Iron Chelator-Mediated Alterations in Gene Expression: Identification of Novel Iron-Regulated Molecules That Are Molecular Targets of Hypoxia-Inducible Factor-1 and p53

Iron deficiency affects 500 million people, yet the molecular role of iron in gene expression remains poorly characterized. In addition, the alterations in global gene expression after iron chelation remain unclear and are important to assess for understanding the molecular pathology of iron deficiency and the biological effects of chelators. Considering this, we assessed the effect on whole genome gene expression of two iron chelators (desferrioxamine and 2-hydroxy-1napthylaldehyde isonicotinoyl hydrazone) that have markedly different permeability properties. Sixteen genes were significantly regulated by both ligands, whereas a further 50 genes were significantly regulated by either compound. Apart from ironmediated regulation of expression via hypoxia inducible factor-1 , it was noteworthy that the transcription factor p53 was also involved in iron-regulated gene expression. Examining 16 genes regulated by both chelators in normal and neoplastic cells, five genes (APP, GDF15, CITED2, EGR1, and PNRC1) were significantly differentially expressed between the cell types. In view of their functions in tumor suppression, proliferation, and apoptosis, these findings are important for understanding the selective antiproliferative effects of chelators against neoplastic cells. Most of the genes identified have not been described previously to be iron-regulated and are important for understanding the molecular and cellular effects of iron depletion. Iron deficiency affects approximately 500 million people. However, despite the enormity of this problem, very little is understood concerning the precise molecular roles played by iron in growth, cell-cycle progression, and apoptosis. Iron plays essential roles in cells, including DNA synthesis and cell cycle control (Buss et al., 2003). It is well known that iron deficiency induced by chelators results in a G1/S arrest and apoptosis (Buss et al., 2003). The best-characterized role of iron in proliferation involves its function in the rate-limiting step of DNA synthesis catalyzed by ribonucleotide reductase (Buss et al., 2003). Iron has also been shown to regulate the expression of a variety of molecules involved in cell-cycle control (e.g., p21, GADD45, p53, cyclin D1, etc.) (Gao and Richardson, 2001; Liang and Richardson, 2003) and metastasis suppression (e.g., N-myc downstreamregulated gene-1; NDRG-1) (Le and Richardson, 2004). Because iron is important for mitochondrial heme and iron sulfur cluster synthesis, it is likely that iron chelation will also affect basic mitochondrial metabolism and function. Iron chelators are well known therapeutics for the treatment of iron-overload disease and some of these agents show potential for cancer therapy (Buss et al., 2003; Whitnall et al., 2006). In general, lipophilic chelators are more effective at inhibiting [H]thymidine incorporation, DNA synthesis, and proliferation than their hydrophilic counterparts (RichThis work was supported by the National Health and Medical Research Council of Australia [Grant 570952 and Senior Principal Research Fellowship]; the Australian Research Council [Grant DP0773027]; the Prostate Cancer Foundation; and the Cancer Institute New South Wales. F.S. and Y.S.R. contributed equally to this study. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.109.061028. ABBREVIATIONS: 311, 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone; DFO, desferrioxamine; IRP, iron-regulatory protein; IRE, ironresponsive element; UTR, untranslated region; HIF-1, hypoxia-inducible factor-1; HUVEC, human umbilical vein endothelial cell; MEF, murine embryonic fibroblast; Tet, tetracycline; RT, reverse transcription; PCR, polymerase chain reaction; Dp44mT, di-2-pyridylketone 4,4-dimethyl-3thiosemicarbazone; APP, amyloid (A4) precursor protein; GDF15, growth differentiation factor 15; FAC, ferric ammonium citrate; CON, control medium alone; TfR1, transferrin receptor 1; BNIP3, BCL2/adenovirus E1B 19-kDa interacting protein 3; CITED2, Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxyl-terminal domain, 2; EGR1, early growth response 1; ERO1L, ERO1-like; GDF15, growth differentiation factor 15; NDRG-1, N-myc downstream regulated gene 1; PNRC1, proline-rich nuclear receptor coactivator 1; PPM1D, protein phosphatase 1D magnesium-dependent, 32 isoform. 