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Identification of Novel Hypoxia Dependent and Independent Target Oncogene (2000) 19, 6297 ± 6305 ã 2000 Nature Publishing Group All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Identi®cation of novel hypoxia dependent and independent target genes of the von Hippel-Lindau (VHL) tumour suppressor by mRNA dierential expression pro®ling Charles C Wyko1, Christopher W Pugh2, Patrick H Maxwell2, Adrian L Harris1 and Peter J Ratclie*,2 1Institute of Molecular Medicine, John Radclie Hospital, Oxford OX3 9DS, UK; 2Wellcome Trust Centre for Human Genetics, Oxford OX3 7BN, UK The von Hippel-Lindau tumour suppressor gene (VHL) Germline mutations in the von Hippel-Lindau targets hypoxia inducible factor (HIF)-a subunits for (VHL) tumour suppressor gene are associated with a ubiquitin dependent proteolysis. To better understand the dominantly inherited cancer syndrome, in which role of this and other putative pathways of gene inactivation or loss of the remaining wild type allele regulation in VHL function we subjected mRNA from as a somatic event results in the formation of typically VHL defective renal carcinoma cells and transfectants hypervascularized neoplasms involving the kidney, re-expressing a wild type VHL allele to dierential retina, pancreas, adrenal gland and central nervous expression pro®ling, and analysed VHL target genes for system (Kaelin and Maher, 1998). In addition, somatic oxygen regulated expression. Among a group of newly inactivation or mutation of both VHL alleles is identi®ed VHL target genes the majority but not all were observed in the majority of sporadic renal cancers regulated by oxygen, indicating that whilst dysregulation (Gnarra et al., 1994). Recent work has connected the of the HIF system makes a dominant contribution to function of the gene product, pVHL, with regulation of alterations in transcription, VHL has other in¯uences on the transcriptional response to hypoxia. pVHL forms patterns of gene expression. Genes newly de®ned as part of a ubiquitin ligase complex (Iwai et al., 1999; targets of the VHL/hypoxia pathway (conditionally Lisztwan et al., 1999) that targets the regulatory a downregulated by VHL in normoxic cells) include subunits of hypoxia inducible factor-1 (HIF-1) for aminopeptidase A, collagen type V, alpha 1, cyclin G2, oxygen dependent proteolysis (Maxwell et al., 1999; DEC1/Stra13, endothelin 1, low density lipoprotein Cockman et al., 2000). HIF-1 is a heterodimeric DNA receptor-related protein 1, MIC2/CD99, and transgluta- binding complex that directs the expression of a large minase 2. These genes have a variety of functions family of hypoxia inducible genes including members relevant to tumour biology. However, not all are involved in glucose transport, glycolysis, and angiogen- connected with the promotion of tumour growth, some esis (Shweiki et al., 1992; Semenza et al., 1994; Ebert et being pro-apoptotic or growth inhibitory. We postulate al., 1995). In VHL defective cells, HIF-a subunits are that co-ordinate regulation as part of the HIF pathway constitutively stabilized leading to activation of the may explain this paradox, and that evolution of anti- transcriptional complex and its target genes irrespective apoptotic pathways may be required for tumour growth of oxygen concentration (Maxwell et al., 1999). under VHL-dysregulation. Our results indicate that it The direct targeting of a hypoxia inducible pathway will be necessary to consider the eects of abnormal that is most clearly linked with the regulation of activity in integral regulatory pathways, as well as the metabolism and angiogenesis is unusual amongst eects of individual genes to understand the role of tumour supressor genes. Though upregulation of abnormal patterns of gene expression in cancer. Onco- angiogenic growth factors provides an explanation for gene (2000) 19, 6297 ± 6305. the angiogenic phenotype of VHL syndrome, the role of HIF activation in other aspects of oncogenesis is less Keywords: VHL; hypoxia; HIF-1a clear. HIF might have other targets that could illuminate such a role, or pVHL might have functions other than HIF regulation that are relevant to its Introduction tumour suppressor function (Ohh et al., 1998; Pause et al., 1998; Iwai et al., 1999). Tumour suppressor genes and oncogenes are often We therefore sought to expand our understanding of involved, directly or indirectly, in the regulation of the patterns of gene expression that are aected by transcription. New opportunities for large-scale analy- pVHL, and to determine the extent to which these sis of the downstream patterns of abnormal gene genes are also subject to regulation by oxygen. We expression (Schena et al., 1995; Iyer et al., 1999) may employed a glass-chip based cDNA array of 9182 therefore provide important insights into the