Histone Deacetylases and Cancer

Home , Acetylation, HDAC9, Histone deacetylase 2, Sulforaphane, SUV39H1

MA Glozak and E Seto

H. Lee Mofﬁtt Cancer Center & Research Institute, Tampa, FL, USA

Histone deacetylases (HDACs) regulate the expression functions of the enzymes they inhibit. In this review, and activity of numerous proteins involved in both cancer we will address the molecular mechanisms by which initiation and cancer progression. By removal of acetyl HDACs act and how these actions relate to cancer. groups from histones, HDACs create a non-permissive chromatin conformation that prevents the transcription of genes that encode proteins involved in tumorigenesis. In addition to histones, HDACs bind to and deacetylate a Chromatin modifications and cancer variety of other protein targets including transcription factors and other abundant cellular proteins implicated in Histones comprise the protein backbone of chromatin. control of cell growth, differentiation and apoptosis. This Histone acetyltransferases (HATs) acetylate the e-amino review provides a comprehensive examination of the tran- group of lysine (K) residues on histones, thereby scriptional and post-translational mechanisms by which neutralizing their positive charge, which diminishes their HDACs alter the expression and function of cancer- ability to bind to negatively charged DNA. In addition, associated proteins and examines the general impact of acetylation of histone H4-K16 specifically disrupts HDAC activity in cancer. the formation of higher-order chromatin structures et al Oncogene (2007) 26, 5420–5432; doi:10.1038/sj.onc.1210610 (Shogren-Knaak ., 2006). An open chromatin configuration provides accessibility for specific tran- Keywords: acetylation; deacetylation; cancer; HDAC; scription factors and the general transcription machin- HDAC inhibitors ery. HDACs remove the acetyl groups, allowing compacted chromatin to reform (Figure 2). Among their many properties, HDACs are required for cell- cycle progression. Indeed, HDAC inhibitors, including NaB, trichostatin A (TSA) and trapoxin, cause mammalian cells to undergo cell-cycle arrest. Using a Introduction trapoxin affinity column, HDAC1 was purified bio- chemically, subsequently cloned and shown to have During the normal lifecycle, a cell encounters numerous HDAC activity (Taunton et al., 1996). In an unrelated opportunities where it must decide whether to prolifer- series of experiments, HDAC2 was cloned on the basis ate, differentiate or die. A defect in any of these of its interaction with the transcriptional activator/ processes may result in cancer, or the uncontrolled repressor YY1 and was shown to have transcriptional growth of the cell. It has been appreciated for decades repressor activity (Yang et al., 1996). As such, the first that histone deacetylases (HDACs) play an important mammalian HDACs were cloned independently, using role in cancer development since the first observation distinct methods. The same roles that HDACs play in that sodium butyrate (NaB), later identified as an influencing cell-cycle progression and repressing tran- HDAC inhibitor, can cause the morphological reversion scription also have important implications in their of the transformed cell phenotype (Ginsburg et al., 1973; connection to cancer. Altenburg et al., 1976; Boffa et al., 1978). Currently, Since the initial cloning of HDAC1 and HDAC2, at HDAC inhibitors are in clinical trials to treat a variety least 16 additional human HDACs have been identified of malignancies. HDAC inhibitors can block cell and are grouped into four classes: class I comprises proliferation, promote differentiation and induce apop- HDAC1–3 and 8, which show similarity to yeast RPD3; tosis, any of which is desirable to thwart the growth of class II comprises HDAC4–7, 9 and 10, which show rogue cells (Figure 1). Despite decades of research, the similarity to yeast HDA1; class III the NAD þ -depen- precise mechanism(s) by which these inhibitors work dent HDACs, comprises SIRT1–7, which show similar- remains far from clear. In many cases, they may ity to yeast SIR2; class IV comprises HDAC11, which influence multiple targets. To understand how HDAC has some features of both class I and II HDACs inhibitors function, we must first understand the (Gregoretti et al., 2004). Class III HDACs are not affected by the cytostatic HDAC inhibitors, but are Correspondence: Dr E Seto, H. Lee Moffitt Cancer Center & Research inhibited by nicotinamide. Institute, SRB 23011, 12902 Magnolia Drive, Tampa, FL 33612, USA. Histone acetylation and deacetylation does not occur E-mail: ed.seto@moffitt.org in a vacuum. Specific patterns of histone acetylation and HDACs and cancer MA Glozak and E Seto 5421 promoters of genes de-repressed by HDAC inhibitors proliferation differentiation often contain hypermethylated DNA, indicating cross- talk between histone acetylation/methylation and DNA methylation. Each of these events can have profound implications for gene expression in normal and cancerous cells. Epigenetic changes, including global DNA hypo- CDKI differentiation factors methylation and promoter CpG island hypermethylation, have long been recognized as characteristics that distinguish cancer cells from their normal counterparts. HDAC HDAC Recently, very specific changes in post-translational modification of histones that characterize cancer cells pro-apoptotic factors have been identified as well. For example, the coupled X loss of acetylation of histone H4-K16 and trimethylation of histone H4-K20 can be considered a common feature of cancer cells as compared to their normal RIP counterparts (Fraga et al., 2005). These changes occur early and accumulate as the cancer progresses. Hypoa- apoptosis cetylation of histone H4-K16 is caused at least in part by diminished recruitment of the MYST (MOZ, Ybf2/Sas3, Sas2 and Tip60) family of HATs (Fraga et al., 2005); proliferation differentiation however, an increase in HDAC recruitment and/or activity cannot be ruled out. Indeed, the class III HDAC SIRT2 shows preference for deacetylation of histone H4-K16. Although this specific deacetylation occurs at the G2/M transition of the cell cycle, it appears to influence the timing of S-phase entry (Vaquero et al., CDKI differentiation factors 2006). In addition to defining cancer cells, specific histone modifications can be used to predict cancer recurrence. Histone H3-K18 acetylation coupled with HDACi HDAC HDAC HDACi histone H3-K4 dimethylation confers lowered risk for prostate cancer recurrence (Seligson et al., 2005). While pro-apoptotic factors these findings presently apply only to prostate cancer, it is likely that similar modifications will be found in other cancer types. In addition, the observation that low RIP acetylation levels correlate with negative outcomes may provide a basis for the clinical use of HDAC inhibitors.

