Epigenetic mechanisms in COPD: implications for pathogenesis and drug discovery.

Expert Opinion on Drug Discovery

ISSN: 1746-0441 (Print) 1746-045X (Online) Journal homepage: http://www.tandfonline.com/loi/iedc20

Epigenetic mechanisms in COPD: implications for pathogenesis and drug discovery Andrea C Schamberger, Nikica Mise, Silke Meiners & Oliver Eickelberg To cite this article: Andrea C Schamberger, Nikica Mise, Silke Meiners & Oliver Eickelberg (2014) Epigenetic mechanisms in COPD: implications for pathogenesis and drug discovery, Expert Opinion on Drug Discovery, 9:6, 609-628, DOI: 10.1517/17460441.2014.913020 To link to this article: https://doi.org/10.1517/17460441.2014.913020

Published online: 21 May 2014.

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Review

Epigenetic mechanisms in COPD: implications for pathogenesis and drug discovery 1.

Introduction

2.

Epigenetics in COPD

3.

Expert opinion

Andrea C Schamberger, Nikica Mise, Silke Meiners & Oliver Eickelberg† Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz Zentrum Mu¨nchen, University Hospital and Ludwig-Maximilians-University, Member of the German Center for Lung Research (DZL), Munich, Germany

Introduction: Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide. The growing burden of COPD is due to continuous tobacco use, which is the most important risk factor of the disease, indoor fumes, occupational exposures and also aging of the world’s population. Epigenetic mechanisms significantly contribute to COPD pathophysiology. Areas covered: This review focuses on disease-relevant changes in DNA modification, histone modification and non-coding RNA expression in COPD, and provides insight into novel therapeutic approaches modulating epigenetic mechanisms. Recent findings revealed, among others, globally changed DNA methylation patterns, decreased levels of histone deacetylases and reduced microRNAs levels in COPD. The authors also discuss a potential role of the chromatin silencing Polycomb group of proteins in COPD. Expert opinion: COPD is a highly complex disease and therapy development is complicated by the fact that many smokers develop both COPD and lung cancer. Of interest, combination therapies involving DNA methyltransferase inhibitors and anti-inflammatory drugs provide a promising approach, as they might be therapeutic for both COPD and cancer. Although the field of epigenetic research has virtually exploded over the last 10 years, particular efforts are required to enhance our knowledge of the COPD epigenome in order to successfully establish epigenetic-based therapies for this widespread disease. Keywords: Biomarker, chronic obstructive pulmonary disease, DNA modification, DNA methyltransferase inhibitors, emphysema, histone deacetylase activators, Polycomb Expert Opin. Drug Discov. (2014) 9(6):609-628

1.

Introduction

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide [1]. The growing burden of COPD is due to the continuous use of tobacco, which is the most important risk factor of the disease, indoor fume, occupational exposures and aging of the world’s population. In addition, genetic susceptibility including epigenetic changes likely plays an underestimated role in disease onset and progression [1]. To date, COPD is not curable, but symptomatic treatment can slow progression of the disease [2]. This review will summarize available evidence for epigenetic alterations in COPD, ranging from DNA and histone modifications to non-coding RNAs (ncRNAs). It will highlight strategies to interfere with de novo or transgenerational epigenetic alterations, leading to or occurring in COPD, in order to identify novel therapeutic targets to combat obstructive lung disease.

10.1517/17460441.2014.913020 © 2014 Informa UK, Ltd. ISSN 1746-0441, e-ISSN 1746-045X All rights reserved: reproduction in whole or in part not permitted

609

A. C. Schamberger et al.

Article highlights. .

. . . .

The growing burden of chronic obstructive pulmonary disease (COPD) is due to the continuous use of tobacco, its most important risk factor. Genetic and epigenetic factors might also be implicated in the pathogenesis of COPD. Cigarette smoke is associated with decreased levels of histone deacetylases. Altered global DNA methylation patterns were found in COPD patients. Further studies are needed to define the COPD epigenome in order to identify potential therapeutic targets.

This box summarizes key points contained in the article.

The burden of COPD The World Health Organization (WHO) estimates, that in 2004, 64 million people had COPD worldwide [3]. COPD prevalence varies among studies, partly due to differences in diagnostic procedures or survey methods. According to the BOLD study, the prevalence of COPD ranges from 5 to 15% worldwide [4], while the PLATINO study revealed prevalences between 7.8 and almost 20% in Latin America with an increased comorbidity score [5,6]. Undoubtedly, COPD ranks as the fourth leading cause of death worldwide, and is projected to become third leading cause of death by 2030 [7]. In 2004, > 3 million people died of COPD, which equals 5% of all deaths globally that year [3]. According to WHO predictions, this number will raise by 30% to 4.6 million people in 2030 [8]. Of note, low- and middle-income countries are at much higher risk, as 90% of the COPD deaths in 2004 occurred in these countries [3]. 1.1

COPD risk factors COPD is due to continued exposure to environmental risk factors. To date, cigarette smoking, including secondhand smoke, is the most-studied and best-defined risk factor for COPD. Numbers on the smoking history of COPD patients vary from 25 to 50%, depending on age and study population [9,10]; up to 50% of elderly smokers have developed COPD [10]. To date, non-smokers have an estimated COPD prevalence of 4.3% throughout all ages [9], but several studies show COPD prevalence in non-smokers above 40 years to be 30% [11]. For example, occupational and/or indoor exposure to vapors, gases, dusts and fumes clearly increases the prevalence to develop COPD in non-smokers [12]. Further, exposure to biomass fuels (coal, straw, animal dung, crop residues and wood) is attributed to COPD development. According to WHO, 35% of COPD cases are related to this indoor air pollution in low- and middle-income countries [13]. Gender also appears to affect COPD susceptibility. Historically, most studies document a higher COPD prevalence among men, but these data probably represent the higher 1.2

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exposure of men to smoke and occupational dusts compared to women [4,5,9]. Recent data suggest an even higher predisposition in women, but this issue needs further investigation [14-16]. Moreover, infections play an important role in both, development and progression of COPD. For example, HIV infection and tuberculosis have been shown to accelerate the onset of smoking-related emphysema and COPD, respectively [17,18]. Another important factor associated with COPD prevalence, morbidity and mortality is age [4,5,9]. The importance of age as a risk factor of chronic diseases rises continuously, as the world’s population is undergoing a dramatic shift toward elderly people [19,20]. Of note, not all people with the same age, gender, infections, or exposures to dusts, biomass fuels or smoke will develop COPD or features of it. There is a marked variability in COPD susceptibility, which can be explained by significant genetic and epigenetic predispositions to disease onset or progression. For example, a well-known genetic risk factor is a-1 antitrypsin (A1AT) deficiency. A1AT is a major circulating inhibitor of serine proteases, particularly of neutrophil elastase. A1AT deficiency results in degradation of lung parenchyma -particularly during periods of inflammation -- leading to severe emphysema, one hallmark feature of COPD [21]. As this monogenic defect accounts only for about 2% of COPD cases [22], it is well conceivable that other, hitherto unknown genetic and epigenetic players contribute to COPD susceptibility. Pathogenesis of COPD Inhalation of dust, smoke or foreign material leads to deposition of particles in the lung. The innate inflammatory immune system provides the primary protection against these continuous insults. A tight epithelial barrier forms the first line of defense, thus keeping foreign material in the bronchial and alveolar space. Efficient mucociliary clearance removes particles out of the respiratory tract in a directed, baso-apical movement. Foreign materials deposited on the bronchial and alveolar cell surface are further cleared by macrophages. In the alveolar space, the surfactant layer transports alveolar macrophages and foreign debris up to the mucociliary clearance apparatus. Part of the surfactant gets reabsorbed into the interstitial space, thus distributing small particulates in the body. If these particles are not cleared by the lymphatic system, they accumulate in the lung and other tissues and are able to activate the innate and adaptive immune systems subsequently [23,24]. Continuous inhalation of toxic gases such as cigarette smoke (CS) or high amounts of environmental and occupational exposures can lead to reduced epithelial barrier formation [25,26] due to high levels of oxidative stress. This results in a persistent innate and adaptive inflammatory response. Infiltrating cells that damage the tissue include polymorphonuclear leukocytes, macrophages, natural killer cells and dendritic cells, which activate the adaptive immune system with CD4+ and CD8+ T-lymphocytes and B-lymphocytes. These responses are linked to tissue-repair and 1.3

Expert Opin. Drug Discov. (2014) 9(6)

Epigenetic mechanisms in COPD

Table 1. FDA-approved medication for COPD*. Medication Bronchodilators

Classification

Drug

b2-agonists (short-acting)

Fenoterol, Levalbuterol, Salbutamol, Terbutaline

b2-agonists (long-acting)

Formoterol, Arformoterol, Indacaterol, Salmeterol, Tulobuterol

Anticholinergics (short-acting)

Ipratropium bromide, Oxitropium bromide

Anticholinergics (long-acting)

Aclidinium bromide, Glycopyrronium bromide, Tiotropium bromide

Methylxantines

Aminophylline, Theophylline

Combination bronchodilator therapy

Fenoterol/Ipratropium bromide, Salbutamol/Ipratropium bromide, Vilanterol/Umeclidium bromide

Corticosteroids

Inhaled corticosteroids

Beclomethasone, Budesonide, Fluticasone

Systemic corticosteroids

Prednisone, Methyl-prednisolone

Combination bronchodilator therapy/ inhaled corticosteroid

Formoterol/Budesonide, Formoterol/Mometasone, Salmeterol/ Fluticasone, Vilanterol/Fluticasone

Phosphodiesterase-4 inhibitors

Roflumilast

*Based on data from [1]. COPD: Chronic obstructive pulmonary disease.

tissue-remodeling processes, including increased mucus production, tissue fibrosis in the peribronchiolar area, dysfunction of alveolar and bronchial epithelial cells, and emphysematous destruction of the gas-exchanging surface of the lung [23,24]. Collectively, these changes lead to progressive airflow limitation in COPD as a result of airway wall thickening, which narrows the lumen, mucus occlusion, pulmonary inflammation and peribronchiolar and interstitial fibrosis. Moreover, alveolar wall destruction decreases the elastic recoil pressure limiting the ability to exhale and thus resulting in a continuous forced expiratory volume in 1 second (FEV1) decline and a reduced FEV1/forced vital capacity ratio [23].

patients with severe A1AT deficiency, but ineffective in elder COPD patients with normal A1AT levels. In this review, we apply the following criteria to critically examine novel and upcoming therapeutic targets of epigenetic control of COPD onset and progression: Descriptive evidence of epigenetic targets will be evaluated with regard to their contribution to disease and analysis in disease-relevant animal models. Moreover, studies with pharmacological inhibition of selected targets in animal models will be highlighted and discussed as potential anti-COPD drug options.

