Cell Cycle

HDAC inhibition has been shown to have antiproliferative effects by inducing cell-cycle arrest in G₁ via up-regulation of cyclin-dependent kinase (CDK) inhibitors or down-regulation of cyclins and CDKs (Chun 2015). HDAC1 and 2 directly bind to the promoters of the p21^WAF1/CIP1, p27^KIP1, and p57^KIP2 genes and negatively regulate their expression (Yamaguchi et al. 2010; Zupkovitz et al. 2010). Loss of HDAC1 and 2 induces expression of CDK inhibitors, leading to a cell-cycle block in G₁. Knockdown of HDAC5 leads to a significant up-regulation of p21 and down-regulation of cyclin D1 and CDK2/4/6, which results in G₁-phase cell-cycle arrest in human HCC cells (Fan et al. 2014). HDAC inhibition might block the cellular G₁/S transition by reactivating Rb function by dephosphorylation and subsequently inhibiting E2F activities in the transcription of genes for G₁ progression. Trichostatin A (TSA) suppressed retinal pigment epithelium (RPE) cell proliferation via a G₁ phase arrest, caused through inhibition of Rb phosphorylation, reduction of cyclinD1/CDK4/6 complexes, and induction of p21 and p27 (Xiao et al. 2014). However, TSA also induced G₁ phase arrest in malignant tumor cells with mutated Rb, indicating an Rb-independent G₁ arrest (Tomosugi et al. 2012).

In addition to controlling the G₁/S transition, HDAC1 knockdown in tumor cells impairs G₂/M transition and inhibits cell growth as evidenced by a reduction of mitotic cells and an increased percentage of apoptotic cells (Senese et al. 2007). HDAC10 regulates the G₂/M transition via modulation of cyclin A2 expression. The effect of HDAC10 on cyclin A2 transcription was dependent on let-7 and HMGA2 (Li et al. 2015b). Consistent with gene knockdown results, inhibition of HDACs by inhibitors including TSA, SAHA, and VPA cause cell-cycle arrest at the G₂/M boundary in a variety of tumor cell lines, supporting pleiotropic roles of HDAC throughout the cell cycle (Juengel et al. 2014).

Besides transcriptional repression of cell-cycle-related genes at the G₁/S and G₂/M cell-cycle checkpoints, HDACs might also regulate cell-cycle progression in a transcription-independent manner. During mitosis, A-kinase-anchoring proteins AKAP95 and HA95 recruit HDAC3 along with Aurora B. In this context, HDAC3 deacetylates histone H3, which in turn allows maximal phosphorylation of Ser10 by Aurora B, leading to HP1β dissociation from mitotic chromosomes. The HDAC3-AKAP95/HA95-Aurora B pathway is required for normal mitotic progression (Li et al. 2006). Indeed, the histone deacetylase inhibitor LBH589 can induce G₂-M cell-cycle arrest and apoptosis in renal cancer cells through degradation of Aurora A and B kinases by targeting HDAC3 and HDAC6 (Cha et al. 2009). Moreover, HDAC3 directly interacts with cyclin A and regulates cyclin A stability by modulating its acetylation status. An abrupt loss of HDAC3 at metaphase facilitates cyclin A acetylation by PCAF/GCN5, which targets cyclin A for degradation. Given that cyclin A is crucial for S phase progression and entry into mitosis, HDAC3 knockdown causes cell accumulation in S and G₂/M phases (Vidal-Laliena et al. 2013). Collectively, HDAC inhibition can arrest the cell cycle at either G₁/S or G₂/M phase, suggesting HDACs as therapeutic targets for abnormal cell growth and proliferation in cancer.

