Von Hippel-Lindau Loss Research Articles

Abstract 773: Identification and characterization of natural product-based inhibitors of hypoxia inducible factor-2 alpha

Abstract Clear cell renal cell carcinoma (RCC) is the most prevalent form of kidney cancer and is frequently associated with loss of von Hippel Lindau (VHL) gene function, resulting in the aberrant transcriptional activation of hypoxia response genes that contribute to tumor growth, angiogenesis and metastasis. Prior studies have shown that normoxic stabilization of hypoxia inducible factor (HIF)-1-alpha alone, while capable of mimicking some aspects of VHL loss, are not sufficient to reproduce tumorigenesis, indicating that it is not the most critical oncogenic substrate of VHL protein. To isolate compounds that selectively modulate HIF2-alpha for use as research tools and drug development leads, a cell-based high throughput screen of the NCI Natural Products Repository was performed. Stably transformed clones of the human renal clear cell carcinoma cell line 786-0 expressing a luciferase reporter construct under the control of the human vascular endothelial growth factor (VEGF) promoter-enhancer were used to identify natural product samples with inhibitory activity and minimal cellular toxicity. Leads from initial high throughput screen were chromatographically separated into component structures yielding several pure compounds with micromolar or submicromolar IC50 values, >80% inhibition and <5% cell toxicity. These compounds were then characterized biologically for their ability to inhibit VEGF protein secretion by intact 786-0 cells; several compounds showed >90% inhibition of VEGF secretion at micromolar doses under normoxic conditions. Studies to establish HIF selectivity and effects on RCC cell proliferation, survival, motility and anchorage independent growth are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 773.

VHL and PTEN loss coordinate to promote mouse liver vascular lesions

Von Hippel-Lindau (VHL) inactivation develops a tumor syndrome characterized by highly vascularized tumors as a result of hypoxia inducible factors (HIF) stabilization. The most common manifestation is the development of hemangioblastomas typically located in the central nervous system and other organs including the liver. PTEN (Phosphatase and tension homologue deleted on chromosome 10) inactivation also upregulates HIF-1alpha and may take part in promoting vascular lesions in tumors. The coordinate effect of loss of these tumor suppressors on HIF levels, and the subsequent effect on vascular lesion formation would elucidate the potential for mechanisms to modify HIF dosage supplementally and impact tumor phenotype. We therefore employed models of somatic conditional inactivation of Vhl, Pten, or both tumor suppressor genes in individual cells of the liver by Cre-loxP recombination to study the cooperativity of these two tumor suppressors in preventing tumor formation. Nine months after tumor suppressor inactivation, Vhl conditional deletion (Vhl (loxP/loxP)) mice showed no abnormalities, Pten conditional deletion (Pten (loxP/loxP)) mice developed liver steatosis and focal nodular expansion of hepatocytes containing lipid droplet and fat. Vhl and Pten conditional deletion (Vhl (loxP/loxP);Pten (loxP/loxP)) mice, however, developed multiple cavernous liver lesions reminiscent of hemangioblastoma. Liver hemangioblastomas in VHL disease may, therefore, require secondary mutation in addition to VHL loss of heterozygosity which is permissive for vascular lesion development or augments levels of HIF-1alpha.

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.

NF-κB–Dependent Plasticity of the Epithelial to Mesenchymal Transition Induced by Von Hippel-Lindau Inactivation in Renal Cell Carcinomas

The critical downstream signaling consequences contributing to renal cancer as a result of loss of the tumor suppressor gene von Hippel-Lindau (VHL) have yet to be fully elucidated. Here, we report that VHL loss results in an epithelial to mesenchymal transition (EMT). In studies of paired isogenic cell lines, VHL silencing increased the levels of N-cadherin and vimentin and reduced the levels of E-cadherin relative to the parental VHL(+) cell line, which displayed the opposite profile. VHL(+) cells grew as clusters of cuboidal and rhomboid cells, whereas VHL-silenced cells took on an elongated, fibroblastoid morphology associated with a more highly invasive character in Matrigel chamber assays. Based on earlier evidence that VHL loss can activate NF-kappaB, a known mediator of EMT, we tested whether NF-kappaB contributed to VHL-mediated effects on EMT. On pharmacologic or molecular inhibition of NF-kappaB, VHL-silenced cells regained expression of E-cadherin, lost expression of N-cadherin, and reversed their highly invasive phenotype. Introducing a pVHL-resistant hypoxia-inducible factor 1alpha (HIF1alpha) mutant (HIFalpha(M)) into VHL(+) cells heightened NF-kappaB activity, phenocopying EMT effects produced by VHL silencing. Conversely, inhibiting the heightened NF-kappaB activity in this setting reversed the EMT phenotype. Taken together, these results suggest that VHL loss induces an EMT that is largely dependent on HIFalpha-induced NF-kappaB. Our findings rationalize targeting the NF-kappaB pathway as a therapeutic strategy to treat renal tumors characterized by biallelic VHL inactivation.

