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 inhibits be directed against non-histone substrates.At least 50 HDAC1 with IC50,at0.3mM, compared to HDAC3 non-histone proteins of known biological function have with an IC50 of about 8 mM, and has little or no

Oncogene Mechanisms of histone deacetylase inhibitors WS Xu et al 5543 Table 2 HDACi in clinical trials Class Compound HDAC Potency Stage of development Company/sponsor Reference targeta (cells)

Hydroxamate SAHA Classes I, II mM FDA approved for Merck Marks and Breslow (Zolinza, CTCL (2007) vorinostat) LBH589 Classes I, II nM Phase I Novartis Bhalla (2005) PXD101 Classes I, II mM Phase II CuraGen, TopoTarget Bhalla (2005) ITF2357 Classes I, II nM Phase I Italfarmaco Garber (2007) PCI-24781 Classes I, II NA Phase I Pharmacyclics Garber (2007) Cyclic FK228 HDAC1, 2 nM Phase II Gloucester Stadler et al.(2006) peptide Pharmaceuticals Benzamide MS-275 HDAC1, 2, 3 mM Phase II Schering AG Bolden et al.(2006) MGCD0103 Class I NA Phase II MethylGene Garber (2007) Aliphatic acid Phenylbutyrate Classes I, IIa mM Phase II NCI Bolden et al.(2006) Valproic acid Classes I, IIa mM Phase II NCI Bolden et al.(2006) AN-9 NA mM Phase II Titan Pharmaceuticals Bolden et al.(2006) Baceca Classes I NA Phase II TopoTarget Garber (2007) Savicol NA NA Phase II TopoTarget Garber (2007)

Abbreviations: CTCL, cutaneous T-cell lymphoma; FDA, Food and Drug Administration; HDACi, histone deacetylase inhibitors; NA, not available; NCI, National Cancer Institute; SAHA, suberoyl anilide hydroxamic acid. aBased on relative sensitivity to histone deacetylase inhibitors. inhibitory effect against HDAC6 and HDAC8 (Hu and number of genes altered increase to certain et al., 2003). Two novel synthetic compounds, SK7041 extent with the increase of time of culture and and SK7068, preferentially target HDAC1 and 2 and concentration of HDACi.Some changes in gene exhibit growth inhibitory effects in human cancer cell expression are probably direct effects of the HDACi lines and tumor xenograft models (Kim et al., 2003a). A on the gene promoter and other secondary and down- small molecule, tubacin, selectively inhibits HDAC6 stream effects.The patterns of alterations of gene activity and causes an accumulation of acetylated a- expression are similar for different HDAC inhibitors, tubulin, but does not affect acetylation of histones, and but show definite differences induced by different agents does not inhibit cell cycle progression (Haggarty et al., in various transformed cells (Glaser et al., 2003; Gray 2003).No other HDACi for a specific HDAC has been et al., 2004; Mitsiades et al., 2004; Peart et al., 2005; reported. Sasakawa et al., 2005). In these several studies, it has been found that HDACi induce about as many genes as are repressed. HDACi selectively alters gene expression The cyclin-dependent kinase (CDK) inhibitor p21 (WAF1/CIP1) is one of the most common genes induced HDACi can affect transcription by inducing acetylation by HDACi (Archer et al., 1998; Richon et al., 2000; of histones, transcription factors and other proteins Sasakawa et al., 2002). HDACi-induced expression of regulating transcription (Table 1) (Glozak et al., 2005; p21 is independent of p53 and correlates with an Marks and Dokmanovic, 2005; Bolden et al., increase in the acetylation of histones associated with 2006; Minucci and Pelicci, 2006).Early differential the p21 promoter region (Richon et al., 2000; Gui et al., display experiments with lymphoid cell lines cultured 2004).In ARP-1 cells, vorinostat caused specific with TSA showed that only 2% of 340 genes examined modifications in the pattern of acetylation and methyla- were altered in their expression, either increased tion of lysines in histones H3 and H4 associated with the or decreased, compared to untreated cells (Van Lint proximal promoter of the p21 gene (Gui et al., 2004). et al., 1996). Recent studies using cDNA arrays showed These changes did not occur in the histones associated as many as 7–10% of genes were altered in their with the promoter region of the expressed p27 (KIP1) or expression in cell lines of leukemia, multiple myeloma, the silent epsilon globin gene in ARP-1 cells, and neither and carcinomas of colon, bladder, kidney, prostate gene was altered in its expression by vorinostat.The and breast, cultured for up to 48 h with butyrate, protein