Targeting Oxidative Stress in Cancer

1. Introduction

2. Histone post-translational

modifications and cancer

3. KATs/HDACs and oxidative

stress

4. Targeting epigenetic

regulators in COPD,

NSCLC and HCC

5. Caveats

6. Conclusions

7. Expert opinion

Review

Targeting oxidative stress incancerMatthew W Lawless, Kenneth J O’Byrne & Steven G Gray††Institute of Molecular Medicine, St James’s Hospital, Trinity Centre for Health Sciences,

Department of Clinical Medicine, Translational Cancer Research Group, James’s Street, Dublin,

Ireland

Importance of the field: Reactive oxygen species (ROS) occur as natural

by-products of oxygen metabolism and have important cellular functions.

Normally, the cell is able to maintain an adequate balance between the for-

mation and removal of ROS either via anti-oxidants or through the use spe-

cific enzymatic pathways. However, if this balance is disturbed, oxidative

stress may occur in the cell, a situation linked to the pathogenesis of many

diseases, including cancer.

Areas covered in this review: HDACs are important regulators of many oxida-

tive stress pathways including those involved with both sensing and coordi-

nating the cellular response to oxidative stress. In particular aberrant

regulation of these pathways by histone deacetylases may play critical roles

in cancer progression.

What the reader will gain: In this review we discuss the notion that targeting

HDACs may be a useful therapeutic avenue in the treatment of oxidative

stress in cancer, using chronic obstructive pulmonary disease (COPD), NSCLC

and hepatocellular carcinoma (HCC) as examples to illustrate this possibility.

Take home message: Epigenetic mechanisms may be an important new

therapeutic avenue for targeting oxidative stress in cancer.

Keywords: cancer, epigenetics, histone deacetylase, oxidative stress

Expert Opin. Ther. Targets (2010) 14(11):1225-1245

1. Introduction

Lung cancer is the leading cause of cancer death worldwide, and is associated withover 1 million deaths annually, and only 13% of lung cancer patients survivemore than 5 years [1]. Estimates for the USA suggest that the incidence and mortal-ity for cancers of the lung and bronchus are expected to be 219,440 and159,390 respectively in 2009 [2]. Lung cancer can be subdivided into two broad cat-egories, NSCLC and small cell lung cancer (SCLC). NSCLC can then be furtherdivided into three major types, squamous cell carcinoma (SCC), adenocarcinomaand large-cell carcinoma. Mortality in lung cancer is high due in part toi) difficulties in detecting it at an early stage and ii) associated resistance to currentlyavailable chemotherapy and radiotherapy regimes.

While lung cancer is often considered to be a preventable as most cases can beattributed to smoking, approximately 25% of all lung cancers worldwide are notcaused by smoking. If considered as a separate entity lung cancer in never smokerswould still rank as the seventh most common cause of cancer death worldwide [1].

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwideand the third leading cause of cancer death [3]. It was estimated that for the year2000, there were 564,000 new cases of HCC, accounting for 7.5% of all cancersin men and 3.5% in women worldwide [4]. Underlying chronic cirrhosis is themajor clinical risk factor for hepatic cancer and is frequently correlated withhepatitis B virus (HBV) or hepatitis C virus (HCV) infection, although cirrhosis

10.1517/14728222.2010.526933 © 2010 Informa UK, Ltd. ISSN 1472-8222 1225All rights reserved: reproduction in whole or in part not permitted

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from non-viral causes such as alcoholism or haemochromatosisare also associated with an elevated risk of HCC [5,6].Oxidative stress has been clearly linked to the development

of cancer including NSCLC [7] and HCC [8]. There are manycancer pathways/genes associated with cellular responses tooxidative stress including genes such as superoxide dismutases(SODs), glutathione peroxidases (GPXs), glucocorticoidreceptors, heme oxygenases (HMOXs), and hypoxia induciblefactor-1alpha (HIF-1a) [9,10]. Emerging evidence now linksthe regulation of many of the pathways associated with theregulation of oxidative stress to epigenetic mechanisms.Epigenetics describes the study of heritable changes in

genome function that occur without a change in DNAsequence. The molecular mechanisms underpinning epige-netics can be divided into four main categories as shownin Figure 1: i) DNA methylation, ii) covalent histone modi-fications, iii) non-covalent mechanisms such as incorpora-tion of histone variants and nucleosome remodeling andiv) non-coding RNAs including microRNAs (miRNAs) [11].There are far-reaching implications of epigenetic research

for human biology and disease, including our understandingof stem cells, ageing and cancer.In the following sections we shall discuss the role that epi-

genetics plays in the regulation of these genes/pathways usingchronic obstructive pulmonary disease (COPD)/NSCLC andHCC to illustrate i) the epigenetic dysregulation associatedwith these diseases and ii) to discuss how targeting theseepigenetic regulators may be an important therapeuticintervention for targeting oxidative stress in cancer.

2. Histone post-translational modificationsand cancer

Post-translational modifications of histones or the ‘histonecode’ has emerged as a major mechanism by which cells regu-late gene expression and cellular function. Aberrant histonepost-translational modifications have now been shown tohave both predictive and prognostic value in various cancerssuch as renal cell carcinoma [12,13], oesophageal cancer [14],gastric cancer [15], breast cancer [16], colorectal cancer andlymphoma [17], prostate cancer [18,19] and NSCLC [9,20].

Furthermore, deregulation of some of the enzymes involvedwith regulating these modifications in a bronchial epithelialcell transformation model suggest that they play importantroles in the transformation process [21]. In addition, strongevidence links aberrant expression of epigenetic regulators,in particular HDACs, to COPD a condition with anincreased risk of developing NSCLC [9].

There is therefore an urgent need to improve our under-standing of the molecular basis of epigenetic mechanisms incancers such as NSCLC and HCC and diseases associatedwith an increased risk of cancer such as COPD.

2.1 HDACs and lysine acetyltransferases (KATs) in

lung and liver organogenesisLiver, pancreas and lung originate from the presumptiveforegut in temporal and spatial proximity, and there is clearevidence that histone deacetylases and lysine acetyltransfer-ases (KATs) play important roles in both lung and liverdevelopment (Table 1). The importance of lysine acetyltrans-ferase activity in both lung & liver development has beenshown from several murine studies, where KATs were foundto be highly expressed in the developing lung and liver, andconfirmation of their importance in these organs’ develop-ment came from knockout studies. While initial knockoutmodels of KAT3A (CBP) and KAT3B (p300) demonstratedembryonic lethality between days 9 and 11.5 of gestation [22],the first data to emerge demonstrating a role for KAT3A(CBP) and KAT3B (p300) in the developing lung camefrom a study by Partanen et al. [23]. Using in situ hybridiza-tion (ISH) KAT3A and KAT3B proteins were expressed inspecific cell types of the developing heart, vasculature, skin,lung and liver during organogenesis. While KAT3A andKAT3B for the most part displayed almost identical expres-sion patterns, in the developing lung KAT3A protein wasfound in the epithelium while KAT3B protein was detectedin the mesenchyme [23]. A separate study examining KATactivity directly from developing tissues found highestHAT activities of KAT3A and KAT3B high in the brainand liver of E14 fetal and newborn mice [24]. Using a gene‘knock-in’ approach to abrogate KAT3A or KAT3B, Ecknerand colleagues found that a single deficient allele of KAT3Aor KAT3B resulted in embryonic or neonatal lethality, andthat formation of the the lung was strongly impaired inKAT3A- and to a lesser extent in KAT3B-mutantembryos [25].

Two knockout mice studies deteremnined that KAT2A(p300/cAMP response element binding protein bindingprotein-associated factor (PCAF)) null animals are viableand fertile with no obvious abnormal phenotype [26,27]. Incontrast, KAT2B (control of amino acid synthesis protein5 (GCN5)) null mice show severe growth retardation at8.5 days post coitum (d.p.c.), and are embryonic lethal. Theembryos lack specific mesodermal-derived structures [26].A role for both KAT2A and KAT2B in lung organogesiswas suggested from the observation that in KAT2A null

Article highlights.

. Oxidative stress occurs in COPD, NSCLC and HCC.

. Epigentic modifiers play important roles in lung and liverorganogenesis.

. Epigenetic modifiers are frequently dysregulated in lungand liver cancer.

. Epigenetic regulators play critical roles in regulatingresponses to oxidative stress.

. Drugs targeting epigenetic regulators may have efficacyin cancers associated with oxidative stress.

This box summarizes key points contained in the article.

Targeting oxidative stress in cancer

1226 Expert Opin. Ther. Targets (2010) 14(11)

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mice, levels of KAT2B are dramatically increased in the lungsin the lungs and liver, whereas in wild-type mice PCAF isdominantly expressed in such tissues [27].

Cong Yan and colleagues studied the expression ofKAT13A (steroid receptor coactivator 1 (SRC1)), KAT3Aand KAT3B in the developing mouse lung. KAT13A wasdetected in epithelial tubules throughout lung developmentand additionally was also observed in certain pulmonary mes-enchymal cells, suggesting potential roles for this KAT in boththe epithelium and the mesenchyme. In contrast, staining forboth KAT3A and KAT3B was present in almost all types ofcells at various stages of lung development [28].

