NovelThioredoxin Inhibitors Paradoxically Increase Hypoxia- Inducible Factor-A Expression but Decrease Functional Transcriptional Activity, DNABinding, and Degradation

Purpose:Hypoxia-inducible factor-a (HIF-a) is a transcription factor that regulates the response to hypoxia. HIF-a protein is found at high levels in many cancers, and the redox protein thioredoxin-1 (Trx-1) increases both aerobic and hypoxia-induced HIF-a. Therefore, Trx-1and HIF-a are attractive molecular targets for novel cancer therapeutics. Experimental Design:We investigated whether two novel anticancer drugs AJM290 and AW464 (quinols), which inhibitTrx-1function, can inhibit the HIF pathway. Results:Treatment of several cancer cell lines with AJM290 orAW464 prevented the hypoxiainduced increase of vascular endothelial growth factor (VEGF) at subtoxic concentrations. AJM290 and AW464 also decreasedVEGF in pVHLmutant renal cell carcinoma cells that constitutively overexpress HIF-a protein. They surprisingly up-regulated HIF-a expression in breast cancer cell lines in normoxia and hypoxia as well as in pVHL mutant cells. In the MDA-MB-468 breast cancer cell line, the compounds inhibited RNA and protein expression of the HIF-a target genes, carbonic anhydrase IX,VEGF, and BNIP3, concordantly with HIF-a up-regulation. Both compounds specifically inhibited HIF-a-dependent induction of hypoxia regulatory elementluciferase and HIF-1ahypoxia regulatory element-DNA binding. To analyze the HIF-1adomain inhibited byAJM290, we transfected cells with plasmids expressing a fusion protein of Gal linked to HIF-1aor HIF-1aCOOH-terminal transactivation domain (CAD) with a Gal4-responsive luciferase reporter gene. AJM290 inhibitedboth the full-lengthHIF-1aandHIF-1aCAD transcriptional activity. Conclusions:AJM290 and AW464 are inhibitors of HIF-1aCAD transcription activity and DNA binding, but they also inhibit degradation of HIF, in contrast to other Trx inhibitors. Hypoxia-inducible factor (HIF) is an a,h heterodimeric transcription factor that directs a broad range of responses in hypoxic cells (1). Both proteins are members of the basic helixloop-helix superfamily of transcription factors in which the basic helix-loop-helix domains bind to DNA (2). HIF-1h is a constitutive nuclear localized subunit that binds to available HIF-a (3). To date, three HIF-a isoforms have been described, with the best characterized being HIF-1a and HIF-2a. In the presence of oxygen, two prolyl sites within a central degradation domain of HIF-a are hydroxylated by a set of closely related Fe and 2-OG-dependent dioxygenases (PHD1-3), which leads to HIF-a degradation via the pVHL E3 ubiquitin ligase complex and the 26S proteasome (4). Limiting oxygen levels or the availability of Fe with iron chelators (5) allows HIF-a to escape proteolysis. In the nucleus, HIF a,h heterodimer interacts with coactivators, such CBP/p300, and becomes transcriptionally active (6). On activation, the HIF-ah complex binds to target genes at sites containing the core recognition sequence 5¶-RCGTG-3¶, also known as the hypoxia regulatory element (HRE; ref. 7), which finally leads to up-regulation of genes involved in angiogenesis, glucose metabolism, and pH regulation (1). The HIF transcription cascade has been shown to contribute to tumor progression and metastasis and plays an important part in the malignant phenotype (8–10). HIF-a is found at increased levels in a wide variety of human primary tumors compared with corresponding normal tissue and increases angiogenesis and other properties that promote increased vascularity and tumor progression (8–10). Besides physiologic hypoxia, genetic abnormalities frequently detected in human cancers, which include key oncogenes (HER-2, FRAP, H-RAS, and SRC) and tumor suppressor genes (pVHL, p53, and PTEN), are also associated with induction of HIF-a activity and expression of HIF-a-inducible genes (11–15). Human Cancer Biology Authors’Affiliations: Cancer Research UK Growth Factor Group,Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford; The WellcomeTrust Centre for Human Genetics, Oxford, United Kingdom; and Cancer Research UK Experimental Cancer Chemotherapy Research Group, University of Nottingham, Nottingham, United Kingdom Received11/1/05; revised 3/15/06; accepted 5/4/06. Grant support: Cancer Research UK and Euroxy EU 6th Framework Grant. 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 with18 U.S.C. Section1734 solely to indicate this fact. Note: AJM290 and AW464 have been licensed to Pharminox and M. Stevens is a share holder in this company. Requests for reprints: Adrian L. Harris, Cancer Research UK Growth Factor Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Headington, Oxford OX3 9DS, United Kingdom. Phone: 44-1865226184; Fax: 44-1865226179; E-mail: [email protected]. F2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-2380 www.aacrjournals.org Clin Cancer Res 2006;12(18) September15, 2006 5384 Research. on August 3, 2017. © 2006 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Due to the involvement of HIF-a in tumor progression and angiogenesis, HIF-a is a promising molecular target for development of cancer therapeutics (16–18). Thioredoxin-1 (Trx-1), which is a ubiquitously expressed small redox protein with a conserved catalytic site (19), has been shown to regulate the activity of enzymes, such as