[CANCER RESEARCH 63, 3524 –3530, July 1, 2003] The Involvement of Hypoxia-inducible Transcription Factor-1-dependent Pathway in Nickel Carcinogenesis 1 Konstantin Salnikow, 2 Todd Davidson, Qunwei Zhang, Lung Chi Chen, Weichen Su, and Max Costa The Nelson Institute of Environmental Medicine, National Institute of Environmental Health Sciences Center and The New York University Cancer Institute, New York University School of Medicine, New York, New York 10016 ABSTRACT Nickel is a potent environmental pollutant in industrial countries. Because nickel compounds are carcinogenic, exposure to nickel represents a serious hazard to human health. The understanding of how nickel exerts its toxic and carcinogenic effects at a molecular level may be important in risk assessment, as well as in the treatment and prevention of occupational diseases. Previously, using human and rodent cells in vitro, we showed that hypoxia-inducible signaling pathway was activated by carcinogenic nickel compounds. Acute exposure to nickel resulted in the accumulation of hypoxia-inducible transcription factor (HIF)-1, which strongly activated hypoxia-inducible genes, including the recently discovered tumor marker NDRG1 (Cap43). To further identify HIF-1-dependent nickel-inducible genes and to understand the role of the HIF-dependent signaling pathway in nickel-induced transformation, we used the Affymetrix GeneChip to compare the gene expression profiles in wild-type cells or in cells from HIF-1 knockout mouse embryos exposed to nickel chloride. As expected, when we examined 12,000 genes for expression changes, we found that genes coding for glycolytic enzymes and glucose transporters, known to be regulated by HIF-1 transcription factor, were induced by nickel only in HIF-1-proficient cells. In addition, we found a number of other hypoxia- inducible genes up-regulated by nickel in a HIF-dependent manner in- cluding BCL-2-binding protein Nip3, EGLN1, hypoxia-inducible gene 1 (HIG1), and prolyl 4-hydroxylase. Additionally, we found a number of genes induced by nickel in a HIF-independent manner, suggesting that Ni activated other signaling pathways besides HIF-1. Finally, we found that in HIF-1 knockout cells, nickel strongly induced the expression of the whole group of genes that were not expressed in the presence of HIF-1. Because the majority of modulated genes were induced or suppressed by nickel in a HIF-1-dependent manner, we elucidated the role of HIF-1 transcription factor in cell transformation. In HIF-1-proficient cells, nickel exposure increased soft agar growth, whereas it decreased soft agar growth in HIF-1-deficient cells. We hypothesize that the induction of HIF-1 transcription factor by nickel may be important during the nickel- induced carcinogenic process. INTRODUCTION The utilization and disposal of nickel (Ni)-containing products in modern industry leads to environmental pollution by both soluble and insoluble forms of Ni. Additionally, combustion of fossil fuel con- tributes significantly to environmental burden, mostly by producing aerosols containing soluble Ni (1). Human exposure to Ni occurs primarily via inhalation and ingestion (2). Cutaneous exposure to Ni causes allergy in the form of contact dermatitis. The chronic exposure to Ni compounds can lead to asthma, inflammation, lung fibrosis, and kidney diseases, but the most serious concerns relate to the carcino- genic activity of Ni (2). Epidemiological studies have clearly impli- cated Ni compounds as human carcinogens based on a higher inci- dence of lung and nasal cancer among Ni mining, smelting, and refinery workers (2, 3). In various animal models, Ni compounds induce tumors at virtually any site of administration (2). Additionally, insoluble Ni compounds such as nickel subsulfide efficiently trans- form rodent and human cells in vitro (2). Based on these observations, the IARC evaluated the carcinogenicity of Ni in 1990 (4). All Ni compounds except for metallic Ni were classified as carcinogenic to humans. The molecular basis of Ni carcinogenesis has been challenging because carcinogenic Ni compounds were weakly mutagenic in most assay systems, even though they were able to produce oxidative DNA damage and inhibit DNA repair activity (2, 5–9). The level of oxida- tive stress induced by Ni in cells is rather weak when compared with other metals; however it depleted glutathione and activated AP1, 3 nuclear factor B, and other oxidatively sensitive transcription factors (10 –13). Recently, new data related to the activation of hypoxic signaling pathways by Ni have emerged (13–15). The response to hypoxia is mediated primarily by the HIF-1 transcription factor. In the presence of oxygen, proline 564 in the HIF-1 subunit is hydroxylated by a prolyl hydroxylase (16). The prolyl hydroxylation of HIF-1 allowed VHL binding, leading to the proteosomal destruction of this protein. The prolyl hydroxylase that fulfills this function requires both oxygen and iron. It is likely that Ni substitutes iron in the prolyl hydroxylase and inactivates the enzyme, thus switching a cell’s metabolism to a state that mimics a state of hypoxia. A first testing of this hypothesis has been initiated by cloning Ni-inducible genes (17). One gene, NDRG1 (Cap43), was found to be highly induced by soluble and insoluble Ni compounds in all tested cell lines (17). In addition to its induction by Ni compounds, NDRG1 (Cap43) was also induced by hypoxia in a HIF-1-dependent manner (18). Moreover, we have shown that acute exposure to Ni activates HIF-1, and in Ni-trans- formed cells, the activity of this transcription factor is significantly elevated (14). Here, using the Affymetrix GeneChip and GeneSpring analysis software, we show that the exposure of cells to Ni triggered the expression of hypoxia-inducible genes involved in glucose trans- port and glycolysis. Other hypoxia-inducible genes up-regulated by Ni in a HIF-dependent manner were BCL-2-binding protein Nip3, EGLN1, HIG1, prolyl 4-hydroxylase, and p125 FAK (19 –24). The induction of ATM, p53, and the p53-dependent genes GADD45 and cyclin kinase inhibitor p21 in a HIF-1-independent manner sug- gests that the p53-dependent pathway was another pathway activated by Ni. The response to the appearance of damaged or unfolded proteins by Ni was HIF-1 independent and can be recognized by the induction of both Chop 10 (GADD153) and chaperone HSP70 (25, 26). Thus, both HIF-dependent and HIF-independent pathways were activated by Ni exposure; however, of 114 genes induced 4-fold by Ni in HIF-1-proficient cells, 85 genes were induced in a HIF- dependent manner, indicating that HIF-1-dependent pathway was a Received 6/14/02; accepted 4/25/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Grants ES00260, ES05512, ES10344, and T32 ES07324 from the National Institute of Environmental Health Sciences; Grant R-827351 from the Environ- mental Protection Agency; and Grant CA16087 from the National Cancer Institute. 2 To whom requests for reprints should be addressed, at Department of Environmental Medicine, National Institute of Environmental Health Sciences and New York University Cancer Institute, New York University, 550 First Avenue, New York, NY 10016. Phone: (845) 731-3516; Fax: (845) 351-2118; E-mail: salnikow@env.med.nyu.edu or salnicow@ncifcrf.gov. 3 The abbreviations used are: AP1, activator protein 1; HIF, hypoxia-inducible tran- scription factor; MEF, mouse embryo fibroblast; EST, expressed sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGF, epidermal growth factor; VEGF, vas- cular endothelial growth factor. 3524