Suppression of Hypoxia-Inducible Factor 2α Restores p53 Activity via Hdm2 and Reverses Chemoresistance of Renal Carcinoma Cells

p53 mutations are rarely detected in clear cell renal cell carcinoma (CCRCC), but, paradoxically, these tumors remain highly resistant to chemotherapy and death receptor-induced death. Here, we show that the accumulation of hypoxia-inducible factor 2alpha (HIF2alpha), a critical oncogenic event in CCRCC following the loss of von Hippel-Lindau (VHL) tumor suppressor protein, leads to Hdm2-mediated suppression of p53. Primary CCRCC specimens exhibiting strong hypoxic signatures show increased levels of activated nuclear phospho-Hdm2(Ser(166)), which is concomitant with low p53 expression. The abrogation of Hdm2-p53 interaction using the small-molecule Hdm2 inhibitor nutlin-3 or the downregulation of HIF2alpha via HIF2alpha-specific short hairpin RNA or wild-type VHL reconstitution restores p53 function and reverses the resistance of CCRCC cells to Fas-mediated and chemotherapy-induced cell death. These findings unveil a mechanistic link between HIF2alpha and p53 and provide a rationale for combining Hdm2 antagonists with chemotherapy for the treatment of CCRCC.

Commentary on Validation of the type 1 insulin-like growth factor receptor as a therapeutic target in renal cancer: Yuen JS, Akkaya E, Wang Y, Takiguchi M, Peak S, Sullivan M, Protheroe AS, Macaulay VM, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Headington, Oxford, United Kingdom

Expression of the type 1 insulin-like growth factor receptor (IGF1R) confers adverse prognosis in clear cell renal cell cancer (CC-RCC). We recently showed that IGF1R expression is inhibited by the von Hippel-Lindau (VHL) tumor suppressor, and the IGF1R is up-regulated in CC-RCC, in which VHL is frequently inactivated. We tested the hypothesis that IGF1R up-regulation mediates resistance to cancer therapeutics, evaluating the effects of IGF1R depletion on sensitivity to cytotoxic drugs, which are ineffective in RCC, and the mammalian target of rapamycin (mTOR) inhibitor rapamycin, analogues of which have clinical activity in this tumor. This study used CC-RCC cells harboring mutant VHL and isogenic cells expressing functional VHL. Cells were transfected with nonsilencing control small interfering RNA (siRNA), or with one of two different IGF1R siRNAs. The more potent siRNA was modified by 2′-O-methyl derivatization for in vivo administration. CC-RCC cells expressing mutant VHL and higher IGF1R were more chemoresistant than cells expressing functional VHL. IGF1R depletion induced apoptosis, blocked cell survival, and sensitized to 5-fluorouracil and etoposide. These effects were significantly greater in CC-RCC cells expressing mutant VHL, supporting the hypothesis that IGF1R up-regulation makes a major contribution to the chemoresistance associated with VHL loss. IGF1R depletion also enhanced sensitivity to mTOR inhibition, at least in part due to suppression of rapamycin-induced Akt activation. Administration of stabilized IGF1R siRNA was shown to sensitize CC-RCC xenografts to rapamycin in vivo. These data validate IGF1R as a therapeutic target in CC-RCC, and support the evaluation of IGF1R-inhibitory drugs in patients with renal cancer.

