Forkhead Transcription Factor 3a (FOXO3a) Modulates Hypoxia Signaling via Up-regulation of the von Hippel-Lindau Gene (VHL).

FOXO3a, a member of the forkhead homeobox type O (FOXO) family of transcriptional factors, regulates cell survival in response to DNA damage, caloric restriction, and oxidative stress. The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of the E3 ubiquitin ligase complex that mediates hypoxia-inducible factor α degradation under aerobic conditions, thus acting as one of the key regulators of hypoxia signaling. However, whether FOXO3a impacts cellular hypoxia stress remains unknown. Here we show that FOXO3a directly binds to the VHL promoter and up-regulates VHL expression. Using a zebrafish model, we confirmed the up-regulation of vhl by foxo3b, an ortholog of mammalian FOXO3a Furthermore, by employing the clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided endonuclease Cas9 (CRISPR/Cas9) technology, we deleted foxo3b in zebrafish and determined that expression of hypoxia-inducible genes was affected under hypoxia. Moreover, foxo3b-null zebrafish exhibited impaired acute hypoxic tolerance, resulting in death. In conclusion, our findings suggest that, by modulating hypoxia-inducible factor activity via up-regulation of VHL, FOXO3a (foxo3b) plays an important role in survival in response to hypoxic stress.

Expression of von Hippel-Lindau tumor suppressor protein (pVHL) characteristic of tongue cancer and proliferative lesions in tongue epithelium.

BACKGROUND: Patients with tongue cancer frequently show loss of heterozygosity (LOH) of the von Hippel-Lindau (VHL) tumor suppressor gene. However, expression of VHL protein (pVHL) in tongue cancer has rarely been investigated and remains largely unknown. We performed immunohistochemical staining of pVHL in tongue tissues and dysplasia, and examined the association with LOH and its clinical significance. METHODS: Immunohistochemical staining of pVHL in formalin-fixed, paraffin-embedded sections of cancerous and other tissues from 19 tongue cancer patients showed positivity for LOH of VHL in four samples, negativity in four samples, and was non-informative in 11 samples. The staining pattern of pVHL was also compared with those of cytokeratin (CK) 13 and CK17. RESULTS: In normal tongue tissues, pVHL staining was localized to the cytoplasm of cells in the basal layer and the area of the spinous layer adjacent to the basal layer of stratified squamous epithelium. Positive staining for pVHL was observed in the cytoplasm of cancer cells from all 19 tongue cancer patients. No differences as a result of the presence or absence of LOH were found. Notably, cytoplasm of poorly differentiated invasive cancer cells was less intensely stained than that of well and moderately differentiated invasive cancer cells. pVHL staining was also evident in epithelial dysplasia lesions with pVHL-positive cells expanding from the basal layer to the middle of the spinous layer. However, no CK13 staining was noted in regions of the epithelium, which were positive for pVHL. In contrast, regions with positive staining for CK17 closely coincided with those positive for pVHL. CONCLUSIONS: Positive staining for pVHL was observed in cancerous areas but not in normal tissues. pVHL expression was also detected in lesions of epithelial dysplasia. These findings suggest that pVHL may be a useful marker for proliferative lesions.

MIR-92 stimulates VEGF by inhibiting von Hippel-Lindau gene product in epithelial ovarian cancer.