Apoptosis

HDACs have been shown to regulate apoptosis in a variety of cancer cells through changing expression of pro- and antiapoptotic proteins. Treatment of tumor cells with HDACi can either directly activate apoptosis through the extrinsic (death receptor)/intrinsic (mitochondria) pathway, or enhance the susceptibility of tumor cells to apoptosis. The extrinsic apoptosis pathway induced by HDACi is via diverse mechanisms including up-regulation of cell surface death receptors and/or ligands including FAS/APO1-FASL, TNF-TNF receptors and TRAIL-TRAIL receptors, reductions in the level of cytoplasmic FLICE-like inhibitory protein (c-FLIP), and enhanced recruitment of DISC formation (Zhang and Zhong 2014). Two nonselective HDACi, vorinostat and panobinostat, have been identified as modulators of FLIP expression in several preclinical cancer models (Bangert et al. 2012). Inhibition of HDAC1, 2, and/or 3, but not HDAC6, is necessary for efficient FLIP down-regulation and caspase-8 activation in NSCLC. HDACi sensitized cancer cells for TRAIL-induced apoptosis in a FLIP- and caspase-8-dependent manner (Riley et al. 2013). Depletion of HDAC2 also synergizes pancreatic cancer cells toward TRAIL-induced apoptosis with an increased expression of TRAIL receptor DR5 (TRAIL-R2) (Schuler et al. 2010).

The intrinsic cell death pathway involves the interplay of the pro- and antiapoptotic Bcl-2 superfamily of proteins, and HDAC inhibition could induce the intrinsic pathway by decreasing the expression of antiapoptotic proteins, and increasing the expression of proapoptotic proteins (Zhang and Zhong 2014). HDAC2 depletion results in regression of tumor cell growth and activation of apoptosis via p53 and Bax activation and Bcl2 suppression in human lung cancer cells (Jung et al. 2012). In gastric cancer cells, HDAC2 knockdown selectively induced the expression of proapoptotic factors Bax, AIF, and Apaf-1, but repressed the expression of antiapoptotic Bcl-2 (Kim et al. 2013). HDAC1 and HDAC8 cooperate to repress BMF (Zhang et al. 2006; Kang et al. 2014). Inhibition of HDAC8 by methylselenopyruvate (MSP), a competitive inhibitor of HDAC8, was sufficient to activate BMF transcription and promote BMF-mediated apoptosis in colon cancer cells (Kang et al. 2014). HDAC3 down-regulates PUMA expression in gastric cancer cells and HDACi, like TSA, promotes PUMA expression through enhancing the binding of p53 to the PUMA promoter (Feng et al. 2013). TSA treatment can also effectively overcome resistance to DNA-damage-induced cell death by reactivating PUMA expression in renal cell carcinoma cells (Zhou et al. 2014).

The relevance of p53 in HDACi-induced apoptosis is controversial. HDACi can activate p53, but does not necessarily require p53 for induction of anticancer action (Sonnemann et al. 2014). Most studies point to a p53-independent action of HDACi because the anticancer effect of HDACi is not influenced by the tumor’s p53 status (Ellis et al. 2009). Other studies, however, suggest an essential role of p53 in the response of tumor cells to HDACi treatment (Bajbouj et al. 2012). Using isogenic HCT-116 colon cancer cell lines with different p53 status, the antitumor effects of vorinostat, apicidin, and VPA were largely independent of p53, whereas entinostat-induced cell death partially depends on p53 (Sonnemann et al. 2014), indicating that HDACi may regulate apoptotic processes via both p53-dependent and independent pathways.

DNA-Damage Response

Numerous studies have shown that HDACs have important roles in DNA-damage repair (DDR) responses because HDACs are critical in modulating chromatin remodeling and maintaining dynamic acetylation equilibrium of DNA-damage-related proteins (Li and Zhu 2014). HDAC1 and HDAC2 are recruited to DNA-damage sites to deacetylate histones H3K56 and H4K16, and facilitate nonhomologous end-joining (NHEJ) (Miller et al. 2010), suggesting a direct role for these two enzymes during DNA replication and double-strand break (DSB) repair. HDAC3 is also associated with DNA-damage control, although it is not localized to DSB DNA-damage sites (Miller et al. 2010). Inactivation of HDAC3 causes genomic instability, and deletion of HDAC3 in the liver leads to hepatocellular carcinoma (Bhaskara et al. 2010). Besides altering histone acetylation status, class I HDACs also regulate other proteins involved in the DNA-damage response, including ATR, ATM, BRCA1, and FUS (Thurn et al. 2013). HDAC inhibition can repress DSB repair and render cancer cells more susceptible to ionizing radiation (IR) and DNA-damaging-agents-induced cell death (Koprinarova et al. 2011).