HDACs have also been shown to play critical roles in liverdevelopment. For instance, HDAC1 and HDAC3 have bothbeen shown to play critical roles in liver development [29-33].Loss of HDAC3 in the liver disrupts lipid and cholesterolhomeostasis, leading to lipid accumulation and a decrease inglycogen storage [30]. In studies of zebrafish developmentusing morpholino based ‘knockout’ strategies HDAC3 wasfound to be specifically required for liver formation whileHDAC1 was more globally required for multiple developmentprocesses including liver/exocrine pancreas formation [29]. In aseparate zebrafish study, HDAC1 was found to critical for reg-ulating distinct steps in endodermal organogenesis. Loss ofHDAC1 resulted in an expansion of the foregut endoderm inthe domain from which the liver and pancreas originate, and

mosaic analyses revealed a cell-autonomous requirement forHDAC1 within the hepatic endoderm [31]. In murine models,a critical balance between HDAC1, CCAAT/enhancer bindingprotein (C/EBP)a and b regulates liver hepatocyte prolifera-tion. In young mice, HDAC1 promotes liver proliferationthrough interactions with C/EBPb [33], whereas in older miceHDAC1 inhibits hepatocyte proliferation through interactionswith C/EBPa [32].

Indirect evidence for the importance of HDACs inliver development has also come from studies using inhi-bitors of HDACs, which have shown that such inhibitorscan differentiate hepatocytes and liver epithelial progenitorcells [34-36].

Other factors known to associate with KATs and HDACshave also been shown to play critical roles in lung and liverdevelopment. For instance, CBP/p300-interacting transacti-vator 2 (CITED2) has been shown to play a critical role innormal liver development [37], while loss of homeodomainprotein (HOP), a protein which can associate with HDAC2,results in defective type 2 pneumocyte development [9].

2.2 HDACs and COPDChronic obstructive pulmonary disease (COPD), represents agroup of disorders that have similar respiratory symptoms --dyspnoea, cough, and sputum production; limited airflow;and chronic inflammation of the lung [38]. Currently COPD

Me

Me

U

SAc

P

KATHDAC

DNMT

KDMKMT

Coactivator–corepressor complexes

B.

A.

D.

ncRNA

Histone PTMS

DNA CpG methylation

C.

Histone variants

Me

MeCP

Figure 1. Basic mechanisms underpinning epigenetic regulation of gene expression. Four main mechanisms have been

elucidated (A) DNA methylation, (B) covalent histone modifications, (C) non-covalent mechanisms such as incorporation of

histone variants and nucleosome remodeling and (D) non-coding RNAs (ncRNAs) including microRNAs (miRNAs).Ac: Acetyl group; DNMT: DNA methyltransferase; KAT: Lysine acetyltransferase; KDM: Lysine demethylase; KMT: Lysine methyltransferase; Me: Methyl group;

MeCP: Methyl CpG binding protein; PTMS: Posttranslational modifications; S: SUMO protein; U: Ubiquitin.

Lawless, O’Byrne & Gray

Expert Opin. Ther. Targets (2010) 14(11) 1227

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represents the fourth leading cause of mortality worldwide,and is responsible for more than 2.5 million deathsannually [39]. Individuals suffering from COPD also have anassociated increased risk of developing lung cancer. A linkbetween HDACs and COPD was found when graded reduc-tions in HDAC activity, reduced levels of HDAC2 proteinand lower mRNA levels for HDACs 2, 5 and 8 were found

in lung tissue specimens of patients with various clinical stagesof COPD [9]. Cigarette smoke has also been shown todecrease the levels of HDAC2 in patients with COPD [9].Support for this has also come from studies showing that oxi-dative stress can decrease HDAC2 levels in a bronchial epithe-lial cell line. Levels of the NAD (+) dependent histonedeacetylase Sirtuin 1 (SIRT1) have also been shown to be

Table 1. KATs, KMTs, and HDACs discussed in this review.

Gene Activity Comments

KAT2A formerly (GCN5) Acetyltransferase Critical for lung and liver development

KAT2B (formerly PCAF) Acetyltransferase Critical for lung development

KAT3A (formerly CBP) Acetyltransferase Critical for lung and liver developmentMutated in lung cancerReduced in hepatocarcinogenesis

KAT3B (formerly p300) Acetyltransferase Critical for lung developmentReduced in hepatocarcinogenesis

KAT6A (formerly MOZ) Acetyltransferase Induced in hepatocarcinogenesis

KAT13A (formerly SRC1) Acetyltransferase Critical for lung development

KAT13D (formerly CLOCK) Acetyltransferase Regulates glucocorticoid receptor activity

PATT1 Acetyltransferase Downregulated in HCC

HDAC1 Deacetylase Elevated mRNA in lung cancerProtein detected in lung cancerCritical for liver developmentHigh expression associated with poor prognosis in liver cancer HCCAltered expression in hepatoblastomaRegulates liver cell proliferation

HDAC2 Deacetylase Association with homeodomain protein involved withpneumocyte developmentReduced levels of HDAC2 protein in lung tissue of patients with COPDProtein detected in lung cancerAltered expression in hepatoblastoma

HDAC3 Deacetylase Elevated protein detected in NSCLC squamous cell carcinomasCritical for liver developmentAltered expression in hepatoblastoma

HDAC5 Deacetylase Reduced mRNA in lung tissue of patients with COPD

HDAC8 Deacetylase Reduced mRNA in lung tissue of patients with COPD

Sirt1 Deacetylase Protein levels are reduced in lung tissue of COPD patients

Sirt2 Deacetylase Decreases ROS

Sirt3 Deacetylase Prevents oxidative-stress-induced apoptosis

Sirt5 Deacetylase Altered mitochobndrial activity

KMT1A (formerly SUV39H1) Methyltransferase Upregulated in cancer cell lines and following immortalization andtransformation of human bronchoepithelial cells

KMT1B (formerly SUV39H2) Methyltransferase Polymorphisms associated with an increased risk for lung cancer

KMT1C (formerly G9a) Methyltransferase Upregulated in cancer cell lines and following immortalization andtransformation of human bronchoepithelial cells

KMT1E (formerly ESET or SETDB1) Methyltransferase Upregulated in cancer cell lines and following immortalization andtransformation of human bronchoepithelial cells

KMT4 (formerly DOT1L) Methyltransferase Upregulated in cancer cell lines and following immortalization andtransformation of human bronchoepithelial cells

KMT6 (formerly EZH2) Methyltransferase Upregulated in cancer cell lines and following immortalization andtransformation of human bronchoepithelial cells

KMT8 (formerly RIZ1) Methyltransferase Polymorphisms associated with an increased risk for lung cancer

SMYD3 Methyltransferase Overexpressed in hepatocellular carcinoma



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reduced in patients with COPD, clearly demonstrating thataberrant HDAC expression is linked to the pathogenesis ofCOPD, and that oxidative stress may be a factor in thisprocess [9].

2.3 Aberrant KAT/HDAC activity in NSCLC and HCCIn NSCLC, elevated levels of HDAC1 mRNA are found inhigher stage (Stage III or IV) cancers [9,20], while other mem-bers of the class I HDACs have also been observed to havealtered expression [9,20]. HDAC3 protein has been observed tobe increased in 92% of the SCC subtype (Table 1) [9,20]. InHCC, it was recently shown that a novel histone acetyltransfer-ase Patt1, was significantly downregulated [40], whileHDAC1 is significantly overexpressed [41]. Altered expressionof class I HDACs have also been observed by us for anotherpediatric liver cancer, hepatoblastoma [42]. KAT6A (formerlyknown as monocytic leukemia zinc finger homolog (MOZ/MYST3)) has also been shown to be upregulated during chem-ically induced hepatocarcinogenesis, while the expression ofKAT3A and KAT3B were decreased [43].

HDACs form large multi-protein complexes to regulategene expression [44]. murine switch-independent 3-associated(mSin3A) is a critical component serving as a scaffold onwhich the multi-component HDAC co-repressor complexassembles has also been observed to have decreased expressionin NSCLC [9,20].

