Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid Research Articles

In vivo redox state of Human thioredoxin and redox shift by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)

The cytosolic thioredoxin (Trx1) system is essential for maintaining a reduced intracellular environment, via reduced Trx1 acting as a general protein disulfide reductase. Trx1 is implicated in cell signaling such as proliferation, DNA synthesis, enzyme activation, cell cycle regulation, transcription, gene activation, and prevention of apoptosis. Human Trx1 contains the active-site cysteines, Cys32 and Cys35, and three additional structural cysteines, Cys62, Cys69, and Cys73, that regulate Trx1 structure and activity via a second disulfide formation, S-glutathionylation or S-nitrosylation.The present study uses an electrophoretic redox Western blot method to analyze the oxidation state of Trx1 in vivo separating the protein-changed isoform following alkylation with iodoacetic acid in 8M urea. Treatment with the histone deacetylase inhibitor SAHA increased Trx1 inhibitor thioredoxin interacting protein (Txnip) levels, decreased Trx1 activity, and switched the Trx1 oxidation state toward a more oxidized one, as a result of complex formation with Trx1, and increased reactive oxygen species (ROS). SAHA is currently in clinical trials for cancer treatment, and one possible mechanism for its anticancer effect is via effects on the Trx1 system. Determining the exact oxidation state of human cytosolic Trx1 may be useful in developing and evaluating cancer drugs and antioxidant agents.

Combinatorial Treatment of DNA and Chromatin-Modifying Drugs Cause Cell Death in Human and Canine Osteosarcoma Cell Lines

Downregulation of microRNAs (miRNAs) at the 14q32 locus stabilizes the expression of cMYC, thus significantly contributing to osteosarcoma (OS) pathobiology. Here, we show that downregulation of 14q32 miRNAs is epigenetically regulated. The predicted promoter regions of miRNA clusters at 14q32 locus showed no recurrent patterns of differential methylation, but Saos2 cells showed elevated histone deacetylase (HDAC) activity. Treatment with 4-phenylbutyrate increased acetylation of histones associated with 14q32 miRNAs, but interestingly, robust restoration of 14q32 miRNA expression, attenuation of cMYC expression, and induction of apoptosis required concomitant treatment with 5-Azacytidine, an inhibitor of DNA methylation. These events were associated with genome-wide gene expression changes including induction of pro-apoptotic genes and downregulation of cell cycle genes. Comparable effects were achieved in human and canine OS cells using the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat) and the DNA methylation inhibitor Zebularine (Zeb), with significantly more pronounced cytotoxicity in cells whose molecular phenotypes were indicative of aggressive biological behavior. These results suggested that the combination of these chromatin-modifying drugs may be a useful adjuvant in the treatment of rapidly progressive OS.

Selective Targeting of the Repressive Transcription Factors YY1 and cMyc to Disrupt Quiescent Human Immunodeficiency Viruses

Quiescent HIV-1 infection of resting CD4(+) T cells is an obstacle to eradication of HIV-1 infection. These reservoirs are maintained, in part, by repressive complexes that bind to the HIV-1 long terminal repeat (LTR) and recruit histone deacetylases (HDACs). cMyc and YY1 are two transcription factors that are recruited as part of well-described, distinct complexes to the HIV-1 LTR and in turn recruit HDACs. In prior studies, depletion of single factors that recruit HDAC1 in various cell lines was sufficient to upregulate LTR activity. We used short hairpin RNAs (shRNAs) to test the effect of targeted disruption of a single transcription factor on quiescent proviruses in T cell lines. In this study, we found that depletion of YY1 significantly increases mRNA and protein expression from the HIV-1 promoter in some contexts, but does not affect HDAC1, HDAC2, HDAC3, or acetylated histone 3 occupancy of the HIV-1 LTR. Conversely, depletion of cMyc or cMyc and YY1 does not significantly alter the level of transcription from the LTR or affect recruitment of HDACs to the HIV-1 LTR. Furthermore, global inhibition of HDACs with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) enhanced the increase in LTR transcription in cells that were depleted of YY1.These findings show that despite prior isolated findings, redundancy in repressors of HIV-1 LTR expression will require selective targeting of multiple restrictive mechanisms to comprehensively induce the escape of quiescent proviruses from latency.