The molecular mechanisms underlying regulation of vascular endothelial growth factor (VEGF) in epithelial ovarian cancer (EOC) remain poorly defined. VEGF, a potent angiogenic factor, is up-regulated in a variety of cancers and contributes to angiogenesis in tumor tissues. The level of VEGF correlates with progression of malignancy. We previously reported that miR-92 is abnormally elevated in the plasma of EOC patients. Here, we tested the hypothesis that miR-92 inhibits von Hippel-Lindau gene product (VHL), a tumor suppressor gene, and in turn de-represses HIF-1α, a known key transcription factor for VEGF, to stimulate VEGF expression. Using a variety of biomedical methods including Western blot, RT-PCR, gene silencing, luciferase assay, and chromatin immunoprecipitation in both surgically-resected specimens and EOC cell culture, we established that EOC cells have elevated levels of HIF-1α and miR-92 expression, but the expression of VHL is reduced. We further demonstrated that miR-92 can target the VHL transcript to repress its expression. We also found that stabilized HIF-1α can form an active complex with transcriptional coactivator p300 and phosphorylated-STAT3 at the VEGF promoter to stimulate its expression. In addition, matrix metalloproteinases MMP-2 and MMP-9 are positively regulated by HIF-1α. These results suggest that miR-92 can potentially be considered as a novel therapeutical target in treatment of EOS.

FAT1 is a novel upstream regulator of HIF1α and invasion of high grade glioma.

The hypoxic microenvironment is an important contributor of glioblastoma (GBM) aggressiveness via HIF1α, while tumour inflammation is profoundly influenced by FAT Atypical Cadherin (FAT1). This study was designed to explore the functional interaction and significance of FAT1 and HIF1α under severe hypoxia-mimicking tumour microenvironment in primary human tumours. We first identified a positive correlation of FAT1 with HIF1α and its target genes in GBM tumour specimens, revealing the significance of the FAT1-HIF1α axis in glioma cells. We found that severe hypoxia leads to an increased expression of FAT1 and HIF1α in U87MG and U373MG cells. To reveal the relevance of FAT1 under hypoxic conditions, we depleted endogenous FAT1 under hypoxia and found a substantial reduction in the expression of HIF1α and its downstream target genes like CA9, GLUT1, VEGFA, MCT4, HK2, BNIP3 and REDD1, as well as a significant reduction in the invasiveness in GBM cells. At the molecular level, under hypoxia the FAT1 depletion-associated reduction in HIF1α was due to compromised EGFR-Akt signaling as well as increased VHL-dependent proteasomal degradation of HIF1α. In brief, for the first time, these results reveal an upstream master regulatory role of FAT1 in the expression and role of HIF1α under hypoxic conditions and that FAT1-HIF1α axis controls the invasiveness of GBM. Hence, FAT1 represents a novel potential therapeutic target for GBM.

Targeting FAK scaffold functions inhibits human renal cell carcinoma growth.

Human conventional renal cell carcinoma (CCC) remains resistant to current therapies. Focal Adhesion Kinase (FAK) is upregulated in many epithelial tumors and clearly implicated in nearly all facets of cancer. However, only few reports have assessed whether FAK may be associated with renal tumorigenesis. In this study, we investigated the potential role of FAK in the growth of human CCC using a panel of CCC cell lines expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene as well as normal/tumoral renal tissue pairs. FAK was found constitutively expressed in human CCC both in culture cells and freshly harvested tumors obtained from patients. We showed that CCC cell growth was dramatically reduced in FAK-depleted cells or after FAK inhibition with various inhibitors and this effect was obtained through inhibition of cell proliferation and induction of cell apoptosis. Additionally, our results indicated that FAK knockdown decreased CCC cell migration and invasion. More importantly, depletion or pharmacological inhibition of FAK substantially inhibited tumor growth in vivo. Interestingly, investigations of the molecular mechanism revealed loss of FAK phosphorylation during renal tumorigenesis impacting multiple signaling pathways. Taken together, our findings reveal a previously uncharacterized role of FAK in CCC whereby FAK exerts oncogenic properties through a non canonical signaling pathway involving its scaffolding kinase-independent properties. Therefore, targeting the FAK scaffold may represent a promising approach for developing innovative and highly specific therapies in human CCC.

