The HIF and other quandaries in VHL disease.

Mutations in VHL underlie von Hippel-Lindau (VHL) disease, a hereditary cancer syndrome with several subtypes depending on the risk of developing certain combination of classic features, such as clear cell renal cell carcinoma (ccRCC), hemangioblastoma and pheochromocytoma. Although numerous potential substrates and functions of pVHL have been described over the past decade, the best-defined role of pVHL has remained as the negative regulator of the heterodimeric hypoxia-inducible factor (HIF) transcription factor via the oxygen-dependent ubiquitin-mediated degradation of HIF-α subunit. Despite the seminal discoveries that led to the molecular elucidation of the mammalian oxygen-sensing VHL-HIF axis, which have provided several rational therapies, the mechanisms underlying the complex genotype-phenotype correlation in VHL disease are unclear. This review will discuss and highlight the studies that have provided interesting insights as well as uncertainties to the underlying mechanisms governing VHL disease.

Juvenile myelomonocytic leukaemia-associated mutation in Cbl promotes resistance to apoptosis via the Lyn-PI3K/AKT pathway.

Juvenile myelomonocytic leukaemia (JMML) is an aggressive myeloproliferative neoplasm in children characterized by granulocyte macrophage colony-stimulating factor (GM-CSF) hypersensitivity and resistance to chemotherapy. We recently identified c-Cbl (henceforth referred to as Cbl) as a GM-CSF receptor (GMR) responsive protein that targets Src for ubiquitin-mediated destruction upon GM-CSF stimulation and showed that a loss of negative regulation of Src is pivotal in the hyperactivation of GMR signalling in JMML cells. However, the mechanism regulating the chemoresistant nature of JMML has remained largely unknown. Here, we show that the JMML-associated Cbl mutant in complex with the Src family kinase Lyn promotes Cbl's adapter function, leading to increased association to PI3K regulatory subunit p85 and Lyn-dependent AKT pro-survival signalling. Notably, molecular or pharmacologic inhibition of the Lyn-PI3K/AKT pathway, but not the Ras/mitogen-activated protein kinase signalling axis, markedly increased the sensitivity of the otherwise chemoresistant Cbl mutant-JMML cells to chemotherapeutic agents currently used in the treatment of JMML patients. These results support the potential translational benefit of combining modalities that inhibit Lyn-PI3K/AKT signalling with traditional antileukaemia agents in the management of JMML.

K63-ubiquitylation of VHL by SOCS1 mediates DNA double-strand break repair.

DNA repair is essential for maintaining genomic stability, and defects in this process significantly increase the risk of cancer. Clear-cell renal cell carcinoma (CCRCC) caused by inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is characterized by high genomic instability. However, the molecular mechanism underlying the association between the loss of VHL and genomic instability remains unclear. Here, we show that suppressor of cytokine signaling 1 (SOCS1) promotes nuclear redistribution and K63-ubiquitylation of VHL in response to DNA double-strand breaks (DSBs). Loss of VHL or VHL mutations that compromise its K63-ubiquitylation attenuates the DNA-damage response (DDR), resulting in decreased homologous recombination repair and persistence of DSBs. These results identify VHL as a component of the DDR network, inactivation of which contributes to the genomic instability associated with CCRCC.

Chemotherapy induces NEDP1-mediated destabilization of MDM2.

MDM2 is an E3 ligase that promotes ubiquitin-mediated destruction of p53. Cellular stresses such as DNA damage can lead to p53 activation due in part to MDM2 destabilization. Here, we show that the stability of MDM2 is regulated by an ubiquitin-like NEDD8 pathway and identify NEDP1 as a chemotherapy-induced isopeptidase that deneddylates MDM2, resulting in MDM2 destabilization concomitant with p53 activation. Concordantly, RNAi-mediated knockdown of endogenous NEDP1 blocked diminution of MDM2 levels and increased chemoresistance of tumor cells. These findings unveil the regulation of MDM2 stability through NEDP1 as a common molecular determinant governing chemotherapy-induced p53-dependent cell death.

HIF alpha 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.