Abstract 158: Regulation of miR-21 Expression by Acetylation in Myocardial Infarction

Cardiovascular diseases are one of the leading causes of morbidity and mortality in the world, underlining the need for innovative therapies and diagnosis. Recent reports have identified microRNAs (miRNAs) as central players in regulating gene expression and showed that several miRNAs are aberrantly expressed in cardiac arrhythmia, hypertrophy, fibrosis, ischemia, vascular atherosclerosis and heart failure. Gene expression is also regulated at the transcriptional level by histone deacetylases (HDACs) under basal and pathological conditions. We previously demonstrated that 1) class I and class II HDACs play an important role in the basal expression and upregulation of the sodium-calcium exchanger (Ncx1) gene in adult cardiomyocytes and pressure-overloaded ventricle and 2) treatment with class I/IIb HDAC inhibitors trichostatin (TSA) or suberoylanilide hydroxamic acid (SAHA) improved ventricular function by suppressing matrix metalloproteinases (MMPs) gene expression in myocardial infarction (MI).Therefore it is possible that protein acetylation regulates the expression of some miRNAs and we hypothesize that HDAC inhibition would attenuate the shift in expression of certain miRNAs that are aberrantly expressed post-MI. In a pilot study, ligation of the left anterior descending (LAD) coronary artery was performed to induce MI with and without SAHA treatment. Because of its misregulation and relevance in cardiac hypertrophy and MI, we examined the expression level of miR-21. qRT-PCR confirmed that miR-21 is increased by 8 fold 7 days post-MI. Interestingly, SAHA treatment significantly attenuated the abnormal expression of miR-21. To our knowledge, it is the first report of the regulation of a miRNA by HDACs in the heart. We anticipate that not only miR-21 but other miRNAs will fall under the same mechanistic control via acetylation. The miR-21 promoter contains binding sites for several transcription factors that get acetylated and we speculate that one or more HDAC mediate the expression of miR-21 by controlling the acetylation state of transcription factors interacting with the miR-21 promoter.

Selenium-containing histone deacetylase inhibitors for melanoma management

Melanoma incidence and mortality rates continue to increase each year. Lack of clinically viable agents, drug combinations, effective targeted delivery approaches and success inhibiting targets in tumor tissue have made this disease one of the most difficult to treat, which makes prevention an important option for decreasing disease incidence and mortality rates. Inhibiting histone deacetylases (HDAC) is an approach currently being explored to more effectively treat melanoma but use for prevention has not been explored. In this study, novel selenium containing derivatives of the FDA approved HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) called 5-phenylcarbamoylpentyl selenocyanide (PCP-SeCN) and Bis{5-phenylcarbamoylpentyl} diselenide (B(PCP)-2Se) were created and efficacy tested for preventing early melanocytic lesion development in skin. Topical application of PCP-SeCN and B(PCP)-2Se inhibited melanocytic lesion development in laboratory-generated skin by up to 87% with negligible toxicological effect. Mechanistically, PCP-SeCN and B(PCP)-2Se inhibited HDAC activity and had new inhibitory properties by moderating Akt activity to induce cellular apoptosis as demonstrated by an increase in the sub-G0-G1 cell population, and cleaved caspase-3 as well as PARP levels. Furthermore, PCP-SeCN and B(PCP)-2Se inhibited cell proliferation by inhibiting cyclin D1 expression and increasing p21 levels. Thus, PCP-SeCN and B(PCP)-2Se are potential melanoma chemopreventive agents with enhanced efficacy compared with SAHA due to new PI3 kinase pathway inhibitory properties.