Among the class II HDACs, HDAC4, HDAC6, HDAC9 and HDAC10, have each been implicated in DNA-damage-repair processes. HDAC4 colocalizes with 53BP to nuclear foci after DSB. Depletion of HDAC4 reduces 53BP1 expression and abrogates the DNA-damage-induced G₂ checkpoint (Kao et al. 2003). HDAC9 and HDAC10 are reported to be required for homologous recombination (HR). Depletion of HDAC9 or HDAC10 inhibits HR and sensitizes cells to mitomycin C treatment (Kotian et al. 2011). The mismatch repair (MMR) system recognizes DNA mismatches that occur during DNA replication or recombination, and corrects these defects to maintain genomic integrity. MutS protein homolog 2 (MSH2), a key DNA mismatch repair protein, is regulated by class IIb HDACs. HDAC6 sequentially deacetylates and ubiquitinates MSH2, causing a cellular tolerance to DNA damage and decreased cellular DNA mismatch repair activities by down-regulation of MSH2 (Zhang et al. 2014). However, the deacetylation of MSH2 by HDAC10 might promote DNA mismatch repair activity (Radhakrishnan et al. 2015).

SIRT1 is a critical component of the DNA-damage response pathway that regulates multiple steps of DDR, including damage sensing, signal transduction, DNA repair, and apoptosis (Gorospe and de Cabo 2008). SIRT1 interacts with and deacetylates several DDR proteins, including Ku70, NBS1, APE1, XPA, PARP-1, TopBP1, and KAP1 (Luna et al. 2013; Li and Zhu 2014; Wang et al. 2014; Lin et al. 2015). SIRT1 antagonizes p53 acetylation and facilitates cancer cells survival after DNA damage (Luo et al. 2001; Vaziri et al. 2001), making SIRT1 a promising target in cancer therapy. However, SIRT1 also plays an essential role in maintaining genome integrity and stability (Wang et al. 2008; Palacios et al. 2010), so the challenge still remains to modulate SIRT1 function in such a manner that it will be beneficial for cancer therapy. Recent research demonstrated that the role of SIRT1 in response to DNA damage requires posttranslational modifications such as site-specific phosphorylation and ubiquitination. HIPK2 interacts and phosphorylates SIRT1 at Ser682 after DNA damage. Phosphorylation of SIRT1 inhibits SIRT1 deacetylase activity on p53, which in turn potentiates apoptotic p53 target gene expression and DNA-damage-induced apoptosis (Conrad et al. 2015). SIRT1 is also ubiquitinated by MDM2 during DDR, and ubiquitination of SIRT1 affects its function in cell death and survival in response to DNA damage (Peng et al. 2015).

SIRT6, also important in DNA repair, was first found to suppress genomic instability by regulating base excision DNA repair (BER) (Mostoslavsky et al. 2006). Recent studies have demonstrated that SIRT6 is involved in homologous recombination (HR) by deacetylating carboxy-terminal binding protein (CtBP) and interacting protein (CtIP) (Kaidi et al. 2010). SIRT6 is rapidly recruited to DNA-damage sites and stimulates DSB repair by mono-ADP-ribosylation of PARP1(Mao et al. 2011). SIRT6 also recruits the ISWI-chromatin remodeler SNF2H to DSBs, and deacetylates histone H3K56, preventing genomic instability through chromatin remodeling (Toiber et al. 2013). Together, results from these studies suggest that at least some of the class III sirtuins (Sir2 proteins) have an equally critical role in DNA-damage response compared to the classical HDACs.