Current therapy of COPD Up to now, there is no cure for COPD. In many advanced stages of COPD, lung transplantation is the only intervention to significantly improve lung function or decrease mortality. After COPD diagnosis, the goal of clinical COPD management is to improve the patient’s functional status and quality of life by preserving residual lung function, improving symptoms and preventing the frequency of exacerbations. Smoking cessation is the first and most important step for patients who smoke. To improve health status and most importantly, symptoms in COPD, a pharmacological therapy is indispensable. Table 1 depicts the most commonly used medication in COPD [1]. Further, health education and exercise programs are necessary modules in COPD therapy. In very severe COPD, long-term administration of oxygen (> 15 h/day) is mostly required. To continuously improve COPD treatment, personalized therapies are therefore needed urgently. For example, A1AT transfer therapy may be a good option for young COPD

Epigenetics is defined as ‘the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence’ [27]. These changes influence gene expression and can be the result of chemical modifications of DNA, histone tails or ncRNAs (Figure 1). Historically, epigenetic modifications were thought to be stable and inert information, passed on to the next generation of cells, for example, X chromosome inactivation and X-linked diseases [28]. But lessons learned from twin studies suggest that epigenetic differences are plastic over time and accumulate during lifetime [29,30]. Epigenetic modifications have now been causally linked to diseases such as autoimmune thyroid disease or asthma [31,32], demonstrating that epigenetic modifications are dynamic information markedly influenced by environmental stimuli, dietary changes, disease or aging. COPD has been shown to accumulate in families [33,34]. Further, there is evidence that the main risk factor for COPD -- cigarette smoking -- is associated with epigenetic changes in the bronchial epithelium [35,36] and that epigenetic

1.4

2.

Epigenetics in COPD


611


Transcriptionally active chromatin

Transcriptionally inactive chromatin

DNA modification

TSS

HDAC MeBP

TSS + DNMT Gene promoter not accessible

Gene promoter accessible

Histone modification

TSS M

A

A M

A

A

TSS M

A

A

M A Gene promoter

A

M

P A

P A A

P

+ HMTS + HDAC

A

M

A

A

RN

M

A

A

TF

1 TSS

Non-coding RNA

TF

M

M

RNA Inc

A

M

M

Gene promoter not accessible Inc

RN

M

P

+ HDMS + HAT

accessible

Inc

DNMT

HP1

A

A

DNMT

2 = HDAC = Unmethylated CpG = HP1 = Methylated CpG = DNMT A = Acetylation TSS = Transcriptional start site M = Methylation TF = Transcription factor P = Phosphorylation IncRNA = Long non-coding RNA = Methyl-CpGPRC = Polycomb repressive comples binding-protein

PRC1 P

PRC2

M

M

M

M

M

3 M

A M

Figure 1. Epigenetic regulation of gene transcription. Three levels of epigenetic regulation of gene expression are displayed: DNA modification (upper panel), histone modification (middle panel) and non-coding RNA (lower panel). Transcriptionally active chromatin with an accessible gene promoter region is illustrated in the left panel. Transcriptionally inactive and packed chromatin, with a non-accessible gene promoter region, or blocked transcription, is depicted in the right panel. DNA modification comprises CpC methylation induced by DNA methyltransferases (DNMTs), resulting in gene inactivation. MethylCpG-binding proteins (MeBP) can recruit histone deacetylases (HDACs) to further silence chromatin. Histone modifications include acetylation (correlating with active transcription), methylation (correlating with inactive transcription) or phosphorylation. Heterochromatin protein 1 (HP1) can bind to methylated histone tails and recruit DNMTs for further gene silencing. Long non-coding RNAs (lncRNAs) are able to activate or inactivate gene transcription. lncRNAs can bind transcription factors (TF) and either recruit them to promoter sequences (left panel) or trap them to block gene transcription (1, right panel). lncRNAs can also recruit DNMTs (2, right panel) or Polycomb repressive complexes (PRC1/2) (3, right panel) to silence chromatin.

pathways regulate airway inflammation [37,38]. Thus, targeting epigenetic mechanisms has the potential to interfere therapeutically with COPD development. Table 2 provides an overview on the main epigenetic modifications encountered in in vitro/in vivo COPD models or COPD patients, which are described in the following sections. Epigenetic-based therapeutics are summarized in Table 3. DNA modification All cells within an organism contain the same DNA sequence. Thus, to ensure the correct transcriptional program in a 2.1

612

specific cell type, the genome is organized into accessible (open) and packed (closed) regions with the help of epigenetic regulation. In higher eukaryotic organisms with complex genomes, DNA methylation is one of such regulatory mechanisms. In mammalian cells, DNA methylation occurs predominantly at cytosine residues in cytosine--phosphate--guanine (CpG) dinucleotides and is catalyzed by the family of DNA methyltransferases (DNMTs). Hypermethylation sequences are associated with transcriptional repression, whereas hypomethylation triggers active gene transcription (Figure 1, upper panel). After replication, DNMT1 methylates



Epithelial cells (CSE)

613

Smoke affects gene transcriptional regulation

" # # #

Previously associated with lung cancer MSK1-dependent regulation of inflammatory response CS- and oxidant-mediated acetylation has implications in endothelial dysfunction; reversible by resveratrol

" H3pS10acK9; " H4K12ac " acetylated lysine

Fibroblasts (CSE)

Endothelial cells (CSE)

H3K27me1; " H3K27me2; # H3K14ac; H3K27ac; # H3K122ac; H3 K122ac + H3R128me; H4K31me2 + H4R35me2

MSK1-dependent regulation of inflammatory response

" H3S10pK9ac; " H4K12ac

" H3K27me3; #H4K16ac; # H4K20me3


Alteration in global DNA methylation

# DNMT1; " DNMT3b

?

# SERPINA1 methylation

Alteration in global DNA methylation

Potential new drug targets

# F2RL3, GPR15 methylation

" DNMT1; # DNMT3b

Cilia-shortening and mucociliary clearance dysfunction

# HDAC6 methylation

[88]

[71]

[41]

[98]

[71]

[41]

[52]

[40,50]

[43,48,49]

[51]

[58]

[57]

[47]

[42]

[40,42,45]

[44]

[41]

[41,58]

Ref.

COPD: Chronic obstructive pulmonary disease; CS: Cigarette smoke; CSE: Cigarette smoke extract; DNMT: DNA methyltransferase; HATs: Histone acetyltransferases; HDACs: Histone deacetylases; MSK1: Mitogen- and stress-activated kinase 1.

Cell culture

Histone modifications

Cell culture

DNA modifying enzymes

Blood DNA (COPD)

Repression of tumor suppressor miR-487b

" miR-487b methylation

Repression of tumor suppressor p16

" p16 promoter methylation

Biopsy DNA (smoker, COPD)

Associated with smoking and age

Repression of tumor suppressor AHRR

" AHRR methylation

Alveolar macrophages, lymphoblast DNA (COPD)

" Runx3 methylation

! biomarker

#" global DNA methylation altered

Small airway, alveolar macrophages, lymphoblast DNA (COPD)

Patient samples

! biomarker DNA methylation changes prior to altered histopathology

#" global DNA methylation altered

Mouse (CS)

Animal models

Global DNA de-methylation in COPD

# NBL2, D4Z4, and LINE-1 methylation

Relevance/main finding

Repression of tumor suppressors RASSF1A, RAR-b, miR-487b

Epigenetic modification

" miR-487b, RASSF1A, RAR-b methylation


Cell culture

DNA modifications

Evidence

Table 2. Main epigenetic modifications encountered in in vitro/in vivo COPD models or COPD patients.


614 Inflammatory gene transcription Inflammatory gene transcription HATs recruitment ! increased inflammatory gene transcription HATs recruitment ! increased inflammatory gene transcription Damage by H3.3 contributes to disease progression

" acetylated H4 " H3S10p " acetylated H4 " acetylated H3 " extracellular H3.3

Lung tissue (healthy smokers)

Lung tissue (COPD)


Induction of senescence via autophagy inhibition CS- and oxidant-mediated SIRT1 reduction has implications in endothelial dysfunction; reversible by resveratrol Theophylline, Nortriptyline, PI3K inhibitors, Curcumin restores HDAC activity and glucocorticoid sensitivity Increased inflammatory gene transcription Correlation with reduced glucocorticoid function ! restored by PI3K inhibition and theophylline SIRT1 activation may be an attractive therapeutic strategy in COPD/emphysema Worse airspace enlargement and more increase in lung compliance compared to WT Increased inflammatory gene transcription

# SIRT6 # SIRT1 # HDAC; # HDAC1; # HDAC2; # HDAC3; # SIRT1; # HDAC activity # HDAC activity; # HDAC2 activity # SIRT1 SIRT1+/# HDAC2 HDAC inhibition

Endothelial cells (CSE)

Macrophages (U937, MonoMac6) (CSE)

Mouse (CS)

Mouse (CS and elastase)

SIRT1+/- mouse (CS and elastase)

Rat (CS)

Rat (TSA)

Emphysema as consequence of increased apoptosis

Oxidant/aldehyde modification mark SIRT1 for proteasomal degradation; counteracted by resveratrol

# SIRT1; # SIRT1 activity


[78]

[68]

[92]

[92]

[79,103]

[69,89,102] [103,106,107] [109]

[88]

[96]

[87]

[97]

[67]

[67]

[68]

[68]

[98]

Ref.


Animal models

Cell culture

Smoke affects gene transcriptional regulation

Rat (CS)

H3K27me2; " H3K36me; " H3K56me2; H3K79ac; " H4K12ac; " H4K20me2; H4K31me2 + H4R35me2; " H4R35me2; H4R36me1;# H3K23me2; #H3R72me2; H4K16ac


" " " " #

Epigenetic modification

Mouse (CS)

Histone modifying enzymes

Patient samples

Animal models

Evidence

Table 2. Main epigenetic modifications encountered in in vitro/in vivo COPD models or COPD patients (continued).



[75,107,111]

Increased inflammatory response; Theophylline restores HDAC activity and glucocorticoid sensitivity # HDAC activity; # HDAC2 levels

Correlates to reduced Nrf2 levels ! impaired antioxidant defense; solithromycin reverses corticosteroid insensitivity

[76,102]

Induction of senescence via autophagy inhibition # SIRT6

# HDAC activity; # HDAC2

[96]

Increased acetylation of RelA/p65 ! SIRT1 regulates NF-kBdependent pro-inflammatory mediators # SIRT1 Lung tissue (COPD, smoker)

Peripheral blood monocytes (COPD)

[80]

Activity and glucocorticoid sensitivity restored by sulforaphane treatment # HDAC; # HDAC2 ; # HDAC2 activity

Alveolar macrophages (COPD)

[77]

Similar inflammatory processes in large and small airways # HDAC2

[89]

[67,76,93]

Decrease mediated by smoke-derived oxidative stress and correlates with disease severity Patient samples

Lung tissue (COPD)

# HDAC2; # SIRT1; # HDAC/SIRT1 activity

Relevance/main finding Epigenetic modification Evidence

Table 2. Main epigenetic modifications encountered in in vitro/in vivo COPD models or COPD patients (continued).

