Epigenetics in Radiation-Induced Fibrosis

REVIEW Epigenetics in radiation-induced ﬁbrosis

C Weigel, P Schmezer, C Plass and O Popanda

Radiotherapy is a major cancer treatment option but dose-limiting side effects such as late-onset fibrosis in the irradiated tissue severely impair quality of life in cancer survivors. Efforts to explain radiation-induced fibrosis, for example, by genetic variation remained largely inconclusive. Recently published molecular analyses on radiation response and fibrogenesis showed a prominent role of epigenetic gene regulation. This review summarizes the current knowledge on epigenetic modifications in fibrotic disease and radiation response, and it points out the important role for epigenetic mechanisms such as DNA methylation, microRNAs and histone modifications in the development of this disease. The synopsis illustrates the complexity of radiation- induced fibrosis and reveals the need for investigations to further unravel its molecular mechanisms. Importantly, epigenetic changes are long-term determinants of gene expression and can therefore support those mechanisms that induce and perpetuate fibrogenesis even in the absence of the initial damaging stimulus. Future work must comprise the interconnection of acute radiation response and long-lasting epigenetic effects in order to assess their role in late-onset radiation fibrosis. An improved understanding of the underlying biology is fundamental to better comprehend the origin of this disease and to improve both preventive and therapeutic strategies.

Oncogene (2015) 34, 2145–2155; doi:10.1038/onc.2014.145; published online 9 June 2014

INTRODUCTION matrix (ECM) architecture in connective tissue and generation of Radiotherapy is a valuable weapon in cancer treatments of an altered cellular phenotype termed myofibroblast, which can different anatomical sites. Despite recent advances in dose develop from various cellular progenitors via transdifferentiation delivery and targeting,1 its use is hampered by acute radiotoxicity processes, such as epithelial–mesenchymal transition or activation – and late complications, such as poor cosmesis, telangiectasia, of fibroblasts.12 14 These events impair tissue elasticity and edema, fibrosis and secondary cancers,2,3 in the co-irradiated ultimately lead to organ failure and death. Fibroblast activation normal tissue. Fibrosis can cause permanent scarring, stiffness and and ECM remodeling are known important components of wound organ malfunction, thus being a severe and dose-limiting side healing and injury response in healthy tissue.15 In contrast to effect. Prediction is strongly wanted by clinicians but efforts healthy wound repair, myofibroblasts remain permanently active to explain it, for example, by genetic variation remained in fibrotic disease16,17 even after repair of the initial tissue damage. inconclusive.4–6 Current research aims to understand the Besides myofibroblasts, a variety of cell types such as endothelial molecular drivers of tissue remodeling7 and cellular radiation and epithelial cells13,18–20 as well as immune cells21 are involved in response.8 Recent advances revealed a prominent role of connective tissue regulation and disease. Various hormones, epigenetic gene regulation in both processes. Epigenetics growth factors and inflammatory mediators act as drivers or describe heritable changes in genome activity without changes suppressors of fibrosis (Figure 1). Cytokines such as transforming in the DNA sequence itself and has emerged as a promising new growth factor-beta 1 (TGFB1),17,18,22–25 connective tissue growth basis for the understanding of common disease conditions.9 factor (CTGF/CCN2)26 and interleukin-6 (IL6)20 induce or sustain Epigenetic mechanisms are diverse, and most research has radiation fibrosis while hepatocyte growth factor,27 interferon focused on DNA methylation, histone modifications and micro- gamma28 and anticoagulant components like thrombomodulin3 RNAs (miRNAs; for detailed reviews, see Esteller10 and Jones11). exert antifibrotic effects. The complexity of radiation fibrosis This review summarizes the current knowledge on epigenetics in becomes also evident with respect to intracellular signaling, where fibrotic disease (Figure 1) and radiation response (Figure 2) and multiple signaling cascades such as TGFB1-associated SMAD points out potential common epigenetic mechanisms in radiation- signaling,17,19,22 integrin signaling and cell adhesion,29 rho/ROCK induced fibrosis. kinase signaling,26 DNA damage response (DDR)7,30 and stress response signals, including c-Jun/activator protein 1,31 peroxisome proliferator-activated receptor (PPARG),32 and antioxidative MOLECULAR PATHOGENESIS OF RADIATION-INDUCED defense like superoxide dismutases,33 mediate stimuli, which FIBROSIS remodel gene expression, cellular morphology, protein turnover The process of radiation-induced fibrosis involves molecular and self-renewal. mechanisms that are common to other fibrotic diseases,3,7,12 At the same time, stress and DDR are crucial aspects of cancer and that are shortly summarized here (Figure 1). Fibrosis is radiotherapy as they determine the efficiency of radiation considered to arise from aberrant remodeling of extracellular treatment in the tumor as well as side effects in the

Department of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany. Correspondence: Dr O Popanda, Department of Epigenomics and Cancer Risk Factors (C010), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. E-mail: [email protected] Received 19 December 2013; revised 17 April 2014; accepted 23 April 2014; published online 9 June 2014 Epigenetics in radiation-induced ﬁbrosis C Weigel et al 2146 co-irradiated normal tissue.34 This comprises both early, transient lesions, including DNA double-strand breaks (DSBs).36 The early and late-onset, long-lasting effects2 (Figure 2). Radiation induces damage response results in a fast activation of DNA repair,36,37 reactive oxygen species,7 aldehydes35 and a variety of DNA survival-modulating pathways such as TGFB1 signaling34 and cell