ATP-dependent SWI/SNF chromatin remodelling com-plexes members have also been shown to be dysregulated inthe lung. In NSCLC cell lines, the SWI/SNF complex hasbeen found to form a larger complex containing neuron-restrictive silencer factor (NRSF) and its co-repressors,mSin3A and RE1-silencing transcription factor (REST) co-repressor 1 (CoREST) and it has been suggested that deregu-lation of NRSF-regulated genes in NSCLC could lead toenhanced tumourigenicity [9,20]. Indeed, expression of theSWI/SNF ATPase subunits, brahma-related gene 1 (BRG1)and brahma (BRM) (BRG1/BRM), have been shown to beeither mutated or lost in approximately 30% of humanNSCLC lines [9]. In primary NSCLC tumours, 10% hadloss of both BRG1 and BRM, correlating with the poorestprognosis [9]. Using multiple tissue arrays 12 core proteinsinvolved with chromatin remodelling machinery were exam-ined in 300 NSCLC samples (150 adenocarcinomas and150 squamous cell carcinomas). Two distinct clusters werefound containing either BRM, integrase interactor 1 (Ini-1),retinoblastoma, mSin3A, HDAC1, and histone acetyltrans-ferase 1 (HAT1), or BRG1, BRG1-associated factor155 (BAF155), HDAC2, BAF170, and retinoblastoma-binding protein p48 (RbAP48) (Table 1) [9]. Positive nuclearBRM (N-BRM) staining was associated with a favourableprognosis in patients with a 5 year-survival of 53.5% com-pared with 32.3% for those patients with tumours that werenegative for N-BRM (p = 0.015). Copositivity for bothN-BRM and nuclear BRG1 had an increased 5 year-survivalof 72% compared with 33.6% (p = 0.013) in patients whose

tumours were found to be positive for either, or negative forboth markers. In contrast, membranous BRM (M-BRM)staining correlated with a poorer prognosis in adenocarci-noma patients with a 5 year-survival of 16.7% comparedwith those without M-BRM staining (38.1%; p = 0.016) [9].

Metastasis-associated protein 1 (MTA-1) has been shownto be significantly elevated in NSCLC and was found to beassociated with both tumour invasiveness and metastasis [9],while overexpression of both MTA-1 and MTA-2 also occursin HCC [45,46]. Both MTA-1 and MTA-2 have been shown tofunctionally associate with histone deacetylases [9], suggestingthat the overexpression of MTA’s may cause aberrantHDAC activity, which may be involved in invasiveness andmetastasis of NSCLC and HCC.

In NSCLC, mutations have been found within the lysine ace-tyltransferase KAT3A in a small subset of patients [9], and poly-morphisms contained within other histone modifying enzymeshave been identified which are associated with an increasedrisk for lung cancer including the lysine methyltransferasesKMT1B (formerly suppressor of variegation 3 -- 9 homolog 2(SUV39H2)) [9], and KMT8 (formerly retinoblastomaprotein-interacting zinc finger protein/PR domain-containingprotein 2 RIZ/PRDM2) (Table 1) [9]. Polymorphisms inanother epigenetic regulator, methyl-CpG binding domain1 (MBD-1) have also been observed to be associated with anincreased risk of developing lung cancer [9]. Intriguingly, thispolymorphism was also associated with significantly higheractivity at the MBD1 promoter. As MBD-1 has been shownto associate with HDACs to repress transcription [9], this sug-gests that the polymorphism may therefore lead to increasednumbers of MBD-1--HDAC complexes forming, resulting inaberrant gene expression leading to NSCLC development.

2.4 Hepatitis, epigenetic regulators and HCCHepatitis infection with either HBV or HCV is a strong riskfactor for the development of HCC [5]. Both HBV andHCV have been shown to interact with epigenetic regulatorsto affect transcriptional regulation. The HCV core protein,a structural protein of HCV virus found to play a majorrole in HCV-induced viral hepatitis, has been shown torecruit the histone acetyltransferases KAT3A/KAT3B (CBP/p300) to regulate transcription of genes such as nucleolarphosphoprotein B23 [47,48]. Oxidative stress induced bychronic HCV infection was found to raise hepatic iron levelsin mice via reduced hepcidin transcription [49]. The mecha-nism by which this occurs has recently been elucidated andfound to occur via increased HDAC activity [50].

Similarly HBV proteins have also been shown to associatewith epigenetic regulators. One of these the HBV virus X pro-tein (HBx), essential for virus replication has been shown tofunctionally associate with both histone acetyltransferases [51]

and histone deacetylases [43,52] to regulate transcription.Indeed the Hepatitis B virus X protein has been shown toregulate both transactivation activity and protein stability ofthe cancer-amplified transcription coactivator also known as



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nuclear receptor coactivator 6 (NcoA6) [53]. NcoA6 is neces-sary for the transcriptional activation of a wide spectrum oftarget genes frequently via association with histone methyl-transferases [54]. It is interesting to note that the genefor NcoA6 is amplified and overexpressed in breast, colonand lung cancers [55]. Hepatitis B virus X protein has alsobeen shown to induce the expression of MTA-1 andHDAC1 [52], and to functionally associate with HDAC1 torepress transcription of IGF binding protein 3 (IGFBP-3) inhepatocellular carcinoma cells [43].

2.5 Non-viral risk factors affecting epigenetic

regulators in HCCAlcohol intake is another associated risk factor for the devel-opment of HCC. A recent study in a rat model demonstratedthat chronic exposure to ethanol resulted in the depletion ofhepatic sirtuin 1 (SIRT1) and altered mitochondrial SIRT5deacetylase activity [56,57].Moreover, in liver the RNA-binding protein CUGBP1

has been found to be involved with HDAC1 and C/EBPbpathway in human tumor liver samples, suggesting thatHDAC1-C/EBPb complexes are involved in the developmentof liver tumors [33].

3. KATs/HDACs and oxidative stress

Oxidative stress is a frequent event in both NSCLC [58] andHCC [59]. There are many pathways involved in the cellularresponse to oxidative stress. Critically, several of these havebeen shown to either be linked with or regulated via HDACs(Table 2). In the following sections we shall describe thosepathways which have been shown to be both altered inNSCLC and HCC, and which have also been associatedwith KATs/HDACs, graphically summarized in Figure 2.

3.1 KEAP1-NRF2-AREA critical oxidative stress protective pathway is the Kelch-like erythroid cell-derived protein with cap ‘n collar homology-associated protein 1 (KEAP1)--nuclear factor-E2-relatedfactor 2 (NRF2)--antioxidant response element (ARE) orKEAP1-NRF2-ARE signalling pathway (Table 2, Figure 3).KEAP1-NRF2-ARE plays important protective roles withinthe lung and liver [9,60], and this pathway has been shown tobe disrupted in NSCLC [61], and significantly disrupted inpatients infected with hepatitis C [62]. Normally NRF2 isfound associated with the cytoplasmic inhibitor, KEAP1.Upon oxidative stress, NRF2 becomes phosphorylated anddissociates from KEAP1, whereupon it translocates to thenucleus and binds to antioxidant response elements (AREs),thereby inducing expression of antioxidant-detoxifying genes,and rescuing cells from oxidative injury [9].Low expression of the oxidative stress sensor KEAP1 is

thought to be an important factor in carcinogenesis. Supportfor this has come from studies in lung cancer where loss ofKEAP1 expression in NSCLC cell lines and primary cancers

is a consequence of increased CpG DNA methylation at theKEAP1 promoter [9]. Loss or reduction of KEAP1 function inNSCLC cell lines activates NRF2 and this activation subse-quently provides cellular growth advantages to the NSCLCcell lines [9]. In COPD, the KEAP1--NRF2--ARE pathway hasalso been shown to be disregulated. However, in COPD, levelsof KEAP1 remain unchanged while significant decreases inNRF2 and DJ-1 protein, a protein which functions to stabilizeNRF2, occur. In the alveolar macrophages from patients withsmoking-related pulmonary emphysema an altered balancebetween the levels of Nrf2/Keap-1--BTB and CNC homology1, basic leucine zipper transcription factor 1 (Bach1) result ina parallel decrease in expression of the important antioxidantproteins heme oxygenase (decycling) 1 (HO-1), GPX2 andNAD(P)H dehydrogenase, quinone 1 (NQO1) [63]. Moreover,expression of HO-1, GPX2 andNQO1was inversely correlatedwith airway obstruction and distension indexes and withincreased macrophage expression of the lipid peroxidationproduct 4 hydroxynonenal (4-HNE). Goven et al. then demon-strated in vitro that the mechanism by which cigarette smokealtered this equilibrium involved the induction of a biphasicHO-1 expression pattern via specific regulation of Nrf2/Keap1-Bach1 through extracellular-signal-regulated kinase(ERK)(1/2) and JNK signalling pathways [64]. Thus, it wouldappear that dysregulation of this critical oxidative stress respon-sive pathway occurs in both COPD and NSCLC (Table 2) [9],and the ERK(1/2) and JNK signalling pathways may be criticalelements involved.

Evidence for a significant role of the KEAP1-NRF2-ARE pathway in hepatocarcinogenesis has come mainlyfrom knockout mouse model studies. Mice deficientfor NRF2, have been shown to be more susceptibleto acetaminophen-induced hepatocellular injury, whileKEAP1 knockout mice have activated NRF2 which conferspotent resistance against this drug [65-67]. However a linkbetween HCV and NRF2 has just emerged which mayhave important implications in hepatitis-induced hepa-tocarcinogenesis. In human hepatoma cells infected withHCV, NRF2 was found to be inducted and translocated tothe nucleus in a time-dependent manner. HCV-mediatedphosphorylation/activation of Nrf2 was mediated by theMAPKs, with enhanced phosphorylation of Akt [68], andconferred a survival advantage on the HCV infected cells.Other studies have also linked the PI3K/Akt [69] andMAPK [70] signalling pathways to the mode of action ofNRF2. This may have critical importance in virus-mediatedhepatocarcinogenesis, whereby the activation of NRF2results in cell survival mechanisms generating conditionsfavourable for liver oncogenesis.

