Target gene selectivity of hypoxia-inducible factor-alpha in renal cancer cells is conveyed by post-DNA-binding mechanisms.

Inactivation of the von Hippel-Lindau tumour suppressor in renal cell carcinoma (RCC) leads to failure of proteolytic regulation of the alpha subunits of hypoxia-inducible factor (HIF), constitutive upregulation of the HIF complex, and overexpression of HIF target genes. However, recent studies have indicated that in this setting, upregulation of the closely related HIF-alpha isoforms, HIF-1alpha and HIF-2alpha, have contrasting effects on tumour growth, and activate distinct sets of target genes. To pursue these findings, we sought to elucidate the mechanisms underlying target gene selectivity for HIF-1alpha and HIF-2alpha. Using chromatin immunoprecipitation to probe binding to hypoxia response elements in vivo, and expression of chimaeric molecules bearing reciprocal domain exchanges between HIF-1alpha and HIF-2alpha molecules, we show that selective activation of HIF-alpha target gene expression is not dependent on selective DNA-binding at the target locus, but depends on non-equivalent C-terminal portions of these molecules. Our data indicate that post-DNA binding mechanisms that are dissimilar for HIF-1alpha and HIF-2alpha determine target gene selectivity in RCC cells.

Identification of novel VHL targets that are associated with the development of renal cell carcinoma.

von Hippel-Lindau (VHL) disease is a dominantly inherited family cancer syndrome characterized by the development of retinal and central nervous system haemangioblastomas, renal cell carcinoma (RCC) and phaeochromocytoma. Specific germline VHL mutations may predispose to haemangioblastomas, RCC and phaeochromocytoma to a varying extent. Although dysregulation of the hypoxia-inducible transcription factor-2 and JunB have been linked to the development of RCC and phaeochromocytoma, respectively, the precise basis for genotype-phenotype correlations in VHL disease have not been defined. To gain insights into the pathogenesis of RCC in VHL disease we compared gene expression microarray profiles in a RCC cell line expressing a Type 1 or Type 2B mutant pVHL (RCC-associated) to those of a Type 2A or 2C mutant (not associated with RCC). We identified 19 differentially expressed novel VHL target genes linked to RCC development. Eight targets were studied in detail by quantitative real-time polymerase chain reaction (three downregulated and five upregulated by wild-type VHL) and for six genes the effect of VHL inactivation was mimicked by hypoxia (but hypoxic-induction of smooth muscle alpha-actin 2 was specific for a RCC cell line). The potential role of four RCC-associated VHL target genes was assessed in vitro. NB thymosin beta (TMSNB) and proteinase-activated receptor 2 (PAR2) (both downregulated by wt pVHL) increased cell growth and motility in a RCC cell line, but aldehyde dehydrogenase (ALDH)1 and ALDH7 had no effect. These findings implicate TMSNB and PAR2 candidate oncogenes in the pathogenesis of VHL-associated RCC.