Multikinase inhibitor Sorafenib and HDAC inhibitor suberoylanilide hydroxamic acid suppress confluent resistance of cancer cells to recombinant protein izTRAIL

Suppression of human tumor cell resistance to TRAIL-induced apoptosis in confluent cultures, using molecular target drugs (sorafenib and SAHA) at non-toxic concentrations was studied. Sorafenib, a multikinase inhibitor, and SAHA, an inhibitor of histone deacetylase, effectively suppressed resistance of confluent human cells derived from the skin carcinoma (A431 cell line) and fibrosarcoma (HT-1080 cell line). The effectiveness of suppression of confluent resistance with these inhibitors for human carcinoma A431 cells was significantly higher than that for the human ovarian carcinoma OVCAR-3 cells. For all cell lines studied, suppression of confluent resistance with SAHA was more effective than when sorafenib was used. The possible reason for increasing tumor cell resistance in confluent cultures and the importance of this phenomenon for understanding drug resistance of cells in the tumor tissue are discussed.

Epigenetic modifiers enhance the synergistic cytotoxicity of combined nucleoside analog-DNA alkylating agents in lymphoma cell lines

Hematopoietic stem cell transplantation is used for treatment of lymphoma. In an attempt to design an efficacious and safe prehematopoietic stem cell transplantation conditioning regimen, we investigated the cytotoxicity of the combination of busulfan (B), melphalan (M), and gemcitabine (G) in lymphoma cell lines in the absence or presence of drugs that induce epigenetic changes. Cells were exposed to drugs individually or in combination and analyzed by the MTT proliferation assay, flow cytometry, and Western blotting. We used ~IC(10) drug concentrations (57 μM B, 1 μM M and 0.02 μM G), which individually did not have major effects on cell proliferation. Their combination resulted in 50% inhibition of proliferation. Reduction to almost half concentration (20 μM B, 0.7 μM M and 0.01 μM G) did not have significant effects, but addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (0.6 μM) to this combination resulted in a marked (~65%) growth inhibition. The cytotoxicity of these combinations correlates with the activation of the ataxia telangiectasia mutated-CHK2 pathway, phosphorylation of KRAB-associated protein-1, epigenetic changes such as methylation and acetylation of histone 3, and activation of apoptosis. The relevance of epigenetic changes is further shown by the induction of DNA methyltransferases in tumor cells with low constitutive levels of DNMT3A and DNMT3B. The addition of 5-aza-2'-deoxycytidine to (BMG+suberoylanilide hydroxamic acid) further enhances cell killing. Overall, BMG combinations are synergistically cytotoxic to lymphoma cells. Epigenetic changes induced by suberoylanilide hydroxamic acid and 5-aza-2'-deoxycytidine further enhance the cytotoxicity. This study provides a rationale for an ongoing clinical trial in our institution using (BMG+suberoylanilide hydroxamic acid) as pre-hematopoietic stem cell transplantation conditioning for lymphoma.

Synergistic inhibition of hepatocellular carcinoma growth by cotargeting chromatin modifying enzymes and poly (ADP-ribose) polymerases

Hepatocellular carcinoma (HCC) is a particularly lethal form of cancer, yet effective therapeutic options for advanced HCC are limited. The poly(ADP-ribose) polymerases (PARPs) and histone deacetylases (HDACs) are emerging to be among the most promising targets in cancer therapy, and sensitivity to PARP inhibition depends on homologous recombination (HR) deficiency and inhibition of HDAC activity blocks the HR pathway. Here, we tested the hypothesis that cotargeting both enzymatic activities could synergistically inhibit HCC growth and defined the molecular determinants of sensitivity to both enzyme inhibitors. We discovered that HCC cells have differential sensitivity to the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and PARP inhibitor olaparib, and identified one pair of cell lines, termed SNU-398 and SNU-449, with sensitive versus resistant phenotype to both enzyme inhibitors, respectively. Coadministration of SAHA and olaparib synergistically inhibited the growth of SNU-398 but not SNU-449 cells, which was associated with increased apoptosis and accumulated unrepaired DNA damage. Multiple lines of evidence demonstrate that the hepatic fibrosis/hepatic stellate cell activation may be an important genetic determinant of cellular sensitivity to both enzymatic inhibitors, and coordinate activation or inactivation of the aryl hydrocarbon receptor (AhR) and cyclic adenosine monophosphate (cAMP)-mediated signaling pathways are involved in cell response to SAHA and olaparib treatment. These findings suggest that combination therapy with both enzyme inhibitors may be a strategy for therapy of sensitive HCC cells, and identification of these novel molecular determinants may eventually guide the optimal use of PARP and HDAC inhibitors in the clinic.