apoptosis Figure 1 (a) HDAC-mediated repression of genes can cause uncontrolled cell growth. HDACs repress the transcription of (1) cyclin-dependent kinase inhibitors (CDKI), allowing continued Histone deacetylation: the classic examples proliferation; (2) differentiation factors, allowing proliferation instead of differentiation; (3) proapoptotic factors, permitting Upon the identification and cloning of HDACs, it was survival. (b) HDAC inhibitors (HDACi) can restore appropriate quickly ascertained that HDACs were recruited to gene expression, preventing the uncontrolled growth of the cell. promoters to repress transcription, thereby counter- HDAC, histone deacetylases. acting the activating effects of HATs. In the simplest view, recruitment of HDACs causes decreased histone acetylation that compacts chromatin and precludes deacetylation are typically influenced by other histone access by the transcription machinery, resulting in modifications. Together, these post-translational mod- transcriptional repression (Figure 2). HDACs act on ifications potentially generate a ‘histone code’ (Jenuwein multiprotein complexes and several distinct complexes and Allis, 2001). For example, histone H3-K9 acetyla- were rapidly identified that clarified the mechanism of tion and H3-K4 methylation are associated with active active repression of transcription. For example, the transcription. In contrast, loss of histone H3-K9 transcription factor Max is the heterodimeric binding acetylation and gain of H3-K9 and H3-K27 methylation partner of both the transcriptional activator Myc and is indicative of heterochromatin (Maison et al., 2002). In the transcriptional repressor Mad. Both Myc–Max and many cases, modifications of a single residue are Mad–Max heterodimers bind the same E box-contain- mutually exclusive. Furthermore, the presence of one ing DNA sequence, but Mad–Max complexes associate modification may set the stage for additional modifica- with the mSin3 scaffold protein that recruits HDAC1 tions at nearby amino acids, thus expanding the coding and HDAC2. Transcriptional repression of Mad–Max information. Adding another layer of complexity, target genes requires HDAC activity (Hassig et al., 1997;

Oncogene HDACs and cancer MA Glozak and E Seto 5422

transcription Ac Ac Ac Ac HAT K K K K KKKK TF Pol transcription II HDAC

Ac Ac Ac Ac HAT KKKK K K K K TF Pol HDAC II transcription

HDACi Figure 2 The balance between histone acetylation and deacetylation determines the level at which a gene is transcribed. Histone acetylation relaxes the chromatin, allowing transcription factor (TF) binding and RNA polymerase II (pol II) recruitment. The addition of HDAC inhibitors (HDACi) has a similar net effect to increasing the amount of HAT activity, resulting in enhanced transcription levels. HDAC, histone deacetylases.

Laherty et al., 1997). In addition, DNA-bound un- liganded nuclear receptors, such as the retinoic acid receptor, bind the co-repressors SMRT and N-CoR. INITIATION These co-repressors bind directly to HDAC3, thereby recruiting HDAC3 to promoters and preventing transcription (Wen et al., 2000; Guenther et al., 2001). cell cycle inhibitors Furthermore, repression of E2F-mediated transcription differentiation by Rb requires HDAC1 and recruitment of HDAC1 coincides with decreased histone acetylation at E2F- apoptosis regulated promoters (Brehm et al., 1998; Luo et al., 1998; Magnaghi-Jaulin et al., 1998). In each of these cases of ‘classic repression’, treatment with an HDAC inhibitor de-represses the promoter in question. Import- antly, repressed genes often are tumor suppressors, cell- cycle inhibitors and differentiation factors or apoptosis inducers. Loss of expression of any combination of these HDACs classes of proteins would be advantageous to a cancerous cell. This offers a clue as to why HDAC inhibitors were initially shown to reverse the transformed cell phenotype and why they may be useful for effective cancer therapy. PROGRESSION

Histone deacetylation and cancer initiation angiogenesis

Histone acetylation and deacetylation affect the expres- invasion sion of numerous genes implicated in oncogenesis adhesion (Figure 3). Countless experiments have been performed, in which cancer cells are treated with HDAC inhibitors migration and gene expression and histone acetylation status are examined. These experiments have provided a basic Figure 3 Histone deacetylation by HDACs inﬂuences the expres- understanding of the mechanisms by which HDACs sion of genes involved in both cancer initiation and progression. modulate the expression of these important genes. See text for details. HDAC, histone deacetylases.

promoter region (Sambucetti et al., 1999; Richon et al., Histone deacetylation and cellular proliferation 2000). Multiple HDACs repress p21 expression in different cell types. In normal development, HDAC1 is The cyclin-dependent kinase inhibitor p21 is one of the most important because targeted disruption of the best studied targets of HDAC inhibitor-mediated HDAC1 causes early embryonic lethality owing to a de-repression. Treatment of many cancer cell types with lack of proliferation caused by increased expression of any of several HDAC inhibitors causes the transcrip- p21. This indicates that other HDACs cannot compen- tional upregulation of this antiproliferative gene. This is sate for the loss of HDAC1 in early embryonic largely independent of p53. Expression of p21 coincides development, despite a concomitant increase in HDAC2 with hyperacetylation of histones H3 and H4 in its and HDAC3 expression (Lagger et al., 2002).