Figure 1. Cellular and molecular signals involved in transdifferentiation of myofibroblasts and fibrogenesis upon radiation exposure. Radiaton fibrosis develops as a response to various stimuli that trigger the generation of permanently active myofibroblasts. Progenitor cells can be different according to the affected organ. Perpetuation of wound-healing response and coagulation after injury, inflammation, release of cytokines and hormones, cell–cell or cell–ECM contact and generation of highly reactive chemical entities can lead to the induction of signaling cascades that result in aberrant gene expression or silencing in affected cells. These events finally result in mutagenesis and/or epigenetic alterations that firmly establish a profibrotic cellular phenotype and ultimately lead to the delayed onset of connective tissue disease after exposure to ionizing radiation. Examples linked to radiation fibrosis are shown. AP1, activator protein 1; EMT, epithelial–mesenchymal transition; HGF, hepatocyte growth factor; ICAM1, intercellular adhesion molecule 1; IFN, interferon; MTD, myofibroblast transdifferentiation; NOS, reactive nitrogen species; PAI-1, plasminogen activator inhibitor-1; ROS, reactive oxygen species; SOD, superoxide dismutase.

Figure 2. Short- and long-term effects of the radiation response at the cellular and tissue levels. The graph shows the onset of radiation- induced effects over time. Early radiation effects are characterized by signaling events such as DDR, including DNA repair and cell cycle arrest, apoptosis and/or necrosis (left). In addition, radiation leads to long-lasting dysregulation in the (epi)genome, which can be passed on to directly affected as well as bystander cells in distant sites. When tissue repair and wound-healing response are completed, an apparently normal tissue phenotype is re-established over time, and dead cells are replaced (middle). However, the alterations in cellular epigenetic and genetic architecture can remain and trigger the onset of late side effects such as ﬁbrosis even years after radiation exposure (right).

Oncogene (2015) 2145 – 2155 © 2015 Macmillan Publishers Limited Epigenetics in radiation-induced fibrosis C Weigel et al 2147 cycle arrest via checkpoint activation leading to reconstitution of an intact genome or induction of apoptosis.34 A defective response may promote cell survival as observed in cancer.34 Late radiation response is characterized by a delayed or transgenerational onset of genomic instability38 and loss of tissue homeostasis and regeneration, which causes tissue dystrophy, chronic pain and radiation fibrosis.2,25 DNA damage can affect both directly irradiated areas and distant sites through soluble factors secreted by irradiated cells (bystander effect).39,40 Although short-term radiation effects can clearly be attributed to direct radiation damage, the trigger of long-lasting effects remains only partially known. Various studies indicate that these effects rely on persistent DNA damage,39,40 (stem) cell loss,41 altered cellular signaling3,40 and genetic42 as well as epigenetic39,42,43 aberrations. Specific investigations of the epigenetic regulation of radiation- induced fibrosis itself are rare, thus the evolving hypotheses presented in the following are still speculative.