MYC directs transcription of MCL1 and eIF4E genes to control sensitivity of gastric cancer cells toward HDAC inhibitors

Histone deacetylases (HDACs) control fundamental physiological processes such as proliferation and differentiation. HDAC inhibitors (HDACi) induce cell cycle arrest and apoptosis of tumor cells. Therefore, they represent promising cancer therapeutics that appear particularly useful in combination therapies. Although HDACi are tested in current clinical trials, the molecular mechanisms modulating the cellular responses toward HDACi are incompletely understood. To gain insight into pathways that limit HDACi efficacy in gastric cancer, we treated a panel of gastric cancer cells with the clinically relevant HDACi suberoylanilide hydroxamic acid (SAHA). We report that higher expression levels of the anti-apoptotic BCL2 family members MCL1 and BCLXL were detectable in cells with high inhibitory concentration 50 (IC50) values for SAHA. Using RNAi, we show that MCL1 and BCLXL lower the efficacy of SAHA. To find strategies to interfere with MCL1 and BCLXL expression, we investigated molecular regulation of both proteins. We show that specific siRNAs against c-MYC as well as pharmacological inhibition of this cancer-relevant transcription factor reduced MCL1 and BCLXL expression. Subsequently, we observed an increase in SAHA efficacy. Our data furthermore demonstrate that two different molecular mechanisms are responsible for the modulation of these factors. Whereas c-MYC controls transcription of MCL1 directly, regulation of BCLXL was due to c-MYC’s capability to regulate the eIF4E gene, which encodes a rate-limiting factor of eukaryotic translation. Our data reveal a new molecular mechanism for how c-MYC controls cell autonomous apoptosis and provide a rationale for a concerted inhibition of HDACs and c-MYC in gastric cancer.

Downregulation of Hypoxia-related Responses by Novel Antitumor Histone Deacetylase Inhibitors in MDAMB231 Breast Cancer Cells

The tumor microenvironment is characterized by a poor circulation which results in the selection of neoplastic cells that can grow or survive under hypoxic conditions. The relationship between hypoxia and histone deacetylase (HDAC) inhibitors has been previously established. In this work we evaluated the effects of novel HDAC inhibitors (the natural peptide FR235222 and three tetrapeptide analogs) in the human breast cancer cell line MDAMB231, cultured under hypoxia (2% O2 ≉ 14 mmHg) or normoxia (20% O2 ≉ 140 mmHg). First, we found that the novel HDAC inhibitors reduced cell proliferation in MDAMB231 cells at an extent which was similar or even higher than that exerted by the classic HDAC inhibitors trichostatin-A and suberoylanilide hydroxamic acid. More interestingly, the antiproliferative effects of the novel HDAC inhibitors were, in general, significantly higher in hypoxic cells than in normoxic controls. Hypoxic MDAMB231 cells expressed high levels of the hypoxia-inducible factor (HIF)-1α and HIF-1α-related genes, such as vascular endothelial growth factor, Bcl-2/E1B 19 kDa interacting protein-3, glucose transporter-1, carbonic anhydrase IX, as determined by Western blot analysis and qRT-PCR. Finally, we found that HIF-1α and HIF-1α-related genes were significantly downregulated by FR235222 and analogs. In conclusion, the identification of novel effects exerted by the HDAC inhibitors, characterized by a strong efficacy in inhibiting the expression of HIF-1α and its related genes, may have important implications in the pharmacological control of several tumors, including breast cancer, characterized by the presence of hypoxia, angiogenesis and metabolic derangements.