The von Hippel-Lindau protein pVHL inhibits ribosome biogenesis and protein synthesis.

pVHL, the product of von Hippel-Lindau (VHL) tumor suppressor gene, functions as the substrate recognition component of an E3-ubiquitin ligase complex that targets hypoxia inducible factor α (HIF-α) for ubiquitination and degradation. Besides HIF-α, pVHL also interacts with other proteins and has multiple functions. Here, we report that pVHL inhibits ribosome biogenesis and protein synthesis. We find that pVHL associates with the 40S ribosomal protein S3 (RPS3) but does not target it for destruction. Rather, the pVHL-RPS3 association interferes with the interaction between RPS3 and RPS2. Expression of pVHL also leads to nuclear retention of pre-40S ribosomal subunits, diminishing polysomes and 18S rRNA levels. We also demonstrate that pVHL suppresses both cap-dependent and cap-independent protein synthesis. Our findings unravel a novel function of pVHL and provide insight into the regulation of ribosome biogenesis by the tumor suppressor pVHL.

von Hippel-Lindau exonic methylation analysis using MALDI-TOF mass spectrometry.

PURPOSE: Aberrant promoter methylation turns off gene expression and is involved in human malignancy. Studies show that first exon methylation has a tighter association with gene silencing compared to promoter methylation or gene mutation. However, to our knowledge the clinical importance of exonic methylation in renal cell carcinoma is unknown. We analyzed renal cell carcinoma for VHL gene exonic methylation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. MATERIALS AND METHODS: In 48 institutionally banked renal cell carcinoma patient tissue samples VHL exon sequencing was done as well as methylation analysis of promoter and exon 1 by mass spectrometry or conventional bisulfite analysis. Methylated human lymphocytic DNA (0% and 100%), nontemplate distilled H2O, and the UOK121 and UOK171 human renal cell carcinoma cell lines served as assay controls. Samples were considered hypermethylated if a CpG site showed greater than 50% methylation. RESULTS: Nine of the 43 patient samples read by our exon 1 assay had methylated VHL exon 1 sites, including 3 showing hypermethylation. The exon 1 methylation assay was robust and reproducible. Samples with exon 1 hypermethylation showed no exonic mutations. All samples assayed at VHL exon 2 were hypermethylated. CONCLUSIONS: To assay renal cell carcinoma tumors for VHL methylation matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is robust and reproducible, and capable of quantifying the methylation status of individual DNA bases. Exon 1 methylation may be an alternate mechanism of VHL gene silencing in renal cell carcinoma in addition to mutation and promoter methylation. Applying this assay in patient populations may allow enhanced diagnosis or tumor typing in the future.

Exposure to cigarette smoke induces overexpression of von Hippel-Lindau tumor suppressor in mouse skeletal muscle.

Cigarette smoke (CS) is a well-established risk factor in the development of chronic obstructive pulmonary disease (COPD). In contrast, the extent to which CS exposure contributes to the development of the systemic manifestations of COPD, such as skeletal muscle dysfunction and wasting, remains largely unknown. Decreased skeletal muscle capillarization has been previously reported in early stages of COPD and might play an important role in the development of COPD-associated skeletal muscle abnormalities. To investigate the effects of chronic CS exposure on skeletal muscle capillarization and exercise tolerance, a mouse model of CS exposure was used. The 129/SvJ mice were exposed to CS for 6 mo, and the expression of putative elements of the hypoxia-angiogenic signaling cascade as well as muscle capillarization were studied. Additionally, functional tests assessing exercise tolerance/endurance were performed in mice. Compared with controls, skeletal muscles from CS-exposed mice exhibited significantly enhanced expression of von Hippel-Lindau tumor suppressor (VHL), ubiquitin-conjugating enzyme E2D1 (UBE2D1), and prolyl hydroxylase-2 (PHD2). In contrast, hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression was reduced. Furthermore, reduced muscle fiber cross-sectional area, decreased skeletal muscle capillarization, and reduced exercise tolerance were also observed in CS-exposed animals. Taken together, the current results provide evidence linking chronic CS exposure and induction of VHL expression in skeletal muscles leading toward impaired hypoxia-angiogenesis signal transduction, reduced muscle fiber cross-sectional area, and decreased exercise tolerance.