Ref.


hemi-methylated CpG dinucleotides in the newly formed strand of DNA. Its function is essential for maintaining DNA-methylation patterns in proliferating cells. DNMT3a and DNMT3b are required for the initiation of de novo methylation in vivo and thus for the establishment of new DNAmethylation patterns. Both DNMT enzyme classes have also been shown to interact with histone deacetylases (HDACs, see Section 2.2.) to repress transcription [39]. Thus, DNA methylation can either block the accessibility of, for example, promoter sequences for transcription factors, or transcription is repressed by methyl-CpG-binding proteins (MeBP), which can interact with HDACs or other corepressor proteins (Figure 1, upper panel). DNA modification in COPD The considerable variability in the susceptibility of smokers to develop COPD may, in part, be explained by alterations in DNA modification. Indeed, large-scale (27,000 genes) genome-wide investigations of DNA methylation marks identified 349 CpG sites to be significantly associated with COPD severity and lung function decline [40]. The CS condensate can induce a variety of CpG targets to be hypoor hypermethylation in human small airways, as well as in bronchial epithelial cells [41]. Further, lymphoblasts and pulmonary macrophages from smokers revealed genome-wide changes in DNA methylation, which were significantly associated with smoking status [42]. Small airways of COPD patients also demonstrated aberrant genome-wide DNA methylation patterns [43]. Altered DNA methylation patterns are evident upon smoke exposure prior to changes in histopathology, as demonstrated in mice [44]. Therefore, DNA methylation induced by a number of risk factors, may prime COPD susceptibility to a second insult. This is supported by findings that prenatal tobacco smoke exposure has been shown to affect global and gene-specific CpG DNA methylation, for example, in detoxification genes [45], and leads to persistent effects such as reduced lung function into adolescence [46]. CS has been demonstrated to alter gene expression via altered DNA methylation in a variety of genes. Heavy smoking has been described to correlate with increased methylation of the p16 promoter [47], or the aryl hydrocarbon receptor repressor, a known tumor suppressor [42]. Expression changes in nuclear factor erythroid 2-related factor 2 (NRF2) and Gprotein-coupled receptor 15 have been associated with smoking and altered DNA methylation in small airway cells and peripheral blood leukocytes, respectively [43,48]. The cg03636183 locus, located in the coagulation factor II receptor-like 3 gene, was significantly correlated with lower methylation in smokers [49]. Interestingly, hypomethylation of the A1AT locus significantly correlated with COPD [40,50]. A study in smoke-exposed mice revealed increased HDAC6 hypomethylation in emphysema, which correlated with autophagy-mediated cilia-shortening and mucociliary clearance dysfunction [51]. Moreover, site-specific methylation 2.1.1


615

616


Rat (CSE)

Mouse (elastase)

Mouse (CSE)

Chinese green tea

No air-space enlargement, no goblet cell hyperplasia

Protective and therapeutic effects on airspace enlargement and increase in lung compliance

SRT1720 (SIRT1 activator)

Protects against airspace enlargement and increase in lung compliance

SIRT1 overexpression

Less emphysema and less inflammatory cells

More potent than resveratrol, better fitness of mice, less neutrophils in BALF

SIRT2172 (SIRT1 activator)

Curcumin

Restoration of corticoid sensitivity and HDAC2 activity

Theophylline

Restored HDAC activity and HDAC2 expression

Curcumin

Suppression of smoke-induced increase in BALF counts

Restoration of HDAC activity and corticosteroid sensitivity

Solithromycin (CEM-101)

Andrographolide

Restoration of corticoid sensitivity

Nortriptyline, PI3K inhibitor = LY294002

De-repression of miR-487b via # methylation ! expression of tumor suppressor miR-487b Restores HDAC1/2 activity

Deoxy-azacytidine

Epithelial cells (CSE, nicotine)

Suppresses NF-kB activation and pro-survival pathways ! anti-inflammatory

Decreases oxidative stress via Nrf2 activation Restores SIRT1 levels Increases acetylated lysine


Theophylline

Epigallocatechin-3-gallate


Macrophages (U937) (CSE)

Resveratrol

Epithelial and endothelial cells (CSE)

Therapeutic

[62]

[92]

[110]

[92]

[93]

[103]

[112]

[109]

[107]

[106]

[102]

[58]

[61]

[116] [87,88] [88]

Ref.

BALF: Bronchoalveolar lavage fluid; COPD: Chronic obstructive pulmonary disease; CS: Cigarette smoke; CSE: Cigarette smoke extract; HDACs: Histone deacetylases; NRF2: nuclear factor erythroid 2-related factor 2.

Animal models

Cell culture

Epigenetic-based therapeutics

Evidence

Table 3. Epigenetic-based therapeutics tested in in vitro/in vivo COPD models or COPD patient samples.


BALF: Bronchoalveolar lavage fluid; COPD: Chronic obstructive pulmonary disease; CS: Cigarette smoke; CSE: Cigarette smoke extract; HDACs: Histone deacetylases; NRF2: nuclear factor erythroid 2-related factor 2.

[105]

Increased HDAC activity in sputum macrophages; reduction of sputum eosinophils Low-dose theophylline combined with corticosteroids

[104]

Increased HDAC activity in sputum macrophages; Improvement of antiinflammatory effects of steroids Patients trial

Low-dose theophylline combined with standard therapy

[117]

SIRT1 activation; decrease of inflammatory response (CCL-2, IL-6, IL-8) Resveratrol in vitro Bronchial smooth muscle cell (COPD)

[103]

Restoration of corticosteroid sensitivity and HDAC2 activity Theophylline, PI3K inhibitor = LY294002 in vitro Peripheral blood monocytes (COPD)

[80]

Increases HDAC activity and glucocorticoid sensitivity NRF2 dependently. Sulforaphane in vitro Lung tissue (COPD)

[102]

Increased HDAC1/2 activity and restored glucocorticoid sensitivity. Theophylline in vitro

[80]

The Nrf2 activator increases HDAC2 activity and promotes the antiinflammatory response Patient samples

Alveolar macrophages (COPD)

Sulforaphane in vitro

Relevance/main finding Therapeutic Evidence

Table 3. Epigenetic-based therapeutics tested in in vitro/in vivo COPD models or COPD patient samples (continued).

Ref.


changes persist even after smoking cessation, which may contribute to the extended risk to develop smoking-related disease [48]. Changes in DNA methylation status after smoke exposure could be explained by altered expression of DNMT. Up to now, this issue has only been partially addressed in the context of lung cancer, but not in COPD. Smokeexposed human bronchial epithelial cells demonstrated decreased DNMT1 and increased DNMT3b levels [41]. In contrast, smoke increased DNMT1 levels in A549 cells, while smoke-mediated downregulation of DNMT3b induced demethylation of prometastatic oncogene synuclein-gamma [52]. In lung tumors patients, DNMT1, DNMT3a and DNMT3b proteins were highly expressed, particularly in smokers [53]. Furthermore, the tobacco-specific carcinogen nicotine-derived nitrosamine ketone (NNK) induced DNMT1 accumulation in the nucleus, accompanied by tumor suppressor gene hypermethylation in mice and lung cancer patients who smoked continuously [54]. A rather new field of epigenetic regulation is represented by the Polycomb group (PcG) of proteins initially discovered in Drosophila melanogaster. In higher eukaryotes, PcG proteins form Polycomb repressive complexes (PRCs), which silence key regulators of development and differentiation. For more details, we would like to refer to recent reviews about Polycomb functions [55,56]. Evidence suggests that unmethylated CpG-rich DNA sequences within Polycomb response elements are involved in the recruitment of PRC2 [55]. Altered CpG methylation upon smoke exposure may affect Polycomb-mediated gene silencing. For example, the Polycomb target gene RUNX3, a tumor suppressor gene, is positively correlated with promoter methylation and smoking history [57]. In addition, it has been shown that expression of the tumor suppressor microRNA (miRNA) miR-487b is epigenetically silenced by CS condensate. CS treatment resulted in PRC-mediated DNA methylation and altered nucleosome positioning within the miR-487b region in lung epithelial and lung cancer cells [58]. Targeting DNA modification for therapy in COPD

2.1.2

As DNA modifications are clearly evident in COPD, therapeutic interventions using DNA modifying ways are possible future options in COPD. For example, nucleoside analogs such as 5-azacytidine and 5-aza-2¢-deoxycytidine act as DNMT inhibitors, by substituting cytidine in the DNA strain. DNMTs use these analogs as substrate analogs. Covalent binding then blocks enzyme activity and results in DNA hypomethylation. The analysis of highly metastatic cells, using genome-wide DNA methylation analysis, revealed that increased DNA methylation correlated with metastatic capacity. Using azacitidine (5-azacytidine), methylation of PRC2 binding sites were reversed to the non-metastatic status [59]. Recent data from an orthotopic


617


lung cancer model in rats showed that aerosolized 5-azacytidine effectively targets the lung and reprograms the lung tumor epigenome, which decreased tumor burden [60]. Azacitidine and decitabine (5-aza-2¢-deoxycytidine) are currently tested in several trials in Phase I and Phase II for the treatment of non-small cell lung cancer (NSCLC). The DNMT inhibitor epigallocatechin 3-gallate (EGCG), a polyphenol from green tea, has been demonstrated to suppress CS condensate-induced NF-kB activation in normal human bronchial epithelial cells, thus acting antiinflammatory [61]. Green tea extracts also ameliorated airspace enlargement and goblet cell hyperplasia in CS-exposed rats [62]. EGCG has been demonstrated to decrease bronchoalveolar lavage fluid (BALF) inflammatory cell numbers and lactate dehydrogenase activity, but not emphysema, in a mouse model of CS exposure. Given the paucity of experimental and clinical evidence of DNMT inhibitors to date, it is imperative to further test their suitability for COPD. Histone modification In contrast to DNA methylation, histone modification is a universal and highly conserved epigenetic regulatory mechanism among eukaryotic organisms from yeast to human. In every nucleus, the DNA strand is wrapped around a protein octamer complex, consisting of two copies each of the core histones (H2A, H2B, H3 and H4), forming the nucleosomes. With help of the linker histone H1, nucleosomes are organized into the higher ordered 30-nm solenoid structures, which are further condensed, finally forming the superstructure of a chromosome. Histone amino-terminal tails, protruding from the nucleosomes, can be modified at specific amino acids (lysine, arginine and serine) with a diverse set of posttranslational modifications (PTMs), such as phosphorylation, acetylation, methylation, ubiquitination or SUMOylation [63]. In addition, HDACs, histone demethylases and phosphatases are able to remove PTMs. Writer enzymes, such as histone acetyltransferases (HATs), histone methyltransferases, kinases and ubiquitin ligases catalyze binding of PTMs to histone tails [63]. The combination of histone modifications in a certain chromatin region determines gene activity, because it translates into either packed heterochromatin or into open euchromatin, which is accessible for the RNA polymerase II and transcription factors (Figure 1, middle panel). To do so, PTMs provide binding sites for other histone-modifying enzymes or ATP-dependent remodeling complexes, which can alter the accessibility of the chromatin [64]. To further silence chromatin regions, proteins like heterochromatin protein 1 (HP1) bind to PTMs and can recruit DNMTs (Figure 1, middle panel) [39]. The most prominent histone modifications are acetylation and methylation of lysine residues in the highly conserved amino termini of histones H3 and H4. In general, increased acetylation correlates with transcriptional activity, whereas decreased acetylation correlates with transcriptional inactivation [64]. Furthermore, methylation of histone H3 lysine 9 strongly correlates with heterochromatin assembly [64]. It is 2.2

618

strictly context-dependent, however, if certain PTMs associate with active or repressed gene expression. For example, di- and tri-methylation of H3 lysine 4 are associated with the stable expression of developmental genes [65]. 2.2.1