Enhancing the anticancer effect of the histone deacetylase inhibitor by activating transglutaminase

Histone deacetylase (HDAC) inhibitors have shown promising anticancer effects in clinical trials. However, a proportion of patients do not respond to HDAC inhibitor therapy. We have previously demonstrated that tissue transglutaminase (TG2) is one of the genes commonly up-regulated by HDAC inhibitors in vitro and in vivo, and that two structurally distinct TG2 protein isoforms, the full-length (TG2-L) and the short form (TG2-S), exert opposing effects on cell differentiation due to difference in transamidation activity. Here we show that the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) transcriptionally activates the expression of both TG2-L and TG2-S, and that up-regulation of TG2-L renders neuroblastoma cells less sensitive to SAHA-induced cytotoxicity. Combination therapy with SAHA and the transamidation activator Naringenin, a natural product found in citrus fruits, synergistically enhanced transamidation activity and SAHA-induced cytotoxicity in neuroblastoma cells, but not in normal non-malignant cells. In tumour-bearing N-Myc transgenic mice, SAHA and Naringenin synergistically suppressed tumour progression. Taken together, our data demonstrate that SAHA-induced TG2-L over-expression renders cancer cells less sensitive to SAHA therapy, and suggest the addition of Naringenin to SAHA and probably also other HDAC inhibitors in future clinical trials in cancer patients.

Transcription factor CCAAT/enhancer-binding protein alpha and critical circadian clock downstream target gene PER2 are highly deregulated in diffuse large B-cell lymphoma

Disturbances of circadian rhythms and mammalian clock genes have been implicated in the etiologies of many chronic illnesses, including cancer. We show that transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha)-regulated PER2 activation is a potential tumor suppressor pathway in diffuse large B-cell lymphoma (DLBCL), one of the commonest types of mature B-cell lymphoma. Expression analysis of human B-cell lymphoma samples including DLBCL (n = 50), mantle cell (n = 21), follicular (n = 25) and Burkitt (n = 18) lymphoma revealed markedly down-regulated CEBPA and PER2 mRNA levels exclusively in DLBCL samples compared to control lymphatic tissue. We demonstrated direct regulation of the circadian core clock gene PER2 by C/EBPalpha in the pro-B cell line Ba/F3, and forced expression of PER2 resulted in decreased proliferation, G0/G1 cell cycle arrest and increased rates of apoptosis. Interestingly, treatment of human DLBCL cell lines with the histone deacetylase-inhibitor suberoylanilide hydroxamic acid (SAHA) significantly increased the expression of C/EBPalpha and Per2, accompanied by cell growth inhibition; in contrast, siRNA knockdown of CEBPA reduced the anti-proliferative effect of SAHA treatment. Our results show for the first time that C/EBPalpha with its associated direct core clock gene target, PER2, are highly deregulated in DLBCL, suggesting an important tumor suppressive pathway in the pathogenesis of this lymphoma entity.

Targeting developmental regulators of zebrafish exocrine pancreas as a therapeutic approach in human pancreatic cancer

SummaryHistone deacetylases (HDACs) and RNA polymerase III (POLR3) play vital roles in fundamental cellular processes, and deregulation of these enzymes has been implicated in malignant transformation. Hdacs and Polr3 are required for exocrine pancreatic epithelial proliferation during morphogenesis in zebrafish. We aim to test the hypothesis that Hdacs and Polr3 cooperatively control exocrine pancreatic growth, and combined inhibition of HDACs and POLR3 produces enhanced growth suppression in pancreatic cancer. In zebrafish larvae, combination of a Hdac inhibitor (Trichostatin A) and an inhibitor of Polr3 (ML-60218) synergistically prohibited the expansion of exocrine pancreas. In human pancreatic adenocarcinoma cells, combination of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and ML-60218 produced augmented suppression of colony formation and proliferation, and induction of cell cycle arrest and apoptotic cell death. The enhanced cytotoxicity was associated with supra-additive upregulation of the pro-apoptotic regulator BAX and the cyclin-dependent kinase inhibitor p21CDKN1A. tRNAs have been shown to have pro-proliferative and anti-apoptotic roles, and SAHA-stimulated expression of tRNAs was reversed by ML-60218. These findings demonstrate that chemically targeting developmental regulators of exocrine pancreas can be translated into an approach with potential impact on therapeutic response in pancreatic cancer, and suggest that counteracting the pro-malignant side effect of HDAC inhibitors can enhance their anti-tumor activity.