apoptosis signal-regulating kinase-1 and protein kinases C a, y, q, and ~ in a redoxdependent manner (20, 21). Trx-1 also increases the DNA binding of redox-sensitive transcription factors, which includes nuclear factor-nB and p53 (22, 23). It has been recently reported that increased expression of Trx-1 in cancer cells increases HIF-a protein levels and transactivating activity under both normoxic and hypoxic conditions (24). Trx-1 expression is highly expressed in several human primary cancers, including colon, cervix, lung, pancreatic, liver, colorectal, and squamous cell cancer (25–30). Several inhibitors of Trx pathway have been developed. PX-12 (Trx-1 inhibitor) and pleurotin and PX478 (Trx-1 reductase inhibitors) have already been shown to downregulate hypoxia-induced increase and constitutive expression of HIF-1a and HIF-1a transcription factor activity (31, 32). Here, we investigated the effects of novel Trx-1 inhibitors AJM290 (indole-substituted quinol) and AW464 (benzothiazole-substituted quinol) on HIF-1a and its downstream targets (Fig. 1A). Both compounds have been shown to have in vitro antitumor activity against colon, renal, and breast cancer cell lines and in vivo antitumor activity in mice bearing breast, colon, and renal xenografts (33–36). Materials andMethods Cell culture and materials. Human breast cancer cell lines MDAMB-468 and MDA-MB-231 and melanoma cell line MDA-MB-435 were maintained in DMEM. The pVHL-deficient RCC4 and 786-0 renal carcinoma cell lines and their counterpart containing a stably transfected pVHL gene were cultured in a-MEM and DMEM, respectively, and maintained in selection with 500 Ag/mL G418. Both a-MEM and DMEM were supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 50 IU/mL penicillin, and 50 Ag/mL streptomycin sulfate. Hypoxic exposures (0.1% O2, 5% CO2, and balance N2) were done in a Heto-Holten CellHouse 170 incubator (RS Biotech, Irvine, Scotland). Cell lines were obtained from the Cancer Research UK. Quinol compounds AJM290 and AW464 were provided by M.F.G. Stevens and proteasome inhibitor Z-Leu-Leu-Leu-Ala (MG132) was from Sigma (Gillingham, United Kingdom). Viability assay. Cells were seeded at 2.5 10 to 10 10 per well 100 AL for 16-hour survival assay and 2.5 10 per well 100 AL for 48-hour survival assay in 96-well plates 24 hours before experimental treatments. Cells were treated with compounds at 0.01 to 250 Amol/L in triplicates and further incubated in hypoxia or normoxia for 16 or 48 hours. Cell viability was measured by measuring metabolic conversion (by viable cells) of the dye MTS Cell Titer 96 AQueous One Solution Cell Proliferation Assay (Promega, Southampton, United Kingdom). In each well of a 96-well plate, 20 AL MTS was added, and plates were incubated for 2 to 4 hours in a cell culture incubator. MTS assay results were read in a 96-well format plate reader by measuring absorbance at 490 nm. Vascular endothelial growth factor ELISA. Vascular endothelial growth factor (VEGF) secretion into the culture medium was measured Fig.1. Chemical structures of AJM290 and AW464 (A), effect of AJM290 and AW464 on MDA-MB-468 cell viability (B), and VEGF expression (C) following16 hours of treatment innormoxic or hypoxic conditions. New Thioredoxin Inhibitors Inhibit Hypoxia Pathway www.aacrjournals.org Clin Cancer Res 2006;12(18) September15, 2006 5385 Research. on August 3, 2017. © 2006 American Association for Cancer clincancerres.aacrjournals.org Downloaded from using DuoSet ELISA Development Human VEGF Immunoassay (R&D Systems, Minneapolis, MN) and 3,3¶,5,5¶-Tetramethylbenzidine Liquid Substrate System for ELISA (Sigma) by following the manufacturers’ protocol. VEGF ELISA assay results were read in a 96-well format plate reader by measuring absorbance at 450 nm with correction at 540 nm. HRE reporter assay. Cells were transfected with 2 Ag/mL HIF-1 reporter plasmid or pGL3 promoter control plasmid and 0.02 Ag/mL phRL-cytomegalovirus (CMV) Renilla luciferase plasmid using Fugene 6 eukaryote transfection reagent kit (Roche, Welwyn Garden City, United Kingdom). The pGL3 firefly luciferase HIF-1 reporter plasmids contained the HRE from phosphoglycerate kinase or carbonic anhydrase IX (CA-IX). The pGL3 SV40 promoter vector was used for control and phRL-CMV Renilla luciferase plasmid was used as control for transfection efficiency (Promega). Twenty-four hours later, cells were exposed to hypoxia for 16 hours as described previously with AJM290 or AW464. Firefly and Renilla luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Western blotting. Whole-cell extracts were made by homogenizing cells in lysis buffer (6.2 mol/L urea, 10% glycerol, 5 mmol/L DTT, 1% SDS + protease inhibitors). Whole-cell extract was separated by 8% or 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane. Primary antibodies used were mouse anti-HIF-1a, rabbit anti-HIF-2a (BD Transduction Laboratories, Lexington, KY), rabbit anti-hydroxylated-HIF-1a (provided by The Wellcome Trust Centre for Human Genetics, Oxford, United Kingdom), mouse anti-CA-IX M75 monoclonal antibody, mouse anti-BNIP3 (Sigma), mouse anti-Hsp70 (Abcam, Cambridge, United Kingdom), mouse anti-Hsp90 (Stressgen, San Diego, CA), and mouse anti-h-tubulin monoclonal antibody (Sigma). Immunoreactivity was visualized with horseradish peroxidase– linked goat anti-mouse or anti-rabbit serum and chemilu-