3.2 Epigenetics and oxidative stress pathways/genes

affected in COPD, NSCLC and HCCIn the previous sections we have tried to demonstrate thatboth epigenetic regulators and oxidative stress pathways aresignificantly disrupted in cancer. In the following sections



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we discuss how these two elements may be linked,whereby aberrant epigenetic regulation is inextricably linkedto the aberrant regulation of appropriate oxidative stressresponse mechanisms.

3.2.1 NRF2As previously discussed the KEAP1--NRF2--ARE pathway isan important anti-oxidative stress pathway. There is nowstrong support linking direct epigenetic regulatory mecha-nisms to the regulation of this cellular response pathway tooxidative stress. In the first instance NRF2 has been shownto be directly acetylated by KAT3A/KAT3B in response tocellular stress. The acetylation occurs within a specificDNA binding domain (Neh1) of NRF2 and this functionsto augment promoter-specific DNA binding of NRF2 toits AREs [71]. Secondly, NRF2 promotes the direct chroma-tin remodelling of its cognate target genes. Two transcrip-tion activation domains, Neh4 and Neh5, associate directlywith the lysine acetyltransferase KAT3B and act synergisti-cally to induce gene expression [9]. Indeed, histone modify-ing regulatory proteins which have been shown to associateand regulate NRF2 transactivation activity include the lysineacetyltransferases KAT3A/KAT3B/KAT2B/KAT13B, andthe arginine methyltransferases coactivator-associated argi-nine methyltransferase 1 (CARM1) and protein argininemethyltransferase 1 (PRMT1) [9].

Under oxidative stress the SWI/SNF complex componentBRG1 also directly interacts with NRF2 to selectively mediateinduction of genes [9].

The NF-kB p65 subunit has been shown to functionallyrepress the NRF2-antioxidant response pathway at the

transcriptional level. In cells where NF-kB and NRF2 weresimultaneously activated, p65 antagonized the transcriptionalactivity of NRF2. The mechanism by which this was achievedinvolved the selective hindrance of KAT3A associationwith p65, which resulted in the selective inactivation ofp65 (Figure 4). This was followed by the recruitment byp65 of HDAC3 (Figure 4) to ARE-dependent promoters torepress transcription of HO-1 [9].

3.2.2 Heme oxygenase 1HO-1 is a cellular stress response gene which has been shownto have cytoprotective effects against oxidative-stress-basedinsults involving anti-oxidative, anti-inflammatory, anti-proliferative and anti-apoptotic effects through the degrada-tion of heme to biliverdin-IXa, carbon monoxide andiron [9].

Expression of HO-1 involves chromatin remodelling, andexposure of cells to heme results in de novo hyperacetylationand hypermethylation of histone H3 at the HO-1 enhancersresulting in its enhanced transcription [9]. Indeed, in a coloncancer cell line the BRG1 catalytic subunit of SWI2/SNF2-like chromatin-remodelling complexes utilisesNRF2 to directly regulate expression of HO-1 in responseto oxidative stress [9], while deletion of the Neh5 domain inNRF2 results in reduced expression of HO-1. This is due toa reduction in the ability of KAT3B and BRG1 to associatewith NRF2, and as a consequence reduces chromatinremodelling activity at the HO-1 promoter [9].

Regulation of HO-1 in the neonatal lung has been shownto involve Bach1 (see below) [72], while ablation of this genein liver cells results in increased expression of HO-1 with a

Table 2. Major pathways discussed, associated epigenetic modifications or enzymes affected and cell function.

Pathway Epigenetic modifications or enzymes affected Cell function

KEAP1--NRF2--ARE DNA methylationLysine acetylation

Oxidative stress sensor

SP-A Histone acetylation Innate host defense

NF-kB Histone acetylationLysine acetyltransferasesHistone deacetylases

Cellular stress responsesInduction of proinflammatory genes

Heme oxygenase 1 Histone acetylationHistone methylationSwi/Snf-like chromatin-remodelling complexesLysine methyltransferases

Cytoprotective cellular stress responses

HIF-1a Histone methylationHistone acetylationLysine acetyltransferasesHistone deacetylases

Regulates responses to oxidative stress

PGC-1a Histone acetylationHistone deacetylasesHistone acetyltransferases

Regulator of antioxidant genesProtects cells from oxidative stress

Glucocorticoids Histone deacetylasesLysine acetyltransferases

Regulate expression of protective factors inoxidative stress



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concommitant amelioration of stress-induced hepaticinjury [73,74].

3.2.3 Bach1Levels of Bach1 have been found to be elevated in whole lungtissue and alveolar macrophages (cytosol and nucleus) frompatients with emphysema compared with those withoutemphysema [9]. Conversely, infection with HCV results inreduced Bach1 expression in liver cells [75]. Bach1 has beenshown to be a repressor of the oxidative stress response inmice [9], and can form a complex containing p53,HDAC1 and the nuclear co-repressor N-CoR to repress targetgenes [9].

3.2.4 HepcidinIt is well established that chronic hepatitis infection is associ-ated with the development of oxidative stress in hepatocytesand this has been proposed to be major mechanism of liverinjury through induction of endoplasmic reticulum stress,the unfolded protein response, oxidative stress, mitochondrialdysfunction and altered growth control [76]. One of the conse-quences of chronic hepatitis infection is the development ofhepatic iron overload, which induces many cellular effectsincluding damage to lysosomes and mitochondria, alteredoxidant defense systems and stimulation of hepatocyte prolif-eration [77], and is associated with a poor prognosis [78] and arisk for progression to HCC [79]. Recent studies have shown

CUGBP1

AKT3

AKT2RBBP4

SETDB1

PRMT1

SUV39H1

ENSG0000

DOT1L

SUV39H2

SMYD3

HDAC8

PRDM2

GCN5L2

ADI1

SMARCA2

SMARCA4

HDAC3

HDAC2

HDAC1

HDAC5

CARM1

NCOR1

NCOR2

RELA

NFKB1

AKT1

JUNHSP90AA1

EZH2

CDKN1BMAPK3

MAPK1

IL1B

HAMP

BACH1

HMOX1

VEGFA

KEAP1

GPX2

GPX1

SOD1

SOD2

MAPK8HIF1A

NR3C1

SIRT1

EP300 SIRT2

XRCC6

SIRT5 SIRT3

CLOCK

GATA6

SFTPA1

GABPA

UCPA

DAXX

UCP2

EPAS1

PPARGC1A

NCOA1FOXOCREBBP

PARK7

HBXIP

NQO1

IL8

Figure 2. Graphical demonstration linking epigenetic regulators, and the various genes mentioned throughout the text. This

figure was generated using STRING 8.2 (http://string.embl.de/).



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Oxidative stress KATs/HDACs

NRF2/KEAP1/ARE NF-κB HIF-1α PGC-1α HO-1Hepcidin

Bach-1 GR

Aberrant activity

Aberrant regulation/activity

COPD/NSCLC/HCC

Epigenetictherapies

Figure 3. Overview of the balance between oxidative stress response mechanisms, critical regulators such as KATs and

HDACs, and the NF-kB pathway in the lung and liver. Perturbations to any of these could lead to aberrant epigenetic

regulation of pathways critical to the cells response to oxidative stress, and may lead to COPD, NSCLC or HCC. For definitions

of abbreviations see text.

Daxx

KAT3A

KAT3B

KAT13A

SMRT

I-κBfamily

BCA3

Daxx

p50

p65

Ac

Ac

S

Gankyrin

HDACBRMS1

HDAC

HDAC

SIRT1

HDAC3

NRF2

KAT3ARestoration ofglucocorticoidsensitivity inCOPD

HDAC2

HDA Cagonist(theophylline) Cigarette smoke

HDAC(1 – 3)

SIRT1

Repression of NRF2-antioxidant response pathways

HDAC2

FOXP3

HDAC

Figure 4. The interplay between KATs/HDACs in the regulation of NF-kB activation. Other abbreviations are defined in

the text.Ac: Acetyl group; BCA3: Breast cancer associated gene 3; BRMS1: Breast cancer metastasis suppressor 1; FOXP3: Forkhead box P3; S: SUMO protein.



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that oxidative stress caused by hepatitis C infection results indecreased hepcidin expression [49], which was functionallyshown to be caused by increased HDAC activity [50].