Histone modifications in COPD Role of histone acetylation/deacetylation in COPD

2.2.1.1

The persistent inflammation in COPD lungs is associated with increased expression of a variety of inflammatory genes encoding cytokines, chemokines or inflammatory mediators. The transcription factors NF-kB and activator protein (AP)-1 are key players for pro-inflammatory gene expression, which is tightly controlled by increased histone acetylation in the respective promoter regions. Activated NF-kB can bind to specific recognition sequences in DNA and interact with large co-activator molecules, such as CREB-binding protein (CBP), p300 or p300/CBP-associated factor, which have intrinsic HAT activities. Thus, activation of NF-kB results in increased histone acetylation contributing to inflammatory gene transcription, as shown for IL-8 [38]. NF-kB has been shown to be upregulated in bronchial biopsies of smokers and COPD patients [66], as well as in total lung homogenates of healthy smokers and COPD patients [67]. CS exposure induces a pro-inflammatory milieu in rat lungs, by activation of NF-kB and AP-1, without affecting inhibitor of kB levels [68]. Further, CS, oxidative stress or TNF-a can activate NF-kB and AP-1 in macrophages and A549 cells, resulting in enhanced IL-8 release [69,70]. Mitogen- and stress-activated kinase 1 (MSK1) is activated by CS in human and mouse primary airway epithelial cells by phosphorylation. MSK1 interacts with the NF-kB family member RelA/p65, leading to activation thereof by phosphorylation and acetylation, which results in PTM of histones, specifically phospho-acetylation of histone H3 and acetylation of histone H4 [71]. The main epigenetic modifications encountered in in vitro/in vivo COPD models or COPD patients are listed in Table 2. HDACs are key negative regulators of pro-inflammatory cytokines [37]. HDAC1 and HDAC2 interact with the RelA/ p65 subunit of NF-kB, thereby blocking transcription of inflammatory genes through histone deacetylation [72]. HDAC3 negatively regulates transcription by deacetylation of RelA itself [73]. Moreover, the phosphorylation status of nuclear NF-kB determines its association with CBP/p300 or HDAC1 [74]. In smokers, COPD patients and ex-smokers with COPD, HDAC activity is decreased in airway biopsies, peripheral blood monocytes and alveolar macrophages [67,75,76]. Diminished HDAC2 levels correlate with disease severity, with lesser reduction of HDAC3 and HDAC5 [67,76]. In smokers with COPD, the reduction in HDAC2 was more prominent in small compared to large airways [77]. Consequently, airway biopsies of COPD patients demonstrated increased histone acetylation in IL-8 promoter regions, promoting the pro-inflammatory milieu [76]. Recent data further demonstrate that HDAC inhibition can cause emphysema and that HDAC-dependent mechanisms contribute to



the maintenance of the adult lung structure [78]. CS exposure mediated decreased HDAC2, but not HDAC1, expression and activity in rat lungs after 3 days [68]. In macrophages, CS exposure let to reduced HDAC activity and decreased HDAC1, HDAC2 and HDAC3 expression levels [69]. The reduced HDAC activity was in part a result of covalent protein modifications by CS components in response to oxidative stress [68,69]. Oxidative stress thus reduces HDAC2 activity and enhances IL-8 gene expression through HDAC2 tyrosine nitration and serine phosphorylation [79], or HDAC2 cysteine S-nitrosylation [80]. In addition, an oxidative stress-dependent increase in PAI-1 levels in the sputum of COPD patients correlated with diminished HDAC2 levels and NF-kB activation, as tested in A549 cells [81]. In sum, the cellular responses to smoke lead to increased acetylation of histone H4, as found in healthy and current smokers, and to increased acetylation of histone H3 in ex-smokers with COPD. Thus, the imbalance between histone deacetylation and acetylation in favor of acetylation in promoters of inflammatory genes may contribute to the enhanced susceptibility of smokers to develop COPD [67]. One important transcription factor protective for oxidative stress is the NRF2 that promotes expression of key antioxidant enzymes. In COPD, NRF2 and DJ-1 levels (DJ-1 stabilizes NRF2 and prevents its proteasomal degradation) were found to be significantly decreased [82,83]. Moreover, decreased DJ-1 levels were demonstrated to be associated with disease severity, due to oxidative modifications leading to proteasomal degradation of DJ-1. Animal experiments using DJ-1 small interfering RNA (siRNA) revealed attenuated CS-induced NRF2 activity and antioxidant gene expression [83]. Sirtuins (SIRT1--SIRT7) are a highly conserved family of NAD+-dependent deacetylases, which are known to regulate lifespan in lower organisms. In mammals, SIRTs have recently been connected to an even wider variety of activities that encompass cellular stress resistance, genomic stability, tumorigenesis or energy metabolism. There is incomplete knowledge about target proteins that are deacetylated by SIRTs, but those described include histones, transcription factors, signaling molecules or other cellular proteins [84,85]. SIRTs are associated with age-related diseases [86] and there is emerging evidence that they also play a role in COPD development and progression. Cellular stress, such as CS, leads to covalent modification of SIRT1 by oxidants/ aldehydes and phosphorylation, which decreases its enzymatic activity and induces its proteasomal degradation [87,88]. Reduced SIRT1 levels in macrophages and lungs of smokers and patients with COPD were associated with increased acetylation of RelA/p65 and IL-8 release, and SIRT1 has been demonstrated to interact with RelA/p65 [89,90]. Thus, SIRT1 appears to play a pivotal role in the regulation of NF-kB-dependent pro-inflammatory conditions. Smoking suppresses SIRT1 expression in large airways. In COPD tissue, suppression of SIRT1 expression was found both in large and small airways [91]. A protective effect of SIRT1 against pulmonary emphysema in a FOXO3-

dependent manner in mice was demonstrated recently [92]. Hence, SIRT1 activation by a small-molecule activator blocked the increase of matrix metalloprotease-9 (MMP9) in a COPD mouse model [93]. The protective effect of SIRT1 in COPD/emphysema development appears to involve alterations of the tissue inhibitor of metalloproteinase (TIMP)-1/ MMP-9 balance with SIRT1-induced deacetylation of TIMP-1 contributing to its increased ability to inhibit MMP-9 [94]. Besides SIRT1, there is limited evidence of other SIRTs involved in COPD. Interestingly, a single-nucleotide polymorphism (rs2241704), located in the flanking regions of the NFKBIB/SIRT2 genes, correlated with COPD-related phenotypes in two cohorts [95]. Recently, SIRT6 levels have been demonstrated to be decreased in lung homogenates from COPD patients. Experiments using human bronchial epithelial cells exposed to CS extract correlated induced senescence to reduced SIRT6 expression [96]. Another mechanism potentially contributing to COPD progression is extracellular, hyperacetylated H3.3, present in the airway lumen and BALF of COPD patients. This histone isoform is resistant to proteasomal degradation and toxic to lung structural cells [97], but its relevance to COPD pathogenesis remains to be elucidated.

Role of histone methylation in COPD The gene regulation with PRCs of development and differentiation through histone modification seems to be even more important than through DNA methylation. For example, it is well-recognized that PRC1 components induce histone H2AK119 ubiquitination, which interferes with transcription elongation by RNA polymerase II. Furthermore, PRC2 components methylate histone H3K27, thereby preventing histone H3K27 acetylation that is associated with an active chromatin state. There is expanding literature about the role of PcG regulation in cancer cells. Hypersilencing of tumor suppressors is thought to be mediated by PcG gain-of-function, while PcG loss-of-function might be involved in T-cell and myeloid leukemia [56]. Concerning COPD, a recent mass spectrometry approach in smoke-exposed mouse lungs identified potentially novel histone marks, including acetylation, mono-methylation and di-methylation, of specific lysine and arginine residues of histones H3 and H4 [98]. Among others, histone H3K27me1 and histone H3K27me2 were only detected in the CS exposure group. This suggests that Polycomb might be involved in specific gene repression, especially after smoke exposure. Moreover, CS was shown to induce Polycomb-mediated repression of Dickkopf-1 in lung cancer cells via increased histone H3K27me3 levels and decreased histone H4K16ac levels, probably regulated by SIRT1 [99]. In accordance with this, SIRT1 and its homolog Sir2 have previously been shown to be part of a PRC complex in human cancer cells and D. melanogaster larvae, respectively [55]. 2.2.1.2


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Targeting histone modification for therapy in COPD

2.2.2

An important anti-inflammatory therapy of patients with COPD is the treatment with inhaled or systemic corticosteroids. However, one major barrier for effective disease management is that a subgroup of COPD patients is resistant to even high doses of corticosteroids. The mechanisms leading to corticosteroid resistance have been extensively reviewed in detail recently [100,101], these include impaired HDAC function. Activated glucocorticoid receptors (GR) bind to co-activators such as CBP or p300/CBP and can inhibit their HAT activity. In addition, GR recruits HDAC2 to the complex, which reverses the acetylation of activated inflammatory genes and switches off inflammatory gene transcription. In COPD, however, decreased levels of HDAC2 impair the anti-inflammatory effect of corticosteroids. Thus, HDAC2 activators have been suggested as beneficial therapeutics to overcome steroid resistance in COPD. Accordingly, epigenetic-based therapeutics are summarized in Table 3. Theophylline has been used to treat airway diseases for many centuries and was recently discovered to act as an HDAC activator. Theophylline restores HDAC activity and steroid response in alveolar macrophages from COPD patients [102]. Low-dose theophylline enhanced the antiinflammatory effect of dexamethasone, with concomitant restoration of HDAC2 activity in CS-exposed mice. The action of theophylline seems to be mediated by direct inhibition of oxidant stress-activated PI3Kd [103]. In patients, low-dose theophylline enhances the anti-inflammatory effects of steroids during exacerbations of COPD (trial registration number: NCT00671151) [104]. Further, a combined treatment with inhaled corticosteroids attenuates airway inflammation in patients with COPD (trial registration number: NCT00241631) [105]. The antidepressant nortriptyline was also found to be a direct PI3Kd inhibitor, resulting in restoration of HDAC activity and prevention of CSE-induced corticosteroid insensitivity in human macrophages [106]. Smokinginduced airway inflammation in mice was inhibited in PI3Kd-mutant or knockout mice and accompanied with restored glucocorticoid function [79]. Thus, PI3Kd inhibitors translate into HDAC activation, which is relevant for therapeutic intervention in COPD. A clinical trial of a potent and highly selective inhaled PI3Kd inhibitor, GSK2269557, for COPD treatment is currently in Phase I (trial registration number: NCT01762878). Further, a novel macrolide/fluoroketolide, Solithromycin (CEM-101), restored corticosteroid sensitivity by inhibition of PI3K signaling at conditions of oxidative stress and was accompanied by restored HDAC activity in human macrophages [107]. Curcumin is a dietary polyphenol that has been reported to possess anti-cancer, anti-inflammatory and anti-oxidant properties [108]. In human macrophages, curcumin reverses CSE-induced serine phosphorylation and ubiquitination of HDAC2, thus preventing HDAC2 proteasomal degradation 620