EPIGENETIC DNA METHYLATION IS INVOLVED IN MYOFIBROBLAST ACTIVATION AND FIBROSIS Epigenetic gene regulation comprises several functional layers, including DNA methylation, histone modifications and miRNAs, which work in close cooperation but are treated separately in the following to facilitate the understanding of these complex Figure 3. Examples of genes that are regulated by epigenetic processes. DNA methylation is a well-described mechanism to mechanisms in radiation-induced myofibroblast formation and regulate gene expression and genomic stability. It nearly fibrogenesis. The three major epigenetic pathways contributing to exclusively affects cytosine at the 5′ position of a guanine causing fibrogenesis are shown. Various epigenetic drivers such as histone- different functional consequences according to the genomic modifying enzymes, proteins involved in establishment and readout location of the CpG dinucleotide. Methylation of CpGs within the of DNA methylation, as well as miRNAs (outer circle) execute epigenetic regulation on downstream target genes (middle circle). promoter region of a gene frequently occurs in CpG-rich regions, These events lead to the final onset of fibrotic phenotypes in various so called CpG islands, and inhibits gene transcription, whereas tissues and different disease contexts. Epigenetic pathways often methylation in the gene body, within intragenic regions and in interact with each other in this process forming a complex network repetitive elements is supposed to contribute to maintenance of of epigenetic regulators, as exemplified by the regulation of genomic integrity.11 MECP2 by miRNAs. HAT, histone acetyltransferase; HDM, histone The cellular methylation pattern is changing physiologically demethylase; HMT, histone methyltransferase. during development and differentiation. Therefore, its maintenance has to be strictly controlled. Impaired DNA methylation at specific gene loci and throughout the genome can result in aging fibrosis-related expression changes were already independently 44 or cancer. In fibrosis, permanent changes in the myofibroblast confirmed for some of these genes. Further genes were also growth program are frequently suggested to be caused by altered regulated by promoter DNA methylation in fibrotic cells from DNA methylation (Figure 3). There are several studies available idiopathic pulmonary fibrosis patients and rodent models for liver 47 47 which reveal (i) that methylation is changed genome wide and at and kidney fibrosis (Table 1, reviewed in Yang et al. and 48 specific genomic sites in fibrotic tissues derived from different Zeisberg and Zeisberg ). In summary, there is sufficient evidence organs, and (ii) that there are fibrosis-related changes in that gene expression changes leading to fibrosis are regulated by methylation enzymes. altered DNA methylation in different organs. Although different genes are reported in different organs, there seem to be some Myofibroblast activation and fibrosis are associated with DNA common features of such epigenetically inactivated genes methylation and gene expression changes as they are mainly involved in the modulation of cell growth or ECM production.48 Furthermore, tumor suppressors involved in Genome-wide methylation patterns were recently established for cell cycle control and apoptosis can be silenced by DNA normal and fibrotic lung tissues from patients with idiopathic 45,49 methylation, thus leading to the perpetuation of cell growth. pulmonary fibrosis, and differential methylation was found in 870 Hence perpetuation of pro-survival signals in the activated out of the 14 000 genes.44 When compared with expression data, myofibroblast can be considered as a process driven by DNA 16 genes showed an inverse correlation of DNA methylation and methylation. gene expression, suggesting a functional impact of the methylation changes. Such results point to the importance of DNA methylation in the pathogenesis of lung fibrosis. This study was Fibrosis is associated with expression changes in enzymes of the confirmed by a second one describing 625 differentially methy- methylation machinery lated CpG islands.45 Many of them were associated with the DNA methyltransferases (DNMTs) methylate cytosine to regulation of genes in apoptosis, morphogenesis and cellular 5-methylcytosine (5mC). DNMT1 is responsible for the main- biosynthetic processes. In a mouse model where liver fibrosis was tenance of methylation following replication; DNMT3A and induced by carbon tetrachloride treatment, high-throughput DNMT3B for de novo methylation during differentiation DNA sequencing revealed reduced methylation throughout the processes.50 Upregulation of DNMT1 was reported in the fibrotic genome that might be crucial for the onset and progression of tissue of skin,51 kidney,49,52 lung,44 and liver.53 Myofibroblast or fibrosis.46 hepatic stellate cell activation was reverted via inhibition of All three studies provided a more detailed validation DNMT1 by the DNA-demethylating drug 5-aza-2′-deoxycytidine of potential candidate genes of fibrosis (Table 1), and (5-aza) or by specific siRNA shutdown.51–54 Profibrotic TGFB1

Table 1. Examples of genes aberrantly methylated in ﬁbrosis and their potential function in radiation response

Gene Fibrotic DNA methylation change in Reference Indication of gene function in radiation response Reference organ ﬁbrotic tissue

PTCH1 Liver Hypermethylated 47 Irradiated PTCH−/+ mice show radiohypersensitivity, bystander 156,157 effect, loss of heterozygosity in radiation-induced tumors PTEN Liver Hypermethylated 53 PTEN expression increases radiosensitivity of tumor cells 158,159 PPARG Liver Hypermethylated 59 Radiation reduces PPARγ, resulting in inﬂammation and immune 144 response in rat colonic mucosa RASAL1 Kidney liver Hypermethylated 49,61 Expression upregulated by benzo[a]pyrene 160 DKK1 Skin Hypermethylated 161 Decrease induces radiosensitivity, increased by DNA damage 162,163 P14ARF Lung Hypermethylated 164 Slight increase after radiation, modulates p53 function 164–166 THY1 Lung Hypermethylated 167 Induced by radiation in human stem cells 168 CDH1 Lung Hypermethylation 169 Reduced after radiation in mouse alveolar epithelium 170 FLI1 Skin Hypermethylation 51 FLI1-1 expression associated with radioresistance in OSCC patients 171 TP53INP1 Lung Hypomethylated 44 Upregulated by radiation in human ﬁbroblasts 172 MMP7 Lung Hypomethylated 44 Upregulated in irradiated ileum 173 SPP1 Liver Hypomethylated 46 Upregulation associated with poor prognosis after radiotherapy 174

