Oncogene (2007) 26, 5541–5552 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW Histone deacetylase inhibitors: molecular mechanisms of action WS Xu1, RB Parmigiani1 and PA Marks Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA This review focuses on the mechanisms of action of histone groups to lysine residues, while HDACs remove the deacetylase (HDAC)inhibitors (HDACi),a group of acetyl groups.In general, acetylation of histone pro- recently discovered ‘targeted’ anticancer agents. There are motes a more relaxed chromatin structure, allowing 18 HDACs, which are generally divided into four classes, transcriptional activation.HDACs can act as transcrip- based on sequence homology to yeast counterparts. tion repressors, due to histone deacetylation, and Classical HDACi such as the hydroxamic acid-based consequently promote chromatin condensation.HDAC vorinostat (also known as SAHA and Zolinza)inhibits inhibitors (HDACi) selectively alter gene transcription, classes I, II and IV, but not the NAD þ -dependent class III in part, by chromatin remodeling and by changes in the enzymes. In clinical trials, vorinostat has activity against structure of proteins in transcription factor complexes hematologic and solid cancers at doses well tolerated by (Gui et al., 2004). Further, the HDACs have many non- patients. In addition to histones, HDACs have many other histone proteins substrates such as hormone receptors, protein substrates involved in regulation of gene expres- chaperone proteins and cytoskeleton proteins, which sion, cell proliferation and cell death. Inhibition of regulate cell proliferation and cell death (Table 1).Thus, HDACs causes accumulation of acetylated forms of these HDACi-induced transformed cell death involves tran- proteins, altering their function. Thus, HDACs are more scription-dependent and transcription-independent properly called ‘lysine deacetylases.’ HDACi induces mechanisms (Marks and Dokmanovic, 2005; Rosato and different phenotypes in various transformed cells, includ- Grant, 2005; Bolden et al., 2006; Minucci and Pelicci, ing growth arrest, activation of the extrinsic and/or 2006). intrinsic apoptotic pathways, autophagic cell death, In humans, 18 HDAC enzymes have been identified reactive oxygen species (ROS)-induced cell death, mitotic and classified, based on homology to yeast HDACs cell death and senescence. In comparison, normal cells are (Blander and Guarente, 2004; Bhalla, 2005; Marks and relatively more resistant to HDACi-induced cell death. Dokmanovic, 2005).Class I HDACs include HDAC1, The plurality of mechanisms of HDACi-induced cell death 2, 3 and 8, which are related to yeast RPD3 deacetylase reflects both the multiple substrates of HDACs and the and have high homology in their catalytic sites.Recent heterogeneous patterns of molecular alterations present in phylogenetic analyses suggest that this class can be different cancer cells. divided into classes Ia (HDAC1 and -2), Ib (HDAC3) Oncogene (2007) 26, 5541–5552; doi:10.1038/sj.onc.1210620 and Ic (HDAC8) (Gregoretti et al., 2004). Class II HDACs are related to yeast Hda1 (histone deacetylase Keywords: histone deacetylase; histone deacetylase 1) and include HDAC4, -5, -6, -7, -9 and -10 (Bhalla, inhibitor; apoptosis; mitotic cell death; senescence; 2005; Marks and Dokmanovic, 2005).This class is angiogenesis divided into class IIa, consisting of HDAC4, -5, -7 and -9, and class IIb, consisting of HDAC6 and -10, which contain two catalytic sites.All class I and II HDACs are zinc-dependent enzymes.Members of a third class, sirtuins, require NAD þ for their enzymatic activity Introduction (Blander and Guarente, 2004) (see review by E Verdin, in this issue).Among them, SIRT1 is orthologous to Acetylation and deacetylation of histones play an yeast silent information regulator 2.The enzymatic important role in transcription regulation of eukaryotic activity of class III HDACs is not inhibited by cells (Lehrmann et al., 2002; Mai et al., 2005). The compounds such as vorinostat or trichostatin A acetylation status of histones and non-histone proteins (TSA), that inhibit class I and II HDACs.Class IV is determined by histone deacetylases (HDACs) and HDAC is represented by HDAC11, which, like yeast histone acetyl-transferases (HATs).HATs add acetyl Hda 1 similar 3, has conserved residues in the catalytic core region shared by both class I and II enzymes (Gao et al., 2002). Correspondence: Dr PA Marks, Cell Biology Program, Memorial HDACs are not redundant in function (Marks and Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA. Dokmanovic, 2005; Rosato and Grant, 2005; Bolden E-mail: [email protected] et al., 2006). Class I HDACs are primarily nuclear in 1These authors contributed