0026-895X/10/7703-443–458$20.00 MOLECULAR PHARMACOLOGY Vol. 77, No. 3 Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics 61028/3564863 Mol Pharmacol 77:443–458, 2010 Printed in U.S.A. 443 at A PE T Jornals on N ovem er 7, 2017 m oharm .aspeurnals.org D ow nladed from ardson et al., 1995). A good example of this is provided by comparing the activity of the tridentate lipophilic ligand 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone (311; Fig. 1A) and the hexadentate hydrophilic compound desferrioxamine (DFO; Fig. 1A). Both are high-affinity iron(III) chelators (Richardson and Bernhardt, 1999) that demonstrate distinct differences in activity (Richardson et al., 1994, 1995; Darnell and Richardson, 1999). In fact, DFO shows limited permeability and iron chelation efficacy, which leads to low antiproliferative efficacy, whereas 311 is membranepermeable and demonstrates marked antiproliferative effects (Richardson et al., 1994, 1995; Darnell and Richardson, 1999). Unlike other cytotoxic chelators, upon binding iron, DFO and 311 do not generate cytotoxic radicals, their effects being due to induction of iron depletion (Chaston et al., 2003). In addition, DFO and 311 up-regulate the iron-regulated gene transferrin receptor 1 (TfR1) (Darnell and Richardson, 1999). Important regulators of intracellular iron status are the iron-regulatory proteins 1 and 2 (IRP1 and -2) that bind conserved iron-responsive elements (IREs) in the 3 and 5 -untranslated regions (UTRs) of mRNAs that play roles in iron metabolism (Sanchez et al., 2007). Previous studies showed that DFO and 311 deplete cellular iron and effectively increase IRP-RNA-binding activity (Darnell and Richardson, 1999). Depending on iron status, the IRPs post-transcriptionally regulate the expression of genes, including TfR1, which is involved in iron uptake, and ferritin Hand L-chain, which play crucial roles in iron storage (Sanchez et al., 2007). However, this is not the only mechanism controlling gene expression in response to iron; the other well known system is mediated by hypoxia-inducible factor-1 (HIF-1 ) (An et al., 1998; Semenza, 1999). The expression of the transcription factor p53 can also be regulated by iron (An et al., 1998; Liang and Richardson, 2003). It is noteworthy that HIF1is thought to stabilize p53 and lead to its up-regulation after iron depletion (An et al., 1998). HIF-1 is activated under hypoxia and/or iron depletion and is composed of two subunits, a constitutively expressed -subunit and the -subunit (Semenza, 1999). UnFig. 1. A, structures of the chelators DFO and 311. B, the effect of various concentrations of 311 (0.25–25 M) or DFO (0.1–250 M) after a 24-h incubation at 37°C on the mRNA expression of seven genes commonly up-regulated by either of these ligands (see Table 2) in MCF-7 breast cancer cells. C, the effect of incubation time (3–24 h at 37°C) with 311 (25 M) or DFO (250 M) on the mRNA expression of the seven genes in B in MCF-7 cells. In B and C, TfR1 expression has been assessed as a positive control for iron depletion. Results in B and C are representative photographs of gels from three separate experiments. 444 Saletta et al. at A PE T Jornals on N ovem er 7, 2017 m oharm .aspeurnals.org D ow nladed from der normal oxygen tension and iron levels, HIF-1 is regulated by prolyl hydroxylase, which allows binding to the von Hippel-Lindau protein. This protein activates ubiquitin E3 ligase, resulting in HIF-1 degradation via the proteasome (Semenza, 1999). Under oxygen and/or iron depletion, prolyl hydroxylase fails to function, leading to HIF-1 accumulation and nuclear translocation, where it binds to HIF-1 to form the HIF-1 complex, which regulates genes, such as the TfR1 (Bianchi et al., 1999), by binding to hypoxia response elements (Semenza, 1999). In this investigation, we examined the effect of iron chelation on global gene expression after incubation of cells with either DFO or 311. These studies were initiated to achieve a more comprehensive understanding of the cellular response to iron depletion, which remains only preliminary. We identified a range of iron-regulated genes that play roles in diverse biological processes, including tumor suppression, proliferation, and apoptosis. Furthermore, this work was designed to also investigate further potential reasons for the selective antiproliferative activity of iron chelators in neoplastic relative to normal cells. Five iron-regulated genes have been identified that were regulated differently in neoplastic cells than in normal cells and could play a role in the selective antitumor effects of chelators. Materials and Methods