induced DNMT1 expression in rat liver stellate cells53 and in lung Radiosensitivity is modulated by an altered methylation status 54 fibroblasts together with alpha smooth muscle actin (ACTA2) In glioblastoma, radiosensitivity was associated with promoter expression. These data support the central role of DNMT1 in methylation of miRNA-21165 or the MGMT DNA repair gene.66 In a modulating the transdifferentiation process. genome-wide comparison of a radiosensitive and a radioresistant Mechanisms involved in enzymatic demethylation such as the non-small cell lung cancer cell line, >1000 gene promoters were ten–eleven translocation (TET) methylcytosine dioxygenases, differentially methylated. Hypermethylated genes were mainly which convert 5mC to oxidized intermediates,55 were shown to enriched in pathways such as cell communication, signal be important in mouse embryonic fibroblasts during differentia- transduction, and cell adhesion, whereas hypomethylated genes tion. First findings indicate a dysregulation of hydroxymethylation were exclusively related to transcriptional regulation.67 In addition, – and TET proteins in profibrotic kidney tissue damaged by the demethylating 5-aza treatment increased radiosensitivity.68 70 ischemia–reperfusion as DNA hydroxymethylation was decreased Although most data are derived from tumor cells, radiation together with TET1/2/3 gene expression.56,57 sensitivity-related methylation differences include genes related A special role in fibrosis is emerging for a member of the to fibrosis (Table 1). These observations suggest similar genes to methylated DNA-binding protein family, MECP2,58 a reader of the be aberrantly methylated in radiation sensitivity as in myofibro- DNA methylation signal. MECP2 expression was selectively blast transdifferentiation. increased in transdifferentiated hepatic stellate cells after induction of liver fibrosis in rats and correlated with the suppression Radiation can change the cellular methylation profile and of several antifibrotic genes, such as PPARG,59,60 RASAL161 and thus gene expression and differentiation 47 PTCH1. Suppression of these genes was reversible by MECP2 Suppression of global methylation was observed after whole-body silencing. For a repressive MECP2 function, cooperation with irradiation, in non-irradiated bystander tissue of the same animal chromatin-modifying enzymes such as the histone methyltrans- or in the off-spring of irradiated mothers.38,39 Global hypomethy- ferase EZH2 or the histone deactelyase/SIN3A complex was lation was reported in various cancer cell lines within days71 or required.59,62 MECP2 function in fibrosis has to be, however, after 20 population doublings.72 Fractionated radiation resulted much more complex, as in bleomycin-induced pulmonary fibrosis not only in lower methylation levels overall but also of specific of rats MECP2 stimulated the expression of the ECM protein gene promoters in cells regrowing after the treatment.43 ACTA2 by binding to a methylated CpG island in its promoter Analyzing normal human bronchial fibroblasts, only a minor region.63 This stimulation was independent of TGFB1-induced number of weak methylation changes were observed 7 days after ACTA2 expression. Only recently, MECP2 was shown to be radiation and the weak changes were not assumed to modify 73 involved in alternative splicing when bound to highly methylated gene expression. Although sometimes contradictory, these exons.64 In summary, MECP2 might be a key regulator of fibrosis, reports give substantial evidence that ionizing radiation (IR) can as it can exert different functions not only according to the modify the methylation pattern over time and affect genes genomic binding site but also according to the context of histone involved in radiation-induced fibrosis. marks and histone-modifying enzymes. Radiation affects the methylation machinery not only in irradiated cells but also in non-irradiated adjacent tissues and THE ROLE OF DNA METHYLATION IN RADIATION-INDUCED progenitor cells FIBROSIS Radiation reduced mRNA and protein levels of DNMT1 as well Radiation causes an enhanced loss of cells and tissue injury in the as global methylation.47,71,74,75 A similar downregulation was co-treated normal tissue (Figure 2), which has to be replaced by described for MECP2.47,75 As fibrosis is a late manifestation of 25 the organ’s restitution capacity in analogy to wound healing. In radiation-induced side effects (Figure 2), late or long-lasting case radiosensitivity is modulated by abnormal epigenetic DNA radiation effects are of special interest. Radiation-induced methylation, the proper regulation of the tissue restitution process genomic instability is persisting over years and after various cell could be affected. Reactive oxygen species caused by inflamma- divisions and even occurs in non-irradiated bystander tissue.42 tion or radiation could be a common trigger mechanism in both These effects might be relevant for fibrosis, which is depending on fibrosis and radiation response. Literature suggests three lines of the microenvironment.76 It was proposed that persistent DNA evidence to support this hypothesis. damage can directly interfere with maintenance of epigenetic

Oncogene (2015) 2145 – 2155 © 2015 Macmillan Publishers Limited Epigenetics in radiation-induced fibrosis C Weigel et al 2149 marks such as DNA methylation,77 linking acute damage stimuli to posttranscriptional level. On the one hand, miRNAs have been later disease onset. In mouse embryonic stem cells, targeted described to contribute to the development of fibrosis in multiple interruption of Dnmt1 and Dnmt3 in irradiated cells completely organs, including the heart, lung, kidney, liver and skin (reviewed eliminated transmission of genomic instability and protected in references 80–87). On the other hand, there is an increasing neighboring cells.78 This effect was not depending on global evidence (i) that miRNAs are crucial factors in the cellular response methylation levels but required a functional Dnmt1.79 Analysis of to IR, (ii) that their own expression is affected by IR in many MECP2 is missing in these two studies. However, regarding the different cell types, and (iii) that they have an important role in the prominent induction of DNMT1 and MECP2 in fibrosis, these modulation of IR response and cellular radiosensitivity (reviewed enzymes could also have a key role in radiation-induced fibrosis. in references 88–90). The role of miRNAs in radiation-induced In summary, there is convincing evidence that DNA methylation fibrosis remains, however, largely unexplored so far. A recent has a role in fibrosis and in the radiation response. There is, synopsis by Bowen et al.87 describes >20 different miRNAs, which however, no obvious common differentially methylated genomic are suggested to be implicated in tissue fibrosis and which are region. This might be due to a lack of adequate models to targeting genes involved in both antifibrotic and profibrotic investigate these two processes together. Most radiation studies effects. Several fibrosis-associated miRNAs, including let7d, are performed in tumor cells with short observation times. Most miR-15b, -19b, -21, -24, -29c, -145 and -155, have been shown to animal studies do not consider radiation-induced fibrosis as an be also deregulated by IR, and they are therefore potential end point. To our knowledge, methylation changes in radiation- candidates involved in the development of IR-induced fibrosis. induced myofibroblast activation have not been addressed up Following investigation of their experimentally validated target to now. genes (miRecords database91), these miRNAs regulate many known key players in the molecular pathways involved in fibrotic disease (Table 2). The widespread role of miRNAs in fibrosis can THE ROLE OF MIRNAS IN RADIATION-INDUCED FIBROSIS further be exemplified by studies linking miR-19,92 miR-200,93 miRNAs represent a class of short non-coding RNAs that miR-21,94 miR-29,95 miR-34,96 miR-13259/miR-30a97 and let-7d98,99 contribute to the regulation of target gene expression at the to changes in expression of their target genes TGFBR2, ZEB1/2,