The role of hypoxia-inducible factors in cancer.

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that mediates the adaptive responses to hypoxia by effecting the transcription of numerous hypoxia-inducible genes. HIF is frequently overexpressed in solid tumors, and the transactivation of HIF targets in transformed cells provides a distinct survival advantage. Accordingly, the upregulation of HIF correlates with increased progression or aggressiveness of the cancer and poor prognosis. In addition to the induction of HIF by hypoxia, its expression is induced by the loss of tumor suppressors VHL, PTEN, TSC1/2, PML, and SDH, as well as by the increased activity of PI3K and/or MAPK signaling pathways, underscoring the significance of HIF in oncogenesis.

Homotypic association between tumour-associated VHL proteins leads to the restoration of HIF pathway.

The von Hippel-Lindau (VHL) tumour suppressor gene encodes a substrate-specifying component of an E3 ubiquitin ligase that targets hypoxia-inducible factor (HIF) alpha subunits for degradation under normoxia. The VHL protein is composed of an N-terminal HIFalpha-binding beta domain and a C-terminal alpha domain, which is necessary and sufficient for the formation of the E3 multiprotein enzyme. A large number of disease-causing mutations in either the alpha or beta domain renders HIFalpha stable irrespective of oxygen tension, leading to the upregulation of numerous HIF-target genes, such as GLUT1 and VEGF. Here, we show that VHL forms a self-associated complex in vivo, but not in vitro, and demonstrate that coexpression of two different VHL missense mutants -- one in the alpha domain and the other in the beta domain -- restores HIF-mediated gene expression profile. These findings indicate that VHL homotypic complexes can function in vivo in a complementary fashion to target HIFalpha for ubiquitin-mediated proteolysis, and potentially explain why VHL-associated tumours with a missense mutation-carrying VHL allele is almost invariably accompanied by a second VHL allele harbouring a gross truncation or deletion.

Molecular targets from VHL studies into the oxygen-sensing pathway.

Inheritance of a faulty von Hippel-Lindau (VHL) tumor suppressor gene is the cause of VHL disease, a rare multisystemic autosomal dominant disorder characterized by the development of hypervascular tumors in a number of organs, including the retina, brain, spine, pancreas, adrenal gland, and the kidney. Recent discoveries have demonstrated that the VHL gene product pVHL serves as a substrate-recognition component of an E3 ubiquitin ligase complex that targets hypoxia-inducible factor (HIF) transcription factor for polyubiquitination and subsequent degradation. Accordingly, tumor cells devoid of functional pVHL show an inappropriate accumulation of HIF, as well as downstream HIF-target genes, such as vascular endothelial growth factor (VEGF), a potent angiogenic factor. Furthermore, HIF has been found to be elevated in many human cancers further underscoring its common significance in oncogenesis. These and other related recent findings have shed significant insight into the mechanisms governing mammalian cellular oxygen homeostasis and how disruptions in this oxygen-sensing pathway can lead to tumorigenesis. Next generation anti-cancer drugs will undoubtedly emerge from our understanding of the molecular pathways governing normal cellular metabolism, growth and differentiation that have gone awry during neoplastic transformation, and studies in VHL disease will serve as one of the proving grounds for the efficacy of 'designer' anti-cancer drugs tailored against the VHL-HIF pathway.

von Hippel-Lindau protein mutants linked to type 2C VHL disease preserve the ability to downregulate HIF.

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germ line mutation of the von Hippel-Lindau tumor suppressor gene (VHL). Tumors observed in this disorder include retinal and central nervous system hemangioblastomas, clear cell renal carcinomas and pheochromocytomas. The VHL gene product, pVHL, is a component of a ubiquitin ligase which targets the transcription factor known as hypoxia-inducible factor (HIF) for degradation in the presence of oxygen. pVHL also plays roles in the control of extracellular matrix formation and cell-cycle exit. Different VHL mutations confer different site-specific risks of cancer. Type 2C VHL mutations confer an increased risk of pheochromocytoma without the other stigmata of VHL disease. Here we report that the products of such type 2C VHL alleles retain the ability to down regulate HIF but are defective for promotion of fibronectin matrix assembly. Furthermore, pVHL L188V, a well studied type 2C mutant, retained the ability to suppress renal carcinoma growth in vivo. These studies strengthen the notion that HIF deregulation plays a causal role in hemangioblastoma and renal carcinoma, and raises the possibility that abnormal fibronectin matrix assembly contributes to pheochromocytoma pathogenesis in the setting of VHL disease.

HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing.

HIF (hypoxia-inducible factor) is a transcription factor that plays a pivotal role in cellular adaptation to changes in oxygen availability. In the presence of oxygen, HIF is targeted for destruction by an E3 ubiquitin ligase containing the von Hippel-Lindau tumor suppressor protein (pVHL). We found that human pVHL binds to a short HIF-derived peptide when a conserved proline residue at the core of this peptide is hydroxylated. Because proline hydroxylation requires molecular oxygen and Fe(2+), this protein modification may play a key role in mammalian oxygen sensing.

Myc-enhanced expression of Cul1 promotes ubiquitin-dependent proteolysis and cell cycle progression.

The c-Myc oncoprotein plays an important role in the growth and proliferation of normal and neoplastic cells. To execute these actions, c-Myc is thought to regulate functionally diverse sets of genes that directly govern cellular mass and progression through critical cell cycle transitions. Here, we provide several lines of evidence that c-Myc promotes ubiquitin-dependent proteolysis by directly activating expression of the Cul1 gene, encoding a critical component of the ubiquitin ligase SCF(SKP2). The cell cycle inhibitor p27(kip1) is a known target of the SCF(SKP2) complex, and Myc-induced Cul1 expression matched well with the kinetics of declining p27(kip1) protein. Enforced Cul1 expression or antisense neutralization of p27(kip1) was capable of overcoming the slow-growth phenotype of c-Myc null primary mouse embryonic fibroblasts (MEFs). In reconstitution assays, the addition of in vitro translated Cul1 protein alone was able to restore p27(kip1) ubiquitination and degradation in lysates derived from c-myc(-/-) MEFs or density-arrested human fibroblasts. These functional and biochemical data provide a direct link between c-Myc transcriptional regulation and ubiquitin-mediated proteolysis and together support the view that c-Myc promotes G(1) exit in part via Cul1-dependent ubiquitination and degradation of the CDK inhibitor, p27(kip1).

Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein.

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome that is characterized by the development of multiple vascular tumors and is caused by inactivation of the von Hippel-Lindau protein (pVHL). Here we show that pVHL, through its beta-domain, binds directly to hypoxia-inducible factor (HIF), thereby targeting HIF for ubiquitination in an alpha-domain-dependent manner. This is the first function to be ascribed to the pVHL beta-domain. Furthermore, we provide the first direct evidence that pVHL has a function analogous to that of an F-box protein, namely, to recruit substrates to a ubiquitination machine. These results strengthen the link between overaccumulation of HIF and development of VHL disease.

Synthetic peptides define critical contacts between elongin C, elongin B, and the von Hippel-Lindau protein.

The von Hippel-Lindau tumor suppressor protein (pVHL) negatively regulates hypoxia-inducible mRNAs such as the mRNA encoding vascular endothelial growth factor (VEGF). This activity has been linked to its ability to form multimeric complexes that contain elongin C, elongin B, and Cul2. To understand this process in greater detail, we performed a series of in vitro binding assays using pVHL, elongin B, and elongin C variants as well as synthetic peptide competitors derived from pVHL or elongin C. A subdomain of elongin C (residues 17-50) was necessary and sufficient for detectable binding to elongin B. In contrast, elongin B residues required for binding to elongin C were not confined to a discrete colinear domain. We found that the pVHL (residues 157-171) is necessary and sufficient for binding to elongin C in vitro and is frequently mutated in families with VHL disease. These mutations preferentially involve residues that directly bind to elongin C and/or alter the conformation of pVHL such that binding to elongin C is at least partially diminished. These results are consistent with the view that diminished binding of pVHL to the elongins plays a causal role in VHL disease.