[109]. It has also been demonstrated that curcumin attenuates elastase- and CS-induced pulmonary emphysema in mice [110]. The effect of curcumin on lung inflammation is currently tested in a clinical trial (trial registration number: NCT01514266). Pharmacological activation of the antioxidant NRF2 using sulforaphane, a small-molecule activator, may be a useful strategy for preventing and treating COPD and is currently tested in a Phase II clinical trial in COPD patients (trial registration number: NCT01335971). Functional HDAC2 prevents NRF2 degradation probably by deacetylation of lysine residues that would otherwise target ubiquitinmediated proteasomal degradation [111]. Decreased HDAC2 activity was associated with S-nitrosylation of the enzyme in peripheral lung tissues and alveolar macrophages from patients with COPD [80]. Of note, sulforaphane treatment denitrosylated HDAC2 with concomitant activation of NRF2 and increased intracellular glutathione levels in alveolar macrophages from CS-exposed mice and COPD patients [80]. Further, sulforaphane treatment restored glucocorticoid resistance in alveolar macrophages. Andrographolide, another antioxidant, possesses anti-oxidative properties against CS-induced lung injury probably via augmentation of NRF2 activity [112]. Chalcones have been reported to possess potent anti-oxidant properties by activating NRF2 in mice and human lung epithelial cells [113] and to block human neutrophil elastase release [114]. Selective activation of SIRT1 using the pharmacological activator SRT1720 has been demonstrated to protect against CS and elastase-induced emphysema in mice [92]. Activation of SIRT1 via SRT2172 significantly inhibited pulmonary neutrophil accumulation, and completely restored exercise tolerance and the fall in oxygen saturation with exercise in a COPD mouse model [93]. Resveratrol, a substance shown to activate SIRT1 [115], exhibits anti-oxidant, anti-inflammatory and anticarcinogenic properties. Resveratrol attenuates CSE-mediated glutathione depletion through reversing CSE-mediated NRF2 carbonylation in A549 cells [116]. Due to a recent study in human airway smooth muscle cells, resveratrol is suggested as an anti-inflammatory therapy alternative to corticosteroids in COPD, particularly in COPD exacerbations [117]. There is emerging evidence, that recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins can be inhibited using synthetic compounds that mimic acetylated histones. Histone acetylation-driven inflammatory gene expression has been shown to be suppressed in macrophages [118]. Furthermore, BET inhibition is demonstrated to be anti-tumorigenic in cancer, including NSCLC and adenocarcinoma [119,120].

Non-coding RNAs ncRNAs are RNAs that are transcribed from DNA and are not translated into proteins. Many of those RNAs are functional and involved in processing and regulation of other RNAs, such as mRNA, rRNA or tRNA. The ncRNAs can 2.3



be divided into two main groups: the long ncRNAs and short ncRNAs. The long ncRNAs were first described during large-scale sequencing of full-length cDNA libraries in the mouse [121]. The functional role of long ncRNAs includes regulation of chromosomal dynamics, telomere biology, as well as subcellular structural organization [122]. Importantly, long ncRNAs are also involved in recruitment of chromatin remodeling complexes to specific genomic loci and can therefore regulate gene expression at the level of chromatin modification (Figure 1, lower panel). Long ncRNAs can, for example, trap transcription factors, recruit DNMTs or PRCs, to promote gene silencing. Further, long ncRNAs can also act as cofactors of transcription factors and interact with basal components of the RNA polymerase II-dependent transcription machinery to promote active gene transcription [123]. Small ncRNAs are RNAs that are < 200 nucleotides in length. There are three major classes of small ncRNAs: miRNA, siRNA and PIWI-interacting RNA (piRNA). Their silencing mechanisms are widely conserved and typically involve proteins of the Argonaute family. Importantly, these small ncRNAs regulate not only gene expression on posttranscriptional and transcriptional levels, but can also interfere with chromatin modifications and organization, which mainly accounts for siRNA and piRNA [124,125]. miRNAs are one member of small ncRNAs approximately 18 -- 25 nucleotides long that are well known for modulating both, gene transcript and protein levels, by either destabilizing mRNA transcripts or directly inhibiting the translation of mRNA targets [126-129]. Thus, strictly speaking most miRNAs are no epigenetic regulators but might be regulated by epigenetic mechanism. A given ncRNA can regulate hundreds of transcripts whose effector molecules can function at various sites within cellular pathways. ncRNAs in COPD Although, miRNAs play an important role in maintaining homeostasis during lung development, recent reports have provided evidence that miRNAs are involved in various aspects of the acquired and innate immune responses, and therefore are indirectly involved in the development of inflammatory lung diseases, including COPD [130-132]. Several studies have identified that CS induces a general downregulation of miRNAs [133-135]. Seventy miRNAs were found differentially expressed in lung tissue of COPD patients and healthy smokers [132,134]. In the same study, the authors identified miRNA expression profiles involved in the regulation of Wnt, TGF-b and focal adhesions pathways, which might be relevant to the pathogenesis of COPD. A general downregulation of the miRNA expression was found in alveolar macrophages derived from smokers compared to non-smokers. Significantly lower expression of miR-452 has been linked to increased expression of matrix metalloproteinase 12 (MMP12), which is known to be an important effector of smoking-related diseases [136,137]. Christenson et al., reported recently that miRNA profiles are altered with regional emphysema severity and can modulate 2.3.1

disease-associated gene expression networks [138]. The authors have found that pathway gene sets involved in cell surface signaling and ECM maintenance were inversely correlated with the expression of multiple upregulated miRNAs. Particularly, this study identified a subset of miRNAs, including miR-30c, miR-181d and miR-638, which expression levels were associated with those of their mRNA targets. They also propose that miR-638 may play an important role in COPD pathology by responding to oxidative stress, and, as such, to contribute to accelerated lung aging. Targeting ncRNAs for therapy in COPD Given the important role of miRNAs in disease development, miRNA profiles may provide a more accurate survival prediction than the expression profiles of protein-coding genes. Recent studies have demonstrated that expression profiles of circulating miRNAs could be promising biomarkers for early disease detection, prognosis or development of new therapeutic targets [139-141]. Last year, Mirna Therapeutics has already initiated a Phase I clinical study of MRX34, the first miRNA designed for human cancer clinical trial (trial registration number: NCT01829971). According to publicly available information, several miRNAs are currently in preclinical development for cancer (miR34 and let-7) and chronic heart failure (miR208/499), and one miRNA (miR-122) in Phase II clinical trials for hepatitis C. Besides, several miRNAs that play important roles in tissue differentiation are often epigenetically downregulated, particularly in tumor tissues. These miRNAs are usually silenced by aberrant DNA or histone methylation or even located in unstable chromosomal regions [142,143]. Thus, miRNA re-expression by treatment with HDAC inhibitors, demethylating compounds or differentiating agents might counteract tumorigenesis. Such epigenetic approaches could have a significant therapeutic value, not only in cancer treatment, but also in other diseases where miRNA expression profile is altered such as COPD. Interestingly, a recent study described that the altered miRNA expression profile in COPD lung fibroblasts could be almost completely reversed by reprogramming these somatic cells to induced pluripotent stem (iPS) cells [144]. Stem cell/progenitor cell maintenance and differentiation are tightly controlled by epigenetic mechanisms, which can be influenced by environmental cues and endogenous factors, as recently reviewed for cardiac progenitor cells [145]. Endothelial progenitor cell dysfunction from smokers and COPD patients has recently been linked to epigenetic regulation [146]. Thus, targeting the progenitor cells with its regenerative capacity or the application of iPS cells with a reprogrammed and ‘non-diseased’ epigenetic status could be a promising future approach for COPD therapy. 2.3.2

3.

Expert opinion

Given the emerging evidence of epigenetic changes in COPD, a clear potential for the development of therapeutic


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Table 4. Clinical trials* for epigenetic therapy in COPD compared to lung cancer. Level of epigenetic regulation DNMTs

Lung cancer

DNMT inhibitors

COPD

n/a

Azacitidine NCT00006019 -- Phase II NCT01928576 -- Phase IIz (recruiting) Decitabine NCT00037817 -- Phase Iz NCT00084981 -- Phase Iz Hydralazine NCT00996060 -- Phase Iz (active, not recruiting) Polyphenon E NCT00707252 -- Phase I/II NCT00363805 -- Phase II HDACs

HDAC inhibitors

HDAC activators

Vorinostat NCT00667082 -- Phase I NCT00481078 -- Phase II

Theophylline NCT00671151 NCT00241631 NCT00634413 NCT00299858 NCT01010178 NCT01415518

Romidepsin NCT00037817 -- Phase Iz NCT00086827 -- Phase II

Phase Phase Phase Phase Phase

II [105] II II/III IV IV

GSK2269557 NCT01762878 -- Phase I

Sodium Phenylbutyrate NCT00006019 -- Phase IIz

Curcumin NCT01514266

Panobinostat NCT01222936 -- Phase II NCT00535951 -- Phase I

Sulforaphane NCT01335971 -- Phase II (ongoing, not recruiting)

Belinostat NCT00926640 -- Phase I (recruiting) Tacedinaline NCT00005093 -- Phase III Chidamide NCT01836679 -- Phase II (recruiting) CUDC-101 NCT01171924 -- Phase I n/a

n/a

*Selected completed or ongoing clinical trials from www.clinicaltrial.gov. z Clinical trial for combination therapy of DNMT inhibitor and HDAC inhibitor. COPD: Chronic obstructive pulmonary disease; DNMT: DNA methyltransferase; HDACs: Histone deacetylases.

622

------

Valproic acid NCT00084981 -- Phase Iz NCT01203735 -- Phase I/II (recruiting)

Entinostat NCT00750698 -- Phase II NCT01928576 -- Phase IIz (recruiting)

Non-coding RNA

[104]



interventions on an epigenetic level has emerged. On the level of DNA modification, recent studies demonstrate an association of global, as well as gene-specific, CpG DNA methylation patterns with COPD. These changes in DNA methylation apparently take place prior to the histopathological changes of COPD and are maintained even after smoking cessation. Changes in DNMT levels likely cause these methylation changes, but this causality remains to be proven. On the level of histone modifications, a reduction of HDAC expression levels and activities was reproducibly found in airway biopsies or macrophages of smokers, COPD patients or ex-smokers with COPD. A range of HDAC activators, which act either through activating the anti-oxidative master regulator NRF2 or inhibiting oxidative stress-activated PI3Kd, are therefore tested in current clinical trials (Table 4). Further evidence suggests that selective SIRT1 activators are beneficial in emphysema models. Changes in histone-modifying enzymes, as well as DNA methylation, in COPD suggest that combination therapies with HDAC activators and DNMT inhibitors are a promising approach for future treatment. Combination therapies are currently successfully used in COPD, for example, using corticosteroids and bronchodilators (Table 1). Of caution, one should not underestimate the potential risks of HDAC activators for the following reasons: COPD patients frequently develop lung cancer, as cigarette smoking is a leading risk factor for both diseases. Enhanced HDAC activity contributes to tumorigenesis, as inhibition of HDACs and SIRTs using specific inhibitors emerged to be beneficial in different types of cancer. Inhibition of SIRT2, for example, has recently been shown to be pro-apoptotic in NSCLC [147]. A large number of HDAC inhibitors are currently in clinical trial for cancer treatment (Table 4) [148]. The HDAC pan-inhibitor vorinostat is FDA-approved since 2006 for the treatment of cutaneous Tcell lymphoma and is currently tested for combination therapy with the proteasome inhibitor NPI-0052 for NSCLC treatment (trial registration number: NCT00667082). Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

2.