complex associated with the proximal promoter TSA, MS-275, vorinostat or FK228 (depsipeptide) using region of the p21 gene contained HDAC1 and -2, Myc, twofold change as the cut-off value (Chambers et al., BAF155, Brg-1, GCN5, P300 and Sp1.Vorinostat 2003; Glaser et al., 2003; Mitsiades et al., 2004; caused a marked decrease in HDAC1 and Myc, and Peart et al., 2005; Sasakawa et al., 2005). The time of recruitment of RNA polymerase II, with little detectable culture, concentration and the HDACi used affect changes in HDAC2 or other proteins in the complex. the number of genes detected with altered transcription. The loss of HDAC1 from the complex was not Short time points (Chambers et al., 2003; Mitsiades associated with a decrease in this protein in the nuclear et al., 2004; Sasakawa et al., 2005) and low concentra- extract (Gui et al., 2004). These findings suggest that the tions (Glaser et al., 2003) cause fewer changes in gene selective alteration of transcription of a gene by HDACi transcription, while the magnitude of change may be determined by the specific composition and

Oncogene Mechanisms of histone deacetylase inhibitors WS Xu et al 5544 configuration of proteins in the transcription factor CDK activity, causing dephosphorylation of retinoblas- complex including the HDACs. toma protein (Rb), which blocks E2F activities in the HDAC activity is required for transcriptional activa- transcription of genes for G1 progression and G1/S tion mediated by signal transducer and activator of transition (Bolden et al., 2006). Transformed cells transcription 5 (STAT5) (Rascle et al., 2003). Inhibiting sensitive to HDACi-induced cell death are generally cell HDAC activity can prevent expression of genes for growth-arrested with increase of p21 expression (Huang which STAT5 is required, and result in repression of and Pardee, 2000; Xu et al., 2006). HDACi can kill both their expression.HDACi-induced transcriptional repres- proliferating and non-proliferating cells (Burgess et al., sion of androgen receptor (AR) results from the 2004).This is in contrast to the action of many induction of a suppressor complex (Wang et al., 2004). chemotherapeutic drugs, which are effective only on proliferating cells. In some cells, the G1 arrest is associated with terminal HDACi-induced antitumor pathways differentiation (Marks et al., 1996). The property of vorinostat to induce differentiation of transformed cells HDACi can induce transformed cell growth arrest, was discovered before identifying its inhibitory activity terminal differentiation, cell death and/or inhibition of against HDACs (Marks and Breslow, 2007). angiogenesis (Figure 1).Normal cells are relatively resistant to HDACi-induced cell death (Burgess et al., HDACi activates the extrinsic apoptotic pathways 2004; Insinga et al., 2005; Ungerstedt et al., 2005). The The extrinsic pathway of apoptosis is initiated by the cell death pathways identified in mediating HDACi- binding of death receptors, including Fas (Apo-1 or CD95), induced transformed cell death include apoptosis tumor necrosis factor (TNF) receptor-1 (TNFR-1), (Rosato and Grant, 2005; Bolden et al., 2006; Minucci TNF-related apoptosis-inducing ligand (TRAIL or and Pelicci, 2006) by the intrinsic (Ruefli et al., 2001) Apo2-L) receptors (DR-4 and -5), DR-3 (Apo3) and and extrinsic pathways, mitotic catastrophe/cell death DR-6, to their ligands, such as FasL, TNF, TRAIL and (Qiu et al., 2000; Dowling et al., 2005; Xu et al., 2005), TL1A (Apo3L), leading to activation of caspase-8 and autophagic cell death (Shao et al., 2004), senescence (Xu caspase-10 (Ashkenazi, 2002).HDACi can upregulate et al., 2005) and reactive oxygen species (ROS)- the expression of both death receptors and their ligands, facilitated cell death (Rosato and Grant, 2005; Unger- in vitro and in vivo, in transformed cells, but not in stedt et al., 2005). The response to HDACi appears to normal cells (Nakata et al., 2004; Insinga et al., 2005). depend, in part at least, on the nature of HDACi, Fas and FasL were induced in human neuroblastoma concentration and time of exposure, and importantly, cells by M-carboxycinnamic acid bihydroxamide (Glick the cell context. et al., 1999), nude mice xenograft of human osteosarco- ma cells by FK228, and mouse model of APL by VPA HDACi induces cell cycle arrest (Insinga et al., 2005). TRAIL