Oncogene HDACs and cancer MA Glozak and E Seto 5423 Like many genes required for proper development, acetylation (Rampalli et al., 2005). In this case, a tumor dysregulated function of HDACs can lead to cancer. As suppressor recruits an HDAC to repress expression of a such, overexpression of HDACs is observed in many proproliferative gene and restrain cell growth. cancer types, with corresponding decreases in p21 Restraint of inappropriate cell growth is critical to expression. For example, prostate cancer cells over- prevent cancer. In normal cells, transforming growth express HDAC1 (Halkidou et al., 2004). Gastric factor-b (TGF-b) inhibits cell growth. In contrast, cancer carcinomas, colorectal carcinomas, cervical dysplasias cells are refractory to the inhibitory effect of TGF-b,which and endometrial stromal sarcomas all overexpress often is due to the loss of TGF-b receptors (TGF-bR). HDAC2 as compared to their normal counterparts TGF-bRII binds TGF-b,andTGF-bRI transduces the (Huang et al., 2005; Song et al., 2005; Hrzenjak et al., signal by activation of Smad family members. While Smad 2006). Increased HDAC2 expression correlates with family members recently were shown to be targets of reduced p21 expression and correspondingly, HDAC2 acetylation themselves, the role of Smad acetylation in knockdown increases p21 expression (Huang et al., cancer remains to be elucidated (Simonsson et al., 2006; 2005; Hrzenjak et al., 2006). Loss of function of the Inoue et al., 2007). Lung cancer cells that do not express tumor suppressor adenomatosis polyposis coli (APC) is TGF-bRII resist endonuclease digestion in the area one mechanism that causes increased HDAC2 levels in surrounding the transcription start site, indicating a closed intestinal mucosa (Zhu et al., 2004). HDAC inhibitors, chromatin conformation. Acetylation of histones H3 and such as valproic acid (VPA), reduce adenoma formation H4 is severely reduced in these cell types. Treatment with in APC mutant mice that express increased levels of TSA or NaB induces expression of TGF-bRII and also HDAC2 (Zhu et al., 2004). Furthermore, reduced tumor increases sensitivity to nuclease digestion, indicative of an formation upon HDAC inhibitor treatment of APC open chromatin conformation (Osada et al., 2001). mutant mice correlates with increased histones H3 and Furthermore, loss of TGF-bRI expression is frequently H4 acetylation in the promoter regions of p21 as well as observed in many cancer types. Treatment of breast cancer the proapoptotic Bax gene (Myzak et al., 2006). cells with HDAC inhibitors increases the levels of HDAC2 knockdown also increases apoptosis. The acetylated histones H3 and H4 at the TGF-bRI promoter critical role of HDAC2 in determining the sensitivity and correspondingly increases the expression of TGF-bRI. of colon cancer cells to HDAC inhibitors is supported Likewise, overexpression of the HAT p300 increases TGF- by sporadic colorectal carcinomas that carry a frame- bRI promoter activity. HDAC inhibitor treatment restores shift mutation encoding truncated, non-functional TGF-b sensitivity by impairing HDAC1 activity asso- HDAC2. These cells express wild-type APC, yet resist ciated with Sp1/Sp3 at the TGF-bRI promoter (Amma- the apoptotic effects of HDAC inhibitor treatment namanchi and Brattain, 2004). By repressing the (Ropero et al., 2006). In addition, APC expression expression of a receptor for a growth-restraining signaling appears to be required for HDAC inhibitor-induced molecule, HDACs render a cancer cell unresponsive to this downregulation of survivin, which sensitizes cells to signal and allow for unfettered cell growth. apoptosis (Huang and Guo, 2006). HDAC3 is also overexpressed in colon cancer and inhibits p21 expression. Similarly, silencing of HDAC3 increases p21 promoter activity and expression (Wilson et al., 2006). HDAC repression of epithelial differentiation HDAC6, a class II HDAC, also represses expression. Runx2 recruits HDAC6 to the p21 promoter in Loss of expression of proliferation-restraining genes is osteosarcoma cells (Westendorf et al., 2002). The one common feature of cancer cells. Inhibition of upregulation of HDACs that repress an important differentiation also causes inappropriate proliferation, growth suppressive gene is an important mechanism to which can lead to cancer. For example, loss of promote cancer cell proliferation. As such, HDAC expression of the GATA family of differentiation factors inhibition can be used to stop the growth of cancer cells. is observed in several cancer types. The mechanism The cell-cycle activator cyclin D1 is frequently over- behind the loss of these critical transcription factors expressed in cancer. Overexpression of this proproli- appears to be cell type dependent. Loss of GATA4 and ferative gene usually is caused by gene ampliﬁcation, but GATA5 expression in gastric and colo-rectal carcinoma can also occur by loss of the tumor suppressor SMAR1, is exclusively due to promoter hypermethylation and not a matrix-associated protein. SMAR1 resides on chro- histone deacetylation (Akiyama et al., 2003). GATA4 mosome 16q24, a region where loss of heterozygosity is and GATA6 are highly expressed in normal ovarian known to deregulate cyclin D1 expression in breast, epithelial cells, but not in ovarian cancer cells. Indeed, prostate and other cancers. Decreased levels of SMAR1 loss of GATA6 expression strongly correlates with correlate with increased cyclin D1 expression. SMAR1 ovarian epithelial morphological transformation and normally binds the cyclin D1 promoter and recruits the de-differentiation. Hypoacetylation of histones H3 and HDAC1/mSin3 repression complex, causing reduced H4, and loss of histone H3-K4 methylation within the cyclin D1 expression through histone deacetylation. GATA4 and GATA6 promoter regions coincide with Correspondingly, SMAR1-expressing cells show redu- lack of GATA expression. Treatment with TSA ced acetylation of histones H3-K9 and H4-K8 in the increases histones H3 and H4 acetylation at these cyclin D1 promoter and surrounding region. In contrast, promoters and increases the promoters’ sensitivity to knockdown of SMAR1 causes an increase in histone DNaseI, indicating an open chromatin conformation.

