The application of histone deacetylases inhibitors in glioblastoma

Histone acetyltransferases are a diverse set of enzymes that can be divided into two groups according to their suspected cellular origin and functions: B-type HATs, in the cytoplasm, likely catalyze acetylation events linked to the transport of newly synthesized histones from the cytoplasm to the nucleus for deposition onto newly replicated DNA [18‐20]. A-type HATs, conversely in nuclear, likely catalyze transcription-related acetylation events [21]. Based on several highly conserved structural motifs of the catalytic domains, HATs contain three major families (Table 1): general control nonderepressible 5 (Gcn5)-related N-acetyltransferases (GNATs) that have members of Gcn5, PCAF, Elp3, Hat1, Hpa2, and Hpa2; MYST family comprised by primarily MOZ, Ybf2 (Sas3), Sas2 and Tip60; and p300/CBP family that consist of p300 and CBP [30]. Besides, there are other proteins like Taf1 as well as many nuclear receptor co-activators that do not contain true consensus HAT domains shown to possess intrinsic HAT activity [31].

The completely catalytic role of HAT families requires certain mechanisms. Respectively, GNAT family employs the kinetic mechanism that requires the formation of a ternary complex (enzyme • acetyl-CoA • H3 histone) before catalysis to guide acetyl transferred from acetyl-CoA to the substrate acceptor without the formation of a covalent enzyme intermediate [18, 32‐35]. MYST family has been shown to possess a ping-pong catalytic mechanism [36], in which the acetyl group from acetyl-CoA is firstly transferred to the cysteine utilizing base glutamate deprotonates the active site cysteine. Then glutamate protonates the leaving cofactor and deprotonates the substrate lysine so that the cysteine can transfer the acetyl group to the lysine [22]. And p300/CBP family, as a represent of ‘orphan class’ of HAT enzymes that do not bind directly to DNA but are recruited to particular promoters through interactions with DNA-bound transcription factors [31], appears to hold a Theorell-Chance mechanism, in which the peptide substrate associates only very transiently with the enzyme with no need for a stable ternary complex, thus leaving as soon as the reaction is complete.

Because of HATs’ catalytic role in histone proteins and even nonhistone proteins, they may be important for normal cell proliferation, growth, and differentiation. Therefore loss or misregulation of these activities may lead to cancer. And several lines of evidence have indicated that HAT is tied to tumor suppression [37].

The discovery of the first HDAC almost accompanied by the first HAT [38]. Afterward, as more HDACs were found, existing 18 genes were subdivided into two types by their dependency on Zn²⁺ and four classes by homology to yeast, resulting in Zinc-dependent enzymes including class I, II and IV and Zinc-independent enzymes composed of class III HDACs (Table 2) [39, 40]. The class I HDAC family consist of HDAC1, 2, 3 and 8, which has similar homology to Rpd-3 yeast transcription factor and generally stay in the nucleus; The class II that shares homology with the yeast Hda1 protein and shuttles between the nucleus and the cytoplasm incorporates class IIa (HDAC4, 5, 7 and 9) and class IIb (HDAC6 and 10); The single HDAC11 belongs to class IV found in the nucleus that has mixed homology between Rpd-3 and Hda1 [41, 42]; And the last class III homologues of the yeast protein Sir2 is comprised of Sirt1-7 and requires NAD⁺ for their activities [43]. Among them, the first three-classes are recognized as ‘classical HDACs’ and common targets for therapy [44].

Stable and closed nucleosomes and chromosomal structures generally impede the access to DNA. But researches, over the past few decades, have revealed that covalent modifications of histone proteins and DNA, such as acetylation, methylation, phosphorylation and citrullination of the histone core, can fundamentally alter the organization and function of chromatin, thus regulating all DNA-based processes, like transcription [45]. Among these covalent modifications, histone acetylation is a major source of dynamic variation in chromatin structure in vivo. Multiple mechanisms of action are involved in the acetylation-dependent disruption of nucleosome array condensation. Two basic connections in chromatin compaction: histone-histone and histone-DNA interactions, are essential to stabilize the condensed chromatin. The octamer of nucleosome core is assembled by a histone (H3-H4)₂ tetramer and two H2A-H2B dimers, around which 146 bp of DNA wraps [5]. This process requires histone-histone interactions and incorporates two steps: the first step leads to the formation of the H2A-H2B dimer and the (H3-H4)₂ tetramer; the second step of assembly is between two H2A-H2B dimers and one (H3-H4)₂ tetramer [46]. Besides, histone-DNA interactions that block hydrophobic histone or charged histone DNA interfaces exist in a side of double-helix DNA facing the core histone octamer and chaperones recognizing specific histone sites [47]. All of these interactions are necessary to assemble into higher-order chromatin structures, which constrain the regulation of DNA.