and its receptor DR-5 HDACi induce cell cycle arrest in both normal and were induced in the mouse model of APL by VPA transformed cells (Marks and Dokmanovic, 2005; (Insinga et al., 2005), and Jurkat human T-cell Ungerstedt et al., 2005). Low concentrations of HDACi lymphoblast leukemia cells and SKW6.4B lymphoblast predominantly induce G1 arrest, while high concentra- cells by LAQ824 (Rosato et al., 2006). HDACi tions induce both G1 and G2/M arrests (Richon et al., upregulated DR-5 in various transformed cells.Expres- 2000).G 1 and G2 arrests are largely associated with sion of TNF-a was upregulated by FK228 in HL-60 and induction of p21, which inhibits CDKs regulating G1 K562 cells.c-FLIP, an inhibitor of the death receptor progression (CDK4/6) and G1/S transition (CDK2), the pathway, was downregulated by HDACi (Nakata et al., activity of proliferating cell nuclear antigen that is 2004; Peart et al., 2005; Rosato and Grant, 2005; required for DNA replication (Vidal and Koff, 2000), Sutheesophon et al., 2005). and cdc2/CDK1 that regulates G2/M transition.Loss of HDACi-induced cell death can be reduced by p21 abolishes HDACi-induced G1 arrest (Archer et al., chimeras of DR5-Fc (anti-TRAIL) or Fas-Fc (anti- 1998; Rosato et al., 2001; Xu et al., 2005). G1 arrest was Fas), monoclonal antibodies against FasL or TRAIL observed in cells without p21 (Hitomi et al., 2003). In and small inhibitor RNA (siRNA) of TRAIL or Fas this case, HDACi may induce other CDK inhibitors that (Nakata et al., 2004; Insinga et al., 2005; Rosato and cause cell cycle arrest.TSA induced G 1 arrest in human Grant, 2005).Taken together, the data indicate that the colon HCT116 p21À/À cells associated with the extrinsic apoptotic pathway can account for HDACi- induction of p15 (INK4b), which is an inhibitor of the induced cell death in many transformed cells. cyclin D-dependent kinases (Hitomi et al., 2003). p27, which inhibits CDK4- and CDK2-containing HDACi activates the intrinsic apoptotic pathways complexes (Vidal and Koff, 2000), was induced by The intrinsic apoptosis pathway is mediated by mito- vorinostat and/or TSA, in leukemia cells K562 and chondria, with the release of mitochondrial inter- LAMA-84 (Nimmanapalli et al., 2003), and breast membrane proteins, such as cytochrome c, apoptosis cancer cells MCF-7 and MDA-MB-231 (Huang and inducing factor (AIF) and Smac, and the consequent Pardee, 2000).In cells cultured with HDACi, the activation of caspases.It is regulated, in part, by pro- increase of the CDK inhibitors and the decrease of and antiapoptotic proteins of bcl-2 family (Jiang and cyclins may act together to account for the reduced Wang, 2004).Activation of the intrinsic apoptotic

Oncogene Mechanisms of histone deacetylase inhibitors WS Xu et al 5545 pathway is a major pathway for HDACi to induce cell prometaphase, followed with aberrant mitosis such as death.By mechanisms that are still not well under- missegregation and loss of chromosomes, resulting in stood, HDACi leads to release of cytochrome c from cell death by either apoptosis or, mitotic cell death/ mitochondrial intermembrane space and activation catastrophe (Qiu et al., 2000; Cimini et al., 2003; Xu caspase-9 (Marks and Dokmanovic, 2005; Bolden et al., 2005). HDACi-induced a-tubulin acetylation does et al., 2006). Overexpression of Bcl-2 or Bcl-XL, not affect mitosis, although a-tubulin is a component of which protect mitochondria, inhibits HDACi-induced mitotic spindle that mediates mitosis.The HDAC6- apoptosis.Inhibition of Bcl-2 by a chemical inhibitor specific inhibitor tubacin induced a-tubulin acetylation, HA14-1 increases HDACi-induced cell death (Xu et al., but did not affect cell cycle progression (Haggarty et al., 2006). 