Oncogene HDACs and cancer MA Glozak and E Seto 5424 Importantly, TSA treatment restores GATA factor Runx1 recruits the histone methyltransferase SUV39H1, expression and the expression of GATA targets, which methylates histone H3-K9, leading to another including the tumor suppressor Dab2 (Caslini et al., mark of inactive chromatin (Reed-Inderbitzin et al., 2006). 2006). ETO also interacts with promyelocytic leukemia The mucin Muc2 plays a role in gastrointestinal cell zinc ﬁnger (PLZF), a member of the BTB/POZ family of differentiation. Loss of expression of this gene is impli- repressor proteins that recruit HDACs and co-repres- cated in pancreatic and colorectal cancer and Muc2-null sors. ETO synergistically acts with PLZF to enhance mice show increased adenoma formation (Velcich et al., transcriptional repression of genes required for differ- 2002). Increased acetylation of histones H3-K9 and H3- entiation in an HDAC-dependent manner (Melnick K27 in the promoter region of Muc2 correlates with et al., 2000). Similarly, ETO binds another BTB/POZ expression of this tumor-suppressor gene. Cells expres- member, BCL6. The BCL6 repressor is frequently sing Muc2 also display histone H3-K4 methylation and expressed inappropriately in B-cell lymphomas and decreased CpG island methylation in the promoter recruits both class I and II HDACs (Lemercier et al., region. TSA treatment of pancreatic cancer cells that do 2002). ETO interacts with DNA-bound BCL6 and not express Muc2 induces histone acetylation and Muc2 enhances repression in an HDAC-dependent manner mRNA and protein expression (Yamada et al., 2006). (Chevallier et al., 2004). In each of these cases, the Regulation of Muc2 by HDACs may be cell type ability of chimeric proteins to inappropriately recruit dependent, because NaB treatment of undifferentiated HDACs to regulatory regions of genes involved in adenocarcinoma cells inhibits Muc2 expression (Augen- differentiation effectively prevents differentiation and licht et al., 2003). This underscores the requirement for allows for continued proliferation of undifferentiated empirical determination of the efﬁcacy of HDAC progenitor cells, resulting in leukemia or lymphoma. inhibitor treatment in any given tumor type.

Histone deacetylation and cancer progression: HDAC repression of hematopoietic differentiation angiogenesis and metastasis

Proper expression of differentiation factors is especially In addition to regulation of genes involved in the genesis important in step-wise differentiation programs such as of cancer, histone acetylation and deacetylation mod- hematopoiesis. The progression from pluripotent stem ulate genes involved in cancer progression (Figure 3). cell to mature hematopoietic cell requires a complex This includes the regulation of angiogenesis that permits interplay of a variety of specific molecules. Blockade at increased tumor growth as well as the regulation of any step along the differentiation pathway may result in adhesion, cell migration and invasion required for the proliferation of leukemic cells. Chromosomal metastasis. Hypoxia, such as encountered in the center translocations are a common hallmark of leukemias of a solid tumor, enhances angiogenesis and also induces and lymphomas. Such translocations often result in the HDAC expression and activity. Overexpression of aberrant recruitment of HDACs to promoters, prevent- HDAC1 represses the tumor suppressors p53 and von ing appropriate gene expression. The critical regulator Hippel–Lindau (VHL) but induces the hypoxia-respon- of definitive hematopoiesis, Runx1 (AML1) is fre- sive genes hypoxia inducible factor-a (HIF-1a) and quently disrupted in leukemia. Runx1-ETO, the product vascular endothelial growth factor (VEGF) and increases of the t(8;21) translocation is frequently observed in angiogenesis. Conversely, HDAC inhibitors de-repress acute myelocytic leukemia, binds HDAC1–3 as well as the tumor suppressors p53 and VHL and repress HIF-1a the co-repressors mSin3a, SMRT and N-CoR (Amann and VEGF, and correspondingly, VEGF signaling. In et al., 2001). In addition, Runx1 binds HDAC5, vivo, hypoxic regions of tumors express increased levels HDAC6 and HDAC9 with varying affinities (Durst of HDAC1 but treatment with TSA inhibits hypoxia- et al., 2003). ETO directly binds HDACs but has no induced angiogenesis (Kim et al., 2001; Deroanne et al., specific DNA binding ability by itself. Since the fusion 2002). Multiple HDAC inhibitors prevent new vessel protein retains the DNA binding domain of Runx1, formation in other models of angiogenesis as well, and Runx1-ETO acts as a dominant negative inhibitor of this is coincident with global hyperacetylation of histone wild-type Runx1 activity. Among the genes that are H4 (Kim et al., 2004; Michaelis et al., 2004). repressed by this chimeric protein is the tumor Metastasis depends on loss of the ability of a cell to suppressor p14ARF, which may extend the lifespan of interact properly with its environment and its neighbor- myeloid progenitor cells due to loss of senescence ing cells. The ability of class I HDACs to regulate (Linggi et al., 2002). Another target of Runx1-ETO extracellular matrix-related genes is highly conserved repression is c-fms, which encodes the colony-stimulat- from Caenorhabditis elegans to humans. HDAC1 ing factor 1 receptor. Cells expressing Runx1-ETO represses cystatin, a peptidase inhibitor that suppresses display decreased amounts of acetylated histones H3- tumor invasion. Knockdown of HDAC1 or overexpres- K9 and H3-K14 at the c-fms intronic regulatory sion of cystatin reduces cellular invasion (Whetstine element, and recruit HDAC1 to this region causing et al., 2005). Similarly, treatment with low doses of decreased expression of this important macrophage HDAC inhibitors reduces v-Fos-transformed fibroblast differentiation factor (Follows et al., 2003). In addition, invasion. HDAC inhibition de-represses several genes in