The packaging of DNA within the tightly folded chromosomes burns a major barrier to transcription. Thus it is important for transcriptional machinery to change the stability and positioning of chromatin structures. Acetylation of the core histone N-termini, as one of the most studied and appreciated modifications, is widely considered to be correlated to the regulation of transcription. Many experiments show that histone acetylation and deacetylation mainly affects gene expression in the following ways (Figure 1).

Firstly, histone acetylation and deacetylation may function by changing the surrounding charge environment of the nucleosome, which will then strengthen or weaken the interactions between proteins related to gene expression and DNA, and in turn lead to altered chromatin architecture [48]. The histone N-terminal tail extending from the nucleosome core is generally rich in hydrophobic amino acids (e.g., lysine, arginine, serine, etc.), among which positively charged lysine residues is most likely to be acetylated by HAT. Then acetylated histone tail through the addition of an acyl group from an acyl-CoA to lysine residues with the positive charge by HATs will be neutralized, thus resulting in a decrease in binding affinity to the DNA backbone and a negatively charged neighboring nucleosome, which subsequently leads to a possibly decreased nucleosome stability, loose chromatin structure and more accessible underlying DNA [49]. On the contrary, HDACs remove the acetyl groups from histones, thereby getting back to the compacted chromatin and transcriptional repression state.

The second way to regulate gene expression by histone acetylation derives from the ‘histone code’ theory that has been recognized by most researches and incorporates ‘writers’, ‘readers’ and ‘erasers’ [4, 50]. It is just like a variety of different passwords when histone tails form a large number of special signals as a result of acetylation and deacetylation on different sites of it. Multiple HATs, as ‘writers’, transfer acetyl groups from acyl-CoA to histone N-terminal tails, producing the code identified by ‘readers’ that almost all contain the bromodomain [51]. Bromodomain, a structurally conserved module present in transcription-associated proteins (or rather more exact, histone chaperones), can recognize and bind to acetylated histones specifically and then changes the remodeling of nucleosomes, subsequently regulating transcription programs [51]. And these ‘readers’, including Spt6, the FACT (facilitates chromatin transcription) complex, anti-silencing function 1 (Asf1) and the chromatin assembly factor 1 (Caf1) complex, generally act by invading the nucleosome gradually and/or interacting with DNA or other proteins to control gene expression indirectly [25]. Once ‘readers’ bind to particular sites of acetylated lysines, the requisite transcription factors would recruit key regulatory DNA sequences (e.g., promoters and enhancers), leading to initiation of transcription. On the contrary, those enzymes that remove acetylated histone marks are termed as ‘erasers’, which prevent contacts between those transcription-associated proteins and histones or DNA [52].

Thirdly, there is one kind of protein called ATP-driven nucleosome remodelers, including the SWI/SNF complex and Chd1 or the RSC complex, expected to exert great effects on chromatin structure and mediate transcription factor binding [47]. These proteins use the energy of ATP hydrolysis to rearrange nucleosomal arrays and introduce superhelical torsion into nucleosomal DNA, which causes nucleosome sliding as well as topological and structural changes of chromatin, ultimately yielding a remodeled nucleosome with accessible DNA sites [52, 53]. Up to now, researches have dictated that these complexes, such as SWI/SNF complex, allow recruitment and DNA binding of TFIID to the TATA box, which is responsible for the initiation of transcription involving the crucial steps of sliding of nucleosome to a new position, changes in histone-DNA interactions and an increase in the association with transcription factors [54]. Additionally, another significant function involved in the transcriptional mechanism mediated by ATP-dependent complexes is that histone acetylation creates recognition sites for bromodomains present in these complexes [4]. In contrast, histone deacetylases associate with corepressor complexes to direct gene-specific transcriptional repression. Hence, the state of acetylation on core histone is critical for the recruitment of transcriptional coactivators by ATP-driven nucleosome, which forms another type of ‘histone code’ similar to chaperones binding to acetylated histone surface. Consequently, it means that chromatin structure and function differ depending on the composition of the histone variants [47].

In addition to these above mechanisms, many ways participate in and assist in transcription regulation by acetylation modification. So it is not just a sample static image of gene regulation, but rather a more dynamic and complex framework for the effects of acetylation on transcription in reality. What’s more, not only histone proteins are targeted by HATs and HDACs, but also are non-histone substrates including transcription factors like E2F,p53 and GATA1 activated by HATs [18, 55], and c-Myc, nuclear factor B (NF-B), Stat3, transcription factor IIE (TFIIE), the retinoblastoma protein, hypoxia-inducible factor 1 (HIF-1), as well as estrogen receptor and the androgen receptor complexes repressed by HDACs [10, 56‐63]. The activities of these transcription factors can be a major determinant of gene activation or inactivation. Moreover, a wealth of studies uncovered the fact that other post-modifications including methylation, phosphorylation and ubiquitylation may have a tremendous impact on the functional activity of acetylation [11]. Once more than one of them acts on the same site on histones, a cross-talk will occur via multiple mechanisms either competitive or cooperative [64].