Validation of the type 1 insulin-like growth factor receptor as a therapeutic target in renal cancer

Expression of the type 1 insulin-like growth factor receptor (IGF1R) confers adverse prognosis in clear cell renal cell cancer (CC-RCC). We recently showed that IGF1R expression is inhibited by the von Hippel-Lindau (VHL) tumor suppressor, and the IGF1R is up-regulated in CC-RCC, in which VHL is frequently inactivated. We tested the hypothesis that IGF1R up-regulation mediates resistance to cancer therapeutics, evaluating the effects of IGF1R depletion on sensitivity to cytotoxic drugs, which are ineffective in RCC, and the mammalian target of rapamycin (mTOR) inhibitor rapamycin, analogues of which have clinical activity in this tumor. This study used CC-RCC cells harboring mutant VHL, and isogenic cells expressing functional VHL. Cells were transfected with nonsilencing control small interfering RNA (siRNA), or with one of two different IGF1R siRNAs. The more potent siRNA was modified by 2'-O-methyl derivatization for in vivo administration. CC-RCC cells expressing mutant VHL and higher IGF1R were more chemoresistant than cells expressing functional VHL. IGF1R depletion induced apoptosis, blocked cell survival, and sensitized to 5-fluorouracil and etoposide. These effects were significantly greater in CC-RCC cells expressing mutant VHL, supporting the hypothesis that IGF1R up-regulation makes a major contribution to the chemoresistance associated with VHL loss. IGF1R depletion also enhanced sensitivity to mTOR inhibition, at least in part due to suppression of rapamycin-induced Akt activation. Administration of stabilized IGF1R siRNA was shown to sensitize CC-RCC xenografts to rapamycin in vivo. These data validate IGF1R as a therapeutic target in CC-RCC, and support the evaluation of IGF1R-inhibitory drugs in patients with renal cancer.

Loss of VHL and Hypoxia Provokes PAX2 Up-Regulation in Clear Cell Renal Cell Carcinoma

The paired box gene 2, PAX2, encodes for a transcription factor that is up-regulated during nephrogenesis and becomes silenced in mature epithelium of the glomeruli, the proximal, and distal tubules. Reactivation of PAX2 has been frequently observed in clear cell renal cell carcinoma (ccRCC), a tumor type characterized by loss of von Hippel-Lindau (VHL) tumor suppressor function. The regulation of PAX2 expression in ccRCC is unknown. We applied reporter gene assays to investigate PAX2 promoter regulation. Furthermore, PAX2 expression was determined in ccRCC cell lines under normoxic and hypoxic condition in a VHL wild-type and mutated background. PAX2 expression was also assessed in 831 human ccRCC and correlated with hypoxia-inducible factor alpha (HIFalpha) and clinical parameters. Here, we show that both loss of VHL protein (pVHL) function and hypoxia leads to strong PAX2 reexpression. Using luciferase reporter gene assays, no induction was obtained in spite of six hypoxia response element motifs identified in the promoter of PAX2. Comprehensive immunohistochemical analyses showed significant correlations between PAX2, HIF1alpha, and HIF2alpha-target CCND1 expression patterns in ccRCC patients. Notably, PAX2 expression was highly associated with early-stage, well-differentiated ccRCC and, consequently, better clinical outcome (P < 0.0001 each). Additional analyses indicated that PAX2 repressor WT1 and cancer-linked hypomethylation are not important for transcriptional regulation of PAX2 in ccRCC. We conclude that in ccRCC, PAX2 reactivation is driven by HIF-dependent mechanisms following pVHL loss.

β-catenin gets jaded and von Hippel-Lindau is to blame

Numerous studies have pointed to interactions between the tumor suppressor von Hippel-Lindau (VHL) and the oncogenic Wnt-beta-catenin signaling cascade; however, the mechanism of this crosstalk has remained elusive. Among other roles, VHL can promote the stabilization of Jade-1. Now, recent findings provide compelling evidence that Jade-1 ubiquitylates beta-catenin, leading to its degradation. Thus, the loss of VHL, as seen in clear cell renal cell carcinoma, could lead to tumor formation through beta-catenin de-repression.