2003).FK228 caused mitotic arrest, but did not inhibit HDACi alter the factors that mediate or regulate the tubulin deacetylase HDAC6. intrinsic apoptosis pathway.Bid cleavage, which can et al initiate the intrinsic pathway (Bolden ., 2006), HDACi induces autophagic cell death and senescence occurred before mitochondrial disruption in CEM cells HeLa cells with Apaf-1 knockout or Bcl-X over- cultured with vorinostat or oxamflatin (Ruefli et al., L expression were induced to autophagic cell death with 2001; Peart et al., 2003). HDACi upregulate proapop- autophagic vacuoles in the cytoplasm, when cultured totic proteins of Bcl-2 family, such as Bim, Bmf, Bax, with vorinostat or butyrate (Shao et al., 2004). In colon et al et al Bak and Bik (Zhang ., 2004; Zhao ., 2005; Xu carcinoma cells, senescence phenotype was observed in et al., 2006). The mechanism of this effect is not vorinostat-induced polyploidy cells associated with understood.It has been shown that vorinostat and mitotic defects (Xu et al., 2005). TSA increase Bim transcription by increasing the activity of E2F1 (Zhao et al., 2005). HDACi decrease antiapoptotic proteins of Bcl-2 family, such as Bcl-2, ROS, thioredoxin and Trx binding protein 2 in Bcl-XL, Bcl-w and Mcl-1 (Zhang et al., 2004; Rosato HDACi-induced cell death et al., 2006; Xu et al., 2006). HDACi decrease the Accumulation of ROS occurs in transformed cells inhibitor of apoptosis (IAP) XIAP by suppressing its cultured with HDACi, such as vorinostat, TSA, transcription (Zhang et al., 2004; Rosato et al., 2006), butyrate or MS-275 (Ruefli et al., 2001; Rosato et al., and survivin by inducing protein degradation (Rosato 2003; Ungerstedt et al., 2005; Xu et al., 2006). et al., 2006). HDACi cause the release of the mitochon- Accumulation of ROS may be important in HDACi- drial intermembrane proteins, cytochrome c, AIF induced cell death.ROS accumulation occurs within 2 h and Smac (Ruefli et al., 2001; Rosato et al., 2006), and of culture with HDACi, before disruption of mitochon- may also increase their levels (Xu et al., 2006). dria.Free radical scavengers such as N-acetylcysteine The effects of HDACi to increase the proapoptotic decrease HDACi-induced apoptosis (Ruefli et al., 2001; proteins and decrease the antiapoptotic proteins are cell Rosato et al., 2003). context dependent.The basal level of these proteins In many transformed cells, ROS-oxidation–reduction vary dramatically in different tumor cells even of pathways are important mechanisms of HDACi- the same type of cancer, for example, prostate, as do induced transformed cell death (Ungerstedt et al., 2005; the changes in these proteins induced by HDACi (Xu Xu et al., 2006). Thioredoxin (Trx) acts as a hydrogen et al., 2006). donor required for activation of many proteins, includ- Using the Eu-myc mouse model of B-cell lymphoma, ing ribonucleotide reductase that is essential for DNA vorinostat was found to selectively induce lymphoma synthesis, and transcription factors, for example, nucle- cell death (in vivo), which was independent of p53 and ar factor kB (NF-kB), and is an antioxidant scavenger death receptor pathways (Lindemann et al., 2007). of ROS (Lillig and Holmgren, 2007).HDACi upregu- Sensititivity to the HDACi was dependent on expression lates the expression of Trx binding protein 2 (TBP2) of the BH3 – only proteins and BID and BIM. (Butler et al., 2002; Xu et al., 2006), which binds and inhibits Trx activity (Nishiyama et al., 1999), and can HDACi induces mitotic cell death cause downregulation of Trx in transformed but not HDACi can induce mitotic defects associated with normal cells (Butler et al., 2002; Ungerstedt et al., 2005). aberrant acetylation of histones in heterochromatin Trx is an inhibitor of apoptosis signal regulating kinase and centromere domains.Newly replicated chromatin 1 (ASK1) (Saitoh et al., 1998). ASK1 promotes contains acetylated histones.In culture with TSA, apoptosis by activation of SET1-JNK and MKK3/ histones in newly synthesized chromatin remain acety- MKK6-p38 signaling cascades, and by enhancing the lated, and this disrupts the structure and function of the expression of proapoptotic protein Bim through a centromere and the pericentric heterochromatin, with positive feedback on E2F1 activity (Tan et al., 2006). loss of binding to heterochromatin binding proteins Inhibition of Trx by TBP2 activates ASK1.Further, (Taddei et al., 2001; Cimini et al., 2003). Histone HDACi increase the expression of ASK1.These effects acetylation interferes with histone phosphorylation and can act together to promote apoptosis.ROS accumula- disrupts the function of mitotic spindle checkpoint tion may also be a consequence of apoptosis.The pan- proteins, such as BubR1, hBUB1, CENP-F and caspase inhibitor Z-VAD-fmk blocked MS-275-induced CENP-E (Dowling et al., 2005; Robbins et al., 2005). apoptosis as well as ROS accumulation in human As a result, the cells show a transient arrest at chronic lymphocytic leukemia cells (Lucas et al., 2004).

