Dead Cells Don’t Form Tumors: HIF-Dependent Cytotoxins

Elimination or reduction of tumor burden is the primary goal of cancer therapy. Strategies to achieve this goal with the fewest adverse effects to the patient are an area of intense investigation. Elevated protein levels of hypoxia-inducible factor (HIF) are commonly found in solid tumors, while rarely found in healthy tissue. Numerous studies have suggested that HIF activity is essential for the development of solid tumors. Thus, inhibition of HIF represents an attractive therapeutic target for eradicating tumors. The search for small molecules that target and inhibit HIF activity is currently underway. We propose an alternate approach: to directly target and kill HIF-activated tumor cells. This approach is advantageous in that cells with activated HIF will be eliminated directly. Specific elimination of HIF-activated cells represents a potential mechanism for inhibiting tumor growth, with the potential advantage of sparing the patient of the normal tissue toxicity associated with current treatment options.     

Dead cells don't form tumors: HIF-dependent cytotoxins

Elimination or reduction of tumor burden is the primary goal of cancer therapy. Strategies to achieve this goal with the fewest adverse effects to the patient are an area of intense investigation. Elevated protein levels of hypoxia-inducible factor (HIF) are commonly found in solid tumors, while rarely found in healthy tissue. Numerous studies have suggested that HIF activity is essential for the development of solid tumors. Thus, inhibition of HIF represents an attractive therapeutic target for eradicating tumors. The search for small molecules that target and inhibit HIF activity is currently underway. We propose an alternate approach: to directly target and kill HIF-activated tumor cells. This approach is advantageous in that cells with activated HIF will be eliminated directly. Specific elimination of HIF-activated cells represents a potential mechanism for inhibiting tumor growth, with the potential advantage of sparing the patient of the normal tissue toxicity associated with current treatment options.     

Inhibiting hypoxia-inducible factor 1 for cancer therapy

Hypoxia has long been recognized as a common feature of solid tumors and a negative prognostic factor for response to treatment and survival of cancer patients. The discovery of hypoxia-inducible factor 1 (HIF-1), a molecular determinant of the response of mammalian cells to hypoxia, has led to the identification of a "molecular target" of hypoxia suitable for the development of cancer therapeutics. Early controversy about whether or not HIF-1 is a good target for therapy has not discouraged academic groups and pharmaceutical companies from actively engaging in the discovery of small-molecule inhibitors of HIF. However, what is the best strategy to inhibit HIF and how HIF inhibitors should be developed for treatment of human cancers is still poorly defined. In this review, aspects related to the identification and early development of novel HIF inhibitors are discussed. Identification and validation of pharmacodynamic end points relevant to the HIF-1 pathway is essential for a rational development of HIF inhibitors. Integration of these biomarkers in early clinical trials may provide valuable information to determine the contribution of HIF inhibitors to response to therapy. Finally, HIF inhibitors should be incorporated in combination strategies to effectively target multiple cellular components of the tumor microenvironment and redundant signaling pathways frequently deregulated in human cancer.     

Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein

The von Hippel-Lindau tumor suppressor protein (pVHL) has emerged as a key factor in cellular responses to oxygen availability, being required for the oxygen-dependent proteolysis of alpha subunits of hypoxia inducible factor-1 (HIF). Mutations in VHL cause a hereditary cancer syndrome associated with dysregulated angiogenesis, and up-regulation of hypoxia inducible genes. Here we investigate the mechanisms underlying these processes and show that extracts from VHL-deficient renal carcinoma cells have a defect in HIF-alpha ubiquitylation activity which is complemented by exogenous pVHL. This defect was specific for HIF-alpha among a range of substrates tested. Furthermore, HIF-alpha subunits were the only pVHL-associated proteasomal substrates identified by comparison of metabolically labeled anti-pVHL immunoprecipitates from proteosomally inhibited cells and normal cells. Analysis of pVHL/HIF-alpha interactions defined short sequences of conserved residues within the internal transactivation domains of HIF-alpha molecules sufficient for recognition by pVHL. In contrast, while full-length pVHL and the p19 variant interact with HIF-alpha, the association was abrogated by further N-terminal and C-terminal truncations. The interaction was also disrupted by tumor-associated mutations in the beta-domain of pVHL and loss of interaction was associated with defective HIF-alpha ubiquitylation and regulation, defining a mechanism by which these mutations generate a constitutively hypoxic pattern of gene expression promoting angiogenesis. The findings indicate that pVHL regulates HIF-alpha proteolysis by acting as the recognition component of a ubiquitin ligase complex, and support a model in which its beta domain interacts with short recognition sequences in HIF-alpha subunits.     

pVHL modification by NEDD8 is required for fibronectin matrix assembly and suppression of tumor development

Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is the cause of the familial VHL disease and most sporadic renal clear-cell carcinomas (RCC). pVHL has been shown to play a role in the destruction of hypoxia-inducible factor alpha (HIF-alpha) subunits via ubiquitin-mediated proteolysis and in the regulation of fibronectin matrix assembly. Although most disease-causing pVHL mutations hinder the regulation of the HIF pathway, every disease-causing pVHL mutant tested to date has failed to promote the assembly of the fibronectin matrix, underscoring its potential importance in VHL disease. Here, we report that a ubiquitin-like molecule called NEDD8 covalently modifies pVHL. A nonneddylateable pVHL mutant, while retaining its ability to ubiquitylate HIF, failed to bind to and promote the assembly of the fibronectin matrix. Expression of the neddylation-defective pVHL in RCC cells, while restoring the regulation of HIF, failed to promote the differentiated morphology in a three-dimensional growth assay and was insufficient to suppress the formation of tumors in SCID mice. These results suggest that NEDD8 modification of pVHL plays an important role in fibronectin matrix assembly and that in the absence of such regulation, an intact HIF pathway is insufficient to prevent VHL-associated tumorigenesis.