HDACs, which have various targets of histone and non-histone proteins, are in the focus of cancer researches due to their pleiotropic effects on genome functions including chromatin assembly, DNA repair, replication and recombination [47, 69, 70], as well as on many biological processes, such as cell proliferation, differentiation, apoptosis, and senescence [71]. Numerous evidence have demonstrated the overexpression of HDACs in diverse types of malignancies such as lung cancer [72], breast cancer [73], and hepatocellular carcinoma [74, 75]. Herein we recapitulate several excellent HDAC carcinogenic ways. For example, high level of HDAC1 has been shown to target the oncosuppressor p53 that mediates cell apoptosis [76], to prevent cells from differentiating thus maintaining an undifferentiated phenotype [77], and to enhance cell proliferation through targeting a subset of cyclin dependent kinase (CDK) inhibitors ( p21 and p27) [65]. Tip60, a DNA damage-response proteins essential for double-strand break (DSB) repair and apoptosis, might be lack of its activities when it is negatively regulated by SIRT1 and HDAC3 deacetylase activity [78‐80], causing accumulation of cell damage and inactivation of the apoptotic program and finally triggering tumorigenesis [81]. The molecular chaperone heat shock protein 90 (Hsp90) is the target of HDAC6, and the involvement of HDAC6 in the Hsp-mediated regulation of VEGFR can result in an increased fibroblast cell migration [82, 83]. Also, HDAC7 overexpression might induce an increased expression of PDGF-B, which lead to angiogenesis and consequently tumor progression [84]. In addition, growing evidence supports the relation between HDACs and immune system function. Researches suggested that HDACs have a role in not only innate immunity, but also adaptive immune systems [85]. For instance, class I HDACs seem to negatively regulate innate immunity through repressing the production of an inflammatory cytokines such as COX2 [86], NF-κB [87], and IFN-β [88], and also crucially influence adaptive immunity. There is a vast array of other roles in the pathogenesis of cancer as well as other complex biological functions by HDACs. A case in point is the repression of tumour suppressor genes like p21 and the upregulation of oncogenes such as BCL2 by HDAC-mediated deacetylation [89]. Also, many studies over the years indicated that overexpression of individual HDACs correlated with poor cancer patient prognosis independent of other variables such as tumor type and disease progression. For example, elevated HDAC2 levels may be of high relevance to worse prognosis in patients with colorectal cancer [90]. Nevertheless, HDAC overexpression is not always a negative prognostic marker. Indeed, downregulated expression of HDAC10 in non-small lung carcinoma cells is reported to be related to poor prognosis in lung cancer patients [91].

Based on the above introduction, HDACs-mediated deacetylation is responsible for altering a large number of genes implicated in tumorigenesis. For an optimal transcription of these genes, proteins should be in an appropriate acetylated state. Hence, inhibition of histone deacetylases as a therapeutic tool in cancer emerged as a novel class of targeted drugs, which exert an antitumor effect in vitro and in vivo including the induction of the growth arrest, differentiation and apoptosis, and the inhibition of angiogenesis, DNA repair and immunomodulatory activities [92]. For instance, shreds of evidence indicate that suberic bishydroxamate (SBHA), a HDAC inhibitor, induces apoptosis by changing the balance between proapoptotic and antiapoptotic proteins in melanoma cells, which means the overexpression of proapoptotic proteins of Bcl-2 family, such as Bim, Bmf, Bax, Bak and Bik, and the repression of anti-apoptotic proteins of Bcl-2 family, such as Bcl-2, Bcl-XL, Bcl-w and Mcl-1 [93]. Also there are some other ways to induce tumour cell death, like autophagy and death receptor pathways. One of the classic HDACis, suberoylanilide hydroxamic acid (SAHA), downregulates AKT-MTOR signaling (a major suppressive cascade of autophagy) that triggers glioblastoma cell death [94]. In addition, HDACis can downregulate the expression of Nanog, a pluripotency regulator that has been shown to promote cancer progression by regulating CSCs (cancer stem cells) [95]. Table 3 provides examples grouped by pathways of alterations of those genes that play a major role in cancer (see details in Table 3).

There are limitations in the efficacy of single HDACis for GBM despite the tolerated toxicities that attribute to the poor pharmacokinetic properties and multiple misregulated growth and survival pathways in GBM. Thus it is rational to believe that the combinational treatment modality may represent an attractive approach to enhance the standard of care in patients with GBM. Many HDAC inhibitors are proved to act synergistically with other chemotherapy drugs, have radiosensitizing effects and enhance immunotherapies. Next, we will give an elaborate list of preclinical and clinical combination studies of HDACis for GBM in Tables 6 and 7 respectively.