Oncogene Mechanisms of histone deacetylase inhibitors WS Xu et al 5546 Antitumor effects of HDAC6 inhibition alterations can be anticipated as a result of HSP90 HDAC6 is an unique HDAC, localized in the cytoplasm, inactivation through HDAC6 inhibition by HDACi, or where it associates with non-histone substrates, its downregulation by HDAC6 siRNA.The effects of such as HSP90 and a-tubulin.It has two catalytic domains inactivation of HSP90 include non-functional gluco- and a ubiquitin binding domain, named BUZ domain corticoid receptor with defects in ligand binding, nuclear (Zhang et al., 2006; Zou et al., 2006). Overexpression translocation and transcription activation (Kovacs of HDAC6 leads to deacetylation of a-tubulin and et al., 2005). increases cell motility (Hubbert et al., 2002; Haggarty AR can be acetylated and become more transcrip- et al., 2003). HDAC6 can not only bind both mono and tionally active, suggesting that some HDACi could poly-ubiquitinated proteins but also promote its own stimulate cell proliferation (Fu et al., 2003). However, mono-ubiquitination.Specific inhibition of HDAC6 activ- HDACi inhibit HDAC6 and consequently HSP90, ity or its downregulation by siRNA increases a-tubulin which hypothetically can overcome the AR activation and HSP90 acetylation, which reduces cellular motility, (as a consequence of its acetylation) by inducing its and induces HSP90 client proteins degradation, degradation.Further, HDACi suppress AR transcrip- cell growth inhibition and cell death (Bali et al., 2005; tion (Wang et al., 2004). These findings suggest that Kovacs et al., 2005). Acetylated HSP90 cannot form HDACi may be promising for the treatment of stable complex with client proteins and its deacetylation hormone-refractory prostate cancer. by HDAC6 is required to regenerate functional HSP90 In human leukemia K562 cells, the knockdown of (Aoyagi and Archer, 2005).HSP90 acetylation is HDAC6 by siRNA or culture with HDACi LAQ824 associated with loss of function, and its client proteins induces acetylation of HSP90 and a-tubulin, and inhibits such as pro-survival and pro-proliferation proteins the binding of HSP90 to ATP (Bali et al., 2005), Akt, Bcr-Abl, c-Raf and ErbB2 can be poly-ubiquitinated disrupting HSP90 chaperone function with client anddegradedviaproteosome(Baliet al., 2005; Chen et al., proteins, inducing poly-ubiquitinalation and partial 2005).Inhibition of HDAC6 by either specific or pan- depletion of Bcr-Abl.Targeted inhibition of HDAC6 HDACi can trigger different mechanisms of cell death. led to polyubiquitylation and depletion of other pro- HDAC6 may also be involved in upregulating p21.Runx2 growth and pro-survival HSP90 client proteins, includ- (Cbfa1, AML-3) is a transcription factor that can bind ing Akt.These studies indicate that inhibition of HDAC6, recruit it to the p21 promoter and repress its HDAC6 leads to disruption of the antiapoptotic Akt expression.Inhibition of HDAC6 activity could result in pathway, either by Akt dephosphorylation and/or by its induction of p21 and, consequently, cell cycle arrest degradation. (Westendorf et al., 2002). Disruption of the aggresome pathway HDAC6 is a Activation of protein phosphatase 1 HDACi can component of the aggresome, a cellular structure that disrupt phosphatase complexes.Recombinant HDAC6 constitutes the major site of degradation for misfolded binds directly to protein phosphatase 1 (PP1) catalytic protein aggregates, both non-ubiquitinated and ubiqui- subunit.TSA disrupts endogenous HDAC6–PP1 com- tinated misfolded proteins (Kawaguchi et al., 2003). plexes (Brush et al., 2004). HDAC1 and 10 are also Direction of misfolded proteins to aggresomes is components of cellular phosphatase complexes.HDACi essential for cell survival, since these proteins are disrupt HDAC–PP1 complexes and activate PP1, which susceptible to forming cytotoxic aggregates that can inactivates Akt by dephosphorylation (Chen et al., interfere with normal cell function.Aggresome forma- 2005).The interactions between HDACs and PP1 tion requires the microtubule network and the micro- provide a mechanism by which HDACi can cause tubule-associated motor, dynein (Johnston et al., 2002). simultaneous changes in cellular protein phosphoryla- HDAC6 can bind p150, a component of dynein motor tion and acetylation, which contribute to the antitumor complex, and act as a bridge