Increased expression of the von Hippel-Lindau gene in the implantation site of human tubal pregnancy.

The objective of this study was to investigate the expression of the von Hippel-Lindau (VHL) gene in tissues of human fallopian tube and tubal pregnancy. Twenty patients undergoing salpingectomy for tubal pregnancy were recruited into the study group. Tissues of tubal pregnancy were separated into both the implantation and non-implantation sites as the implantation group and the non-implantation group, respectively. Samples of ampullary fallopian tube during mid-secretory phase were collected from twenty patients with benign uterine disease as the control group. Immunohistochemistry, real-time reverse transcription polymerase chain reaction, and Western blotting analysis were performed to detect expressions of VHL mRNA and protein. The results showed that VHL immunostaining appeared in the cytoplasm of tubal epithelial cells. Expression of VHL mRNA in the implantation group was higher than that in the non-implantation group or the control group (P < 0.01). Intensity of VHL protein in the implantation group was increased compared with that in the non-implantation group (P < 0.05) or in the control group (P < 0.01). There was no difference on expressions of VHL mRNA and protein between the non-implantation group and the control group (P > 0.05). In conclusion, VHL mRNA and protein are present in human tubal tissues. The VHL gene expression is increased in the implantation site of tubal pregnancy, and locally elevated expression of the VHL gene might be associated with human tubal pregnancy.

Von Hippel-Lindau gene product modulates TIS11B expression in renal cell carcinoma: impact on vascular endothelial growth factor expression in hypoxia.

TIS11B belongs to a group of RNA-binding proteins (including TIS11/tristetraprolin and TIS11D) that share characteristic tandem CCCH-type zinc-finger domains and can be rapidly induced by multiple stimuli. TIS11B has been shown to regulate vascular endothelial growth factor (VEGF) mRNA stability in adrenocorticotropic hormone-stimulated primary adrenocortical cells. TIS11B has also been documented as a negative regulator of VEGF during development, but nothing has yet been reported in the context of human cancers. The Von Hippel-Lindau (VHL) tumor suppressor protein regulates VEGF gene expression at both the transcriptional and post-transcriptional levels in normoxia. However, whether it can do so in hypoxia is still unclear. Here, we report a unique regulatory function of VHL in VEGF expression in hypoxia that is mediated through modulation of TIS11B protein levels in renal cancer cells. In normoxia, we detected increased expression of the microRNA hsa-miR-29b in the VHL-overexpressing renal cancer cell line 786-O. We also show that this increased expression of hsa-miR-29b decreased TIS11B protein expression by post-transcriptional regulation in normoxia. In contrast, in hypoxia, increased TIS11B expression paralleled an increased TIS11B mRNA stability in VHL-overexpressing 786-O cells. This VHL-mediated TIS11B up-regulation in hypoxia may be important for TIS11B-regulated gene expression: we observed a down-regulation of VEGF mRNA in hypoxia in VHL-overexpressing cells compared with parental 786-O cells, and this effect was reversible by silencing TIS11B expression.

Utility of pVHL, maspin, IMP3, S100P and Ki67 in the distinction of autoimmune pancreatitis from pancreatic ductal adenocarcinoma.

