Histone Deacetylase (HDAC) Inhibitors: Current Evidence for Therapeutic Activities in Pancreatic Cancer

HDAC Classification and Mechanism of Action

The primary role of HDACs is to oppose the activity of histone acetyltransferases (HATs) balancing the level of acetylation of the histones. HDACs are enzymes that catalyze the removal of the acetyl modification on lysine residues of proteins, including the core nucleosome histones H2A, H2B, H3 and H4. We recognize four classes of these enzymes: class I includes HDAC 1, 2, 3 and 8 (nuclear localization), class II includes HDAC 4, 5, 6, 7, 9 and 10 (exonuclear and nuclear localization), class III includes sirtuins (SIRT1-7) and class IV includes only HDAC 11, which exhibits features of both classes I and II. Classes I, II and IV HDACs share homology in both structure and sequence and require a Zinc (Zn⁺) ion for their catalytic activity. Class III sirtuins share no similarities in their three-dimensional model or sequence with class I, II or IV HDACs and require a nicotinamide adenine dinucleotide (NAD⁺) ion for their catalytic activity (Table I) (10). HDACs are localized either in cytoplasm or nucleus (11).

HDACs modulate the most important cellular processes (Table II) (8). It is known that the balance of acetylation of nucleosomal histones regulates the transcription of many genes. The repression of gene transcription is induced by a condensed chromatin structure associated with hypoacetylation of histones, whereas acetylated histones are associated with a less condensed chromatin structure and activation of transcription. Apart from histones, it has been shown that other acetylated proteins are substrates for the HDACs. These include NF-YA, p53 and GATA-1. There exists increasing evidence that alterations in HAT and HDAC activity occur in cancer. HAT activity can be disrupted by translocation, over-/ under-expression or mutation in a wide spectrum of cancers, including those of an epithelial and a hematological origin.

An imbalance in histone acetylation may lead to changes in chromatin structure and transcriptional deregulation of genes involved in the control of cell cycle progression, differentiation and apoptosis. Restraining HDAC activity and preventing histone deacetylation may favor their hyperacetylation, which leads to the unfolding of previously condensely ordered chromosome regions and promotes transcription factors combined with DNA. Thus, genes, which are inhibited, can now be expressed. If these genes belong to the family of oncogenes, then, cell proliferation is promoted and a tumor may arise. Therefore, a defect in the acetylation machinery appears to result in alterations of acetylation and perhaps the development of cancer. HDAC enzymes remove the acetyl group from the histone (hypoacetylation), thereby decreasing the space between the nucleosome and the DNA wrapped around it, thus lessening transcription factor access and leading to transcriptional repression (12). HDACs have been found to relate to aberrant transcription factors. As expected, it has been recently shown that HDACs/SIRTs are regulators of growth, differentiation and cell death (apoptosis). Inhibitors of these enzymes can induce arrest of growth, differentiation and apoptosis of many cultural transformed cells (13).

Indeed, modulation of expression levels of genes encoding HDACs (over- and/or under-expression) has been reported for different types of cancers. For example, over-expression of HDAC1 has been reported in gastric and HDAC2 and 3 in colorectal cancer. Additionally, the HDAC5 gene reveals decreased transcription in colorectal cancer. Regarding class III HDACs, SIRT8 was found over-expressed in thyroid cancer, while SIRT2 gene expression is down-regulated in human gliomas (14). Furthermore, it has been observed that HDAC7 is over-expressed in pancreatic adenocarcinoma and HDAC6 is over-expressed in cutaneous T cell lymphoma (11, 15).

The catalytic domain of the HDAC is formed by a stretch of 390 aminoacids containing a set of conserved residues. The active site of the enzyme consists of a curved tubular pocket with a wider bottom. The removal of an acetyl group occurs via a charge-relay system, an important component of which is the zinc-binding site at the bottom of the pocket. The presence of a zinc ion at this site is an important factor in the mechanism of action of HDAC inhibitors (13, 16).

HDAC Inhibitors as Anticancer Agents

With the finding that HDAC inhibition is the cause of the selective differentiation of malignant cells, the field of HDACIs was deeply studied (17-19). Extensive research showed that HDACIs are chemical compounds that may block tumor cell proliferation by restoring the balance of histone acetylation, thus resulting in proper gene expression. These compounds are potent inducers of growth arrest, apoptosis and/or differentiation of transformed cells in vitro and in vivo. They can also urge cancer cells to apoptosis, whereas normal cells are relatively resistant to HDACI-induced cell death (20).

