Class II Histone Deacetylases Are Associated with VHL-Independent Regulation of Hypoxia-Inducible Factor 1A David Z. Qian, 1 Sushant K. Kachhap, 1 Spencer J. Collis, 1 Henk M.W. Verheul, 1 Michael A. Carducci, 1 Peter Atadja, 2 and Roberto Pili 1 1 The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Johns Hopkins School of Medicine, Baltimore, Maryland and 2 Novartis Research Institute, Cambridge, Massachusetts Abstract Hypoxia-inducible factor 1A (HIF-1A) plays a critical role in transcriptional gene activation involved in tumor angiogen- esis. A novel class of agents, the histone deacetylase (HDAC) inhibitors, has been shown to inhibit tumor angiogenesis and HIF-1A protein expression. However, the molecular mecha- nism responsible for this inhibition remains to be elucidated. In the current study, we investigated the molecular link between HIF-1A inhibition and HDAC inhibition. Treatment of the VHL-deficient human renal cell carcinoma cell line UMRC2 with the hydroxamic HDAC inhibitor LAQ824 resulted in a dose-dependent inhibition of HIF-1A protein via a VHL- independent mechanism and reduction of HIF-1A transcrip- tional activity. HIF-1A inhibition by LAQ824 was associated with HIF-1A acetylation and polyubiquitination. HIF-1A immunoprecipitates contained HDAC activity. Then, we tested different classes of HDAC inhibitors with diverse inhibitory activity of class I versus class II HDACs and assessed their capability of targeting HIF-1A. Hydroxamic acid derivatives with known activity against both class I and class II HDACs were effective in inhibiting HIF-1A at low nanomolar concentrations. In contrast, valproic acid and trapoxin were abletoinhibitHIF-1A onlyatconcentrationsthatareeffective against class II HDACs. Coimmunoprecipitation studies showed that class II HDAC4 and HDAC6 were associated with HIF-1A protein. Inhibition by small interfering RNA of HDAC4 and HDAC6 reduced HIF-1A protein expression and tran- scriptional activity. Taken together, these results suggest that class II HDACs are associated with HIF-1A stability and providearationalefortargetingHIF-1A with HDAC inhibitors against class II isozymes. (Cancer Res 2006; 66(17): 8814-21) Introduction Hypoxia-inducible factor 1a (HIF-1a) is an important transcrip- tion factor regulating gene expression in erythropoiesis, angiogen- esis, and glycolytic metabolism (1). Under normoxic conditions, HIF-1a is hydroxylated by prolyl hydroxylase domain oxygenases. The hydroxylated proline serves as a recognition signal for E3 ubiquitin ligase VHL complex binding and subsequent targeting for polyubiquitination and proteasomal degradation. Under hypoxic conditions, the oxygen-dependent prolyl hydroxylase domains are not active and HIF-1a is not hydroxylated for VHL binding and degradation. Stable HIF-1a translocates into the nucleus, dimerizes with HIF-1h, and activates the transcription of target genes. During cancer progression, inefficient tumor vasculature ham- pers oxygen delivery and produces intermittent hypoxia. As a result, hypoxic tumor cells accumulate HIF-1a protein, which in turn transactivates genes promoting adaptation, survival, angio- genesis, and metastases. In several human tumor types, HIF-1a can also be stabilized under normoxic conditions due to the abnormal oncogenic signaling pathways (1). More than 50% of renal cell carcinomas have von Hipple-Lindau (VHL) gene inactivation by deletion, mutation or an epigenetic mechanism (2). HIF-1a is constitutively accumulated in renal cell carcinoma both in cell lines and in tumors. Reintroduction of wild-type VHL can significantly reduce the HIF-1a protein level and impair tumor growth in vivo (2, 3). In addition, targeting the chaperone function of heat shock protein 90 (Hsp90; i.e., geldanamycin) can trigger a VHL-independent HIF-1a degradation pathway under both normoxic and hypoxic conditions (4). In addition to proline hydroxylation, HIF-1a can also be acetylated at Lys 532 by the acetyltransferase ARD1 (5). The acetylation promotes HIF-1a interaction with VHL and subsequent degradation. Histone acetylation is important for DNA chromatin structure and gene transcription regulation. Nonhistone protein acetylation has been shown to regulate protein function and stability. The reversible acetylation of histone and nonhistone proteins at the lysine residue is controlled by histone deacetylases (HDAC) and histone acetyltransferases (6–8). HDAC isozymes can be catego- rized into three classes: class I includes HDAC1, HDAC2, HDAC3, and HDAC8. All class I HDACs are localized in the nucleus and act as transcriptional corepressors by deacetylation of chromatin histone proteins and other DNA binding proteins. Class II includes HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10. Most of them can be regulated and shuttled between the cytoplasm and the nucleus in response to various signal transduction stimuli. In addition, class II HDACs exert their transcriptional corepressor functions by interacting with (or deacetylating) other corepressors or direct binding to (and sequestering) sequence-specific tran- scriptional factors such as MEF2, Runx3, and nuclear factor nB (NF-nB; refs. 8, 9). Class III HDACs are Sir2 family deacetylases, which deacetylate many nonhistone proteins but need NAD for their activities (8). HDAC inhibitors are a new class of anticancer agents designed to inhibit HDACs and to allow histone acetyltransferases to induce histone acetylation and, consequently, gene transcription by opening the chromatin structure. Treatment of transformed cells with HDAC inhibitors results in growth inhibition (10). This phenotype can be due to gene transcriptional activation and/or other unknown mechanisms related to HDAC inhibition. HDAC Note: D.Z. Qian and S.K. Kachhap contributed equally to this work. Requests for reprints: Roberto Pili, Baunting-Blaustein Cancer Research, Building 1M52, 1650 Orleans Street, Baltimore, MD 21231. Phone: 410-502-7482; Fax: 410-614- 8160; E-mail: rpili@jhmi.edu. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-4598 Cancer Res 2006; 66: (17). September 1, 2006 8814 www.aacrjournals.org Research Article Downloaded from http://aacrjournals.org/cancerres/article-pdf/66/17/8814/2553486/8814.pdf by guest on 26 June 2022