Oncogene HDACs and cancer MA Glozak and E Seto 5425 this model system, including RYBP, STAT6 and a review see Glozak et al., 2005). As the list of non-histone protocadherin. Overexpression of any of these genes proteins grows almost daily, it is becoming obvious that inhibits invasion, but it is unclear whether the role of acetylation and deacetylation may rival phosphorylation HDAC activity is direct or indirect (McGarry et al., and dephosphorylation as post-translational modifica- 2004). tions that influence the activity of many proteins Class I HDACs also directly regulate the expression (Kouzarides, 2000). Importantly, many of the proteins of E-cadherin, a gene important for cell–cell adhesion. modulated by acetylation play key roles in oncogenesis Loss of E-cadherin expression causes epithelial invasion, and cancer progression. a necessary first step in metastasis. The repressor Snail recruits HDAC1 and HDAC2 and the co-repressor mSin3A to the E-cadherin promoter, repressing E- cadherin expression. Snail overexpression correlates Deacetylation of transcription factors with an increase in hypoacetylated histones H3 and H4 at the E-cadherin promoter, as well as increased Transcription factors comprise one of the largest families methylated histone H3-K9 and decreased methylated of proteins modulated by acetylation status. Acetylation histone H3-K4. Each of these modifications is consistent and deacetylation affect many properties of transcription with repressed gene expression. TSA treatment abolishes factors, including, but not limited to, their ability to bind Snail-mediated repression (Peinado et al., 2004). In DNA and activate transcription. Indeed, the tumor addition, combined treatment of prostate cancer cells suppressor p53 is the founding member of the collection with a peroxisome proliferator-activated receptor-g of non-histone targets of acetylation and deacetylation. (PPARg) agonist and the HDAC inhibitor VPA reduces Depending on the trigger, activation of p53 can prompt the invasiveness of these cells coincident with increased cell-cycle arrest or induce apoptosis. Acetylation of p53 expression of E-cadherin mRNA and protein. HDAC3 by p300/CBP at K305, K370, K372, K373, K381 and and PPARg together bind to the E-cadherin promoter, K382 and by PCAF at K320 increases its DNA-binding and repression in this case is mediated by HDAC3. In ability and consequently increases its ability to activate the presence of the combination of a PPARg agonist and transcription of its target genes (Gu and Roeder, 1997; an HDAC inhibitor, HDAC3 and PPARg are no longer Sakaguchi et al., 1998; Liu et al., 1999; Wang et al., 2003; bound to the promoter, histone H4 is hyperacetylated at Luo et al., 2004). Recently, the role of site-specific p53 the promoter and E-cadherin is expressed (Annicotte acetylation in transcriptional regulation has become the et al., 2006). Since E-cadherin expression often is lost in target of intense scrutiny. Activation of p53 by different prostate cancer, combination therapy may reduce types of damage causes different acetylation events. For metastatic potential of prostate cancer. example, DNA damage caused by alkylating agents HDAC inhibitors downregulate expression of the induces acetylation of K320, which activates genes with chemokine receptor CXCR4 in acute lymphoblastic high-affinity p53 binding sites, including p21,and leukemia (ALL) cells. This reduces the migration that promotes cell-cycle arrest. In contrast, DNA damage targets the ALL cells to the spleen, liver, lymph nodes caused by topoisomerase inhibition induces acetylation of and brain, all of which express high levels of the K373, which activates genes with low-affinity p53 sites, chemoattractant stromal cell derived-factor (Crazzolara such as Bax, and promotes apoptosis. These differential et al., 2002). HDAC inhibitors also increase expression acetylation patterns affect subsequent p53 phosphoryla- of the intercellular adhesion molecule ICAM1 on tion, which enhances the nuclear localization of p53. In tumor-derived endothelial cells. This enhances the addition, acetylation of K373 stabilizes the interaction of ability of lymphocytes to adhere to endothelial cells, p53 with HDAC1 and SIRT1 (Knights et al., 2006). allowing for better tumor infiltration by the lympho- SIRT1 binds to and deacetylates p53 at K382, reducing cytes. The ICAM1 promoter from tumor-derived its activator function (Luo et al., 2001; Vaziri et al., 2001). endothelial cells contains hypoacetylated histone H3 Abrogation of acetylation of the K320 homolog in mice and hypomethylated histone H3-K4. Treatment with (K317) enhances p53-mediated apoptosis after DNA HDAC inhibitors and methyltransferase inhibitors damage by inducing expression of proapoptotic genes reverses these modifications (Hellebrekers et al., 2006). (Chao et al., 2006). Tip60 and hMOF, members of the MYST family of HATs, acetylate p53 following DNA damage (Sykes et al., 2006; Tang et al., 2006). Acetylation by MYST family members occurs specifically on K120 Deacetylation of non-histone targets implicated in cancer and is required for inducing apoptosis, but is dispensable for initiating cell-cycle arrest. Intriguingly, K120R muta- Over the past decade, it has become increasingly apparent tions have been identified in human cancer. Since arginine that histones are not the only targets of acetylation and cannot be acetylated and because acetylation is required deacetylation. Indeed, a plethora of non-histone proteins for p53 to activate the proapoptotic genes Bax and are also regulated by the reversible actions of HATs and PUMA, cells carrying this mutation resist apoptosis, a HDACs (Figure 4). Acetylation and deacetylation of distinct advantage for a cancer cell. non-histone proteins have pleiotrophic effects on protein Treatment of lung cancer cells with the HDAC function, including modulation of protein–protein inter- inhibitor depsipeptide causes the specific acetylation of actions, protein stability and subcellular localization (for p53 at K373 and K382, which recruits p300 and