WHO. Global Initiative for Chronic Obstructive Lung Disease (GOLD). Global Strategy for Diagnosis, Management, and Prevention of COPD. 2013. Available from: http://www. goldcopd.org/uploads/users/files/ GOLD_Report_2013_Feb20.pdf [cited 13 January 2014] WHO. Chronic obstructive pulmonary disease (COPD). 2013. Fact sheet N 315. Available from: http:// www.who.int/mediacentre/factsheets/ fs315/en/ [cited 13 January 2014]

Thus, HDAC activators for COPD treatment should be used with caution to avoid facilitating carcinogenesis in these patients. Descriptive data suggest that enhanced methylation is involved both in the pathogenesis of COPD and lung cancer. As such, DNMT inhibitors (e.g., azacitidine), which are in clinical trial for NSCLC treatment, could present as a promising combination therapy for COPD on top of existing regimes. The feasibility of this concept urgently requires experimental validation in appropriate animal models. The value of miRNAs as potential therapeutic targets in different diseases is nowadays widely recognized. However, serious challenges represent the absence of an effective delivery system, as well as safety of these molecules in vivo. Table 4 displays drugs currently used in clinical trials for cancer, as well as COPD. This table makes it evident that research of epigenetics in COPD is still in its infancy, despite the fact that HDAC activators are developed for COPD treatment. We are still lacking detailed knowledge about the underlying mechanism of DNMTs, HDACs or ncRNAs in COPD onset and progression. Thus, studies unraveling the causal contributions of these epigenetic regulators in COPD animal models, as well complex phenotypic assays are needed to further define the potential of epigenetic modifiers in COPD in the future.

Declaration of interest The authors are supported by the Helmholtz Zentrum Mu¨nchen. O Eickelberg is the Chairman of Comprehensive Pneumology Center (CPC), Director of Institute of Lung Biology and Disease (iLBD) and Vice Chairman of the German Center for Lung Research (DZL). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

3.

WHO. The global burden of disease: 2004 update. 2013. Available from: http://www.who.int/healthinfo/ global_burden_disease/ GBD_report_2004update_full.pdf [cited 13 January 2014]

6.

Lopez Varela MV, Montes de Oca M, Halbert R, et al. Comorbidities and health status in individuals with and without COPD in five latin american cities: the PLATINO study. Arch Bronconeumol 2013;49(11):468-74

4.

Buist AS, McBurnie MA, Vollmer WM, et al. International variation in the prevalence of COPD (the BOLD Study): a population-based prevalence study. Lancet 2007;370(9589):741-50

7.

WHO. World health statistics 2008. 2008. Available from: http://www.who. int/whosis/whostat/EN_WHS08_Full.pdf [cited 11 September 2012]

8.

5.

Menezes AM, Perez-Padilla R, Jardim JR, et al. Chronic obstructive pulmonary disease in five Latin American cities (the PLATINO study): a prevalence study. Lancet 2005;366(9500):1875-81

WHO. Projections of mortality and causes of death, 2015 and 2030. Available from: http://www.who.int/ entity/healthinfo/global_burden_disease/ GHE_DthGlobal_Proj_2015_2030.xls [cited 14 January 2014]


623


9.

Halbert RJ, Natoli JL, Gano A, et al. Global burden of COPD: systematic review and meta-analysis. Eur Respir J 2006;28(3):523-32

10.

Lundback B, Lindberg A, Lindstrom M, et al. Not 15 but 50% of smokers develop COPD? -- Report from the obstructive lung disease in Northern Sweden studies. Respir Med 2003;97(2):115-22

11.

Salvi SS, Barnes PJ. Chronic obstructive pulmonary disease in non-smokers. Lancet 2009;374(9691):733-43

12.

Toren K, Jarvholm B. Occupational exposure to vapors, gases, dusts and fumes and mortality in relation to chronic obstructive pulmonary disease among Swedish construction workers - a longitudinal cohort study. Chest 2013. [Epub ahead of print]

13.

14.

15.

16.

17.

18.

624

19.

20.

United Nations DoEaSA, Population Division. World Population Prospects: the 2012 Revision, Volume I: Comprehensive Tables ST/ESA/SER.A/ 336. 2013. Available from: http://esa.un. org/wpp/Documentation/pdf/ WPP2012_Volume-I_ComprehensiveTables.pdf [cited 18 February 2014] United Nations DoEaSA, Population Division. World Population Prospects: the 2012 Revision, Volume II: Demographic Profiles. ST/ESA/SER.A/ 345. 2013. Available from: http://esa.un. org/wpp/Documentation/pdf/ WPP2012_Volume-II-DemographicProfiles.pdf [cited 18 February 2014]

31.

Brix TH, Hegedus L. Twin studies as a model for exploring the aetiology of autoimmune thyroid disease. Clin Endocrinol (Oxf) 2012;76(4):457-64

32.

Runyon RS, Cachola LM, Rajeshuni N, et al. Asthma discordance in twins is linked to epigenetic modifications of T cells. PLoS One 2012;7(11):e48796

33.

Givelber RJ, Couropmitree NN, Gottlieb DJ, et al. Segregation analysis of pulmonary function among families in the Framingham Study. Am J Respir Crit Care Med 1998;157(5 Pt 1):1445-51

34.

Tager I, Tishler PV, Rosner B, et al. Studies of the familial aggregation of chronic bronchitis and obstructive airways disease. Int J Epidemiol 1978;7(1):55-62

35.

Belinsky SA, Palmisano WA, Gilliland FD, et al. Aberrant promoter methylation in bronchial epithelium and sputum from current and former smokers. Cancer Res 2002;62(8):2370-7

21.

Foreman MG, Campos M, Celedon JC. Genes and chronic obstructive pulmonary disease. Med Clin North Am 2012;96(4):699-711

Ezzati M, Hoorn SV, Lopez AD, et al. Comparative quantification of mortality and burden of disease attributable to selected risk factors. Chapter 4. In: Lopez AD, Mathers CD, Ezzati M, Jamison DT, Murray CJL, editors. Global burden of disease and risk factors. Oxford University Press and The World Bank, Washington, DC; 2006

22.

Stoller JK, Aboussouan LS. Alpha1-antitrypsin deficiency. Lancet 2005;365(9478):2225-36

23.

Hogg JC, Timens W. The pathology of chronic obstructive pulmonary disease. Annu Rev Pathol 2009;4:435-59

36.

24.

Tuder RM, Petrache I. Pathogenesis of chronic obstructive pulmonary disease. J Clin Invest 2012;122(8):2749-55

Barnes PJ. Reduced histone deacetylase in COPD: clinical implications. Chest 2006;129(1):151-5

37.

Lopez Varela MV, Montes de Oca M, Halbert RJ, et al. Sex-related differences in COPD in five Latin American cities: the PLATINO study. Eur Respir J 2010;36(5):1034-41

25.

Schamberger AC, Mise N, Jia J, et al. Cigarette smoke-induced disruption of bronchial epithelial tight junctions is prevented by transforming growth factor-beta. Am J Respir Cell Mol Biol 2013. [Epub ahead of print]

Adcock IM, Tsaprouni L, Bhavsar P, Ito K. Epigenetic regulation of airway inflammation. Curr Opin Immunol 2007;19(6):694-700

38.

Barnes PJ, Adcock IM, Ito K. Histone acetylation and deacetylation: importance in inflammatory lung diseases. Eur Respir J 2005;25(3):552-63

39.

Li E. Chromatin modification and epigenetic reprogramming in mammalian development. Nat Rev Genet 2002;3(9):662-73

40.

Qiu W, Baccarelli A, Carey VJ, et al. Variable DNA methylation is associated with chronic obstructive pulmonary disease and lung function. Am J Respir Crit Care Med 2012;185(4):373-81 A large-scale analysis suggesting DNA methylation as biomarker for chronic obstructive pulmonary disease (COPD).

Silverman EK, Weiss ST, Drazen JM, et al. Gender-related differences in severe, early-onset chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2000;162(6):2152-8 Foreman MG, Zhang L, Murphy J, et al. Early-onset chronic obstructive pulmonary disease is associated with female sex, maternal factors, and African American race in the COPDGene Study. Am J Respir Crit Care Med 2011;184(4):414-20

26.

Heijink IH, Brandenburg SM, Postma DS, van Oosterhout AJ. Cigarette smoke impairs airway epithelial barrier function and cell-cell contact recovery. Eur Respir J 2012;39(2):419-28

27.

Russo VEA, Martienssen RA, Riggs AD. Epigenetic mechanisms of gene regulation. Cold Spring Harbor Laboratory Press; Plainview, NY; 1996

28.

Puck JM, Willard HF. X inactivation in females with X-linked disease. N Engl J Med 1998;338(5):325-8

..

Talens RP, Christensen K, Putter H, et al. Epigenetic variation during the adult lifespan: cross-sectional and longitudinal data on monozygotic twin pairs. Aging Cell 2012;11(4):694-703

41.

Liu F, Killian JK, Yang M, et al. Epigenomic alterations and gene expression profiles in respiratory epithelia exposed to cigarette smoke condensate. Oncogene 2010;29(25):3650-64

42.

Monick MM, Beach SR, Plume J, et al. Coordinated changes in AHRR methylation in lymphoblasts and

Gingo MR, Morris A, Crothers K. Human immunodeficiency virusassociated obstructive lung diseases. Clin Chest Med 2013;34(2):273-82

29.

Lee CH, Lee MC, Lin HH, et al. Pulmonary tuberculosis and delay in anti-tuberculous treatment are important risk factors for chronic obstructive pulmonary disease. PLoS One 2012;7(5):e37978

30.

Fraga MF, Ballestar E, Paz MF, et al. Epigenetic differences arise during the lifetime of monozygotic twins. Proc Natl Acad Sci USA 2005;102(30):10604-9



pulmonary macrophages from smokers. Am J Med Genet B Neuropsychiatr Genet 2012;159B(2):141-51 43.

44.

45.

.

46.

47.

48.

49.

50.

51.

.

Vucic EA, Chari R, Thu KL, et al. DNA methylation is globally disrupted and associated with expression changes in COPD small airways. Am J Respir Cell Mol Biol 2013. [Epub ahead of print] Phillips JM, Goodman JI. Inhalation of cigarette smoke induces regions of altered DNA methylation (RAMs) in SENCAR mouse lung. Toxicology 2009;260(1-3):7-15 Breton CV, Byun HM, Wenten M, et al. Prenatal tobacco smoke exposure affects global and gene-specific DNA methylation. Am J Respir Crit Care Med 2009;180(5):462-7 In utero tobacco smoke exposure triggers life-long effects through altered DNA methylation patterns. Hollams EM, de Klerk NH, Holt PG, Sly PD. Persistent effects of maternal smoking during pregnancy on lung function and asthma in adolescents. Am J Respir Crit Care Med 2014;189(4):401-7 Georgiou E, Valeri R, Tzimagiorgis G, et al. Aberrant p16 promoter methylation among Greek lung cancer patients and smokers: correlation with smoking. Eur J Cancer Prev 2007;16(5):396-402 Wan ES, Qiu W, Baccarelli A, et al. Cigarette smoking behaviors and time since quitting are associated with differential DNA methylation across the human genome. Hum Mol Genet 2012;21(13):3073-82 Breitling LP, Yang R, Korn B, et al. Tobacco-smoking-related differential DNA methylation: 27K discovery and replication. Am J Hum Genet 2011;88(4):450-7 Siedlinski M, Klanderman B, Sandhaus RA, et al. Association of cigarette smoking and CRP levels with DNA methylation in alpha-1 antitrypsin deficiency. Epigenetics 2012;7(7):720-8 Lam HC, Cloonan SM, Bhashyam AR, et al. Histone deacetylase 6-mediated selective autophagy regulates COPDassociated cilia dysfunction. J Clin Invest 2013;123(12):5212-30 Epigenetically regulation of histone deacetylase 6 (HDAC6) triggers cilia dysfunction in COPD.

