Normoxic Stabilization of Hypoxia-Inducible Factor-1A by Modulation of the Labile Iron Pool in Differentiating U937 Macrophages: Effect of Natural Resistance–Associated Macrophage Protein 1 Helen J. Knowles, 1 David R. Mole, 2 Peter J. Ratcliffe, 2 and Adrian L. Harris 1 1 Cancer Research UK Molecular Oncology Laboratory, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital and 2 Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford, United Kingdom Abstract Hypoxia-inducible factor (HIF) is a transcription factor with major roles in many cellular and systemic responses to hypoxia. Activation of HIF pathways under hypoxia is mediated by suppression of the Fe 2+ - and O 2 -dependent HIF hydroxylase enzymes that normally inactivate HIFA subunits. Mechanisms underlying induction of HIF in normoxic con- ditions are less clearly understood. In human cancers, infiltrating macrophages show up-regulation of HIF and it has recently been shown that normoxic expression of HIF-1A is essential for macrophage function. Here, we report studies of HIF-1A induction following phorbol-12-myristate 13-ace- tate (PMA)–induced differentiation of monocytic U937 and THP1 cells. HIF-1A was markedly up-regulated under nor- moxia in this setting and this involved failure of HIF-1A prolyl hydroxylation despite the presence of O 2 . Fluorescence measurements showed that differentiation was associated with marked reduction of the labile iron pool. Both the reduction in labile iron pool and the up-regulation of HIF-1A were suppressed by RNA interference–mediated down- regulation of the iron transporter natural resistance– associated macrophage protein 1. Up-regulation of HIF-1A following PMA-induced differentiation was also abolished by addition of Fe 2+ or ascorbate. These results indicate that physiologic changes in macrophage iron metabolism have an important effect on HIF hydroxylase pathways and suggest means by which the system could be manipulated for therapeutic benefit. (Cancer Res 2006; 66(5): 2600-7) Introduction Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of a hypoxia-inducible a-subunit and a constitu- tively expressed h-subunit. Under hypoxic conditions, stabilization of HIFa results in formation of an active complex that regulates expression of genes, including angiogenic factors, glycolytic enzymes, and survival factors (1). Under normoxic conditions, HIFa is posttranslationally hydroxylated by the prolyl hydroxylase domain enzymes (PHD1-3), targeting it for interaction with the von-Hippel Lindau (VHL) E3 ubiquitin ligase complex and rapid proteasomal degradation (2, 3). These enzymes have an absolute requirement for O 2 and are therefore inactive under hypoxic conditions, allowing HIFa protein to accumulate (2, 3). The PHD enzymes also require 2-oxoglutarate, Fe 2+ , and ascorbate as cofactors. Accumulating evidence indicates that limiting availabil- ity of these cofactors may also activate the HIF response by impairing hydroxylation. We recently reported that ascorbate or iron supplementation can significantly blunt the HIF transcrip- tional response and ablate normoxic induction of HIF-1a by enhancing hydroxylase activity under conditions of growth factor or oncogenic activation of HIF (4). Hypoxia is a fundamental microenvironmental component of solid tumor tissue and hypoxic induction of HIF-1a is evident in a range of cancers (5, 6). Many solid tumors are also characterized by the presence of an inflammatory infiltrate. High levels of macrophage infiltration have been associated with poor prognosis in cancers of the breast, cervix, and bladder (7). In breast cancer, high macrophage indices have been correlated with poor relapse- free and overall survival (8) and have been positively associated with angiogenic variables, such as increased blood vessel density (8) and high tumor vascular endothelial growth factor expression. In other cancer types, including prostate, non–small cell lung, glioma, and ovarian cancers, the association between macrophage infiltration and prognosis is less clear (7). HIF-positive tumor- associated macrophages have been described in cancers of the breast, lung, ovary, prostate, and pancreas (6, 9). Currently, only two studies have investigated macrophage HIF expression and prognosis. High macrophage expression of HIF-2a in breast cancer correlated strongly with high levels of angiogenesis and moder- ately with overall survival (10). In bladder cancer, a positive association was observed with both histologic grade and pathologic stage (11). Recent experiments in vivo have shown HIF-1a expression to be essential for normal macrophage function. Conditional ablation of HIF-1a in murine myeloid cells greatly inhibited homotypic adhesion, motility, and invasion of ex vivo peritoneal macrophages (12). In vivo macrophage infiltration, edema formation, and tissue destruction was significantly reduced in murine models of acute skin and chronic joint inflammation (12). The authors showed that HIF-1a is essential for the maintenance of intracellular energy homeostasis in macrophages. Even under normoxic tissue culture conditions, HIF-1a null macrophages had ATP levels reduced by >80% (12). This suggests a substantial role for HIF in macrophage physiology in a range of microenvironmental conditions and raises questions as to the mechanism of activation and functional importance of the response. There is now substantial evidence for HIF induction by nonhypoxic stimuli, especially oncogenic mutation (ras, src, and PTEN) and growth factor stimulation (insulin, insulin-like growth Requests for reprints: Adrian L. Harris, Cancer Research UK Molecular Oncology Laboratory, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom. Phone: 44-1865-222457; Fax: 44-1865-222431; E-mail: harrisa@cancer.org.uk. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-2351 Cancer Res 2006; 66: (5). March 1, 2006 2600 www.aacrjournals.org Research Article Research. on June 12, 2015. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from