3.2.5 HIF-1aHIF-1a is associated with both oxidative stress mediated geneexpression responses [80] and the activation of the unfoldedprotein response in endoplasmic reticulum stress [81]. Formost solid tumours regions of hypoxia are common includingNSCLC [82], and HCC [83], but hypoxia also occurs inCOPD [84].Molecular targeting of HIF-1a by siRNA in a NSCLC

model prolonged mouse survival by reducing cancer cellproliferation, angiogenesis and apoptosis, and other cellularresponses in the lung tumour tissue [9].Furthermore, in the bronchial epithelium of COPD

patients with subepithelial fibrous remodelling, it was shownthat HIF-1a was expressed in epithelial cells associated withincreased progression of structural changes, increased reticularbasement membrane (RBM) thickness, and with a reductionin the number of blood vessels in the subepithelium [9].A positive correlation between upregulation of HIF-1a,VEGF and DNA oxidation and overgeneration of reactiveoxygen species (ROS) was found in healthy volunteers placedunder conditions of hypoxia-induced oxidative stress [9].In hepatitis, HCV infection leads to the stabilization of

HIF-1a mediated via oxidative stress induced by HCV geneexpression [85], while HBV virus protein X has also beenshown to stabilise HIF-1a [86].HIF-1a plays many roles in cancer. It can directly regulate

the expression of epigenetic regulators such as lysine demethy-lases [9]. Moreover it can associate in complexes containing theepigenetic machinery to regualte gene expression. Forinstance, MTA-1 has been shown to both stabilize HIF-1aand enhance tumour metastasis via angiogenesis throughdirect interactions with HDACs [9,20].KATs have also been shown to interact with HIF-1a and

regulate gene expression during hypoxia [9]. The overall inter-actions with both KATs and HDACs appear to be essentialfor hypoxia-driven transcription. In mice engineered to carrydeletions in the domains of HIF-1a which associate withthese proteins, the domains necessary for KAT3A andKAT3B interactions with HIF-1a were found to be indis-pensable for HIF-1a transactivation function, while a similareffects were observed for the domain required for HIF-1aassociation with HDACs [9]. Critically, for some targetHIF-1a responsive genes both KAT and HDAC activitywere required for greater than 90% of the genes response.This suggests that functional interactions between KATs,HDACs and HIF-1a are essential for nearly all HIF-1aresponsive transcription in response to hypoxia [9].The activities of HIF-1a and HIF-2a have now been

shown to be directly regulated by post-translational modifica-tions whereby HIF-1a and HIF-2a are stabilized throughlysine acetylation [9,87]. Indeed targeting this acetylation

through inhibitors of HDACs have been shown to both desta-bilize HIF-1a and reduce angiogenesis [9], indicating that oneway to target oxidative stress in cancers may be to destabiliseHIF-1a/HIF-2a via targeting histone deacetylases.

3.2.6 PPAR-g coactivator 1a (PGC-1alpha)Originally isolated from a brown fat cDNA library, PPAR-gcoactivator 1a (PGC-1a) was initially associated with adap-tive thermogenesis. However this protein has important rolesin many other cellular pathways including adipocyte cell fatedecision, glucose homeostasis, hepatic gluconeogenesis, andmitochondrial oxidative metabolism. PGC-1 mediates theregulation of several crucial elements of energy metabolism,and key targets of PGC-1 in mitochondrial biogenesis areNRF1 and NRF2.

PGC-1a acts as a transcriptional co-regulator associatingwith both KATs and HDACs [9,88] to regulate anti-oxidantgene expression (Table 2). Under stress conditions PGC-1aincreases the expression of major antioxidant enzymes includ-ing copper/zinc SOD (SOD1), manganese SOD (SOD2),catalase and glutathione peroxidase (GPx1) [9]. PGC-1a canregulate the expression of uncoupling protein 2 (UCP2) anduncoupling protein 3 (UCP3), both of which are now under-stood to be able to mitigate ROS formation. NO, a criticalregulator of oxidative stress, protects mitochondria by affect-ing PGC-1a expression. As a consequence this causes changesin the expression of various mitochondrial ROS detoxificationsystem proteins.

3.2.7 Glucocorticoids and their receptorsA standard treatment for patients with acute exacerbations ofCOPD is glucocorticoid therapy [9]. However, a large propor-tion of patients subsequently go on to develop resistance or areduced sensitivity to these agents [9]. Binding of glucocorti-coids to the glucocorticoid receptor (GR) results in its translo-cation from the cytoplasm to the nucleus where it functions tosuppress the expression of pro-inflammatory genes. Inpatients with NSCLC high levels of GR expression havebeen detected in approximately 50% of NSCLC and are asso-ciated with a better progression-free survival, while GRexpression was observed in approximately 93% of HCCs [9].

GR activity has now been shown to be directly regulated byKATs. Acetylation of the GR by KAT13D at multiple lysineresidues prevents the GR from binding to its DNA recogni-tion sequences [89], while HDACs (in particular HDAC2)function to remove acetylation on the GR [9]. Furthermore,HDAC3 has also been shown to associate with SMAD6 inthe liver to suppress GR-mediated transcriptional activity [90].

GR-mediated responses can sometimesd require HDAC1to act as a transcriptional coactivator (Table 2), in thatHDAC1 activity is required to induce the induction ofsome genes expression by GR [9].

Oxidative stress has also been implicated in affecting gluco-corticoid sensitivity in patients with COPD. One potentialmechanism to explain this may be that levels of HDAC2 are



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decreased in the lungs of COPD patients (Table 1), and as aconsequence, loss of GR acetylation induces glucocorticoidinsensitivity toward NF-kB-mediated gene expression [9].Indeed, restoration of HDAC2 by overexpression inglucocorticoid-insensitive alveolar macrophages from patientswith COPD is able to restore glucocorticoid sensitivity [9].

One of the genes negatively regulated by glucocorticoids issurfactant protein A (SP-A), which has a role in regulatingNF-kB in airway epithelial cells [9]. GR regulates the expres-sion of the SP-A gene via chromatin remodelling and assem-bly of complexes containing either HDACs or KATs tofunctionally regulate transcription of this gene. SP-A is alsoregulated by the transcription factor GATA-6, which formsa complex with HDAC2 and natural killer cell-associatedantigen 2 homeobox 1 (Nkx2.1) to negatively regulateSP-A expression [9].

SP-A has been shown to be a biomarker with elevatedexpression in patients with COPD and in patients whosmoke [9]. As levels of HDAC2 have been shown to bedecreased in the lungs of patients with COPD, this mightprovide a reason for why SP-A is elevated in patients withCOPD [91,92]. It has been reported that accumulation ofinhibitor of NF-kB A (I-kBA), a paramount regulator ofNF-kB activity is an important contributor to the anti-inflammatory effects of SP-A [9]. Within macrophages isolatedfrom the lung environment, SP-A decreases the phosphoryla-tion of I-kBA, resulting in reduced nuclear translocation ofthe NF-kB p65 subunit dampening inflammatory responsesin the cells [9].

Overexpression of SP-A occurs in a proportion of NSCLCtissues [93] and in NSCLC patient serum [94,95]. Furthermorein NSCLC cell lines treated with HDAC inhibitors expressionof SP-A is downregulated [96].

The UCP3 gene, another protective factor in oxidativestress is also regulated at the transcriptional level throughglucocorticoid-mediated chromatin remodelling. In musclecells, activation of UCP3 expression is mediated viaKAT3B, while its transcription is repressed or switched offvia SIRT1 [9].

Acetylation would appear to be an importanty element inthe regulation of the GR itself. HDAC6 has been shown tobe important in the maturation of GR via heat shock protein90 (Hsp90). When cells have reduced or absent HDAC6,Hsp90-dependent maturation of the GR is dramaticallyaltered, with reduced ligand binding, nuclear translocation,and aberrant transcriptional activation [9], primarily as a con-sequence of the cells inability to assemble stable GR/hsp90 heterocomplexes [97].

GR-mediated repression of genes regulated by the pro-inflammatory cytokine IL-1b involve the activities of KATsand HDACs. Dexamethasone, a synthetic glucocorticoidinhibits IL-1b-induced acetylation at histone H4 lysine 8 atits cognate target promoters in a concentration-dependentmanner using either of two mechanisms. GR may directlyinhibit KAT3A lysine acetyltransferase activity [9], or it may

recruit HDAC2 to the p65--KAT3A complex to repress genetranscription [9].

3.2.8 Oxidative stress, acetylation and NF-kB

activationThroughout the previous sections inflammtion and gene tran-scription mediated via oxidative stress has frequently beenlinked to NF-kB. Activation or regulation of NF-kB is a crit-ical mediator of pro-inflammatory cascade signalling path-ways [98]. The NF-kB/Rel family consists of five members,but the most common form of NF-kB is a heterodimericprotein comprised of a p50 and a p65 (RelA) subunit.