The therapeutic effect of histone deacetylase inhibitor PCI-24781 on gallbladder carcinoma in BK5.erbB2 mice

Gallbladder carcinoma (GBCa), a type of biliary tract cancer (BTC), has proven challenging to treat, demonstrating the need for more effective therapeutic strategies. In our current study, we examined the therapeutic effects of the histone deacetylase (HDAC) inhibitor PCI-24781 against GBCa that developed in BK5.erbB2 mice. PCI-24781 [50 mg/kg/day] and control solutions were administered to BK5.erbB2 mice for 4 weeks. The therapeutic effect of PCI-24781 was evaluated by ultrasound biomicroscopy (USBM) throughout the experiment and histological analyses at the end of the experiment. To investigate potential mechanisms underlining the therapeutic effects of PCI-24781 on GBCa in BK5.erbB2 mice, PCI-24781-treated gallbladders were subjected to Western blot and RT-PCR analysis. The inhibitory effect of PCI-24781 on the growth of BTC cells was compared to the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and gemcitabine. To study the role of miRNAs in GBCa tumorigenesis, the expression profile of 368 miRNAs in GBCas from BK5.erbB2 (both treated and untreated) and wild type mice was analyzed. Treatment of BK5.erbB2 mice with PCI-24781 for 1 month prevented 79% of GBCa cases from progression and showed a clinical effect in 47% of cases. We also confirmed a potent inhibitory effect on tumor cell growth in human BTC cell lines treated with PCI-24781. This effect was associated with downregulation of ErbB2 mRNA and ErbB2 protein/activity and upregulation of acetylated histone and acetylated tubulin. Treatment with PCI-24781 resulted in decreased expression of Muc4, an intramembrane ligand for ErbB2, in BTC cells. PCI-24781 had more effects on growth inhibition of BTC cells than SAHA. In addition, PCI-24781 effectively inhibited the growth of gemcitabine-resistant cells. miRNA profiling revealed that the expression of several miRNAs was significantly altered in GBCa in the BK5.erbB2 mouse compared to normal gallbladder, including upregulated miR21, which was downregulated by PCI-24781. These results indicate that PCI-24781 potently inhibits the growth of BTC cells by decreasing ErbB2 expression and activity as well as regulating altered miRNA expression. PCI-24781 may have a potential value as a novel chemotherapeutic agent against human BTC in which ErbB2 is overexpressed.

HDAC inhibitor SAHA normalizes the levels of VLCFAs in human skin fibroblasts from X-ALD patients and downregulates the expression of proinflammatory cytokines in Abcd1/2-silenced mouse astrocytes

X-adrenoleukodystrophy (X-ALD) is a peroxisomal metabolic disorder caused by mutations in the ABCD1 gene encoding the peroxisomal ABC transporter adrenoleukodystrophy protein (ALDP). The consistent metabolic abnormality in all forms of X-ALD is an inherited defect in the peroxisomal β-oxidation of very long chain FAs (VLCFAs >C22:0) and the resultant pathognomic accumulation of VLCFA. The accumulation of VLCFA leads to a neuroinflammatory disease process associated with demyelination of the cerebral white matter. The present study underlines the importance of a potent histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA) in inducing the expression of ABCD2 [adrenoleukodystrophy-related protein (ALDRP)], and normalizing the peroxisomal β-oxidation, as well as the saturated and monounsaturated VLCFAs in cultured human skin fibroblasts of X-ALD patients. The expression of ELOVL1, the single elongase catalyzing the synthesis of both saturated VLCFA (C26:0) and monounsaturated VLCFA (C26:1), was also reduced by SAHA treatment. In addition, using Abcd1/Abcd2-silenced mouse primary astrocytes, we also examined the effects of SAHA in VLCFA-induced inflammatory response. SAHA treatment decreased the inflammatory response as expression of inducible nitric oxide synthase, inflammatory cytokine, and activation of NF-κB in Abcd1/Abcd2-silenced mouse primary astrocytes was reduced. These observations indicate that SAHA corrects both the metabolic disease of VLCFA as well as secondary inflammatory disease; therefore, it may be an ideal drug candidate to be tested for X-ALD therapy in humans.