Table 2. Overview of radiation-responsive miRNAs with impact in ﬁbrosis

MicroRNA Experimentally validated target genes

let-7d DAD1, DAD3R, DICER1, EIF4G2, RABGAP1L, SMOX mir-15b BCL2, BMI1, CCNE1, MKK4, RECK mir-19b CTGF, ESR1, FMR1 mir-21 ACTA2, APAF1, BMPR2, BTG2, CDC25A, CDK6, CDKN1A, CFL2, E2F1, FAM3C, FAS, GLCCI1, HIPK3, IL6R, JAG1, LRRFIP1, MARCKS, MTAP, NFIB, NTF3, PDCD4, PELI1, PPARA, PRRG4, PTEN, RECK, RP2, SERPINB5, SESN1, SGK3, SLC16A10, SOCS5, SOX5, STAT3, TGFBR2, TIMP3, TPM1 mir-24 NOTCH1, SLITRK1 mir-29c CDC42, COL15A1, COL1A1, COL1A2, COL3A1, COL4A1, COL4A2, DNMT3A, DNMT3B, FBN1, FUSIP1, LAMC1, PIK3R1, TDG mir-145 ACBD3, C11orf58, CBFB, CCDC25, CCNA2, CLINT1, FBXO28, FSCN1, IGF1R, IRS1, KRT7, MUC1, MYC, PARP8, PPP3CA, RASA1, RTKN, SOX9, USP46 mir-155 AGTR1, BACH1, CCND1, CEBPB, CTLA4, CYR61, ETS1, FADD, FOXO3, IKBKE, JARID2, KGF, LDOC1, TAB2, MATR3, MECP2, MEIS1, MYD88, RHOA, SHIP, SMAD2, SOCS1, SPI1, TM6SF1

Table 3. Overview of genes and their associated changes in histone marks during ﬁbrosis and their potential contributions to radiation-induced ﬁbrosis

Gene Fibrotic organ Change of histone mark in ﬁbrotic tissue Reference Indication of gene function in radiation Reference response

PPARG Liver H3K27me3 increase, 59 Decreased after radiation 144,145 H3K4me3 decrease, EZH2, HP1alpha ACTA2 Liver ASH1L, 106 Increase after whole liver irradiation 146 H3K4me3 increase TGFB1 Liver, kidney ASH1L, H3K4me3 increase, 106,108 Increase in radiation-induced fibrosis, 146,175,176 H2A.Z increase including liver fibrosis MMP9 Liver HDAC4, SIRT1, H4ac decrease 118,149 Increase in irradiated liver 177 CTGF/CCN2 Kidney H3K9me2/3 decrease, 107 Increase associated with radiation-induced 18,175,178 H3K4me3 increase by SETD7 fibrosis and radiation nephropathy SERPINE1/PAI-1 Kidney H3K9me2/3 decrease, 107 Increase associated with radiation-induced 18,139,175 H3K4me3 increase by SETD7 fibrosis, including kidney fibrosis IL6 Liver H3K9ac increase by SIRT6 deficiency, 21 Increase in irradiated liver and serum .20,179–181 H3K4me3 increase CCL2 Kidney, liver H3K4me3 and H2A.Z increase, SETD1A 21,108 Increase in irradiated liver and serum 180,182 and BRG1 H3K9ac increase by SIRT6 deficiency COL3A1 Kidney H3K4me3 and H2A.Z increase, SETD1A 108 Increase associated with radiation 178,179 and BRG1 nephropathy and irradiated lung injury COL1A1 Liver ASH1L, H3K4me3 increase, SETD7, 106,107 Increase in various types of radiation fibrosis 3 H3K4me1 increase, H3K9me3 decrease SNAI1 Mammary epithelial Increase via H3K27me3 removal by 112 Increase in radiation-induced lung fibrosis 183 cells KDM6B