Metastasis

Epithelial-to-mesenchymal transition (EMT) is a major process in cancer cell invasion and metastasis, and emerging studies have demonstrated the key role of HDACs in EMT regulation in a variety of cancer contexts. EMT is characterized by the loss of epithelial cell markers, namely, epithelial-cadherin (CDH1), and several transcriptional repressors of CDH1 have been identified, including Snail, Slug, Twist, ZEB1, and ZEB2. A mechanism of their action involves recruitment of HDACs to the CDH1 promoter resulting in deacetylation of H3 and H4 histones. Snail recruits HDAC1/2 and Sin3A complex to the CDH1 promoter for histone deacetylation, and the snail/HDAC1/HDAC2 repressor complex contributes to CDH1 silencing in the metastasis of pancreatic cancer (von Burstin et al. 2009). Moreover, the complex of snail/HDAC1/HDAC2 is required for EZH2-mediated CDH1 repression in nasopharyngeal carcinoma cells (Tong et al. 2012). Recruitment of HDACs to the CHD1 promoter is also regulated by ZEB1 in human pancreatic cancer cells (Aghdassi et al. 2012). Given that ZEB1 could also alter the splicing of CDH1 exon 11, a recent study revealed a dual effect of ZEB1 and its interacting class I HDACs; the decrease in CDH1 is the result of a combination of transcriptional inhibition and aberrant splicing (Liao et al. 2013). Treatment of cells with the HDACs inhibitor, LBH589 (panobinostat), induces CDH1 expression, and represses EMT and metastasis in triple-negative breast cancer (TNBC) cells. Besides preventing ZEB-mediated repression of CDH1 by inhibiting the class I HDACs corepressors, the effect of LBH589 is partially mediated by inhibition of ZEB expression (Rhodes et al. 2014). The expression of ZEB1 is also reduced in pancreatic cancer cells after mocetinostat treatment that sensitizes the undifferentiated, ZEB1-expressing cancer cells for chemotherapy (Meidhof et al. 2015). These findings indicate the therapeutic potential of inhibition of class I HDACs in targeting EMT and metastasis of cancer cells.

The role of SIRT1 in EMT regulation depends on the tumor type. In prostate cancer cells, SIRT1 induces cell migration in vitro and metastasis in vivo by cooperating with ZEB1 to suppress CDH1 transcription (Byles et al. 2012). A recent study reveals a vital role of MPP8-SIRT1 interaction in CDH1 silencing (Sun et al. 2015). The deacetylation of MPP8 at K439 by SIRT1 increases MPP8 protein stability, and MPP8 in turn facilitates SIRT1 recruitment to the CDH1 promoter for H4K16 deacetylation by regulating SIRT1-ZEB1 interaction. Thus, disruption of MPP8-SIRT1 interaction derepresses CDH1 expression and reduces cell motility and invasiveness in prostate cancer cells (Sun et al. 2015). In breast cancer, SIRT1 overexpression is associated with decreased miR-200a. miR-200a negatively regulated SIRT1 expression and reduced EMT (Eades et al. 2011). miR-204 also inhibits EMT in gastric cancer and osteosarcoma cells by directly targeting and repressing SIRT1 at the posttranscriptional level (Shi et al. 2015b). However, an opposite role of SIRT1 in cancer metastasis was indicated by the demonstration that SIRT1 reduces EMT in breast epithelial cells by deacetylating Smad4 and repressing the effect of TGF-β signaling on MMP7 (Simic et al. 2013). A similar mechanism is also found in oral squamous cell carcinoma (OSCC) cells, suggesting the key role of the SIRT1/Smad4/MMP7 pathway in EMT process (Chen et al. 2014).