An initial indication that oxidative stress affects NF-kB acti-vation within the lung came from reports demonstrating thatoxidative stress and TNF-a activated NF-kB/AP-1 in a lungcancer cell line model, resulting in both histone acetylation atthe IL-8 promoter and enhanced release of this proinflamma-tory cytokine from the cells [9]. It is now well established thatactivation of NF-kB is heavily reliant on KATs and HDACs(Figure 4). KAT3B and KAT3A act as key coactivators inregulating NF-kB induction of gene expression [9,20,88], throughassociations with the p65 subunit, while KAT13A potentiatesNF-kB transactivation through association the p50 subunit(Figure 4) [9,20,88]. In the lung, oxidative-stress-induced expres-sion of proinflammatory genes requires NF-kB recruitment ofKAT3A or KAT3B [9,20]. Once KATs were found to associatewith NF-kB, interactions between NF-kB and HDACs weresubsequently identified as necessary to repress or controlNF-kB-regulated genes (Figure 4) [9,20,88].

Cytoplasmic sequestration of NF-kB is an important ele-ment in the regulation of NF-kB driven responses. Forinstance sequestration of p65 by IkBa (IKBA) causes thetranslocation of the nuclear co-repressor (N-CoR) to the cyto-plasm [9]. Silencing mediator for retinoic acid and thyroidhormone receptor (SMRT) is another transcriptional core-pressor which NF-kB utilises to repress transcription throughassociation with HDACs [9]. When IKBA sequesters p65 inthe cytoplasm SMRT and N-CoR are also translocated tothe cytoplasm resulting in upregulation of gene transcription.This could be inhibited in a dose-dependent mannerby KAT3A, further implicating a role for lysine acetylation inthis process [9]. It was later demonstrated that IKBA results inNF-kB mediated transcription by phosphorylating (SMRT)leading to the exchange of transcriptional co-repressor(HDAC3) for coactivator (KAT3B) complexes facilitatingKAT3B to acetylate RelA/p65 [9].

NF-kB activation can also be inhibited by the binding of aprotein, death-domain associated protein (Daxx), to the regionof p65 that gets physically acetylated by KAT3B/KAT3A [9].This may prevent acetylation of p65 by direct interference, butDaxx itself has also been shown to directly associate withHDAC2 [9], and so may represent a pathway whereby KATsand HDACs act to acetylate/deacetylate critical lysines onNF-kB subunits. In this context, small ubiquitin-like modifier(SUMO) modification of KAT3A has been revealed to



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negatively modulate its transcriptional potential by recruiting aDaxx complex containing HDAC2 (Figure 4) [9].Cigarette smoke, a critical element in airway induced oxi-

dative stress may activate NF-kB through posttranslationalmodification of HDAC1, HDAC2 and HDAC3 proteinand reduction in HDAC activity (Figure 4) [9]. The class IIIHDAC, SIRT1, has also been shown to regulate NF-kBtransactivation [9]. Cigarette smoke downregulates the expres-sion of SIRT1 in the lung, resulting in increased acetylation ofp65 and activation of NF-kB-mediated proinflammatorycytokine release (Figure 4) [9].A large literature relating to how lysine acetylation regu-

lates NF-kB exists, and for a detailed review of the impor-tance of KATs and HDACs in the regulation of NF-kBthe reader is directed to the following reviews [98,99].

4. Targeting epigenetic regulators in COPD,NSCLC and HCC

Targeting epigenetic regulators is an attractive possibility forpharmacological intervention in disease. In NSCLC, twosuch epigenetic avenues have been extensively studied. Theseare i) targeting DNA methylation via DNA methyltransferase(DNMT) inhibitors (DNMTi), and ii) targeting histone acet-ylation via histone deacetylase inhibitors (HDi). DNA CpGmethylation is a common event in lung cancer [9,20], andmany in vitro studies involving DNMTi have demonstratedthe reactivation of critical genes in NSCLC in response toDNMTi treatment [9]. DNA CpG hypermethylation hasbeen shown to downregulate expression of KEAP1 in NSCLCcell lines and tumour tissues, and treatments with theDNMTi 5-aza-2¢-dexoycytidine (Decitabine) can reactivateits expression [100].

4.1 Current clinical trials involving epigenetic

therapies in COPD, NSCLC and HCCA large number of clinical trials in patients with COPD,NSCLC and HCC examining epigenetic therapies have eitherbeen completed or are still underway (Table 3), and as theseand other trials continue, side effects and dose-limiting toxicities of these potential therapies will reveal them-selves. For the most part many HDi are first generation inhib-itors which have well-tolerated safety profiles [20,88]. Some ofthese epigenetic inhibitors have received FDA approval forthe treatment of various diseases including Zolinza� (suberoy-lanilide hydroxamic acid, a HDi), Dacogen� (5-aza-2¢-deoxy-cytidine, a DNMTi) and Vidaza� (5-Azacytidine, aDNMTi). For brevity, we have provided a list of currentor completed clinical trials in COPD, NSCLC and HCCas found on the NIH clinical trials web site (http://clinicaltrials.gov/).

4.2 Theophylline: -- new use for an old drugA novel new development in relation to epigenetictargeting of COPD comes from the finding that

theophylline can act as an agonist of HDACs [101]. Aspatients suffering from COPD have decreased HDAC levelsand activity [9], several clinical trials are ongoing to assessthe clinical efficacy of this drug in both COPD andNSCLC (Table 3).

Intriquingly, theophylline may also have a therapeuticbenefit in the treatment of chronic viral hepatitis. Forinstance, in a chronic HBV infection model, theophyllinewas found to suppress both HBV infection and replica-tion [102]; indicating that such a strategy might be of use intargeting HBV.

Hepatitis has also been shown to affect asthmatic patientresponses to inhaled corticosteroid therapy [103]. In thisregard, a patient who was being treated with theophylline totreat chronic asthma was subsequently found to have chronicHCV. When interferon was administered to treat the HCV,the frequency of bronchial asthma attack was found to gradu-ally decrease and culminated with sustained virologicalresponse (SVR), and asthmatic control without the need fortheophylline [104]. This may indicate that combined theophyl-line/interferon may have some therapeutic benefit in hepatitisinfected individuals. As a caveat, it must be noted that hepati-tis infection and treatment with interferon have been shownto alter theophylline metabolism and clearance [105,106], whilean early report suggested that theophylline may in fact inducehepatitis [107].

4.3 Dietary KAT/HDAC inhibitors: potential

chemopreventative agents against cancer?Dietary interventions with bioactive compounds that canaffect epigenetic regulators may prove to have a critical rolein targeting oxidative stress and preventing the developmentof cancer. In the following sections we describe some of thecurrent knowledge surrounding these bioactive compoundsin relation to targeting oxidative stress pathways andpotentially preventing cancer.

4.3.1 SulforaphaneFound in broccoli, L-sulforaphane (SFN) was first recog-nised as a compound that protected cells against oxidativestress via activation of antioxidant enzymes [108]. Consider-able attention has focused on the use of SFN as a ‘blocking’agent, in this regard particularly in relation to its abilityto activate the NRF2/KEAP1 pathway, but SFN also hasbeen shown to have chemoprevntative properties utilisingnumerous other mechanisms in this regard including inhi-bition of Phase I antioxidant enzymes, cell cycle arrest andapoptosis, inhibition of tubulin polymerization, activationof checkpoint 2 kinase, inhibition of inflammation viainterference with NF-kB, inhibition of signalling pathwaysvia disruption of interactions with AP-1, and most recentlyvia its ability to function as a histone deacetylaseinhibitor [9,109-111].

This compound may therefore have efficacy in treatingconditions associated with oxidative stress, particularly within



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Table 3. Clinical trials involving epigenetic therapies in COPD, NSCLC and HCC.

Company Intervention Phase Clinical trial

identifier*

Ottawa Health Research InstituteOntario Thoracic Society

Theophylline III NCT00299858

University of GlasgowGlaxoSmithKlineChest, Heart & Stroke ScotlandChief Scientists Office

Theophylline versusRosiglitazone

III NCT00119496

Hospital Universitari Son DuretaFondo de Investigacion Sanitaria (FIS)Sociedad Espanola de Neumologıa yCirugıa Toracica

Theophylline I? NCT00671151

National Cancer Institute of Canada Theophylline III NCT00003684Argenta Discovery Ltd Theophylline & Budesonide II NCT00634413NCI -- Center for Cancer Research-Medical OncologyNational Cancer Institute (NCI)

Decitabine and FR901228 I NCT00041158

Arthur G. James Cancer Hospital & Richard J. SoloveResearch InstituteNational Cancer Institute (NCI)

Decitabine and Valproic Acid I NCT00084981

NCI -- Center for Cancer Research-Medical OncologyNational Cancer Institute (NCI)

Decitabine I NCT00019825

Sidney Kimmel Comprehensive Cancer CenterNational Cancer Institute (NCI)

Azacitidine and MS-275 I/II NCT00387465

Memorial Sloan-Kettering Cancer CenterNational Cancer Institute (NCI)

Phenylbutyrate and Azacitidine II NCT00006019

H. Lee Moffitt Cancer Center and Research InstituteGenentechNovartis

LBH589 & Erlotinib I/II NCT00738751

NCI - Center for Cancer Research-Medical OncologyNational Cancer Institute (NCI)