Mechanism of action of everolimus in renal cell carcinoma

The mammalian target of rapamycin (mTOR) is a strategic target for cancer therapy. This serine/threonine kinase is involved in the integration of signals from numerous growth factors and nutrients and regulates the mRNA translation of proteins that mediate cell growth, proliferation, metabolism, and angiogenesis. The pathways which impact mTOR signaling are dysregulated in many cancers, including renal cell carcinoma (RCC). In addition, for many patients, the pathogenesis of RCC is related to loss of function of the von Hippel-Lindau (VHL) gene, resulting in inappropriate accumulation of the hypoxia-inducible factor, HIF-1α. The heterodimer of HIF-1α and HIF-1β forms HIF-1, a transcription factor that regulates the production of mitogenic factors including vascular endothelial growth factor (VEGF), platelet-derived growth factor-β (PDGF-β), and transforming growth factor-α (TGF-α). Because mTOR regulates the production of HIF-1α, increased mTOR signaling in RCC may enhance the tumorigenic effects of loss of VHL. Therefore, inhibition of mTOR is particularly relevant in RCC. mTOR is located downstream of other therapeutic targets such as VEGF (target of bevacizumab) and VEGF receptor tyrosine kinases (targets of sorafenib and sunitinib), which provides opportunities for combination therapy and therapy in patients refractory to single agent treatments. Everolimus (RAD001), a synthetic, orally bioavailable analog of rapamycin, is a specific inhibitor of mTOR currently being developed for RCC and other types of cancer. This article will summarize the involvement of mTOR in RCC and the identified mechanisms by which mTOR inhibitors exert their antineoplastic effects, with a focus on everolimus.

Von Hippel-Lindau Tumor Suppressor Gene Loss in Renal Cell Carcinoma Promotes Oncogenic Epidermal Growth Factor Receptor Signaling via Akt-1 and MEK-1

ObjectivesClear-cell renal cell carcinoma (RCC) is the most prevalent form of kidney cancer and is frequently associated with loss of von Hippel-Lindau (VHL) gene function, resulting in the aberrant transcriptional activation of genes that contribute to tumor growth and metastasis, including transforming growth factor-α (TGF-α), a ligand of the epidermal growth factor receptor (EGFR) tyrosine kinase. To determine the functional impact of EGFR activation on RCC, we suppressed critical components of this pathway: EGFR, Akt-1, and MEK-1. MethodsStable transfection of RCC cells with plasmids bearing shRNA directed against each of these genes was used to individually suppress their expression. Transfectants were characterized for growth and invasiveness in vitro and tumorigenesis in vivo. ResultsRCC cell transfectants displayed significantly reduced growth rate and matrix invasion in vitro and RCC tumor xenograft growth rate in vivo. Analysis of tumor cells that emerged after extended periods in each model showed that significant EGFR suppression was sustained, whereas Akt-1 and MEK-1 knock-down cells had escaped shRNA suppression. ConclusionsEGFR, Akt-1, and MEK-1 are individually critical for RCC cell invasiveness in vitro and tumorigenicity in vivo, and even partial suppression of each can have a significant impact on tumor progression. The emergence of transfectants that had escaped Akt-1 and MEK-1 suppression during tumorigenicity experiments suggests that these effectors may each be more critical than EGFR for RCC tumorigenesis, consistent with results from clinical trials of EGFR inhibitors for RCC, where durable clinical responses have not been seen.

Dominant-Negative HIF-3α4 Suppresses VHL-Null Renal Cell Carcinoma Progression

The most prevalent mutations associated with the development of clear-cell renal cell carcinoma (CC-RCC) are the loss-of-function mutations of von Hippel-Lindau (VHL) tumor suppressor gene. These mutations invariably result in an inappropriate accumulation of HIF-α due to a failure of VHL as a substrate-recognition component of an E3 ubiquitin ligase complex to target HIFα for oxygen-dependent ubiquitin-mediated destruction. Stabilization of HIF-2α, but not HIF-1α, is the critical oncogenic event upon the functional loss of VHL in the development of CC-RCC. Here, we show that HIF-3α4, an alternatively spliced variant of human HIF-3α with similar domain structure as the murine inhibitory PAS protein (IPAS), forms an abortive transcriptional complex with HIF-2α, prevents the engagement of HIF-2 to the hypoxia-responsive elements (HREs) located in the promoter/enhancer regions of hypoxia-inducible genes. In addition, the re-expression of HIF-3α4 in VHL-null 786-O CC-RCC cells via adenovirus decreases the endogenous expression of HIF-2-driven gene expression and suppresses the growth of 786-O tumor xenografts in SCID mice. These results suggest that HIF-3α4 is a naturally occurring dominant-negative HIF-3α splice isoform with tumor suppressive activity and support the targeted delivery of HIF-3α4 as a potential therapeutic option to curtail HIF-dependent tumor progression.