Oncogene HDACs and cancer MA Glozak and E Seto 5426 Ac Ac Ac Ac HAT K K K K KKKK TF Pol transcription II translation HDAC HAT HDAC

Ac Ac Altered protein-protein interaction K K Altered transcriptional activation Altered DNA binding ability Altered stability Altered subcellular localization

Figure 4 HATs and HDACs modify not only histones, but also other proteins as well. The acetylation status of histones determines whether a gene will be transcribed. After the protein is produced, it may also be the target of post-translational modiﬁcations by HATs and HDACs. The acetylation status of a protein inﬂuences a number of the protein’s functional properties. HAT, histone acetyltransferases; HDAC, histone deacetylases.

increases expression of p21 (Zhao et al., 2006). On the regulated transcriptionally by histone acetylation, in- other hand, treatment of prostate cancer cells with the dicating multiple levels where deacetylation can act. The HDAC inhibitors TSA or CG-1521 differentially functions of the Runx family of transcription factors are stabilizes the acetylation of K382 or K373, respectively. regulated by acetylation and deacetylation. Runx1, a Each of these distinct acetylation events recruit mutually critical regulator of definitive hematopoiesis may act as exclusive co-activator complexes and only acetylation of a transcriptional activator or repressor. In addition to K373 in this case is sufficient to assemble the basal its ability to inappropriately recruit HDACs to chro- transcriptional machinery on the p21 promoter (Roy matin when expressed as the Runx1-ETO fusion as and Tenniswood, 2007). Thus, treatment of different discussed above, Runx1 must be acetylated to function cancer cells with different HDAC inhibitors each can as a transcriptional activator. p300 acetylates Runx1 at have unique outcomes. K24 and K43, and mutation of these lysines impairs the Acetylation and deacetylation may also modulate transforming ability of Runx1 (Yamaguchi et al., 2004). p53’s stability. Since many of the lysines that are Another Runx family member, Runx3, is required for acetylated are also sites of ubiquitination, acetylation T-cell development and acts as a gastric tumor suppressor. can protect p53 from ubiquitination and subsequent Upon TGF-b stimulation, p300 binds to and acetylates degradation by the proteasome. The E3 ubiquitin ligase Runx3 at K148, K186 and K182. HDAC4 and HDAC5, Mdm2 recruits HDAC1 to p53, and deacetylation and to a lesser extent, HDAC1 and HDAC2 reduce this promotes ubiquitination and degradation (Ito et al., acetylation. Acetylation enhances Runx3 stability by 2002). The importance of this in vivo, however, is preventing ubiquitination by Smurf ubiquitin ligases (Jin currently a matter of debate due to recent studies et al., 2004). Paradoxically, Runx3 can act as an indicating that acetylation may not be required for oncogene, based on its overexpression in basal cell p53 stability. Replacement of the C-terminal lysines carcinomas. The effect of acetylation in this setting, with arginines in knock-in mice showed that p53 half- however, remains unknown (Salto-Tellez et al., 2006). life is normal, although p53 activation following DNA BTB/POZ repressor proteins that recruit HDACs and damage differs (Feng et al., 2005; Krummel et al., 2005). co-repressors to chromatin, including BCL6 and PLZF, Such findings underscore the importance of verifying are targets of protein acetylation. Acetylation by p300 at in vitro results with in vivo experiments as results may K376, K377 and K379 inhibits the ability of BCL6 to vary based on the experimental system utilized. bind to HDAC2, thereby inhibiting its ability to repress In addition to p53, many other transcription factors transcription. Both TSA and nicotinamide enhance are regulated by acetylation and deacetylation. Interest- BCL6 acetylation levels, indicating both class I/II and ingly, many transcription factors that are acetylated are III HDACs can deacetylate BCL6. Such treatment leads