under- unbiased genes and ESTs to compare the gene lying processes which promote the development of expression pro®le in stable transfectants of the renal cancer. cell carcinoma (RCC) line, RCC4, that were either defective or competent for pVHL. Regulation by pVHL was con®rmed by ribonuclease protection assay *Correspondence: PJ Ratclie both in the original pair of RCC4 cell lines, and a Received 7 August 2000; revised 10 October 2000; accepted 10 second pair of transfectants based on another VHL October 2000 defective RCC line, 786-0. We demonstrate the mRNA differential expression profiling to identify VHL target genes CC Wykoff et al 6298 reproducibility of the screen, and show that for the for 30 genes showing the greatest induction or large majority of genes identi®ed by the screen, repression by transfection of VHL in the ®rst screen dierential expression could be con®rmed by ribonu- plotted against expression data for the same cDNAs in clease protection assay. For about 50% of the the second screen. There was a good correlation, identi®ed genes, a similar pattern of dierential though quantitative dierences between the screens in expression in relation to VHL status was apparent in the amplitude of expression were commonly observed. both cell backgrounds. The majority of these genes were also regulated by hypoxia in multiple non-renal RNAse protection analysis of VHL dependent gene cell types, therefore de®ning several new classes of expression in different renal cell carcinoma cell lines hypoxia/pVHL responsive genes. In addition, other genes were de®ned whose expression was responsive to GEM analysis identi®ed 22 genes that were consis- pVHL but not to hypoxia, indicating that pVHL may tently repressed by VHL in both screens by an in¯uence additional pathways of gene expression that average of twofold or greater. For these genes, are not regulated by hypoxia. riboprobe templates were constructed to permit accurate quanti®cation of mRNA regulation by ribonuclease protection assay (RPA). Two dierent sets of renal cell carcinoma transfectants were studied, Results the original pair of RCC4 and RCC4/VHL transfec- tants, and a similar pair of 786-0 transfectants Analysis of VHL dependent expression by gene expression expressing vector backbone alone or wild type human microarray pVHL (786-0 and 786-0/VHL). Results are summar- To examine the eects of the tumour suppressor gene ized in Table 1a. Of the 22 genes identi®ed by GEM VHL on global patterns of gene expression we screening, 20 were con®rmed by RPA as showing a employed an mRNA dierential screen of 9182 unique clear reduction in expression in RCC4/VHL cells clones using a Gene Expression Microarray (GEM) versus RCC4 cells, whereas two genes were not based on competitive hybridization expression pro®ling expressed at levels quanti®able by RPA. Interestingly, (Incyte Genomics). Of the 9182 cDNA clones, 8481 are for many genes the amplitude of dierential mRNA unique annotated genes. We compared mRNA from expression demonstrated by RPA was substantially stable transfectants of the pVHL de®cient renal underestimated by GEM analysis. Of the 20 genes carcinoma cell line RCC4 expressing vector backbone showing repression by re-expression of VHL in RCC4, alone (RCC4) or wild type human pVHL (RCC4/ seven were also repressed by re-expression of VHL in VHL). To test the reproducibility of the screen, the 786-0, ®ve were not expressed at levels quanti®able by procedure was carried out on a second independently RPA in 786-0 cells, and eight were expressed but not derived pair of mRNA samples extracted from RCC4 in¯uenced by re-expression of VHL in this back- and RCC4/VHL cells. Figure 1 shows expression data ground. Figure 1 Reproducibility of the GEM data sets. Dierential expression ratios are shown for the 30 genes showing either the greatest induction or the greatest repression following re-expression of wild type VHL in RCC4 cells as de®ned by the ®rst GEM screen. Data from this screen (GEM1) are plotted against expression data for the same genes in the second GEM (GEM2). Positive numbers indicate induction of expression following re-expression of VHL. Negative numbers indicate repression of expression following re-expression of VHL Oncogene mRNA differential expression profiling to identify VHL target genes CC Wykoff et al 6299 Table 1 a Analysis of candidate VHL-repressible genes Fold regulation Accession number Gene RCC4 array RCC4 RPA 786-0 RPA Y00749 endothelin 1 2.7 47 21 AB004066 DEC1 2.2 5.7 8.7 AI061430 MIC2 2.6 2.5 3.2 NM004613 transglutaminase 2 2.7 12 35 X56134 vimentin 2.7 2 2 Y00757 secretory granule neuroendocrine prot 1 6.9 45 14 AL047358 spermidine N1-acetyltransferase 2.5 3.3 3.9 NM002332 LRP1 2.5 11 NE M76729 collagen type V, alpha 1 2.3 21 NE AI271688 cyclin G2 2.4 8.3 NE L12468
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