52.

53.

54.

55.

..

56.

..

57.

Liu H, Zhou Y, Boggs SE, et al. Cigarette smoke induces demethylation of prometastatic oncogene synucleingamma in lung cancer cells by downregulation of DNMT3B. Oncogene 2007;26(40):5900-10 Lin RK, Hsu HS, Chang JW, et al. Alteration of DNA methyltransferases contributes to 5’CpG methylation and poor prognosis in lung cancer. Lung cancer 2007;55(2):205-13 Lin RK, Hsieh YS, Lin P, et al. The tobacco-specific carcinogen NNK induces DNA methyltransferase 1 accumulation and tumor suppressor gene hypermethylation in mice and lung cancer patients. J Clin Invest 2010;120(2):521-32 Schwartz YB, Pirrotta V. A new world of Polycombs: unexpected partnerships and emerging functions. Nat Rev Genet 2013;14(12):853-64 An excellent review about polycomb function. Simon JA, Kingston RE. Occupying chromatin: polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put. Mol Cell 2013;49(5):808-24 An excellent review about polycomb function. Wolff EM, Liang G, Cortez CC, et al. RUNX3 methylation reveals that bladder tumors are older in patients with a history of smoking. Cancer Res 2008;68(15):6208-14

58.

Xi S, Xu H, Shan J, et al. Cigarette smoke mediates epigenetic repression of miR-487b during pulmonary carcinogenesis. J Clin Invest 2013;123(3):1241-61

59.

Hascher A, Haase AK, Hebestreit K, et al. DNA methyltransferase inhibition reverses epigenetically embedded phenotypes in lung cancer preferentially affecting polycomb target genes. Clin Cancer Res 2014;20(4):814-26

60.

Reed MD, Tellez CS, Grimes MJ, et al. Aerosolised 5-azacytidine suppresses tumour growth and reprogrammes the epigenome in an orthotopic lung cancer model. Br J Cancer 2013;109(7):1775-81

61.

Syed DN, Afaq F, Kweon MH, et al. Green tea polyphenol EGCG suppresses cigarette smoke condensate-induced NF-kappaB activation in normal human bronchial epithelial cells. Oncogene 2007;26(5):673-82 Expert Opin. Drug Discov. (2014) 9(6)

62.

.

Chan KH, Ho SP, Yeung SC, et al. Chinese green tea ameliorates lung injury in cigarette smoke-exposed rats. Respir Med 2009;103(11):1746-54 Antioxidants are protective for cigarette smoke-induced COPD.

63.

Helin K, Dhanak D. Chromatin proteins and modifications as drug targets. Nature 2013;502(7472):480-8

64.

Grewal SI, Moazed D. Heterochromatin and epigenetic control of gene expression. Science 2003;301(5634):798-802

65.

Feil R, Fraga MF. Epigenetics and the environment: emerging patterns and implications. Nat Rev Genet 2011;13(2):97-109

66.

Di Stefano A, Caramori G, Oates T, et al. Increased expression of nuclear factor-kappaB in bronchial biopsies from smokers and patients with COPD. Eur Respir J 2002;20(3):556-63

67.

Szulakowski P, Crowther AJ, Jimenez LA, et al. The effect of smoking on the transcriptional regulation of lung inflammation in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2006;174(1):41-50

68.

Marwick JA, Kirkham PA, Stevenson CS, et al. Cigarette smoke alters chromatin remodeling and induces proinflammatory genes in rat lungs. Am J Respir Cell Mol Biol 2004;31(6):633-42

69.

Yang SR, Chida AS, Bauter MR, et al. Cigarette smoke induces proinflammatory cytokine release by activation of NF-kappaB and posttranslational modifications of histone deacetylase in macrophages. Am J Physiol Lung Cell Mol Physiol 2006;291(1):L46-57

70.

Rahman I, Gilmour PS, Jimenez LA, MacNee W. Oxidative stress and TNF-alpha induce histone acetylation and NF-kappaB/AP-1 activation in alveolar epithelial cells: potential mechanism in gene transcription in lung inflammation. Mol Cell Biochem 2002;234-235(1-2):239-48

71.

Sundar IK, Chung S, Hwang JW, et al. Mitogen- and stress-activated kinase 1 (MSK1) regulates cigarette smokeinduced histone modifications on NF-kappaB-dependent genes. PLoS One 2012;7(2):e31378

72.

Ashburner BP, Westerheide SD, Baldwin AS Jr. The p65 (RelA) subunit

625


of NF-kappaB interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression. Mol Cell Biol 2001;21(20):7065-77 73.

Chen L, Fischle W, Verdin E, Greene WC. Duration of nuclear NFkappaB action regulated by reversible acetylation. Science 2001;293(5535):1653-7

74.

Zhong H, May MJ, Jimi E, Ghosh S. The phosphorylation status of nuclear NF-kappa B determines its association with CBP/p300 or HDAC-1. Mol Cell 2002;9(3):625-36

75.

76.

..

77.

78.

79.

80.

.

81.

82.

626

Chen Y, Huang P, Ai W, et al. Histone deacetylase activity is decreased in peripheral blood monocytes in patients with COPD. J Inflamm 2012;9:10 Ito K, Ito M, Elliott WM, et al. Decreased histone deacetylase activity in chronic obstructive pulmonary disease. N Engl J Med 2005;352(19):1967-76 This article demonstrates for the first time decreased HDAC activity in COPD patients. Isajevs S, Taivans I, Svirina D, et al. Patterns of inflammatory responses in large and small airways in smokers with and without chronic obstructive pulmonary disease. Respiration 2011;81(5):362-71

equilibrium in pulmonary emphysema. Thorax 2008;63(10):916-24 83.

84.

..

85.

..

Houtkooper RH, Pirinen E, Auwerx J. Sirtuins as regulators of metabolism and healthspan. Nat Rev Mol Cell Biol 2012;13(4):225-38 This is an excellent review about the role and function of sirtuins. Lavu S, Boss O, Elliott PJ, Lambert PD. Sirtuins -- novel therapeutic targets to treat age-associated diseases. Nat Rev Drug Discov 2008;7(10):841-53

87.

Caito S, Rajendrasozhan S, Cook S, et al. SIRT1 is a redox-sensitive deacetylase that is post-translationally modified by oxidants and carbonyl stress. FASEB J 2010;24(9):3145-59

88.

Marwick JA, Caramori G, Stevenson CS, et al. Inhibition of PI3Kdelta restores glucocorticoid function in smokinginduced airway inflammation in mice. Am J Respir Crit Care Med 2009;179(7):542-8

89.

Malhotra D, Thimmulappa RK, Mercado N, et al. Denitrosylation of HDAC2 by targeting Nrf2 restores glucocorticosteroid sensitivity in macrophages from COPD patients. J Clin Invest 2011;121(11):4289-302 Smoke-induced covalent modification of HDACs reduce its activity.

90.

Goven D, Boutten A, Lecon-Malas V, et al. Altered Nrf2/Keap1-Bach1

Finkel T, Deng CX, Mostoslavsky R. Recent progress in the biology and physiology of sirtuins. Nature 2009;460(7255):587-91 This is an excellent review about the role and function of sirtuins.

86.

Mizuno S, Yasuo M, Bogaard HJ, et al. Inhibition of histone deacetylase causes emphysema. Am J Physiol Lung Cell Mol Physiol 2011;300(3):L402-13

To M, Takagi D, Akashi K, et al. Sputum plasminogen activator inhibitor1 elevation by oxidative stress-dependent nuclear factor-kappaB activation in COPD. Chest 2013;144(2):515-21

Malhotra D, Thimmulappa R, Navas-Acien A, et al. Decline in NRF2-regulated antioxidants in chronic obstructive pulmonary disease lungs due to loss of its positive regulator, DJ-1. Am J Respir Crit Care Med 2008;178(6):592-604

91.

92.

Arunachalam G, Yao H, Sundar IK, et al. SIRT1 regulates oxidant- and cigarette smoke-induced eNOS acetylation in endothelial cells: role of resveratrol. Biochem Biophys Res Commun 2010;393(1):66-72 Rajendrasozhan S, Yang SR, Kinnula VL, Rahman I. SIRT1, an antiinflammatory and antiaging protein, is decreased in lungs of patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2008;177(8):861-70 Yeung F, Hoberg JE, Ramsey CS, et al. Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase. EMBO J 2004;23(12):2369-80 Isajevs S, Strazda G, Kopeika U, Taivans I. Different patterns of lung sirtuin expression in smokers with and without chronic obstructive pulmonary disease. Medicina (B Aires) 2012;48(10):552-7 Yao H, Chung S, Hwang JW, et al. SIRT1 protects against emphysema via FOXO3-mediated reduction of


.

premature senescence in mice. J Clin Invest 2012;122(6):2032-45 Activation of SIRT1 may be an attractive therapeutic strategy in COPD/emphysema.

93.

Nakamaru Y, Vuppusetty C, Wada H, et al. A protein deacetylase SIRT1 is a negative regulator of metalloproteinase-9. FASEB J 2009;23(9):2810-19

94.

Yao H, Hwang JW, Sundar IK, et al. SIRT1 redresses the imbalance of tissue inhibitor of matrix metalloproteinase-1 and matrix metalloproteinase-9 in the development of mouse emphysema and human COPD. Am J Physiol Lung Cell Mol Physiol 2013;305(9):L615-24

95.

Bakke PS, Zhu G, Gulsvik A, et al. Candidate genes for COPD in two large data sets. Eur Respir J 2011;37(2):255-63

96.

Takasaka N, Araya J, Hara H, et al. Autophagy induction by SIRT6 through attenuation of insulin-like growth factor signaling is involved in the regulation of human bronchial epithelial cell senescence. J Immunol 2014;192(3):958-68

97.

Barrero CA, Perez-Leal O, Aksoy M, et al. Histone 3.3 participates in a selfsustaining cascade of apoptosis that contributes to the progression of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2013;188(6):673-83

98.

Sundar IK, Nevid MZ, Friedman AE, Rahman I. Cigarette smoke induces distinct histone modifications in lung cells: implications for the pathogenesis of COPD and lung cancer. J Proteome Res 2014;13(2):982-96 A bottom-up mass spectrometry approach identifies novel distinct posttranslational modifications after smoke exposure, which may serve as usable biomarkers.

..

99.

Hussain M, Rao M, Humphries AE, et al. Tobacco smoke induces polycombmediated repression of Dickkopf-1 in lung cancer cells. Cancer Res 2009;69(8):3570-8

100. Barnes PJ, Adcock IM. Glucocorticoid resistance in inflammatory diseases. Lancet 2009;373(9678):1905-17 101. Barnes PJ. Corticosteroid resistance in patients with asthma and chronic obstructive pulmonary disease. J Allergy Clin Immunol 2013;131(3):636-45


102.

Cosio BG, Tsaprouni L, Ito K, et al. Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages. J Exp Med 2004;200(5):689-95

112.