MS-275 I NCT00020579

Memorial Sloan-Kettering Cancer CenterMerck

Vorinostat I NCT00588055

Merck VorinostatCisplatinPemetrexed

I NCT00106626

Merck Vorinostat I NCT00632931Merck Vorinostat II NCT00126451Merck Vorinostat I/II NCT00251589Merck Vorinostat I NCT00127127Merck Vorinostat I NCT00373490Merck Vorinostat

GemcitabineCisplatin

I NCT00423449

Merck VorinostatCarboplatinPaclitaxel

I NCT00424775

Institut Claudius RegaudMerck Sharp & Dohme-Chibret

VorinostatVinorelbine

I NCT00801151

Titan Pharmaceuticals Pivanex II NCT00073385Dartmouth-Hitchcock Medical CenterMerck

Vorinostat ? NCT00735826

University of Michigan Cancer Center VorinostatDocetaxel

I NCT00565227

Fred Hutchinson Cancer Research CenterNational Cancer Institute (NCI)

Vorinostat, Paclitaxel, andRadiation therapy

I/II NCT00662311

Nereus Pharmaceuticals, Inc. NPI-0052 and Vorinostat I NCT00667082Penn State University Carboplatin, Paclitaxel, Bevacizumab

and VorinostatI NCT00702572

California Cancer ConsortiumNational Cancer Institute (NCI)

Carboplatin and Paclitaxel With orWithout Vorinostat

II NCT00481078

*As found on clinicaltrials.gov (http://clinicaltrials.gov).



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the setting of the lung. For instance, loss of the DJ-1 protein isassociated with decreased levels of NRF2 in patients withCOPD [9]. If DJ-1 expression is silenced in in vitro models,treatment with SFN can upregulate expression of NRF2-mediated antioxidant enzymes [9]. SFN has also been shownto cause apoptosis by various mechanisms in NSCLC celllines, including disruption of microtubule polymerisation,cell cycle arrest or by sensitizing cells to TNF-related apopto-sis-inducing ligand (TRAIL) [9]. Furthermore. treatment withSFN of mice exposed to tobacco carcinogens resulted in asignificant inhibition in progression to adenocarcinomas [9].

4.3.2 CurcuminAnother bioactive compound known to target epigeneticregulators is curcumin (diferuloylmethane). Derived fromturmeric, curcumin has been shown to exert potent anti-inflammatory and antioxidant effects [9]. As such this com-pound has been suggested to be a dietary interventionoption in the treatment of inflammatory lung disease [9],and as an ‘anti-cancer’ agent in NSCLC [9]. It as also beentested for efficiacy in the prevention of HCC [112]. In clinicaltrials this compound is well tolerated and lacks significanttoxicity [9].

Table 3. Clinical trials involving epigenetic therapies in COPD, NSCLC and HCC (continued).

Company Intervention Phase Clinical trial

identifier*

University of Erlangen-Nurnberg Sorafenib + LBH589 I NCT00823290Yale UniversityMerck

VorinostatRadiotherapy

I NCT00821951

University of Wisconsin, MadisonMillennium Pharmaceuticals, Inc.Merck

VorinostatBortezomib

II NCT00798720

University of Wisconsin, MadisonNational Cancer Institute (NCI)

VorinostatBortezomib

I NCT00227513

University of Wisconsin, MadisonNational Cancer Institute (NCI)

Vorinostat II NCT00138203

Memorial Sloan-Kettering Cancer CenterNational Cancer Institute (NCI)

VorinostatAlvocidibOther: pharmacological study

I NCT00324480

Princess Margaret Hospital, CanadaNational Cancer Institute (NCI)

VorinostatCapecitabine

I NCT00121277

UPMC Cancer CentersNational Cancer Institute (NCI)

Vorinostat I NCT00499811

H. Lee Moffitt Cancer Center andResearch InstituteNational Cancer Institute (NCI)

VorinostatDoxorubicin

I NCT00331955

M.D. Anderson Cancer CenterNational Cancer Institute (NCI)

Vorinostat gemcitabinehydrochloride

I NCT00243100

Roswell Park Cancer InstituteNational Cancer Institute (NCI)

Vorinostat fluorouracilLeucovorin calciumOxaliplatin

I NCT00138177

University of Colorado at DenverMerckBayer

VorinostatSorafenib

I NCT00635791

Stanford UniversityNational Comprehensive Cancer Network

VorinostatRadiation

I NCT00946673

University of Virginia VorinostatVelcade

I NCT00731952

Massachusetts General Hospital Curcumin Pharmacokinetics NCT00181662Hadassah Medical Organization Curcumin Pharmacokinetics NCT00542711Medical University of Vienna Curcumin I NCT00895167Barbara Ann Karmanos Cancer InstituteNational Cancer Institute (NCI)

CurcuminGreen tea extractPolygonum cuspidatum extractSoybean extract

NCT00768118

Sidney Kimmel ComprehensiveCancer CenterNational Cancer Institute (NCI)

Broccoli sprout extract NCT00255775

Johns Hopkins University Broccoli sprout extract NCT00994604

*As found on clinicaltrials.gov (http://clinicaltrials.gov).



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Critically, curcumin affects epigenetic mechanisms byinhibiting both KATs, and HDACs [9]. This may result inthe upregulation of curcumin and also affects the expressionof anti-oxidant responsive genes such as HO-1 andNRF2 in breast cancer cells, and therefore may also inducesuch defences in COPD, NSCLC and HCC 9. Furthermore,curcumin restores HDAC2 levels, and steroid sensitivity inmonocytes exposed to cigarette smoke raising the notionthat this compound may have efficacy in the treatment ofCOPD. Intriguingly, curcumin and L-sulphoraphane syner-gistically downregulate pro-inflammatory markers such asIL-1, while synergistically increasing levels of HO-1 raisingthe possibility that these compounds could used as be combi-natorial agents in the treatment of cancers such as NSCLCand HCC [9].

One of curcumins’ known modes of action involves inhi-bition of NF-kB mediated responses, either by either pre-venting the phosphorylation of IkBa, or by preventing itsdegradation, and essentially reducing nuclear translocationof NF-kB. As curcumin also inhibits KATs it may also bepresumed that NF-kB activation and translocation andNF-kB-mediated chromatin remodelling will also beaffected by this associated loss of KAT activity, particularlyin light of the important roles of KATs/HDACs in regulat-ing NF-kB (Figure 4) [9]. The inhibition of NF-kB by curcu-min subsequently leads to a reduction of pro-inflammatorycytokines such as CXCL1 and CXCL2, or upregulationof regulatory genes controlling pro-inflammatory genessuch as mitogen-activated protein kinase phosphatase-5(MKP5) [9], and has also been shown to have proven efficacyin ameliorating the oxidative stress associated with a mousemodel of steatohepatitis through inhibition of NF-kBactivation [113].

Obviously, the differences observed concerning both themechanism of action and effects of curcumin in clinical trialsmay in part stem from either the concentrations/doses usedand/or the cells targeted. This may have profound effects onthe potential clinical utility of this compound, and willrequire further evaluation.

4.4 Can dietary epigenetic inhibitors work in the

clinical setting?Initial epidemiological evidence suggested a link between die-tary isothiocyanate (of which sulforaphane is a member)intake and a reduced risk of developing lung cancer [9]. Strongevidence is now emerging within the clinical setting that broc-coli or cruciferous vegetable consumption has a protectivebenefit in smokers, specifically in former smokers. In aplacebo-controlled dose-escalation trial to evaluate the effectsof sulforaphane, on the expression of glutathione-S-transferaseM1 (GSTM1), glutathione- S -transferase P1 (GSTP1),NADPH quinone oxidoreductase (NQO1), and HO-1 inthe upper airway of human subjects, using nasal lavage andRT-PCR, significant increases in expression of all thePhase II enzymes compared with baseline were observed for

the sulforaphane-treated patients, whereas in the controlsreceiving non-sulforaphane-containing alfalfa sprouts, nochanges to Phase II enzymes were observed [9]. In a similarhospital-based, case-controlled study with lung cancer casesand controls matched on the basis of smoking status it wasdetermined that among smokers, the protective effect of cru-ciferous vegetable intake ranged from a 20% reduction inrisk to a 55% reduction depending on the type of vegetableconsumed and the duration and intensity of smoking. In par-ticular, it was found that the consumption of raw cruciferousvegetables was associated with a risk reduction of lung cancerfor current smokers. Additionally, in patients with actual can-cer, the strongest reduction was found for patients with eithersquamous or small-cell carcinoma, the two subtypes moststrongly associated with heavy smoking [9]. Finally, a smallcrossover study designed to examine the protective effect ofbroccoli intake in smokers and non-smokers, found that whilebroccoli intake (200 g/day) provided significant protectionagainst oxidative-stress-induced DNA damage, no alterationsto HDAC activity occurred over the trial period [114].

These results clearly indicate that dietary sulforaphaneintake may have an important chemopreventative role inNSCLC, and may also be important in the treatmentof COPD.