© 2015 Macmillan Publishers Limited Oncogene (2015) 2145 – 2155 Epigenetics in radiation-induced fibrosis C Weigel et al 2150 SPRY1, COL1A1, VEGFA/B, MECP2 and HMGA2, respectively activation during myofibroblast activation and can be induced by (Figure 3). Many of these targets are critical components of profibrotic stimuli.106–108 In contrast, knockout of DOT1L, the only cellular processes involved in fibrosis. This underlines a key role for known H3K79 methyltransferase, led to the development of miRNAs, and their dysregulation can be triggered by profibrotic cardiomyopathy and heart fibrosis.121 The H3K27 methyltransfer- factors, such as TGFB1 and inflammation.95 In line with these ase EZH2 showed an age-dependent increase in kidney tissue and examples, total disruption of miRNA homeostasis via conditional colocalized with increased collagen deposition.109 In addition, knockout of the miRNA processing RNase DICER1 equally induced histone variant H2A.Z incorporation108 and histone demethylase a fibrosis-like phenotype.100 The actual contribution of miRNAs in KDM6B112 have also been associated with fibrosis and epithelial– radiation fibrosis is, however, still largely speculative and needs to mesenchymal transition, respectively. The role of individual be further elucidated both in cellular systems and in vivo, for histone-modifying enzymes in radiation fibrosis has only been example, in a murine radiation-induced fibrosis model that closely assessed indirectly, for example, via unspecific HDAC-inhibition 101,102 resembles the clinical appearances in humans. studies,103 and conclusions regarding their importance in radiation-induced fibrosis therefore remain speculative. Still, data from stimulus-independent experiments on fibrogenesis21,119,121 HISTONE MODIFICATIONS INVOLVED IN FIBROTIC DISORDERS 112 AFTER RADIOTHERAPY and use of ubiquitous activators like TGF-beta1 suggest a general contribution of these enzymes to fibrotic disease, fi Histone modi cations are major determinants of constitutive and including radiation fibrosis. In fact, there is increasing evidence inducible gene expression. In concordance with the transcriptional that epigenetic components effective in fibrosis might, at the fi deregulation of many genes, interference with histone modi ca- same time, be central players in radiation response and radiation fi 103 tion patterns was shown to affect radiation brosis in the skin. fibrosis, as outlined below. Histone modifications altered in fibrosis include various lysine fi modi cations and histone variants (Table 3), but the role of fi individual histone modifications in radioprotection103,104 and Histone modi cations and associated proteins affect radiation fibrogenesis remain ambiguous and none of the reported resistance and subsequent diseases alterations in histone modifications could be identified as a Histone modifiers have received much attention with regard to strictly antifibrotic or profibrotic factor. For example, reduction of their function as radioprotectors and/or radiosensitizers. For gene-repressive H3K27 trimethylation was shown to exacerbate example, data from non-cancerous cells show that the polycomb fibrosis via induction of previously silenced profibrotic genes, such repressive complex protein BMI1,122 HDACs103,104 or the histone as FOSL2.105 In contrast, inhibition of the H3K27 methyltransferase acetyltransferase MOF123 have important roles in radiation EZH2 also induced attenuation of fibrosis and re-expression of the response and radioprotection. Knowing that fibrosis is initiated antifibrotic PPARG gene.59 Given the pleiotropic functions of by tissue damage and apoptosis,7,30 it can be assumed that histone modifiers in cellular biology, these seemingly contra- altered radiosensitivity leads to differential susceptibility to dicting findings underline the necessity to identify specific radiation fibrosis and thereby provides a potential link between profibrotic and antifibrotic target site signatures of aberrant epigenetic regulation, disease risk and radiation fibrosis. histone modifications throughout the genome. fi Several histone modi cation changes were already linked to Radiation-induced DDR requires changes in histone modifications individual genes (Table 3; Figure 3). Profibrotic genes such as – Systematic studies of histone marks revealed the contribution of collagens,106 109 ACTA2,106 TGFB1,106,108,110 SERPINE1/PAI-1,107,111 histone modifications to DNA repair processes directly following CTGF107 and the epithelial–mesenchymal transition marker radiation exposure.124,125 DNA repair requires decondensation of SNAI1112 are targeted directly by histone-modifying enzymes via chromatin and assembly of repair complexes at the sites of DNA introduction of activating histone marks or removal of repressing 36 marks at their gene promoter site. These results show that both damage. This process is mediated by the interdependent generation or removal of histone marks and by nucleosome increases and decreases of epigenetic marks at specific gene – remodeling120,124,126 134 linked to the recruitment of histone- loci can disrupt tissue homeostasis. It can be expected that 135 fi modifying enzymes together with the DSB repair machinery. altered epigenetic modi cations do not only represent aberrant fi regulation but also antifibrotic mechanisms that are induced in Among the chromatin modi cations associated with IR damage, response to damaging stimuli. A recent remarkable example of phosphorylation of histone H2A.X has a critical role and links fi 37 such a counter-regulatory event is the antifibrotic regulation of radiation damage to epigenetic histone modi cations. Several proteins of DSB repair such as the MRN complex,135 ATM,130,136 H3K27 trimethylation and histone variant H2A.Z at the PPARG 127 120 131 110 Ku70, CHK1 or MDC1, directly interact with histone- promoter. 135 130 120 Apart from site-specific changes in histone modifications, modifying enzymes, such as TIP60, MOF, SETMAR or with fi 127 the responsible histone-modifying enzymes were shown to be histone modi cations themselves. Disruption of histone mod- fi fi ifications results in decreased DSB repair,127,130,137 increased dysregulated and to have either pro brotic or anti brotic 131,137 138 functions (Table 3). Recent studies have mainly focused radiation susceptibility and cancer development. The on histone acetyltransferases, histone deacetylases (HDACs), general role of DDR in fibrotic disorders remains, however, methyltransferases and histone demethylases. The histone ambiguous as findings link defects of key repair components to 30,139 140 acetyltransferase p300 is an important downstream mediator both antifibrotic as well as profibrotic effects. It was shown 30 of TGFB1-induced effects.113–115 In fibrosis, p300 is increased that ATM-deficient mice develop less fibrosis. In contrast, together with global H3K9 acetylation.116 In addition, the BET conditional ATR knockout was associated with exacerbation of bromodomain proteins BRD2 and BRD4 are known readers of age-related heart and kidney fibrosis.141 Although ATM directly histone acetylation at induced gene promoter sites and their interacts with histone-modifying enzymes,136 ATR phosphorylates expression is altered in radiation fibrosis-resistant and susceptible downstream regulators involved in histone modification, such mouse models.117 BRD-inhibition can counteract fibrosis as CHK1.120 It can be hypothesized that IR affects ATM- and induction by the radiomimetic drug bleomycin.111 Upregulation or ATR-regulated histone-modifying pathways, and the downstream downregulation of HDACs like HDAC4,118 SIRT6/721,119 and the effects directly influence fibrosis development in an organ- HDAC1/2-containing NuRD complex120 were reported. Histone specific141 and context-dependent way as exemplified by the methyltransferases such as ASH1L, KMT2E, SETD1A106,108 and requirement of an exogenous profibrotic stimulus in an ATM SETD7107 induce increased H3K4 trimethylation, trigger gene knockout setting.30