HIFα expression in VHL-deficient renal cancer cells is dependent on phospholipase D

Loss of the von Hippel-Lindau (VHL) tumor suppressor gene contributes to proliferative disorders including renal cell carcinoma. The consequence of VHL loss is increased levels of hypoxia-inducible factor-alpha (HIFalpha), which is targeted for proteolytic degradation by the VHL gene product pVHL. HIF is a transcription factor that increases the expression of factors critical for tumorigenesis in renal cell carcinoma. We report here another regulatory component of HIFalpha expression in renal cancer cells. Phospholipase D (PLD), which is commonly elevated in renal and other cancers, is required for elevated levels of both HIF1alpha and HIF2alpha in VHL-deficient renal cancer cells. The induction of both HIF1alpha and HIF2alpha by hypoxic mimetic conditions was also dependent on PLD in renal cancer cells with restored pVHL expression. The effect of PLD activity upon HIFalpha expression was at the level of translation. PLD activity also provides a survival signal that suppresses apoptosis induced by serum deprivation in the renal cancer cells. Suppression of HIF2alpha has been shown to reverse tumorigenesis with renal cancer cells. The finding here that HIF2alpha expression is dependent on PLD in renal cancer cells suggests that targeting PLD signals may represent an alternative therapeutic strategy for targeting HIF2alpha in renal cancers where HIF2alpha is critical for tumorigenesis and elevated PLD activity is common.

Pseudohypoxic Pathways in Renal Cell Carcinoma

Mutations of the von Hippel-Lindau (VHL) or fumarate hydratase (FH) genes lead to morphologically different renal cell carcinomas with distinct clinical courses and outcomes. The VHL protein is a part of an ubiquitin ligase complex that targets proteins for proteosomal degradation. FH is one of the mitochondrial enzymes of the Kreb's cycle. Despite two different functionalities and cellular locations, loss of either VHL or FH products has been shown to alter expression levels of hypoxia-inducible factors (HIF-1alpha and HIF-2alpha) and their downstream targets. HIF proteins are key regulators of oxygen homeostasis. Tight regulation of HIF allows for cell survival and growth at the time of hypoxic stress. HIF acts via transcriptional regulation of vascular endothelial growth factor, platelet derived growth factor, endothelial growth factor receptor, glucose transporter protein 1, erythropoietin, and transforming growth factor-alpha. Loss of VHL or FH is thought to result in a pseudohypoxic state so that cellular response pathways mediated by HIF are activated despite normal oxygen conditions. Understanding of these pseudohypoxic pathways has provided a better appreciation of the molecular mechanisms of carcinogenesis in addition to providing a rationale for targeted therapeutic approaches.

Loss of Secreted Frizzled-Related Protein-1 Expression in Renal Cell Carcinoma Reveals a Critical Tumor Suppressor Function

In this issue of Clinical Cancer Research , Gumz et al. report on the loss of secreted frizzled-related protein-1 (sFRP-1) in clear cell renal cell carcinoma (RCC; ref. [1][1]). sFRP-1 is a member of a family of proteins that bind Wnts and can inhibit Wnt signaling during embryonic development and

Targeting the Loss of the von Hippel-Lindau Tumor Suppressor Gene in Renal Cell Carcinoma Cells

Late-stage clear cell renal carcinoma poses a formidable clinical challenge due to the high mortality rate associated with this disease. Molecular and genetic studies have identified functional loss of the von Hippel-Lindau (VHL) gene as a frequent and crucial event in the development of the malignant phenotype of clear cell renal carcinomas. Loss of VHL function thus represents a pathognomonic molecular defect for therapeutic exploitation. The objective of this study was to evaluate the possibility of targeting VHL loss through pharmacologic means. Chromomycin A3 (ChA3) was identified through in silico analysis of existing publicly available drug profiles from the National Cancer Institute as an agent that seemed to selectively target VHL-deficient clear cell renal carcinoma cells. Genotype-selective toxicity was first determined through short-term viability assays and then confirmed with clonogenic studies. Coculture of fluorescently labeled VHL-deficient and VHL-positive cells showed discriminate killing of the VHL-deficient cells with ChA3. Mechanistically, overexpression of hypoxia-inducible factor (HIF)-2alpha in VHL-positive clear cell renal carcinoma cells phenocopied loss of VHL with respect to ChA3 toxicity, establishing ChA3 as a HIF-dependent cytotoxin. This study shows the feasibility of selectively targeting the loss of the VHL tumor suppressor gene in clear cell renal carcinoma for potential clinical benefit and may have greater ramifications in the development of new targeted therapies for the treatment of cancer and other genetic diseases.