The von Hippel-Lindau tumour suppressor protein: new perspectives.

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations of the VHL tumour suppressor gene. The VHL gene product, pVHL, forms multiprotein complexes that contain elongin B, elongin C and Cul-2, and negatively regulates hypoxia-inducible mRNAs. pVHL is suspected to play a role in ubiquitination given the similarity of elongin C and Cul-2 with Skp1 and Cdc53, respectively. pVHL can also interact with fibronectin and is required for the assembly of a fibronectin matrix. Finally, pVHL, at least indirectly, plays a role in the ability of cells to exit the cell cycle. Thus, pVHL is a tumour suppressor protein that regulates angiogenesis, extracellular matrix formation and the cell cycle.

pVHL19 is a biologically active product of the von Hippel-Lindau gene arising from internal translation initiation.

The von Hippel-Lindau (VHL) gene encodes a protein consisting of 213 amino acid residues with an apparent molecular mass of 30 kDa (pVHL30). Here we show that cells also produce a VHL protein (pVHL19) that appears to arise as a result of internal translation from the second methionine within the VHL ORF. pVHL30 resides primarily in the cytosol, with less amounts found in the nucleus or associated with cell membranes. In contrast pVHL19, in biochemical fractionation experiments, is equally distributed between the nucleus and cytosol and is not found in association with membranes. pVHL19, like pVHL30, can bind to elongin B, elongin C, and Hs-Cul2 in coimmunoprecipitation assays and can inhibit the production of hypoxia-inducible proteins such as vascular endothelial growth factor (VEGF) and GLUT1 when reintroduced into renal carcinoma cells that lack a wild-type VHL allele. Thus, cells contain two biologically active VHL gene products.

The von Hippel-Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix.

Fibronectin coimmunoprecipitated with wild-type von Hippel-Lindau protein (pVHL) but not tumor-derived pVHL mutants. Immunofluorescence and biochemical fractionation experiments showed that fibronectin colocalized with a fraction of pVHL associated with the endoplasmic reticulum, and cold competition experiments suggested that complexes between fibronectin and pVHL exist in intact cells. Assembly of an extracellular fibronectin matrix by VHL-/- renal carcinoma cells, as determined by immunofluorescence and ELISA assays, was grossly defective compared with VHL+/+ renal carcinoma cells. Reintroduction of wildtype, but not mutant, pVHL into VHL-/- renal carcinoma cells partially corrected this defect. Finally, extracellular fibronectin matrix assembly by VHL-/- mouse embryos and mouse embryo fibroblasts (MEFs), unlike their VHL+/+ counterparts, was grossly impaired. These data support a direct role of pVHL in fibronectin matrix assembly.

Functions of the von Hippel-Lindau tumour suppressor protein.

Von Hippel-Lindau disease (VHL) is caused by germline mutations in the VHL tumour suppressor gene. Tumour development in this setting is due to loss or inactivation of the remaining wild-type VHL allele. The VHL gene product (pVHL) resides primarily in the cytoplasm. A frequently mutated region of pVHL can bind to complexes containing elongin B, elongin C and Cul2. Loss of pVHL leads to an inappropriate accumulation of hypoxia-inducible mRNAs, such as the mRNA encoding vascular endothelial growth factor (VEGF), under normoxic conditions. This finding is most likely to account for the hypervascular nature of VHL-associated neoplasms. Current studies are focussed on understanding if and how binding to elongins and Cul2 is linked to the ability of pVHL to regulate hypoxia-inducible mRNAs. In this regard, it is perhaps noteworthy that elongin C and Cul2 are homologous to yeast proteins Skp1 and Cdc53. These latter proteins participate in the formation of complexes that target certain proteins for ubiquitination.

Regulation of hypoxia-inducible mRNAs by the von Hippel-Lindau tumor suppressor protein requires binding to complexes containing elongins B/C and Cul2.