Morphology plays an important role in the distinction of autoimmune pancreatitis (AIP) from pancreatic ductal adenocarcinoma (PDAC). However, we aimed to determine the utility of immunohistochemical tumor markers to contribute in the distinction of these entities. In surgical specimens with AIP (n = 20), PDAC (n = 20) and normal pancreas (n = 20), the expression of pVHL, maspin, IMP3, S100P and Ki67 was examined. We evaluated intralobular reactive ducts / acinoductal metaplasia (ILDs) and extralobular ducts (ELDs) in AIP, neoplastic glands in PDAC, and ductal epithelium in the normal pancreas, using a five-tiered scoring system. The Ki67 hot spot index (Ki67-HSPI) was determined manually and using automated digital imaging analysis of virtual double stains of Ki67 and CK8. Besides, sequential dual-immunohistochemical staining of maspin/pVHL, maspin/IMP3 and Ki67/maspin was performed in a subset of the specimens. Strong overexpression of IMP3, maspin, S100P and Ki67 and loss of pVHL was observed in PDAC compared to AIP and normal pancreas. In AIP however, focal and weak aberrant expression was observed with the following proportions in ILDs/ELDs: pVHL in 45 %/85 %, maspin in 30 %/70 %, IMP3 in 55 %/5%, S100P in 10 %/35 % and Ki67-HSPI >20 % in 15 %/70 %. At least two markers were aberrantly expressed in ILDs/ELDs in 45 %/60 %. The aberrant expression was more pronounced in type 2 AIP compared to type 1. In conclusion, our data indicate that pVHL, maspin, IMP3, S100P and Ki67 can be focal and weak aberrantly expressed in AIP. However, when used as a panel, these markers seem to be useful for the differentiation of AIP from PC.

Transcriptomic expression levels of the VHL, TIMP-3, and RASSF1A genes in renal tumors.

OBJECTIVE: In this study, we aimed to investigate the relation between the mRNA expression levels of VHL, TIMP-3 and RASSF1A genes, and the histopathological and clinical characteristics of patients with renal tumors. PATIENTS AND METHODS: Radical nephrectomy specimens of cases presented without neoadjuvant treatment were confirmed to be cancerous, non-cancerous, benign, and healthy after removal from separate localizations. A total of 69 patients with kidney tumors (138 tissue samples) were included in the study group. RNA isolation, reverse transcriptase PCR (RT-PCR), and quantitative real time PCR (qPCR) were performed, and the GAPDH gene was used to normalize mRNA levels. RESULTS: In the RCC cancerous tissue, TIMP-3 levels increased 1.3 times and RASSF1A levels increased 1.4 times compared to the corresponding levels in non-cancerous tissues, and there was no statistically significant difference in these values. On the other hand, VHL gene expression levels in cancerous tissue were 2.8 times higher than in matched adjacent non-cancerous tissues (p < 0.05). In the case of oncocytomas, TIMP-3 levels were found to be 3.2 times higher, RASSF1A levels 3.8 times higher, and VHL levels 2.2 times lower than the corresponding levels in healthy tissues (p < 0.05). CONCLUSIONS: The roles of VHL, TIMP-3, and RASSF1A mRNA expression in contributing to the development of renal tumors could not be clearly established. Further studies are therefore required to elucidate the mechanisms underlying renal tumors.

Diagnostic utility of S100P and von Hippel-Lindau gene product (pVHL) in pancreatic adenocarcinoma-with implication of their roles in early tumorigenesis.

Recently, we demonstrated von Hippel-Lindau gene product (pVHL) was expressed in normal pancreatic ducts but absent in pancreatic ductal adenocarcinoma (PDA). Previous studies have suggested the diagnostic value of S100P, S100A4, and S100A6 in PDA. In this study, we evaluated pVHL, S100P, S100A4, and S100A6 as potential markers for PDA, pancreatic intraepithelial neoplasia (PanIN), ampullary adenocarcinoma (AAD), and cholangiocarcinoma (CC). Immunostains were performed on 56 PDA cases, 20 AAD cases, and 28 CC cases using antibodies against pVHL, S100P, S100A6, and S100A4. Western blots were also performed on 2 cases of PDA and the matching non-neoplastic pancreatic tissues. Of the 56 PDA cases, immunoreactivity for S100P, S100A6, and S100A4 was observed in 56, 55, and 41 cases, respectively. Non-neoplastic ductal epithelium was negative for S100P in all cases. Ninety percent of PanINs were also positive for S100P. pVHL was not detected in all PDAs and 96% of PanINs by immunohistochemistry. S100P, S100A4, and S100A6 were present in a significant number of AADs and CCs; and pVHL expression was observed in 25% of AADs and 21% of CCs. Our data indicate that (1) S100P and pVHL are a pair of sensitive and specific markers for identifying primary PDA and PanIN; (2) up-regulation of S100P and down-regulation of pVHL may play a role in early tumorigenesis in PDA; and (3) the 4 markers studied have limited value in differentiating among PDA, AAD, and CC.