Oncogene HDACs and cancer MA Glozak and E Seto 5427 to cell-cycle arrest and apoptosis of B-cell lymphoma activity is tightly regulated by acetylation and deacetyla- cells. In addition, mutation of BCL6 that mimics tion at multiple levels. The ramifications of these acetylation reduces the transforming ability of BCL6 modifications depend on the cell type and which lysine (Bereshchenko et al., 2002). PLZF is acetylated by p300 residue is modified. The transcription factor NF-kB at K562, K565, K647, K650 and K653 in zinc fingers six comprises a heterodimer of RelA/p65 and p50 or p52 and nine. Acetylation has no effect on the ability of subunits. In resting cells, NF-kB resides in the cyto- PLZF to recruit HDACs, but it does increase the ability plasm, associated with its repressor, IkB. Upon stimula- of PLZF to bind DNA and therefore enhances tion, IkB kinase (IKK) phosphorylates IkB, causing its transcriptional repression of growth promoting genes. ubiquitination and degradation by the proteasome. Free Mutation of acetylation sites severely impairs the ability NF-kB translocates to the nucleus, where the p65 of PLZF to suppress cell growth (Guidez et al., 2005). subunit is acetylated by p300/CBP or PCAF. Acetyla- B-cell follicular lymphomas, characterized by the tion of p65 at K218, K221 and K310 by p300 enhances t(14;18) translocation, overexpress the antiapoptotic the DNA binding and transcriptional activation ability gene Bcl-2 as a consequence of the translocation. of NF-kB and prevents its association with IkB, which Treatment of lymphoma cells with HDAC inhibitors prevents nuclear export (Chen et al., 2001, 2002). In this causes a significant decrease in Bcl-2 expression and case, deacetylation by HDAC3 promotes NF-kBand induces apoptosis. The transcription factors Sp1 and C/ IkB binding, facilitating nuclear export (Chen et al., EBPa, both of which can be acetylated, regulate Bcl-2 2001). SIRT1 also deacetylates p65 at K310, leading expression. TSA treatment increases the acetylation to decreased expression of NF-kB target genes levels of Sp1 and C/EBPa; however, it decreases their (Yeung et al., 2004). The targets activated by binding to their cognate promoters, inhibiting transcrip- NF-kB include antiapoptotic genes such as inhibitors tion. TSA treatment also decreases the interaction of of apoptosis (Yeung et al., 2004; Hoberg et al., 2006) Sp1 and C/EBPa with HDAC2 and reduces the amount and Bcl-xL (Chen et al., 2000). Therefore, repression of HDAC2 bound to the Bcl-2 promoters. Overexpres- by HDACs would be advantageous to downregulate sion of HDAC2, but not HDAC1 or HDAC3, increases these genes and promote apoptosis of cancerous cells. Bcl-2 expression. Correspondingly, overexpression of Indeed, treatment of osteosarcoma cells with topoi- HDAC2 decreases the amount of acetylated Sp1 and somerase inhibitors such as doxorubicin induces p65 to enhances DNA binding (Duan et al., 2005). In this case, associate with HDAC1–3 leading to repression of HDAC2 is required to promote hypoacetylation of antiapoptotic genes, including Bcl-xL, and correspond- transcription factors, which allows for promoter binding ingly, decreased histone H3 acetylation at the Bcl-xL and activation of an antiapoptotic gene. As such, promoter (Campbell et al., 2004). In addition, doxor- HDAC inhibitors promote lymphoma cell death. ubicin treatment of breast cancer cells inhibits the GATA factors are another family of transcription fa- p65 acetylation, reducing its affinity for binding DNA ctors modulated by protein acetylation and deacetylation. (Ho et al., 2005). Each of the hematopoietic GATA factors (GATA1–3) is HDACs are responsible for the basal repression acetylated. Acetylation of GATA1 by CBP/p300 within mediated by NF-kB p50 or p52 homodimers, which the zinc fingers is required for chromatin binding in vivo recruit SMRT/N-CoR–HDAC3 complexes. De-repression, and is critical for its ability to activate transcription of caused by the exchange of co-repressor complexes for genes required for terminal differentiation (Boyes et al., co-activator complexes, requires phosphorylation of 1998; Hung et al., 1999; Lamonica et al., 2006). SMRT. This releases HDAC3, allowing recruitment of Deacetylation by HDAC5 represses GATA1-mediated p300 to acetylate p65 at K310, thus activating transcrip- transcription, but the association of GATA1 and tion (Hoberg et al., 2006). In contrast, acetylation of p65 HDAC5 decreases during differentiation (Watamoto at K122 and K123 by p300 or CBP reduces the DNA- et al., 2003). DNA-bound, acetylated GATA1 is targeted binding affinity of NF-kB allowing binding to IkBand by MAPK-dependent phosphorylation for ubiquitination removal from the nucleus (Kiernan et al., 2003). and subsequent degradation. In this way, a cell can Upstream of NF-kB activation is the signal transdu- attenuate its response to growth factors by removing a cers and activators of transcription (STAT) family of transcription factor once its job is complete (Hernandez- signaling molecules, which play critical roles in onco- Hernandez et al., 2006). Acetylation of GATA2 is genesis. Acetylation regulates both STAT1 and STAT3. required for its DNA binding and transcription activity Dimerization of STAT3 depends on acetylation of K685 (Hayakawa et al., 2004). In addition, mutation of by CBP/p300, which enhances both DNA binding and GATA3 that prevents acetylation prolongs the survival transactivation. Deacetylation, primarily by HDAC3, of T cells and impairs T-cell homing (Yamagata et al., inhibits transcription of STAT3 target genes including 2000). Given the importance of GATA factors in growth-promoting genes such as cyclin D1 and anti- hematopoietic differentiation, the dysregulation of their apoptotic genes such as Bcl-xL (Wang et al., 2005; Yuan function by deacetylation may directly contribute to et al., 2005). In addition, activation of STAT3 by hematopoietic malignancy by maintenance of highly acetylation enhances the proteolytic cleavage of NF-kB proliferative, undifferentiated precursor cells. p100 to p52. In prostate cancer cells, increased amounts Nuclear factor-kB (NF-kB) plays an important role in of p52 allows for cell survival following chemotherapy the development of many human cancers (Basseres and (Nadiminty et al., 2006). In the above scenarios, Baldwin, 2006). The activity and duration of NF-kB upregulation of HDAC activity would be advantageous

