Abstract
“The characterization to date of the multiple zinc-dependent HDACs and discovery of HDACi represent encouraging progress toward a new,potentially effective, therapy for cancers and other disease.”
Histone deacetylase inhibitors (HDACi) represent promising potential as a novel strategy of targeted anti-cancer agents for therapy of hematologic and solid tumors, as well as several non-hematological diseases. During the past decade, in particular, there has been a marked increase in efforts to better understand their biological roles and therapeutic value Citation[1–4].
In humans there are 18 HDACs, classified based on their homology to yeast proteins. A total of 11 of these HDACs are zinc-dependent enzymes and are divided into three classes: class I (HDACs 1, 2, 3 and 8), class IIa (HDACs 4, 5, 7 and 9), class IIb (HDACs 6 and 10) and class IV (HDAC 11) Citation[5,6]. Class III HDACs (sirtuins 1–7) are NAD+-dependent enzymes. HDACs were thought to be primarily involved in regulation of gene expression, largely by altering the structure of the chromatin and proteins associated with DNA epigenetic regulation, which is the regulation of gene expression without changes in DNA sequence Citation[7,8]. It is now well established that the zinc-dependent HDACs have many nonhistone protein targets. Indeed, phylogenetic analysis of bacterial HDAC relatives indicates that the evolution of zinc-dependent HDACs preceded those of histones Citation[9].
There is much still to be learned about the biological role of the 11 zinc-dependent HDACs. The present evidence indicates that these HDACs are not redundant in their biological activity. Analysis of lysine acetylation by high-resolution mass spectrometry identified 3600 acetylated lysines in 1750 proteins, of which the HDACi vorinostat (suberoylanilide hydroxamic acid or SAHA) altered only approximately 10% of the acetylated sites Citation[10].
Among the many proteins that are substrates of the HDACs, are proteins that have a role in regulating gene expression and cellular pathways critical to cell proliferation, cell death and cell migration Citation[1]. HDACi, such as vorinostat, have been shown to induce cell cycle arrest, disrupt chromosome arrangement, activate chromosome degradation, inactivate heat shock protein chaperone complexes with consequent apoptotic cell death, induce factors that lead to both extrinsic and intrinsic apoptosis, alter activity of DNA repair enzymes, induce DNA double strand breaks (DSB) and alter angiogenesis Citation[6–8,11]. The pattern of HDACi induced transformed cell death appears to depend, in part, on the molecular defects present in the target cancer cells.
An important question in the HDAC field is whether isoform-specific HDACi represent potentially better therapeutic agents than the present spectrum of pan-HDACi in clinical trials. The development of HDAC isoform-specific inhibitors will provide a better understanding of the role of individual HDACs, as well as potential new opportunities for developing useful drugs.
Histone deacetylase 6, which has two catalytic sites and a ubiquitin binding site, is unique among the 11 zinc-dependent enzymes. HDAC6 has specific substrates which include α-tubulin, cortactin, transmembrane proteins, HSP90 and peroxiredoxins Citation[12–15]. HDAC6, in addition to its activity as a deacetylase, has a role in protein–protein interactions, particularly in aggresome formation in degradation pathways of misfolded proteins Citation[16]. These properties of HDAC6 demonstrate that the development of an isoform-specific HDAC6 inhibitor could have useful therapeutic applications.
An important step towards a better understanding of the role of the different HDAC isoforms has been the progress in solving the crystalline structure of the catalytic site of a histone deacetylase-like protein Citation[17]. More recently, the crystal structures of the catalytic site of HDAC4, 7 and 8 have been solved Citation[18]. The studies of the catalytic domains of HDAC4 and 7 showed a difference with the class I HDACs, suggesting a molecular basis for the relatively low enzymatic activity of these HDACs for targets such as the histones.
Normal cells are relatively resistant to HDACi-induced cell death, up to ten-times more than transformed cells. Recently, we have gained some insight into this important issue. Vorinostat induces DSB with normal and transformed cells within 30–60 min of culture with the inhibitor. Normal cells, but not transformed cells, can repair the DNA DSB without detectable loss of viability Citation[19]. The mechanism of vorinostat-induced DNA DSB is not known; however, it is associated with accumulation of acetylated histones, which may alter chromatin structure and expose DNA to various potential toxic agents, for example, reactive oxygen species. Alternatively, vorinostat causes cell cycle arrest in the G1/early S-phase and transformed cells with multiple molecular defects may not repair the breaks occurring as DNA is replicated in the early S-phase.
Differences between normal and transformed cell sensitivity to HDACi have also been described in the redox pathway Citation[20]. Vorinostat, for example, generates reactive oxygen species in some transformed cells and upregulates the expression of thioredoxin binding protein (TBP-2) in transformed but not normal cells Citation[20,21]. Inhibition of Trx by binding to TBP-2 activates ASK1, which, in turn, facilitates apoptosis of transformed cells Citation[22].
There are implications of these preclinical findings for clinical trial protocols. For example, intermittent dosing of HDACi may minimize toxic side effects. These findings also suggest that antioxidants should not be taken with HDACi.
There still remains much to be learned as to the best dosing of HDACi in the therapy of malignanices. Currently, there are some 20 or more structurally different HDACi in clinical trials, either as monotherapy or in combination therapy for many different hematologic and solid neoplasms Citation[2,5,23]. It remains to be determined which cancers are likely to be responsive to which combination therapeutic protocol. Importantly, biomarkers that may inform us of therapeutic response need to be identified. The characterization to date of the multiple zinc-dependent HDACs and discovery of HDACi represent encouraging progress toward a new, potentially effective, therapy for cancers and other disease.
Acknowledgements
The authors thank Joann Perrone and Mabel Miranda for their assistance in the preparation of this manuscript.
Financial & competing interests disclosure
These studies were supported, in part, by the National Institute of Cancer Grant P30CA08748-44, The David Koch Foundation, The CapCure Foundation, and Experimental Therapeutics at Memorial Sloan–Kettering Cancer Center. Memorial Sloan-Kettering Cancer Center and Columbia University hold patents on suberoylanilide hydroxamic acid (SAHA, vorinostat) and related compounds that were exclusively licensed in 2001 to ATON Pharma, a biotechnology start-up that was wholly acquired by Merck, Inc., in April 2004. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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