In Vivo PET Imaging of Histone Deacetylases by 18F-Suberoylanilide Hydroxamic Acid (18F-SAHA)

Histone deacetylases (HDACs) are a group of enzymes that modulate gene expression and cell state by deacetylation of both histone and non-histone proteins. A variety of HDAC inhibitors (HDACi) have already undergone clinical testing in cancer. Real-time in vivo imaging of HDACs and their inhibition would be invaluable; however, the development of appropriate imaging agents has remained a major challenge. Here, we describe the development and evaluation of (18)F-suberoylanilide hydroxamic acid ((18)F-SAHA 1a), a close analogue of the most clinically relevant HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). We demonstrate that 1a has near identical biochemical activity profiles to that of SAHA and report findings from pharmacokinetic studies. Using a murine ovarian cancer model, we likewise show that HDAC inhibitor target binding efficacy can be quantitated within 24 h of administration. 1a thus represents the first (18)F-positron emission tomography (PET) HDAC imaging agent, which also exhibits low nanomolar potency and is pharmacologically analogous to a clinically relevant HDAC inhibitor.

An “Exacerbate-reverse” Strategy in Yeast Identifies Histone Deacetylase Inhibition as a Correction for Cholesterol and Sphingolipid Transport Defects in Human Niemann-Pick Type C Disease

Niemann-Pick type C (NP-C) disease is a fatal lysosomal lipid storage disorder for which no effective therapy exists. A genome-wide, conditional synthetic lethality screen was performed using the yeast model of NP-C disease during anaerobiosis, an auxotrophic condition that requires yeast to utilize exogenous sterol. We identified 12 pathways and 13 genes as modifiers of the absence of the yeast NPC1 ortholog (NCR1) and quantified the impact of loss of these genes on sterol metabolism in ncr1Δ strains grown under viable aerobic conditions. Deletion of components of the yeast NuA4 histone acetyltransferase complex in ncr1Δ strains conferred anaerobic inviability and accumulation of multiple sterol intermediates. Thus, we hypothesize an imbalance in histone acetylation in human NP-C disease. Accordingly, we show that the majority of the 11 histone deacetylase (HDAC) genes are transcriptionally up-regulated in three genetically distinct fibroblast lines derived from patients with NP-C disease. A clinically approved HDAC inhibitor (suberoylanilide hydroxamic acid) reverses the dysregulation of the majority of the HDAC genes. Consequently, three key cellular diagnostic criteria of NP-C disease are dramatically ameliorated as follows: lysosomal accumulation of both cholesterol and sphingolipids and defective esterification of LDL-derived cholesterol. These data suggest HDAC inhibition as a candidate therapy for NP-C disease. We conclude that pathways that exacerbate lethality in a model organism can be reversed in human cells as a novel therapeutic strategy. This "exacerbate-reverse" approach can potentially be utilized in any model organism for any disease.

Abstract 1641: Targeting developmental regulators of zebrafish exocrine pancreas as a therapeutic approach in human pancreatic cancer

Abstract We aim to test the hypothesis that histone deacetylases (HDACs) and RNA polymerase III (Polr3) cooperatively control exocrine pancreatic growth, and combined inhibition of HDACs and POLR3 produces enhanced growth suppression in pancreatic cancer. This hypothesis is based on the observations that HDACs and POLR3 play vital roles in fundamental cellular processes, and deregulation of these enzymes has been implicated in malignant transformation. Using chemical inhibitors of HDACs and POLR3, we examined the effect on exocrine pancreas during morphogenesis, and then translated the developmental findings to human pancreatic adenocarcinoma cultured cells. Hdacs and Polr3 are required for exocrine pancreatic epithelial proliferation during morphogenesis. In zebrafish larvae, combination of a Hdac inhibitor (Trichostatin A) and an inhibitor of Polr3 (ML-60218) synergistically prohibited expansion of exocrine pancreas. In human pancreatic adenocarcinoma cells, combination of the clinical HDAC inhibitor suberoyl-anilide hydroxamic acid (SAHA) and ML-60218 produced enhanced suppression of colony formation and proliferation, and induction of cell cycle arrest and apoptotic cell death. The enhanced cytotoxicity was associated with supra-additive induction of a pro-apoptotic regulator and the cyclin-dependent kinase inhibitors. tRNAs have been shown to have pro-proliferative and anti-apoptotic roles, and SAHA-stimulated expression of tRNAs was reversed by ML-60218. These findings demonstrate that chemically targeting developmental regulators in zebrafish can be translated into a potential therapeutic approach in human cancer, and suggest that counteracting the pro-malignant side effect of HDAC inhibitors can enhance their anti-tumor activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1641. doi:10.1158/1538-7445.AM2011-1641

