poor prognosis and cancer-related death, development of therapeutic modalities that can control RCC and improve patient survival is urgently needed. We determined whether blockade of the vascular endothelial growth factor-receptor 2 (VEGF-R2) signaling pathway inhibits the growth of human renal cell carcinoma cells in the kidney and bone of nude mice. Male nude mice implanted with 1x10(6) RBM1-IT4 cells in the kidney or in the tibia were treated with oral administrations of TSU-68, anti-VEGF-R2 tyrosine kinase inhibitor beginning 5 days after implantation. The tumor incidence, tumor weight and bone destruction were determined at twelve weeks after commencing the therapy. VEGF production by RCCs was determined by ELISA and alterations in VEGF production related with genetic instability were also analysed. VEGF-R expression of mouse osteoclast precursors (mOCPs) and human umbilical vascular endothelial cell (HUVEC) was determined by RT-PCR and Western immunoblotting. in vitro, the effects of TSU-68 on the cellular proliferation of HUVEC, normal human renal proximal tubule epithelial cell (RPTEC) and mOCPs were determined. RBM1-IT4 cells had loss of heterozygosity and frame shift mutation on chromosome 3p, inactivating the von Hippel-Lindau (VHL) tumor suppressor gene and resulting in the production of relatively higher levels of VEGF than the RCCs without VHL mutation. TSU-68 significantly inhibited the growth of RBM1-IT4 in the kidney (p<0.05). In contrast, TSU-68 did not inhibit the growth of RBM1-IT4 in the tibia or bone lysis. Although HUVEC, RPTEC and mOCPs expressed VEGF-R2, TSU-68 directly inhibited the VEGF-stimulated cell growth of HUVEC and RPTEC but not the mOCPs in vitro. These data indicate that the VEGF-VEGF-R2 pathway is not required for survival of the osteoclasts and anti-VEGF-R2 therapy did not contribute to the suppression of metastatic RCC growth in the bone.     

The von Hippel-Lindau tumor suppressor gene protects cells from UV-mediated apoptosis

The familial cancer syndrome, von Hippel-Lindau (VHL) disease, characterized by a predisposition to renal cell carcinoma and certain other tumor types, is caused by mutational inactivation of the VHL tumor suppressor gene. Loss of VHL gene function is detected also in the vast majority of sporadic renal cell carcinomas. Previous reports have determined a protective role for VHL in response to serum withdrawal and glucose deprivation. In this study, the effect of UV irradiation on VHL-negative and VHL-positive renal carcinoma cells was examined. VHL-negative 786-O renal carcinoma cells underwent apoptosis following UV irradiation. In contrast, reintroduction of wild-type VHL expression protected 786-O cells from UV-mediated cell death. p53 and Bax levels were equivalent in VHL-negative and VHL-positive 786-O cells. Strikingly, cyclin-dependent kinase inhibitors p21 and p27 underwent proteasome-dependent degradation in VHL-negative 786-O cells following UV treatment. However, p21 and p27 protein levels were stable in VHL-positive cells. Also, levels of the anti-apoptotic proteins, Bcl-2 and Bcl-xL were elevated in VHL-positive cells, consistent with the protection from apoptotic stimuli. UV treatment led to increased S phase in VHL-negative, but not VHL-positive cells. Thus, following UV irradiation, diminution of p21 and p27 levels resulted in a hyperproliferative state in VHL-negative cells, leading to apoptosis. These results suggest that loss of VHL function promotes apoptosis and may provide selective pressure toward cells that are able to escape apoptosis, leading to tumorigenesis.