5. Caveats

Despite all the evidence that indicates that epigenetic mecha-nisms may be important therapeutic targets in the treatmentof oxidative-stress-based conditions within the lung, thereare some caveats which require consideration. For instance,in the lungs of rats treated with sulforaphanes glucosinolateprecursor (glucoraphanin), levels of Phase I carcinogen-activating enzymes, including activators of carcinogenic poly-cyclic aromatic hydrocarbons (PAHs) have been shown to bestrongly induced, suggesting that regular administration ofglucoraphanin could actually increase rather than decreaselung cancer risk, especially in smokers as these individualswould be exposed to tobacco-smoke-related mutagens andcarcinogens [9].

Overexpression of MMP9 is reported to be involved in thepathogenesis of COPD, NSCLC and HCC. In COPD, oxi-dative stress has been shown to reduce the levels of SIRT1,resulting in elevation of MMP9 expression [115]. Indeed, a spe-cific activator of SIRT1 (SRT1720), has been shown todecrease the expressions of marker genes for oxidativestress [116]. As such agonists of HDACs may prove to be ofgreater benefit than inhibitors.

Issues of bioavailability may also be encountered in relationto efficacy of dietary epigenetic interventions in the treatmentof COPD and NSCLC. For instance it is well established thatcurcumin is not easily bioavailable. In a recent Phase II clini-cal trial in patients with advanced pancreatic cancer, eventhough patients received up to 8 g of curcumin by mouthdaily until disease progression, with restaging every 2 months,



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the bioavailable levels of curcumin achieved in plasma werewithin the range of 22 -- 41 ng/ml [117]. However, curcuminbioavailability has been increased through heat treatment [118],or by the synthesis of novel derivatives [119], and should lead tobetter compounds for use in the clinical setting. In contrastthe bioavailability of SFN is increased if cruciferous vegetablesare eaten raw rather than heat-treated [120]. It is clear that fur-ther efforts are required to understand the bioavailability andpharmacokinetics of these dietary-derived natural com-pounds. For instance, in a recent study in human volunteersit was found that repeated ingestion of broccoli did not leadto higher plasma levels of SFN [121].Synthetic HDi have also been shown to have issues pertain-

ing to bioavailability and clearance. A recent study found thatthere was high clearance of a synthetic HDi in the lung due tooxidation [122].Finally, while HDi may be beneficial for the treatment of

disease, they may not be of great use in targeting oxidative-stress-induced disease. In this regard, they have been shownto induce apoptosis in cancer cells by inducing oxidativestress [9].Lung cancer and COPD are leading causes of morbidity

and mortality in the USA and worldwide. They share a com-mon environmental risk factor in cigarette smoke exposureand a genetic predisposition represented by the incidence ofthese diseases in only a fraction of smokers. The presence ofCOPD increases the risk of lung cancer up to 4.5-fold. How-ever, in addition one outstanding issue concerns the fact that asmall number of patients who develop lung cancer or COPDare non-smokers raising the possibility that there may bei) completely different diseases which will require differenttreatments or ii) genetic/epigenetic susceptibilities or environ-mental exposures that could drive similar changes in ROSstress and epigenetic modifications without the need for ciga-rette smoke exposure. We feel that currently we are not yet ina position to distinguish between these two possibilities. At arecent workshop held by the National Heart, Lung, andBlood Institute (NHLBI) and the National Cancer Institute(NCI), it was decided that future studies should have as theirmain objectives: i) to clarify common epidemiological charac-teristics of lung cancer and COPD; ii) to identify sharedgenetic and epigenetic risk factors; iii) to identify and validatebiomarkers, molecular signatures, and imaging-derived meas-urements of each disease; and iv) to determine common anddisparate pathogenetic mechanisms [9].Another area of concern pertains to the different effects of

cigarette smoke on cells. This may be due to different celltypes, different degrees of anti-oxidant capacity in the differ-ent cell types or are other factors such as chemical and envi-ronmental carcinogens may also play important roles?Indeed, some chemical carcinogens such as Benzo[a]pyrene(B[a]P) are present in both the environment and cigarettesmoke, and this compound has been shown to increase cellu-lar proliferation in lung cancer cells [9]. Small airways inpatients with COPD also have evidence for proliferative

responses, particularly with respect to smooth muscle and toepithelial cells [9]. Clearly, future studies will be required tomolecularly dissect the genetic epigenetic and molecularalterations underpinning these differential responses.

6. Conclusions

Current knowledge demonstrates that that the hypoxic envi-ronment encountered in COPD, NSCLC and HCC resultsin the development of oxidative stress. Throughout thisreview we have attempted to demonstrate how epigeneticsmay be a critical mechanism by which cells regulate oxidativestress responsive genes and pathways. A fine balance betweenthe epigenetic regulatory mechanisms and their intended tar-gets to both dampen pro-inflammatory conditions and con-trol cellular oxidative stress pathways exists (summarizedin Figures 1 and 2), which if disturbed may disrupt epigeneticcontrol and subsequently to exacerbations of pre-existing con-ditions (e.g.,: COPD; hepatitis), which in turn may resultin tumourigenesis.

We have discussed the therapeutic aspects of targeting epi-genetic regulatory mechanisms for the treatment of COPD,NSCLC and HCC, either via pharmacological interventionor through the use of dietary chemopreventatives. The avail-able data would indicate that there is compelling evidencefor further studies into epigenetic intervention strategies totreat diseases associated with oxidative stress in particular todetermine whether or not to use inhibitors or agonists ofspecific HDACs to target oxidative stress in cancer.

7. Expert opinion

The key central finding of the current field is that HDACsmay be critical proteins in all aspects pertaining to oxidativestress pathways in cancer.

The key weakness currently is that the presently developedHDi tend to be pan-specific. At present we do not haveenough knowledge concerning which HDACs may be criticaltargets and at which time-points in disease progression theymay best be targeted. Also while a lot of evidence suggeststhat HDi may be excellent candidates for targeting oxidativestress pathways, they have also been shown to reactivateBRM, but inhibit its function, and functional activity isonly restored once the HDi is removed [9]. Therefore, itmay be important to design staggered therapeutic inter-ventions with HDi to allow the critical cellular functions ofreactivated genes to take effect.

The potential to target oxidative stress may allow us to pro-actively target HDACs to prevent cancer progression in at riskpatients, and furthermore may add to the pharmacologicalarmamentarium used by oncologists to treat patients withcancer.

The ultimate goal of this field is to more precisely deter-mine the role epigenetic regulators and in particular HDACsplay in the regulation of oxidative-stress pathways in cancer,



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HP-PI-1

Highlight

with a view to specifically targeting these pathways by directintervention through HDi.

Unfortunately, we don’t yet know precisely the fullmechanisms underpinning epigenetic regulation of oxidativestress pathways. This will require substantially more researchto molecularly delineate the functional elements and his-tone/protein post-translational modifications involved. Simi-larly to the ‘histone code’ there may be a ‘protein code’ ofregulatory modifications that will need deciphering inthe future.

In this regard, in the coming years we see exciting newdevelopments involving such areas. Proteomic and epige-nomic methodologies are rapidly enhancing how we under-stand the molecular mechanisms by which epigeneticregulatory proteins regulate gene expression through chroma-tin remodelling and via modulation of various protein’smolecular activities.

The precise molecular dissection of the mechanims utilisedby HDACs to regulate cellular events such as the oxidative-stress response, will provide exciting new vistas in phar-macological intervention strategies to target oxidative stress.This will be clinically important as medicine strives to usetranslationally based research to target disease.

The advantage of HDACs for novel drug design centresupon their highly conserved deacetylase domains. This willallow the development of highly effective inhibitors. Howeverit may be necessary to incorporate other regions of eachHDAC into inhibitor design in order to achieve specificity.

Dietary intervention that can actively target oxidative stressthrough inhibition of HDACs is an attractive proposal. Theworld faces a continuously increasing burden of medical carecosts. Hence, cancer chemoprevention through diet mayplay an increasingly important role, and the incorporationof agents targeting HDACs into dietary requirements mayprove to be essential to reduce this burden.

Acknowledgements

We recognize the many outstanding published contributionof investigators in the field that could not be included inthis review due to the space and citation limitations.

Declaration of interest

The authors state no conflict of interest and have received nopayment in preparation of this manuscript.

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Xenobiotica 2009;39(10):722-37

AffiliationMatthew W Lawless1, Kenneth J O’Byrne2 &

Steven G Gray†2

†Author for correspondence1Mater Misericordiae University

Hospital -- University College Dublin,

Centre for Liver Disease,

School of Medicine and Medical Science,

Dublin, Ireland2Institute of Molecular Medicine,

St James’s Hospital,

Trinity Centre for Health Sciences,

Department of Clinical Medicine,

Translational Cancer Research Group,

Room 2.103, James’s Street,

Dublin 8, Ireland

Tel: +0035 318963620;

E-mail: [email protected]



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Targeting Oxidative Stress in Cancer

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