Abstract 2627: Antitumor effect in human lung cancer by a combination treatment of novel histone deacetylase inhibitors: SL142 or SL325 and retinoic acids

Abstract Histone deacetylase (HDAC) inhibitors arrest cancer cell growth and cause apoptosis with low toxicity thereby constituting a promising treatment for cancer. In this study, we investigated the anti-tumor activity in lung cancer cells of the novel cyclic amide-bearing hydroxamic acid based HDAC inhibitors SL142 and SL325. In A549 and H441 lung cancer cells both SL142 and SL325 induced more cell growth inhibition and cell death than the hydroxamic acid-based HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). Moreover, the combination treatment using retinoid drugs ATRA or 9-cis RA along with SL142 or SL325 significantly induced more apoptosis and suppressed colony formation than the single use of either. The expression of the retinoic acid receptors RAR α, RAR β, RXR α and RXR β were unchanged with the treatment. However a luciferase reporter construct (pGL4. RARE 7x) containing seven tandem repeats of the retinoic acid responsible element (RARE) generated significant transcriptional activity after the combination treatment of retinoic acids and SL142 or SL325 in H441 lung cancer cells. Moreover, apoptosis-promoting Bax expression and caspase-3 activity was increased after the combination treatment. These results suggest that the combination treatment of SL142 or SL325 with retinoic acids exerts significant anti-tumor activity and is a promising therapeutic candidate to treat human lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2627. doi:10.1158/1538-7445.AM2011-2627

Abstract LB-353: Targeting pancreatic cancer stem cells by epigenetic regulation of miR-34a

Abstract MicroRNA-34a (miR-34a) is a transcriptional target of p53 that is down-regulated in pancreatic cancer. This study aimed to investigate the functional significance of miR-34a on growth of pancreatic cancer stem cells through its epigenetic restoration by chromatin modulators, demethylating agent 5-Aza-2′-deoxycytidine (5-Aza dC) and HDAC inhibitor Suberoylanilide hydroxamic acid (Zolinza/vorinostat/SAHA). Re-expression of miR-34a in pancreatic cancer stem cells and pancreatic cancer cell lines on treatment with 5-Aza dC and SAHA strongly inhibited the cell proliferation, cell cycle progression, self renewal, epithelial to mesenchymal transition and invasion. miR-34a upregulation of by 5-Aza dC and SAHA in pancreatic cancer stem cells (PCSC's) also induced apoptosis by activating caspase 3/7. Modulation of miR-34a expression inhibited the protein expression of its putative target genes Bcl-2, CDK6, Cdc25, SIRT1, which was rescued by inhibiting miRNA-34a in the presence of miR-34a antagomir. miR-34a upregulation also induced acetylated p53, p21WAF1, p27, and PUMA protein levels in PCSC's. Interestingly treatment of PCSC'c with 5-Aza dC and SAHA resulted in the inhibition of epithelial -mesenchymal transition by downregulating N-Cadherin, Zeb-1, a crucial inducer of EMT, Slug and Snail expression and also by transcriptional upregulation of E-Cadherin. SAHA retards in vitro cancer stem cell migration and invasion by the inhibition of Notch signaling pathway. Inhibition of miR-34a by antogomiR abrogates the effects of SAHA and 5-Aza dC, suggesting that SAHA and 5-Aza dC regulate stem cell characteristics in PCSC's through miR-34a. The present study thus demonstrates the role of miR-34a as a critical regulator of pancreatic cancer progression. The restoration of miR-34a expression by 5-Aza dC and SAHA in human primary pancreatic cancer stem cells and cell lines will not only provide mechanistic insight and therapeutic targets for pancreatic cancer but also promising reagent to boost patient response to existing chemotherapies or as standalone cancer drug by the elimination of the cancer stem cells characteristics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-353. doi:10.1158/1538-7445.AM2011-LB-353