The field of HDACIs is moving into a new phase of evolution. The exponential growth in the level of research activity surrounding the HDACIs witnessed over the past decade has now started to achieve successful results in clinical practice, particularly in the field of oncology. Although the study of HDACIs is a new field, an impressive body of data describes the ability of these molecules to modulate a wide variety of cellular functions, including cell differentiation and proliferation, apoptosis, cytoskeletal modifications and angiogenesis (8, 21).

HDACIs are grouped into four main classes based on their structure (hydroxamic acid, cyclic tetrapeptide, benzamide, aliphatic acid) (22). Trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA) and sodium butyrate (NaB) are commonly studied HDACIs (23). These inhibitors induce cell growth arrest and apoptosis in a broad spectrum of transformed cells. Because of these characteristics, HDACIs are being tested in clinical trials for cancer therapy (24).

Several HDACIs are already used in clinical trials and have shown significant activity against a spectrum of both hematological and solid tumors at doses that are well-tolerated by patients. Two HDACIs, SAHA and romidepsin, have been approved by FDA for the treatment of cutaneous T-cell lymphoma (8, 23). Other HDACIs in clinical trials include LAQ-824, a hydroxamate, and a sulfonamide derivative called PXD-101. Most compounds inhibit class I and class II HDACs at nanomolar concentrations. The cyclic peptides are the most structurally complex group of HDACIs. The natural product depsipeptide (also known as FK-228) is currently being tested in phase I and II clinical trials. Other HDACIs include the aliphatic acids, such as phenylbutyrate and its derivatives valproic acid and MS-275, which are in phase I and II clinical trials (25, 26).

However, HDACIs as monotherapeutics are only effective in a defined subset of hematological tumors. There is considerable evidence showing that rational- and molecular-defined HDACI-based combination therapies are more useful for the treatment of solid cancers. Overall, the field of HDACIs is relatively new and unexplored. Early successful results, such as those with SAHA and romidepsin, have set the bar high for possible future drugs and revealed the wide potential of the activity of the HDACs' (7).

HDAC Inhibitors in Pancreatic Adenocarcinoma

Pancreatic cancer cells are highly effective in escaping cell death by up-regulation of pro-survival and inhibition of pro-death pathways. Progress in the therapy of pancreatic cancer may come from many areas, including a better understanding of survival mechanisms that allow pancreatic cancer cells to escape cell death. Cell transformation to tumor cells involves the silencing of tumor suppressor epigenetic alteration, which includes DNA hyper-/hypo-methylation of promoter CpG islands and/or changes in associated post-translational histone modifications leading to transcriptional repression (27).

HDACI-based combinations are particularly important in the treatment of PDAC. A recent phase II clinical trial disproved the effectiveness of the weak HDACI CI-994 combined with the current standard chemotherapy, which includes GEM (20). Recently, it was shown that specific depletion of HDAC2, but not HDAC1, sensitizes PDAC cells towards tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis suggesting a new therapeutic strategy (28). TRAIL is included in the tumor necrosis factor (TNF) superfamily and is responsible for the induction of apoptosis of many cancer cell lines (23).

HDAC inhibitors induce apoptosis in pancreatic adenocarcinoma cell lines irrespective of their intrinsic resistance to conventional anti-neoplastic agents. This is a result of a serine protease-dependent and caspase-independent mechanism. Initially, HDACIs increase BAX protein levels without affecting BCL-2 levels. Then, the apoptosis-induced factor (AIF) and Omi/HtrA2 are released from the mitochondria inducing programmed cell death.

Several HDACIs have been studied against pancreatic cancer cell lines. TSA can strongly inhibit the cellular growth of pancreatic cancer cell lines. The mechanism underlying this effect consists of cell-cycle arrest at the G₂ phase and apoptotic cell death. HDACI FK228 strongly inhibits the growth of five human pancreatic adenocarcinoma cell lines through cell-cycle arrest and apoptosis; this is followed by caspase-3 activation, surviving degradation and p21WAF1 cleavage. FK228 seems to be clinically useful for the treatment of refractory pancreatic cancers (9).

