Current Pharmaceutical Design, 2009, 15, 3867-3877 3867 1381-6128/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd. Kinases as Upstream Regulators of the HIF System: Their Emerging Potential as Anti-Cancer Drug Targets Elitsa Y. Dimova 1 *, Carine Michiels 2 and Thomas Kietzmann 1* 1 Fachbereich Chemie, Abteilung Biochemie, Universität Kaiserslautern, D-67663, Germany; 2 Laboratory of Biochemistry and Cellular Biology, University of Namur-FUNDP, 61 rue de Bruxelles, 5000 Namur, Belgium Abstract: The hypoxia-inducible factor-1 (HIF-1) is a key regulator in the mammalian response to oxygen deficiency under both physiological and pathological conditions such as cancer. A number of studies indicated an association between tumor hypoxia, increased hypoxia-inducible factor (HIF-1 ) levels and a poor prognosis. The HIF-1 regulation in response to hypoxia occurs primarily on the level of protein stability due to posttranslational hydroxylation. However, HIF -subunits also respond to various growth factors, hormones, or cytokines under non-hypoxic conditions implicating the involvement of different kinase pathways in their regulation thereby increasing the interest in HIF-1 as a new drug target. Herein, we review current knowledge about phosphorylation-dependent HIF-1 regulation, HIF-1 protein-protein interactions and subcellular localization with emphasis on new therapeutic strategies targeting the HIF pathway. Keywords: Phosphorylation, HIF-1, hypoxia, kinase, MAPK pathway, PI3K/PKB pathway. INTRODUCTION In most western countries cancer is a disease with a high mortality rate. Cancer cells often arise in a multistep process due to oncogenic mechanisms such as mutations, overex- pression or rearrangements of proto-oncogenes or tumor- suppressor genes as well as due to viral infection. The outcome of these events is linked to the activation of different signal transduction pathways that ultimately lead to the carcinogenic process accompanied with continuous proliferation and uncontrolled growth. Hypoxia is also a major feature of cancer and almost all solid tumors contain hypoxic regions in which O 2 concentrations are greatly reduced compared to the surrounding normal tissue. In addition, a number of studies indicated that various tumor entities are accompanied with increased levels of hypoxia- inducible factor (HIF-1 ) and that these tumors are associated with a poor prognosis. Further, HIF -subunits also respond to growth and coagulation factors, hormones, cytokines or stress factors under non-hypoxic conditions implicating the involvement of different kinase pathways in their regulation. Altogether this increased the interest in HIF -subunits, especially HIF-1 , as a drug target and new specific therapeutic strategies targeting the HIF pathway are intensively investigated. The family of hypoxia-inducible factors which are key regulators in the mammalian response to oxygen deficiency contains three -subunits (HIF-1 , HIF2 and HIF-3 ) and three -subunits (HIF-1 , ARNT2 and ARTN3), respec- tively which may give rise to different HIF heterodimers from which HIF-1 is the best characterised (for review see [1]). HIF-1 comprises of an oxygen-induced HIF- 1 and a constitutively expressed HIF-1 subunit (also known as arylhydrocarbon receptor-nuclear translocator, *Address correspondence to these authors at the Department Chemistry/ Biochemistry, Erwin-Schrödinger Strasse 54, D-67663 Kaiserslautern, Germany; Fax: 49-631- 205-3419; E-mail: dimova@chemie.uni-kl.de; tkietzm@gwdg.de ARNT) which binds to the hypoxia-responsive element (HRE) consensus sequence 5´-BACGTSSK-3´ (B=G/C/T; S=G/C; K=G/T) [2] within promoters or enhancers of about 100 hypoxia-responsive genes among them VEGF, PAI-1 and others (reviewed by [3]). The HIF-1 subunit is a protein of 826 amino acids (aa) with an approximate molecular weight of 120 kDa [4]. It contains two nuclear localisation sequences responsible for translocation of HIF-1 to the nucleus under hypoxia; they are localised N-terminal (aa 17-33) and C-terminal (aa 718- 721) [5]. The transactivity is determined by two trans- activation domains (TAD); N-terminal (TAD-N, aa 531-575) and C-terminal (TAD-C, aa 786-826) [6-8]. The residues between aa 576-785 contain an inhibitory domain; its deletion was shown to increase transactivation under nor- moxia [6]. An oxygen-dependent degradation domain (ODDD, aa 401-603), partially overlapping the TAD-N is a target for posttranslational modifications and has an impact on the hypoxia-regulated stabilisation of HIF-1 [9]. The posttranslational stabilization of HIF-1 protein appears to be the major regulatory pathway although regulation at the transcriptional and translational level has also been evidenced to play a role [10-15]. The major posttranslational modification appears to be the oxygen-dependent hydroxylation [16, 17] of two specific proline residues, Pro 402 and Pro 564, within the ODDD [17, 18] and of an asparagine residue, N 803, within the TAD-C of human HIF-1 [19]. Hydroxylation of the proline residues within the ODDD allows binding of the von Hippel- Lindau tumor suppressor protein (pVHL) as part of an E3 ubiquitin ligase complex which subsequently leads to ubiquitination and proteasome-dependent degradation of HIF-1 [20-27]. In addition, other posttranslational modi- fications such as phosphorylation, acetylation, S-nitrosy- lation and SUMOylation can modulate HIF-1 trans- criptional activity, protein stabilization, protein-protein inter- action, and cellular localization. Although not considered as crucial as for many other transcription factors, phospho-