Oncogene (2015) 2145 – 2155 © 2015 Macmillan Publishers Limited Epigenetics in radiation-induced fibrosis C Weigel et al 2151 Radiation and fibrogenesis affect common histone-modifying involved in fibrogenesis are still rare and are urgently needed to enzymes and target genes unravel the molecular mechanisms of fibrosis, better understand Although the precise downstream target genes regulated by its origin and improve therapeutic strategies. histone-modifying enzymes often remain unknown, there are In addition, most investigations currently assess epigenetic examples linking epigenetic components to radiation-induced changes at gene promoter sites. Recent studies have, however, changes in the expression of specific genes.122,142 Alteration of shown that other gene-regulatory elements such as enhancers are radiation-responsive histone marks like H3K79 dimethylation126 equally affected by epigenetic modification, for example, DNA and H4K16 acetylation143 resulted in global changes of the methylation,153 and the contribution of these epigenetic events transcriptional landscape, including genes involved in apoptosis in fibrogenesis deserves further attention. With the rise of and DNA repair. Furthermore, analysis of gene expression changes epigenome-wide technologies154,155 and a deepened understand- showed that radiation fibrosis-related genes are affected by ing of the molecular events in fibrosis and radiation response, epigenetic changes in histone modifications during fibrogenesis. the stage is set for the investigation of epigenomics in radiation- Examples include PPARG,144,145 ACTA2146 and others (Table 3; induced fibrosis and for novel approaches in prevention, risk Figure 3). Importantly, the direction of gene expression changes assessment and ultimately reversal of this adverse reaction. and the anatomical site of fibrosis are often identical in radiation fi and brotic response indicating common gene expression CONFLICT OF INTEREST patterns. Interestingly, several key components of the epigenetic histone- The authors declare no conflict of interest. modifying machinery, including p300,113–115,147 EZH2,59,109,148 DOT1L,121,126,137 SIRT1,147,149 KDM6B112,122 and HDAC4,71,118,150 ACKNOWLEDGEMENTS have also been reported in the context of both radiation response We apologize to all researchers whose relevant contributions were not cited due to and connective tissue disorders. These findings provide potential space limitations. This work was supported by a grant from the Deutsche Krebshilfe, links between radiation exposure and subsequent accumulation of project number 109394. CW holds a stipend from the Helmholtz International epigenetic changes in radiation-induced fibrosis. Graduation School for Cancer Research.