between the dynein motors activity of HDACi. and the ubiquitination process, directing the poly- ubiquitinated proteins to aggresome.HDAC6 has a high affinity for ubiquitin molecule (due to the presence Disruption of the function of chaperonin HSP90 HDA- of the ZnF-UBP or BUZ domain) and is involved in the Ci can cause accumulation of acetylated HSP90 through transport of poly-ubiquitinated proteins (Boyault et al., HDAC6 inhibition, with consequent inactivation of 2006).HDAC6 deacetylase activity is important for et al HSP90 (Bali ., 2005). As indicated above, this transport of misfolded poly-ubiquitinated proteins to chaperone protein is essential for the stability the aggresome, and loss of HDAC6 function makes cells and function of many client proteins, including steroid more sensitive to misfolded protein stress induced by hormone receptors and protein kinases, that are crucial protease inhibitor and, as a consequence, cell death for numerous cell signaling processes and cellular (Kawaguchi et al., 2003). homeostasis (Solit and Rosen, 2006).Recent studies have demonstrated both a direct physical interaction between HDAC6 and HSP90, and HDAC6 as a HDACi inhibits angiogenesis regulator of HSP90 activity, through its deacetylation HDACi can block tumor angiogenesis by inhibition (Bali et al., 2005; Kovacs et al., 2005). Considering of hypoxia inducible factors (HIF) (Liang et al., the number of HSP90 client proteins, many molecular 2006).HIF-1 and HIF-2 are transcription factors for

Oncogene Mechanisms of histone deacetylase inhibitors WS Xu et al 5547 angiogenic genes (Brown and Wilson, 2004).The oxygen including chemotherapeutic drugs, new targeted ther- level can control HIF activity through two mechanisms. apeutic reagents and radiation, by various mechanisms, First, under normoxic conditions, HIF-1a binds to von some unique for particular combinations (Rosato and Hippel–Lindau protein (pVHL) and is degraded by the Grant, 2004; Bhalla, 2005; Marks and Dokmanovic, ubiquitination–proteasome system.Second, HIF activ- 2005; Bolden et al., 2006). ity depends on its transactivation potential (TAP), HDACi have shown synergy with chemotherapeutic which is affected by the interaction with the coactivator agents, such as antimetabolites 5-ﬂuorodeoxyuridine p300/CBP among others.This complex can be disrupted and gemcitabine, antitubule agents docetaxel and by Factor Inhibiting HIF (FIH).Hypoxic conditions epothilone B, topoisomerase (Topo) II inhibitors activate HIF through repression of the hydroxylases doxorubicin, epirubicin, VP-16 (etopside) and ellipticine responsible for HIF degradation and loss of function. (Munster et al., 2001; Kim et al., 2003b), and DNA Hypoxia is a common event in tumors.Hypoxic crosslinking agent cisplatin (Kim et al., 2003b). The conditions can induce transcription activation of synergistic effects may depend on the sequence of HDAC1, -2 and -3 in transformed cells (Liang et al., drug administration.For example, prior treatment 2006).These class I HDACs downregulate expression of with HDACi induced chromatin decondensation and p53 and pVHL, which reduces FIH expression and increased Topo IIb/DNA cleavable complex formation, consequently activates HIF-1a and promotes angiogen- resulting in synergy of HDACi plus Topo II inhibitors esis (Liang et al., 2006). (Marchion et al., 2005). The reverse order of adminis- TSA, vorinostat, FK228, butyrate and LAQ824 were tration of the drugs resulted in antagonistic effects, found to repress angiogenesis in vitro and in vivo, and or had no more effect than each drug alone (Kim et al., reduce expression of pro-angiogenesis factors, including 2003b).Further, pretreament with HDACi had HIF-1a and VEGF (Deroanne et al., 2002; Bolden et al., more effect (four times) than the reverse (1.8 times) in 2006; Liang et al., 2006). HDACi can induce HIF-1a the combination with cisplatin, although the reverse degradation by acetylation at Lys532, leading to the was not antagonistic (Kim et al., 2003b). HDACi have interaction with, and ubiquitination by pVHL (Jeong also been reported to have synergy with transcription et al., 2002). Further, HDACi can induce HIF-1a modulator all-trans retinoid acids, vitamin D3 and its degradation in a VHL-independent mechanism (Kong analogs, DNA demethylating agent 5-aza- et al., 2006). Class II HDACs, HDAC4 or HDAC6, 20deoxycytidine, abl