The ability of HDACIs to synergize with classic chemotherapeutic agents, as well as newer signal transduction pathway modulators and angiogenesis inhibitors, has become a highly appreciated quality. In contrast to the current wave of targeted-therapies, the utility of HDACIs could span multiple cancers and be used alongside a broad range of therapeutics. This is a similar function to that advanced for lead apoptosis modulators. However, the additional effects of HDACIs on cell-cycle progression and angiogenesis should make HDACIs the partner of choice in combinatorial approaches to cancer (21).

According to a recent survey, histone modification levels indicate that patients who suffered from pancreatic cancer and were treated with adjuvant chemotherapy were more or less likely to derive a significant survival benefit from adjuvant fluorouracil when compared with GEM. Although the differences were moderate requiring further validation, they increase the possibility that histone modification levels could serve as predictive biomarkers for adjuvant treatment.

In human pancreatic adenocarcinoma cell lines, TSA and SAHA induce cell death by apoptosis and are caspase-independent (29). Other investigations have shown that TSA can synergize with GEM (30) or the proteasome inhibitor PS-341 (31) to induce apoptosis of human pancreatic cancer cells. Interestingly, it has been demonstrated that Class III HDACIs, such as sirtinol and nicotinamide, may also induce pancreatic cell death. Despite that, whether these HDACIs are efficient in vivo anticancer drugs has yet to be proved (20).

Belinostat and panobinostat are in the same group of HDACIs as TSA and SAHA (hydroxamic acid HDACIs). Chien et al. show that belinostat and panobinostat may be antitumor agents for some human pancreatic cancer cells potentially able to suppress proliferation of human pancreatic cells by multiple molecular pathways (32, 33).

The anticancer actions of HDACIs are also attributed to transcriptional inhibition of certain genes that are mainly related to histone acetylation. N-myc downstream regulated gene 1 (NDRG1) has been described as a potential tumor suppressor gene in various human cancers and may be associated with tumor's grade of aggressiveness and possibility for metastasis. NDRG1 expression in pancreatic cancer is linked to cellular differentiation and patient survival. NDRG1 promoter contains a large CpG island augmenting the possibility that it could be silenced by DNA hypermethylation in cancer. Several HDACIs enhance NDRG1 expression in human colon cancer cell lines. In a mouse embryonic cell line, TSA was shown to repress the inhibition of NDRG1 by N-myc/Max, thus potentially enhancing NDRG1 transcription too. It became clear, moreover, that NDRG1 expression can be regulated by the pharmacologic inhibition of DNA methylation and histone deacetylation in pancreatic cancer cells (26).

As has also been demonstrated, under hypoxic conditions, various cell lines in vitro (malignant and primary) exhibit increased expression of HDAC1, HDAC2 and HDAC3 mRNA, as well as HDAC1, HDAC2 and HDAC3 protein levels. Furthermore, experiments showed that over-expression of HDAC1 mediated the reduction of the p53 and pVHL expression. The suppression of these two antioncogenes resulted in the over-expression of HIF-1a and VEGF, which was inversed by the use of TSA. Mahon et al. also proved that the reduction in p53 and pVHL expression resulted in reduction of factor inhibiting HIF-1a (FIH) as well, thus allowing the stimulation of angiogenesis in endothelial cells. This means that HDACs indirectly regulated HIF-1a activity under hypoxic conditions. With the use of several methods, it was shown that HDACIs could lead to increased interaction and degradation of HIF-1a by Hsp70, thus inhibiting tumor angiogenesis (34).

Numerous studies have been conducted for the identification of potential biomarkers, such as HDAC and SIRT genes, which are highly expressed in pancreatic cancer compared to normal pancreas. So far, an increased number of SIRT5 mRNA transcripts have been observed in most of the pancreatic adenocarcinoma samples. Approximately 81% of the PDAC tissue samples displayed an increased expression of HDAC7 mRNA transcripts and its corresponding protein. Although it is difficult at this stage to determine whether the up-regulation of HDAC7 in PDAC is a cause or a consequence of malignant progression, its over-expression in cancerous tissues and not in their normal counterparts is a promising field of future research for new approaches in the design of antipancreatic cancer therapy (14, 15).

Finally, of crucial importance is the fact that many clinical studies, in which HDACIs are used, have not shown any antitumor effect because of the HDAC pleiotropic activities. Peulen et al. suggest that simultaneous inhibition of cyclooxygenase-2 and HDAC may have a better result than the exclusive use of HDACIs (15). In addition, there is a high rate of combining HDACIs with other anticancer agents to improve the therapeutic result in various cancer types (35-40) or combining inhibitors of HDACs from different classes (41).