Acute inactivation of the VHL gene contributes to protective effects of ischemic preconditioning in the mouse kidney.

BACKGROUND/AIMS: The von Hippel-Lindau (VHL) protein functions as an E3 ubiquitin ligase, controlling the stability of hypoxia-inducible factor (HIF). Preinduction of HIF-1alpha before pathological insult activates a self-defense mechanism and suppresses further aggravation of organ or cellular injury by ischemia. We investigated whether acute inactivation of the VHL gene might play a role in the response of mice to ischemic renal injury. METHODS: We generated tamoxifen-inducible conditional VHL knockout (VHL-KO) mice to inactivate the VHL gene in an acute manner during renal ischemia-reperfusion injury (IRI) induced by bilateral clamping of kidney arteries. Renal IRI is characterized by renal dysfunction and tubular damage. RESULTS: After the procedure of IRI, blood urea nitrogen (BUN) and creatinine (CRN) levels in control mice were significantly higher (BUN, 138.10 +/- 13.03 mg/dl; CRN, 0.72 +/- 0.16 mg/dl) than in VHL-KO mice (BUN, 52.12 +/- 6.61 mg/dl; CRN, 0.24 +/- 0.04 mg/dl; BUN: p < 0.05; CRN: p < 0.05). Histologically, tubular injury scores were higher in control mice than in VHL-KO mice (p < 0.05). CONCLUSION: We suggest that the acute inactivation of the VHL gene contributes to protective effects of ischemic preconditioning in renal tubules of the mouse.

[Inactivation of the VHL gene in sporadic clear cell renal cancer].

Renal cell carcinoma is the most common variant of the kidney cancer, which accounts approximately 75% patients with this disease. The majority of those tumors are characterized by inactivation of the VHL gene suppressor as a result of mutations, allelic deletions and/or methylation. We have conducted the complex molecular-genetic analysis of 64 samples obtained from patients with the clear cell renal cancer. VHL mutations were detected by single strand conformation polymorphism and subsequent sequencing, loss of heterozygosity was analyzed using two STR-markers, methylation was tested by methylsensitive polymerase chain reaction. All revealed variations were statistically analyzed in respect to the parameters of primary tumors in various groups of patients. Seventeen VHL somatic mutations were detected, 12 from which were described for the first time. Allelic deletions of VHL were found in 31.6%, and methylation--in 7.8% samples of the renal cancer. As a whole, VHL inactivating events were presented in 46.9% cases of disease, in 51.7% -among renal cancer patients with first stage. We have not observed any association of mutations, loss of heterozygosity and methylation with clinical-pathological parameters of disease. Results of this investigation specify for expediency of further studies of molecular genetics aberrations in the VHL gene. Perhaps, it would promote renal cancer molecular markers evaluation, for example, a determination of suppressor genes methylated in renal cancer.

[Hydroxysafflor yellow A up-regulates HIF-1alpha via inhibition of VHL and p53 in Eahy 926 cell line exposed to hypoxia].

In present study, we investigated the mechanism of regulating HIF-1alpha expression by hydroxysafflor yellow A (HSYA) in Eahy 926 cell line under 1% O2 hypoxia. Eahy 926 cells were incubated with HSYA (100, 10 and 1 micromol x L(-1)) under hypoxia for the indicated time after treatment. Cell proliferation rate was detected using MTT assays. VHL and p53 location and protein expression were analyzed by immunocytochemical stain. HIF-1alpha, VHL and p53 mRNA expression were detected by RT-PCR. Protein expression of HIF-1alpha, VHL and p53 were assayed by Western blotting method. HSYA at 100 micromol x L(-1) increased Eahy 926 cells proliferation rate under hypoxia. HIF-1alpha mRNA and protein expression were up-regulated in the presence of HSYA. VHL, p53 mRNA and protein expression decreased significantly after 8 hours of treatment under hypoxia. HSYA protected Eahy 926 cells from hypoxia, and up-regulated HIF-1alpha expression partially via its inhibition of VHL and p53 expression.