The von Hippel-Lindau tumor suppressor gene product represses oncogenic beta-catenin signaling in renal carcinoma cells.

Loss of von Hippel-Lindau (VHL) tumor suppressor gene function occurs in familial and most sporadic clear cell renal cell carcinoma (RCC), resulting in the aberrant expression of genes that control cell proliferation, invasion, and angiogenesis. The molecular mechanisms by which VHL loss leads to tumorigenesis are not yet fully defined. VHL loss has been shown to allow robust RCC cell motility, invasiveness, and morphogenesis in response to hepatocyte growth factor (HGF) stimulation, processes that are known to contribute to tumor invasiveness and metastatic potential. Among the most likely intracellular mediators of these HGF-driven activities is beta-catenin, a structural link between cadherens and the actin cytoskeleton, as well as a gene transactivator. We show that reconstitution of VHL expression in RCC cells repressed HGF-stimulated beta-catenin tyrosyl phosphorylation, adherens junction disruption, cytoplasmic beta-catenin accumulation, and reporter gene transactivation in RCC cells. Ectopic expression of a ubiquitination-resistant beta-catenin mutant specifically restored HGF-stimulated invasion and morphogenesis in VHL-transfected RCC cells. VHL gene silencing in non-RCC renal epithelial cells phenotypically mimicked VHL loss in RCC, and HGF-driven invasiveness was blocked by the expression of a dominant-negative mutant of Tcf. We conclude that, unlike many other cancers, where HGF pathway activation contributes to malignancy through the acquisition of autocrine signaling, receptor overexpression, or mutation, in RCC cells VHL loss enables HGF-driven oncogenic beta-catenin signaling. These findings identify beta-catenin as a potential target in biomarker and drug development for RCC.

VHL and p53: Tumor Suppressors Team Up to Prevent Cancer

In a recent issue of Molecular Cell, Roe et al. (2006) report that the von Hippel-Lindau (VHL) protein is a positive regulator of p53, thus providing insight into the mechanisms by which VHL loss of function leads to cancer.

The Tumor Suppressor von Hippel-Lindau Gene Product and Metastasis: New Thoughts on an Old Molecule

In this issue of The American Journal of Pathology, long-time von Hippel-Lindau (VHL) pioneer William G. Kaelin with Nakamura and colleagues1 demonstrates that cells lacking wild-type VHL are defective in the expression and secretion of the glycoprotein clusterin. This phenomenon was observed in cells capable of ubiquitylating hypoxia-inducible factor (HIF) and possessing Type 2C VHL mutants. The authors also found that pVHL-defective renal carcinoma cells overexpress insulin-like growth factor binding protein-3 (IGBP3) and plasminogen activator inhibitor type-1 in a HIF-dependent manner. Previous reports have described pVHL modulating other genes in a HIF-independent manner to promote its tumor suppressor function; however, the overexpression of clusterin by pVHL raises several questions and requires further discussion.

Interplay between VHL/HIF1α and Wnt/β-catenin pathways during colorectal tumorigenesis

Activation of the Wnt signaling pathway initiates the transformation of colorectal epithelial cells, although the transition to metastatic cancer requires angiogenesis. We have investigated the expression of the von Hippel-Lindau (VHL) tumor suppressor in the intestines from humans and mice. Here, we show that VHL expression is regulated by TCF4 and is restricted to the proliferative compartment at the bottom of intestinal crypts. Accordingly, VHL is completely absent from the proliferative intestinal pockets of Tcf4(-/-) perinatal mice. We observed complementary staining of the hypoxia-inducible factor (HIF) 1alpha to VHL in normal intestinal epithelium as well as in all stages of colorectal cancer (CRC). To the best of our knowledge, this is the first report demonstrating the presence of nuclear HIF1alpha in normoxic healthy adult tissue. Although we observed upregulated levels of VHL in very early CRC lesions from sporadic and familial adenomatous polyposis patients - presumably due to activated Wnt signaling - a clear reduction of VHL expression is observed in later stages of CRC progression, coinciding with stabilization of HIF1alpha. As loss of VHL in later stages of CRC progression results in stabilization of HIF, these data provide evidence that selection for VHL downregulation provides a proangiogenic impulse for CRC progression.