Oncogene HDACs and cancer MA Glozak and E Seto 5428 to treat cancer. On the other hand, in other tumor types, Cellular migration is an important determinant of acetylated STAT1 preferentially binds p65. This en- cancer metastasis. HDAC6 plays a critical role in cell hances the nuclear export of p65 and prevents expres- motility as a tubulin deacetylase. In contrast to stable sion of antiapoptotic NF-kB target genes. CBP microtubules that are highly acetylated, dynamic micro- acetylates STAT1 at K410 and K413, making melanoma tubules are largely hypoacetylated. HDAC6 directly cells susceptible to apoptosis (Kramer et al., 2006). In a deacetylates a-tubulin at K40, allowing tubulin depoly- situation such as this, inhibition of HDAC activity can merization that is necessary for cell migration. Over- be advantageous in cancer treatment. This exemplifies expression of HDAC6 causes a-tubulin hypoacetylation the need for empirical testing to determine the role of and depolymerization, promoting cell movement (Hub- deacetylation in any given process. bert et al., 2002; Matsuyama et al., 2002; Zhang et al., 2003). The class III HDAC SIRT2 also deacetylates a-tubulin at K40 (North et al., 2003). HDAC6 is unique, in that it has two HDAC domains and both of which are Deacetylation of other cellular proteins with roles in required for deacetylation because mutation in the cancer catalytic core of either HDAC domain destroys HDAC activity (Zhang et al., 2006). Estrogen induces HDAC6 In addition to transcription factors, acetylation and in breast cancer cells leading to increased migration. deacetylation regulate other classes of cellular proteins Treatment with antiestrogens inhibits migration. In that have roles in cancer development and progression. addition, treatment with tubacin, an HDAC6 inhibitor, Ku70 is a multifunctional protein regulated by acetyla- inhibits the estrogen-induced migration (Saji et al., tion. Ku70 binds the proapoptotic protein Bax, 2005). HDAC6 also enhances lymphocyte migration. sequestering Bax in the cytoplasm. CBP and PCAF Interestingly, while HDAC6 protein is required, HDAC acetylate Ku70 at K539 and K542. This releases Bax, activity is not. Instead, HDAC6 may act as a scaffold to allowing it to translocate to the mitochondria and assemble the complexes required for rapid lymphocyte initiate apoptosis. Both class I/II and III HDACs movement (Cabrero et al., 2006). Thus, HDAC inhibi- deacetylate Ku70, which serves to keep Ku70–Bax tion is an important means of preventing cell migration complexes in the cytosol (Cohen et al., 2004b). As such, and consequently, metastasis. treatment with HDAC inhibitors promotes Bax-dependent apoptosis in several cell types, including neuroblastoma (Cohen et al., 2004a; Subramanian et al., 2005). Ku70 and Ku80 play a critical role in DNA repair Conclusions and future directions as the DNA-binding subunits of DNA-dependent protein kinase. Although the mechanism is unclear, Acetylation and deacetylation are important mechan- HDAC inhibitors decrease the overall levels of Ku70 isms to regulate the activity of histones and a plethora of and Ku80 in melanoma cells, which diminishes DNA other proteins. Early studies envisaged that HDACs had repair, thus enhancing the radiation sensitivity of the roles in the development and progression of human melanoma cells (Munshi et al., 2005). cancer, and recent studies overwhelmingly support these Acetylation modulates the activity of the molecular predictions. The potential use of HDAC inhibitors in chaperone HSP90. Multiple client proteins must interact the clinic to treat cancer and other disorders is exciting, with the HSP90 complex to achieve their mature but should be approached with caution. Considering the conformation and localization. Acetylation of HSP90 at pleiotropic cellular effects of acetylation and deacetyla- K294 inhibits its ability to form these complexes with tion and because new targets of acetylation and clients and co-chaperones (Scroggins et al., 2007). This deacetylation are being identified at a remarkable rate, prevents activation or enhances degradation by ubiquiti- it becomes even more critical to understand the nation of client proteins. HDAC6 binds to and deacety- ramifications of these modifications on protein function lates HSP90, allowing for maturation or stabilization of its to foresee potential off-target effects. client(s). As such, treatment with HDAC inhibitors can Acknowledgements promote the degradation of oncogenic client proteins such as BCR-ABL or Runx1-ETO (Bali et al., 2005; Kovacs We apologize to all investigators whose works were not cited et al., 2005; Yang et al., 2007). In addition, coupling in this article due to space limitations. Work in our laboratory HDAC inhibitor treatment with HSP90 antagonists may is supported by grants from the National Institutes of Health prove even more beneficial for cancer treatment. and an endowment from the Kaul Foundation.

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