Angiogenesis

Tumor growth and metastasis depend on angiogenesis. Angiogenesis is triggered by hypoxia or hypoxic microenvironment, and the cellular response to hypoxia is primarily regulated by the transcription factor hypoxia-inducible factors 1 α (HIF-1α). Many HDACs are associated with HIF-1α activity as cell treatment with HDACi causes HIF-1α degradation and functional repression. HDAC1 and HDAC4 directly deacetylate HIF-1α and block degradation of the protein (Yoo et al. 2006; Geng et al. 2011). Instead of regulating HIF-1α acetylation, HDAC5 and HDAC6 facilitate HIF-1α maturation and stabilization by deacetylating its chaperones, HSP70 and HSP90 (Kong et al. 2006; Chen et al. 2015). Inhibition of HDAC5 and 6 results in hyperacetylation of these chaperones, accumulation of the immature HIF-1α complex, and degradation of HIF-1α by the 20S proteasome. HDAC4, HDAC5, and HDAC7 increased transcriptional activity of HIF-1α by promoting its association with p300 (Kato et al. 2004; Seo et al. 2009). In contrast, SIRT1-mediated deacetylation of HIF-1α at Lys674 inhibits HIF-1α activity by blocking p300 recruitment. The suppression of SIRT1 under hypoxic conditions provides a positive feedback loop that maintains a high level of HIF-1 activity (Lim et al. 2010).

Although the antiangiogenic activity of HDAC inhibition has been demonstrated to be associated with decreased expression of proangiogenic genes, the specific effect of individual HDAC enzymes on angiogenic gene expression is controversial. KLF-4 recruits HDAC2 and HDAC3 at the VEGF promoter and represses its transcription. The up-regulation of VEGF in cancer is associated with loss of KLF-4-HDAC-mediated transcriptional repression (Ray et al. 2013). HDAC5 is another negative regulator of angiogenesis by repressing proangiogenic gene expression, such as FGF2 or Slit2, in endothelial cells (Urbich et al. 2009). Recent studies demonstrated a dual role of HDAC6 in angiogenesis. HDAC6 promotes angiogenesis by deacetylating cortactin in endothelial cells, thereby regulating endothelial cell migration and sprouting (Kaluza et al. 2011). Another study reported the antiangiogenic effect of HDAC6 in HCC, as depletion of HDAC6 facilitates angiogenesis by up-regulating the expression of HIF-1α and VEGFA (Lv et al. 2015). HDAC7 is crucial in maintaining vascular integrity and endothelial angiogenic functions, such as tube formation, migration and proliferation (Turtoi et al. 2012). HDAC9 positively regulates endothelial cell sprouting and vascular growth by the repression of the miR-17-92 cluster, which reduces the expression of proangiogenic proteins (Kaluza et al. 2013). Taken together, HDACs play important roles in angiogenesis by modulating a multitude of pro- and antiangiogenic factors, indicating that they are potential targets for antiangiogenesis in cancer therapy.

Nonselective Broad-Spectrum HDAC Inhibitors

The most extensively studied and commonly used HDACi are nonselective HDACi. For example, vorinostat (SAHA), a hydroxamate class agent, was the first histone deacetylase inhibitor approved by the Food and Drug Administration (FDA) to treat patients with cutaneous T-cell lymphoma (CTCL). Like TSA, SAHA inhibits all zinc-dependent HDACs in low nanomolar ranges, although more recent studies suggest that most hydroxamate-based HDACi have a weak effect on class IIa enzymes (Bradner et al. 2010). Preclinical studies have demonstrated that SAHA induces apoptosis and cell-cycle arrest, and reduces the proliferation and metastatic potential of tumor cells. SAHA also sensitized tumor cells to chemotherapy and/or radiotherapy (Shi et al. 2014; Xue et al. 2015). In addition to SAHA, two hydroxamate-based nonselective HDACi, belinostat (Beleodaq/PXD101) and panobinostat (LBH-589), were recently approved by the FDA to treat peripheral T-cell lymphomas (PTCL) and multiple myeloma, respectively. Both of these drugs are also under investigation in combination therapies for solid tumors.

Selective HDAC Inhibitors

To date, there are relatively few highly selective HDACi, but compounds with proposed selectivity for several class I and class II HDACs have been developed (Table 3).

This work is supported in part by National Institutes of Health (NIH) Grants R01CA169210 and R01CA187040 to E.S.