The von Hippel-Lindau tumor suppressor protein (pVHL) binds to elongins B and C and posttranscriptionally regulates the accumulation of hypoxia-inducible mRNAs under normoxic (21% O2) conditions. Here we report that pVHL binds, via elongin C, to the human homolog of the Caenorhabditis elegans Cul2 protein. Coimmunoprecipitation and chromatographic copurification data suggest that pVHL-Cul2 complexes exist in native cells. pVHL mutants that were unable to bind to complexes containing elongin C and Cul2 were likewise unable to inhibit the accumulation of hypoxia-inducible mRNAs. A model for the regulation of hypoxia-inducible mRNAs by pVHL is presented based on the apparent similarity of elongin C and Cul2 to Skp1 and Cdc53, respectively. These latter proteins form complexes that target specific proteins for ubiquitin-dependent proteolysis.

New insights into the regulation of ICAM-1 gene expression.

Cell-cell adhesion is critical in the generation of effective immune responses and is dependent upon the expression of a variety of cell surface receptors. Intercellular adhesion molecule-1 (ICAM-1; CD54) is an inducible cell surface glycoprotein expressed at a low level on a subpopulation of hematopoietic cells, vascular endothelium, fibroblasts, and certain epithelial cells. However, its expression is dramatically increased at sites of inflammation, providing important means of regulating cell-cell interactions and thereby inflammatory responses. Inasmuch the modulation of ICAM-1 expression during inflammation by pharmacologic agents might be very attractive for medical treatment, the intracellular regulatory elements and signaling pathways underlying the inducible expression of ICAM-1 by proinflammatory cytokines remain largely unknown. In this review, a novel posttranscriptional regulation of ICAM-1 gene expression by two inflammatory mediators, interferon-gamma and phorbol myristate acetate, and the possible role of the serine/threonine phosphorylation pathway in the cycloheximide-induced ICAM-1 message stabilization are discussed in light of our current understanding of ICAM-1 gene regulation during an inflammatory response.

Regulation of ICAM-1 mRNA stability by cycloheximide: role of serine/threonine phosphorylation and protein synthesis.

Cycloheximide is a protein synthesis inhibitor that superinduces the expression of many genes by preventing the degradation of otherwise labile mRNAs. In some genes this depends on the presence of the AUUUA destabilizing multimers in the 3'UTR. We examined the effect of cycloheximide on the murine intercellular adhesion molecule-1 (ICAM-1; CD54) gene expression in several cell lines including A20 (B cell lymphoma), T28 (T cell hybridoma), P388D1 (monocytic cell), SVEC4-10 (lymphoid endothelial cell), and ICAM-1-transfected murine fibroblast L cells. Cycloheximide was indeed able to dramatically increase the accumulation of ICAM-1 mRNA in all the cell lines examined except T28, and this seemed to be due to the stabilization of the ICAM-1 mRNA as indicated by the half-life analysis. To determine whether this effect is dependent on the 3'UTR containing the AUUUA sequences, L cells were transfected with either the full-length ICAM-1 cDNA or a truncated form lacking the AUUUA sequences in the 3'UTR (ICAM-1Delta3). There was no discernible difference in the effect of cycloheximide on ICAM-1 mRNA accumulation or half-life between the two types of transfected cells. The effect of cycloheximide on ICAM-1 mRNA was markedly suppressed by serine/threonine (ser/thr) kinase inhibitors, H-7 and staurosporine, whereas the ser/thr phosphatase inhibitor, okadaic acid, augmented the cycloheximide effect. Inhibitors of protein tyrosine kinases and phosphatases had no effect. Unexpectedly, the level of cell surface ICAM-1 as well as de novo synthesis of ICAM-1 in SVEC4-10 and the ICAM-1-transfected L cells were also upregulated by cycloheximide, whereas the overall protein synthesis in these cells was profoundly inhibited, suggesting that ICAM-1 protein synthesis in these cells escapes the translational inhibition by cycloheximide. These results suggest that the stabilization of ICAM-1 mRNA by cycloheximide is independent of its translational inhibition and that ser/thr phosphorylation of unidentified protein(s) seems to play a crucial role in this effect.