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.

Primary cilium formation requires von hippel-lindau gene function in renal-derived cells.

Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene, VHL, occurs in the majority of renal clear cell carcinomas (RCC). VHL's function, regulating the degradation of hypoxia-inducible factor alpha (HIFalpha) subunits, explains the angiogenic nature of these tumors, but not tumor initiation. Because the development of renal cysts precedes tumor formation, and because the dysfunction of primary cilium is a common pathogenic mechanism in polycystic kidney diseases, we determined whether kidney-derived VHL- cells required VHL for the generation of cilium. Ectopic expression of VHL in RCC(VHL-) cells induced increased polarization and primary cilium formation. Cilium formation correlated directly with the expression of both wild-type VHL isoforms and a VHL mutant not associated with RCC development, whereas expression of RCC-associated VHL mutants did not support ciliogenesis. Requirement of VHL for ciliogenesis was independent of HIFalpha abundance. These data indicate separable independent functions for VHL (HIFalpha degradation and differentiation) and suggest a mechanism whereby disruption of both functions is required for renal carcinogenesis.

Regulation of E-cadherin expression by VHL and hypoxia-inducible factor.

Mutations in von Hippel-Lindau tumor suppressor gene (VHL) underlie the VHL hereditary cancer syndrome and also occur in most sporadic clear cell renal cell cancers (CCRCC). Currently, the mechanism(s) by which VHL loss of function promotes tumor development in the kidney are not fully elucidated. Here, we show that VHL inactivation in precancerous lesions in kidneys from patients with VHL disease correlates with marked down-regulation of the intercellular adhesion molecule E-cadherin. Moreover, in VHL-defective cell lines (RCC4 and RCC10) derived from sporadic CCRCC, reexpression of VHL was found to restore E-cadherin expression. The product of the VHL gene has multiple reported functions, the best characterized of which is its role as the recognition component of an ubiquitin E3 ligase complex responsible for mediating oxygen-dependent destruction of hypoxia-inducible factor-alpha (HIF-alpha) subunits. We show that HIF activation is necessary and sufficient to suppress E-cadherin in renal cancer cells. Given the fundamental role of E-cadherin in controlling epithelial behavior, our findings give insight into how VHL inactivation/HIF activation may lead to kidney cancer and also indicate a mechanism by which reduced oxygenation could alter E-cadherin expression in other cancers and influence normal homeostasis in other epithelia.

Hypoxia-inducible factor-1-dependent repression of E-cadherin in von Hippel-Lindau tumor suppressor-null renal cell carcinoma mediated by TCF3, ZFHX1A, and ZFHX1B.

A critical event in the pathogenesis of invasive and metastatic cancer is E-cadherin loss of function. Renal clear cell carcinoma (RCC) is characterized by loss of function of the von Hippel-Lindau tumor suppressor (VHL), which negatively regulates hypoxia-inducible factor-1 (HIF-1). Loss of E-cadherin expression and decreased cell-cell adhesion in VHL-null RCC4 cells were corrected by enforced expression of VHL, a dominant-negative HIF-1alpha mutant, or a short hairpin RNA directed against HIF-1alpha. In human RCC biopsies, expression of E-cadherin and HIF-1alpha was mutually exclusive. The expression of mRNAs encoding TCF3, ZFHX1A, and ZFHX1B, which repress E-cadherin gene transcription, was increased in VHL-null RCC4 cells in a HIF-1-dependent manner. Thus, HIF-1 contributes to the epithelial-mesenchymal transition in VHL-null RCC by indirect repression of E-cadherin.