kinase inhibitor imatinib (Gleevec, physically associate with HIF-1a, and their selective STI571) in both imatinib-sensitive and imatinib-resis- inhibition by siRNA induced HIF-1a degradation (Qian tant chronic myelogenous leukemia (CML) cells, HSP90 et al., 2006). Moreover, HIF-1a binds to HSP90, and inhibitor 17-ally-amino-demethoxy geldanamycin, pro- HDACi can disrupt HSP90 chaperone function, expos- teasome inhibitor bortezomib (PS-341), trastuzumab ing HIF-1a to proteasomal degradation.These observa- (herceptin), which is a monoclonal antibody against tions suggest that HDAC6 should be inhibited for an Her-2/neu (erbB2) receptor, and radiotherapy effective abolishment of HIF-1a function. (Nimmanapalli et al., 2003; Marks and Dokmanovic, Another mechanism by which HDACi disrupt HIF- 2005; Bolden et al., 2006). 1a function is through repression of its TAP.Low doses Upregulation of death receptors and/or reducing the of HDACi, which did not induce HIF-1a degradation, inhibitory regulators of death receptor pathway by did repress HIF-1a TAP under both normoxic and HDACi sensitize tumor cells to TRAIL (Bolden et al., hypoxic conditions (Fath et al., 2006). This repression 2006).HDACi also achieve synergy with TRAIL by occurs due to the targeting of HIF-1a/p300 complex by simultaneous activation of the intrinsic and the extrinsic HDACi.HDACi could also decrease HIF-1 a activation apoptotic pathways, without changing the expression of by inhibiting HDAC7 activity, since under hypoxic TRAIL receptors or the inhibitory protein c-FLIP conditions, HDAC7 translocates to the nucleus, where it (Rosato and Grant, 2004). can interact with HIF-1a and increase its transcriptional Many kinase inhibitors, including CDK inhibitor activity (Kato et al., 2004). ﬂavopyridol, phosphatidylinocitol 3 kinase inhibitor HDACi inhibit angiogenesis by preventing endothe- LY294002, FLT3 inhibitor, PKC412 and MEK1/2 lial cells from responding to the angiogenic inhibitor PD184352, suppress HDACi-mediated p21 stimulus generated by VEGF (Deroanne et al., 2002). induction, and potentiate the cell killing effect of TSA and vorinostat inhibit VEGF-induced expression HDACi.Blocking NF- kB activation by I Kappa B of VEGF receptors and neuropilin-1, and induction of Alpha (IkBa) phosphorylation inhibitor Bay 11-7082 semaphoring III expression in endothelial cells.The markedly increase HDACi-induced apoptosis (Alme- anti-angiogenic effects of HDACi can contribute their nara et al., 2002; Rosato and Grant, 2005). antitumor activities.These observations support the use of combination therapies with VEGF inhibitors. Clinical development of HDACi

Combination of HDACi with other antitumor agents The studies with tumor bearing animals and clinical trials of HDACi have been extensively reviewed The HDACi have shown synergistic or additive anti- elsewhere (Rosato and Grant, 2004; Bhalla, 2005; tumor effects with a wide range of antitumor reagents, Marks and Dokmanovic, 2005; Bolden et al., 2006;

Oncogene Mechanisms of histone deacetylase inhibitors WS Xu et al 5548 Marks and Breslow, 2007) (Table 2).Vorinostat is the apoptosis and other forms of cell death.Indeed, the fact first of the new HDACi to be approved by Food and that HDACs have histone and multiple nonhistone Drug Administration for the clinical use in cancer protein substrates suggests these enzymes should be patients, namely the treatment of cutaneous T-cell referred to as ‘lysine deacetylases’.HDACi can lymphoma (CTCL) (Duvic et al., 2007; Garber, 2007). cause transformed cells to undergo growth arrest, In a phase II study with orally administered vorinostat differentiation and/or cell death.Normal cells are on 33 previously treated patients with refractory relatively resistant to HDACi.HDACi are selective cutaneous T-cell lymphoma, partial response were in altering gene expression, which may reflect, in part, observed in eight patients (24.2%) and 14 of 31 the proteins composing the transcription factor evaluable patients (45.2%) had pruritis relief (Duvic complex to which HDACs are recruited.Both et al., 2007). altered gene expression and changes in non-histone At least 14 different HDACi are in some phase proteins caused by HDACi-induced acetylation play of clinical trials as monotherapy or in combination a role in the antitumor activity of HDACi.This with retinoids, taxols, gemcitabine, radiation, etc, in