Guan SP, Tee W, Ng DS, et al. Andrographolide protects against cigarette smoke-induced oxidative lung injury via augmentation of Nrf2 activity. Br J Pharmacol 2013;168(7):1707-18

123. Mariner PD, Walters RD, Espinoza CA, et al. Human Alu RNA is a modular transacting repressor of mRNA transcription during heat shock. Mol Cell 2008;29(4):499-509

103.

To Y, Ito K, Kizawa Y, et al. Targeting phosphoinositide-3-kinase-delta with theophylline reverses corticosteroid insensitivity in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2010;182(7):897-904

113.

Kumar V, Kumar S, Hassan M, et al. Novel chalcone derivatives as potent Nrf2 activators in mice and human lung epithelial cells. J Med Chem 2011;54(12):4147-59

124. Moazed D. Small RNAs in transcriptional gene silencing and genome defence. Nature 2009;457(7228):413-20

114.

Hwang TL, Leu YL, Kao SH, et al. Viscolin, a new chalcone from Viscum coloratum, inhibits human neutrophil superoxide anion and elastase release via a cAMP-dependent pathway. Free Radic Biol Med 2006;41(9):1433-41

115.

Sinclair DA, Guarente L. Small-molecule allosteric activators of sirtuins. Annu Rev Pharmacol Toxicol 2014;54:363-80

104.

.

105.

106.

107.

108.

109.

Cosio BG, Iglesias A, Rios A, et al. Low-dose theophylline enhances the antiinflammatory effects of steroids during exacerbations of COPD. Thorax 2009;64(5):424-9 Low-dose theophylline reduces lung inflammation in COPD. Ford PA, Durham AL, Russell RE, et al. Treatment effects of low-dose theophylline combined with an inhaled corticosteroid in COPD. Chest 2010;137(6):1338-44 Mercado N, To Y, Ito K, Barnes PJ. Nortriptyline reverses corticosteroid insensitivity by inhibition of phosphoinositide-3-kinase-delta. J Pharmacol Exp Ther 2011;337(2):465-70 Kobayashi Y, Wada H, Rossios C, et al. A novel macrolide/fluoroketolide, solithromycin (CEM-101), reverses corticosteroid insensitivity via phosphoinositide 3-kinase pathway inhibition. Br J Pharmacol 2013;169(5):1024-34 Aggarwal BB, Harikumar KB. Potential therapeutic effects of curcumin, the antiinflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplastic diseases. Int J Biochem Cell Biol 2009;41(1):40-59

116.

117.

Kode A, Rajendrasozhan S, Caito S, et al. Resveratrol induces glutathione synthesis by activation of Nrf2 and protects against cigarette smoke-mediated oxidative stress in human lung epithelial cells. Am J Physiol Lung Cell Mol Physiol 2008;294(3):L478-88 Knobloch J, Wahl C, Feldmann M, et al. Resveratrol attenuates the release of inflammatory cytokines from human bronchial smooth muscle cells exposed to lipoteichoic Acid in chronic obstructive pulmonary disease. Basic Clin Pharmacol Toxicol 2014;114(2):202-9

118.

Nicodeme E, Jeffrey KL, Schaefer U, et al. Suppression of inflammation by a synthetic histone mimic. Nature 2010;468(7327):1119-23

119.

Lockwood WW, Zejnullahu K, Bradner JE, Varmus H. Sensitivity of human lung adenocarcinoma cell lines to targeted inhibition of BET epigenetic signaling proteins. Proc Natl Acad Sci USA 2012;109(47):19408-13

Meja KK, Rajendrasozhan S, Adenuga D, et al. Curcumin restores corticosteroid function in monocytes exposed to oxidants by maintaining HDAC2. Am J Respir Cell Mol Biol 2008;39(3):312-23

120.

110.

Suzuki M, Betsuyaku T, Ito Y, et al. Curcumin attenuates elastase- and cigarette smoke-induced pulmonary emphysema in mice. Am J Physiol Lung Cell Mol Physiol 2009;296(4):L614-23

121.

Okazaki Y, Furuno M, Kasukawa T, et al. Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs. Nature 2002;420(6915):563-73

111.

Mercado N, Thimmulappa R, Thomas CM, et al. Decreased histone deacetylase 2 impairs Nrf2 activation by oxidative stress. Biochem Biophys Res Commun 2011;406(2):292-8

122.

Amaral PP, Mattick JS. Noncoding RNA in development. Mamm Genome 2008;19(7-8):454-92

Shimamura T, Chen Z, Soucheray M, et al. Efficacy of BET bromodomain inhibition in Kras-mutant non-small cell lung cancer. Clin Cancer Res 2013;19(22):6183-92


125. Castel SE, Martienssen RA. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nat Rev Genet 2013;14(2):100-12 .. This excellent review summarizes the current view of small RNA action in the nucleus. 126. Ambros V. The functions of animal microRNAs. Nature 2004;431(7006):350-5 127. Pillai RS, Bhattacharyya SN, Artus CG, et al. Inhibition of translational initiation by Let-7 MicroRNA in human cells. Science 2005;309(5740):1573-6 128. Nottrott S, Simard MJ, Richter JD. Human let-7a miRNA blocks protein production on actively translating polyribosomes. Nat Struct Mol Biol 2006;13(12):1108-14 129. Petersen CP, Bordeleau ME, Pelletier J, Sharp PA. Short RNAs repress translation after initiation in mammalian cells. Mol Cell 2006;21(4):533-42 130. Oglesby IK, McElvaney NG, Greene CM. MicroRNAs in inflammatory lung disease -- master regulators or target practice? Respir Res 2010;11:148 131. O’Neill LA, Sheedy FJ, McCoy CE. MicroRNAs: the fine-tuners of Toll-like receptor signalling. Nat Rev Immunol 2011;11(3):163-75 132. Ezzie ME, Crawford M, Cho JH, et al. Gene expression networks in COPD: microRNA and mRNA regulation. Thorax 2012;67(2):122-31 133. Van Pottelberge GR, Mestdagh P, Bracke KR, et al. MicroRNA expression in induced sputum of smokers and patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2011;183(7):898-906 134. De Flora S, Balansky R, D’Agostini F, et al. Smoke-induced microRNA and related proteome alterations. Modulation by chemopreventive agents. Int J Cancer 2012;131(12):2763-73

627


135. Izzotti A, Calin GA, Steele VE, et al. Relationships of microRNA expression in mouse lung with age and exposure to cigarette smoke and light. FASEB J 2009;23(9):3243-50 136. Hunninghake GM, Cho MH, Tesfaigzi Y, et al. MMP12, lung function, and COPD in high-risk populations. N Engl J Med 2009;361(27):2599-608 137. Graff JW, Powers LS, Dickson AM, et al. Cigarette smoking decreases global microRNA expression in human alveolar macrophages. PLoS One 2012;7(8):e44066 138. Christenson SA, Brandsma CA, Campbell JD, et al. miR-638 regulates gene expression networks associated with emphysematous lung destruction. Genome Med 2013;5(12):114 139. Leidinger P, Keller A, Borries A, et al. Specific peripheral miRNA profiles for distinguishing lung cancer from COPD. Lung cancer 2011;74(1):41-7 140. Akbas F, Coskunpinar E, Aynaci E, et al. Analysis of serum micro-RNAs as potential biomarker in chronic obstructive pulmonary disease. Exp Lung Res 2012;38(6):286-94 141. Sanfiorenzo C, Ilie MI, Belaid A, et al. Two panels of plasma microRNAs as non-invasive biomarkers for prediction of

628

recurrence in resectable NSCLC. PLoS One 2013;8(1):e54596 142. Calin GA, Sevignani C, Dumitru CD, et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci USA 2004;101(9):2999-3004 143. Davalos V, Moutinho C, Villanueva A, et al. Dynamic epigenetic regulation of the microRNA-200 family mediates epithelial and mesenchymal transitions in human tumorigenesis. Oncogene 2012;31(16):2062-74 144. Basma H, Gunji Y, Iwasawa S, et al. Reprogramming of COPD lung fibroblasts through formation of induced pluripotent stem cells. Am J Physiol Lung Cell Mol Physiol 2014;306(6):L552-65 145. Zhou Y, Kim J, Yuan X, Braun T. Epigenetic modifications of stem cells: a paradigm for the control of cardiac progenitor cells. Circ Res 2011;109(9):1067-81 146. Paschalaki KE, Starke RD, Hu Y, et al. Dysfunction of endothelial progenitor cells from smokers and chronic obstructive pulmonary disease patients due to increased DNA damage and senescence. Stem Cells 2013;31(12):2813-26


147. Hoffmann G, Breitenbuecher F, Schuler M, Ehrenhofer-Murray AE. A novel SIRT2 inhibitor with p53-dependent pro-apoptotic activity in non-small-cell lung cancer. J Biol Chem 2014;289(8):5208-16 148. Giannini G, Cabri W, Fattorusso C, Rodriquez M. Histone deacetylase inhibitors in the treatment of cancer: overview and perspectives. Future Med Chem 2012;4(11):1439-60

Affiliation Andrea C Schamberger1, Nikica Mise1, Silke Meiners1 & Oliver Eickelberg†1,2 MD † Author for correspondence 1 Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz Zentrum Mu¨nchen, University Hospital and LudwigMaximilians-University, Member of the German Center for Lung Research (DZL), Max-LebschePlatz 31, 81377 Munich, Germany 2 Chairman of Comprehensive Pneumology Center (CPC), Director of Institute of Lung Biology and Disease (iLBD), Vice Chairman of the German Center for Lung Research (DZL), Max-Lebsche-Platz 31, 81377 Munich, Germany Tel: +0049(89)31874666; Fax: +0049(89)31874661; E-mail: oliver.eickelberg@helmholtz-muenchen. de

Epigenetic drug discovery for Alzheimer's disease.

Epigenetic approaches for bipolar disorder drug discovery.

Challenges and opportunities in epigenetic drug discovery.

Epigenetic Mechanisms Involved in Huntington's Disease Pathogenesis.

Antimicrobial interactions: mechanisms and implications for drug discovery and resistance evolution.

Epigenetic assays for chemical biology and drug discovery.

Novel aspects of pathogenesis and regeneration mechanisms in COPD.

Epigenetic mechanisms of neuroplasticity and the implications for stroke recovery.

Epigenetic mechanisms involved in melanoma pathogenesis and chemoresistance.

Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis.

Developing models for cachexia and their implications in drug discovery.

Epigenetic mechanisms in the pathogenesis of diabetic retinopathy.

Dynamic bias and its implications for GPCR drug discovery.

Mechanisms, assessment and therapeutic implications of lung hyperinflation in COPD.

Epigenetic regulation of peroxiredoxins: Implications in the pathogenesis of cancer.

Epigenetic mechanisms underlying the pathogenesis of neurogenetic diseases.

Systematic discovery of drug interaction mechanisms.

Potential relevance of microRNAs in inter-species epigenetic communication, and implications for disease pathogenesis.

Epigenetic modifications in fibrotic diseases: implications for pathogenesis and pharmacological targets.

Implications of the E-selectin S128R mutation for drug discovery.

Reactive dirty fragments: implications for tuberculosis drug discovery.

Evidence for contribution of epigenetic mechanisms in the pathogenesis of systemic mast cell activation disease.

Structural Mechanisms and Drug Discovery Prospects of Rho GTPases.

Transport mechanisms at the pulmonary mucosa: implications for drug delivery.