equally to this work. localization and ubiquitously expressed, while class II Mechanisms of histone deacetylase inhibitors WS Xu et al 5542 Table 1 Nonhistone protein substrates of HDACs (partial list)a Function Proteins DNA binding p53, c-Myc, AML1, BCL-6, E2F1, E2F2, transcriptional factors E2F3, GATA-1, GATA-2, GATA-3, GATA-4, Ying Yang 1 (YY1), NF-kB (RalA/p65), MEF2, CREB, HIF-1a, BETA2, POP-1, IRF-2, IRF-7, SRY, EKLF Steroid receptors Androgen receptor, estrogen receptor a, glucocorticoid receptor Transcription Rb, DEK, MSL-3, HMGI(Y)/HMGA1, coregulators CtBP2, PGC-1a Signaling mediators STAT3, Smad7, b-catenin, IRS-1 DNA repair enzymes Ku70, WRN, TDG, NEIL2, FEN1 Nuclear import Rch1, importin-a7 Chaperone protein HSP90 Structural protein a-Tubulin Inflammation mediator HMGB1 Viral proteins E1A, L-HDAg, S-HDAg, T antigen, Figure 1 Multiple HDACi-activated antitumor pathways.See text HIV Tat for detailed explanation of each pathway.HDAC6, histone deacetylase 6; HIF-1a, hypoxia-induced factor-1a; HSP90, heat- shock protein 90; PP1, protein phosphatase 1; ROS, reactive Abbreviation: HDACs, histone deacetylases. aSee text for references. oxygen species; TBP2, thioredoxin binding protein 2; Trx, thioredoxin; VEGF, vascular endothelial growth factor. HDACs can be primarily cytoplasmic and/or migrate between the cytoplasm and nucleus and are tissue- been identified, which may be acetylated and substrates restricted in expression. of HDACs (Table 1) (Glozak et al., 2005; Marks and The structural details of the HDAC–HDACi inter- Dokmanovic, 2005; Rosato and Grant, 2005; Bolden action has been elucidated in studies of a histone et al., 2006; Minucci and Pelicci, 2006; Zhao et al., deacetylase-like protein from an anerobic bacterium with 2006).In addition, two recent proteomic studies TSA and vorinostat (Finnin et al., 1999). More recently, the identified many lysine-acetylated substrates (Iwabata crystal structure of HDAC8–hydroxamate interaction has et al., 2005; Kim et al., 2006). In view of all these been solved (Somoza et al., 2004; Vannini et al., 2004). findings, HDACs may be better called ‘N-epsilon-lysine These studies provide an insight into the mechanism of deacetylase’.This designation would also distinguish deacetylation of acetylated substrates.The hydroxamic acid them from the enzymes that catalyse other types of moiety of the inhibitor directly interacts with the zinc ion at deacetylation in biological reactions, such as acylases the base of the catalytic pocket. that catalyse the deacetylation of a range of Na-acetyl This review focuses on the molecular mechanisms amino acids (Anders and Dekant, 1994). triggered by inhibitors of zinc-dependent HDACs in Non-histone protein targets of HDACs include tumor cells that explain in part: (I) the effects of these transcription factors, transcription regulators, signal compounds in inducing transformed cell death and (II) transduction mediators, DNA repair enzymes, nuclear the relative resistance of normal and certain cancer cells import regulators, chaperone proteins, structural to HDACi induced cell death.HDACi, for example, the proteins, inflammation mediators and viral proteins hydroxamic acid-based vorinostat (SAHA, Zolinza), are (Table 1).Acetylation can either increase or decrease the promising drugs for cancer treatment (Richon et al., function or stability of the proteins, or protein–protein 1998; Marks and Breslow, 2007).Several HDACi are in interaction (Glozak et al., 2005). These HDAC sub- phase I and II clinical trials, being tested in different strates are directly or indirectly involved in many tumor types, such as cutaneous T-cell lymphoma, acute biological processes, such as gene expression and myeloid leukemia, cervical cancer, etc (Bug et al., 2005; regulation of pathways of proliferation, differentiation Chavez-Blanco et al., 2005; Kelly and Marks, 2005; and cell death.These data suggest that HDACi could Duvic and Zhang, 2006) (Table 2).Although consider- have multiple mechanisms of inducing cell growth arrest able progress has been made in elucidating the role of and cell death (Figure 1). HDACs and the effects of HDACi, these areas are still in early stages of discovery. HDACi HDAC substrates HDACi have been discovered with different structural characteristics, including hydroximates, cyclic peptides, Recent phylogenetic analyses of bacterial HDACs aliphatic acids and benzamides (Table 2) (Miller et al., suggest that all four HDAC classes preceded the 2003; Yoshida et al., 2003; Marks and Breslow, 2007). evolution of histone proteins (Gregoretti et al., 2004). Certain HDACi may selectively inhibit different This suggests that the primary activity of HDACs may HDACs.For example, MS-275 preferentially