is reflected in the different inducer-activated anti- patients with hematologic and solid tumors, including tumor pathways in transformed cells (Figure 1). cancer of lung, breast, pancreas, renal and bladder, The functions of HDACs are not redundant.Thus, melanoma, glioblastoma, leukemias, lymphomas, a pan-HDAC inhibitor such as vorinostat may multiple myeloma (see National Cancer Institute web- activate more antitumor pathways and have thera- site for CTEP clinical trials, ctep.cancer.gov or clinical- peutic advantages compared to HDAC isotype-specific trials.gov, and website of companies developing inhibitors. HDACi; Table 2). Almost all cancers have multiple defects in the expression and/or structure of proteins that regulate cell proliferation and death.Compared to other antitumor reagents, the plurality of action of HDACi The resistance to HDACi potentially confers efficacy in a wide spectrum of cancers, which have heterogeneity and multiple defects, Development of resistance to HDACi is a major both among different types of cancer and within concern as with any new antitumor therapy.In different individual tumors of the same type.The preclinical studies, resistance to HDACi-induced multiple defects in a cancer cell may be the reason for transformed cell death was observed in human bladder transformed cells being more sensitive than normal cells carcinoma cells (T24) and prostate cancer cells to HDACi.Thus, given the relatively rapid reversibility (PC3) (Butler et al., 2000; Richon et al., 2000; Xu of vorinostat inhibition of HDACs, normal cells may be et al., 2006). Although vorinostat achieved 24.2% able to compensate for HDACi-induced changes more response rate in a phase II trial on CTCL, a considerable effectively than cancer cells. proportion of patients with CTCL did not respond HDACi have synergistic or additive antitumor effects well (Duvic et al., 2007). Resistance has been observed in with many other antitumor reagents – suggesting that clinical trials with other HDACi in different tumors. combination of HDACi and other anticancer agents The basis of resistance to HDACi is not well under- may be very attractive therapeutic strategies for using stood.High levels of Bcl-2 (Pommier et al., 2004), these agents.Complete understanding of the mechan- Trx (Powis et al., 2000) and peroxiredoxins (Chung isms underlying the resistance and sensitivity to HDACi et al ., 2001) have been associated with resistance has obvious therapeutic importance.Targeting resistant of transformed cells to chemotherapy, and may factors will enhance the antitumor efficacy of HDACi. play a role in the resistance to HDACi.Upregulation Identifying markers that can predict response to HDACi of Trx protects normal cells against HDACi-induced is a high priority for expanding the efficacy of these cell death (Ungerstedt et al., 2005). Overexpression novel anticancer agents. of Bcl-2 blocks HDACi-induced transformed cell death (Mitsiades et al., 2003). Peroxiredoxins reduce ROS generation (Kang et al., 1998) and may protect transformed cells from HDACi-induced cell death, Acknowledgements which is strongly associated with ROS production (Rosato and Grant, 2005).Resistance to FK228 The studies reported in this paper from the authors’ laboratory have been supported, in part, by grants from the National has been associated with multiple drug resistance, Institute of Health (P30CA08748-41), Jack and Susan Rudin upregulation of and efflux by P-gp (MDR1) (Glaser, Foundation, David H Koch Foundation, and the Prostate 2006).The MDR1-mediated resistance does not affect Cancer Research Award, Experimental Therapeutics Center at vorinostat (Ruefli et al., 2002). Memorial Sloan-Kettering Cancer Center and the DeWitt Wallace Research Fund.MSKCC and Columbia University jointly hold patents on hydroxamic acid-based polar compounds, including vorinostat (SAHA), that were exclusively Conclusions and perspectives licensed to Aton Pharma Inc., a biotechnology company that was acquired by Merck Inc.in April 2004.PAM was a founder HDACs have multiple substrates involved in many of Aton and has a financial interest in Merck’s further biological processes, including proliferation, differentiation, development of vorinostat.

Oncogene Mechanisms of histone deacetylase inhibitors WS Xu et al 5549 References

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