CONCLUSION AND FUTURE DIRECTIONS REFERENCES 1 Thariat J, Hannoun-Levi JM, Sun Myint A, Vuong T, Gerard JP. Past, present, and There is accumulating evidence that alterations in epigenetic fi 10 marks have a considerable role both in fibrosis development and future of radiotherapy for the bene t of patients. Nat Rev Clin Oncol 2013; : 52–60. radiation response. Importantly, epigenetic changes are long-term 2 Sperk E, Welzel G, Keller A, Kraus-Tiefenbacher U, Gerhardt A, Sutterlin M et al. determinants of gene expression and can therefore support those Late radiation toxicity after intraoperative radiotherapy (IORT) for breast cancer: mechanisms that induce and perpetuate fibrogenesis even in the results from the randomized phase III trial TARGIT A. Breast Cancer Res Treat absence of the initial damaging stimulus as it is the case in 2012; 135:253–260. radiation fibrosis. By now, most studies use well-characterized 3 Yarnold J, Brotons MC. Pathogenetic mechanisms in radiation fibrosis. Radiother inducers of fibrosis, mostly in short-term treatments, or they Oncol 2010; 97:149–161. investigate a reversible phenotype,110 which stands in strong 4 Popanda O, Marquardt JU, Chang-Claude J, Schmezer P. Genetic variation in 667 – contrast to the chronic, slowly developing and irreversible normal tissue toxicity induced by ionizing radiation. Mutat Res 2009; :58 69. fi 5 Chang-Claude J, Ambrosone CB, Lilla C, Kropp S, Helmbold I, von Fournier D et al. character of brosis in a clinical setting. It is not clear whether Genetic polymorphisms in DNA repair and damage response genes and late short-term treatments induce durable epigenetic alterations or normal tissue complications of radiotherapy for breast cancer. Br J Cancer 2009; only acute responses, and only very few studies focus on long- 100: 1680–1686. 110 109 term, transgenerational or aging-related epigenetic effects in 6 Andreassen CN, Alsner J. Genetic variants and normal tissue toxicity after fibrosis development and susceptibility. Similarly, most studies on radiotherapy: a systematic review. Radiother Oncol 2009; 92:299–309. radiation response appreciate only reversible DDR-associated 7 Cheresh P, Kim SJ, Tulasiram S, Kamp DW. Oxidative stress and pulmonary changes in epigenetic marks. These studies again focus on fibrosis. Biochim Biophys Acta 2013; 1832: 1028–1040. 8 Amundson SA, Bittner M, Fornace Jr. AJ. Functional genomics as a window on short-term effects centered on DSB repair and cell cycle control 22 – while only a few studies link these acute epigenetic perturbations radiation stress signaling. Oncogene 2003; : 5828 5833. 9 Bird A. Perceptions of epigenetics. Nature 2007; 447:396–398. to durable regulatory changes. This is particularly true for radiation 10 Esteller M. Non-coding RNAs in human disease. Nat Rev Genet 2011; 12:861–874. markers like H2A.X phosphorylation, which deserve a re- 11 Jones PA. Functions of DNA methylation: islands, start sites, gene bodies evaluation in the light of their potential role as gene regulatory and beyond. Nat Rev Genet 2012; 13:484–492. histone marks. Again, future work must reveal the interconnection 12 Zvaifler NJ. Relevance of the stroma and epithelial-mesenchymal transition of acute radiation response and long-lasting epigenetic effects in (EMT) for the rheumatic diseases. Arthritis Res Ther 2006; 8: 210. order to assess their role in late-onset of radiation fibrosis. 13 Krenning G, Zeisberg EM, Kalluri R. The origin of fibroblasts and mechanism of For example, promising murine models that closely resemble the cardiac fibrosis. J Cell Physiol 2010; 225: 631–637. clinical appearance of human radiation-induced fibrosis are 14 Hinz B, Phan SH, Thannickal VJ, Galli A, Bochaton-Piallat ML, Gabbiani G. The 101,102 myofibroblast: one function, multiple origins. Am J Pathol 2007; 170: 1807–1816. already available and have to be analyzed for long-term 15 Bielefeld KA, Amini-Nik S, Alman BA. Cutaneous wound healing: recruiting epigenetic alterations. developmental pathways for regeneration. Cell Mol Life Sci 2013; 70: 2059–2081. The concepts of fibrogenesis are currently built largely on the 16 Wynn TA. Cellular and molecular mechanisms of fibrosis. J Pathol 2008; 214: classification of signaling events and protein factors as either 199–210. profibrotic or antifibrotic mediators. Although this view has been 17 Pohlers D, Brenmoehl J, Loffler I, Muller CK, Leipner C, Schultze-Mosgau S et al. confirmed in many clinical and experimental settings of fibrosis, it TGF-beta and fibrosis in different organs - molecular pathway imprints. Biochim should only be considered as a model system. Factors such as Biophys Acta 2009; 1792:746–756. TGFB1 can have pleiotropic as well as cell- and organ-specific 18 Kruse JJ, Floot BG, te Poele JA, Russell NS, Stewart FA. Radiation-induced acti- 151 fi 17 vation of TGF-beta signaling pathways in relation to vascular damage in mouse effects, which can both support or suppress brosis. It is 171 – fi kidneys. Radiat Res 2009; : 188 197. probably due to this complexity why extensive anti brotic drug 19 Milliat F, Francois A, Isoir M, Deutsch E, Tamarat R, Tarlet G et al. Influence of research has only yielded a limited set of therapeutics that endothelial cells on vascular smooth muscle cells phenotype after irradiation: 152 successfully reached clinical application. Unbiased screening implication in radiation-induced vascular damages. Am J Pathol 2006; 169: approaches for the identification of genes and drug targets 1484–1495.

© 2015 Macmillan Publishers Limited Oncogene (2015) 2145 – 2155 Epigenetics in radiation-induced fibrosis C Weigel et al 2152 20 Gaugler MH, Vereycken-Holler V, Squiban C, Vandamme M, Vozenin-Brotons MC, 46 Komatsu Y, Waku T, Iwasaki N, Ono W, Yamaguchi C, Yanagisawa J. Global analysis Benderitter M. Pravastatin limits endothelial activation after irradiation and of DNA methylation in early-stage liver fibrosis. BMC Med Genomics 2012; 5:5. decreases the resulting inflammatory and thrombotic responses. Radiat Res 47 Yang JJ, Tao H, Huang C, Shi KH, Ma TT, Bian EB et al. DNA methylation and 2005; 163: 479–487. MeCP2 regulation of PTCH1 expression during rats hepatic fibrosis. Cell Signal 21 Xiao C, Wang RH, Lahusen TJ, Park O, Bertola A, Maruyama T et al. Progression of 2013; 25:1202–1211. chronic liver inflammation and fibrosis driven by activation of c-JUN signaling in 48 Zeisberg EM, Zeisberg M. The role of promoter hypermethylation in fibroblast Sirt6 mutant mice. J Biol Chem 2012; 287: 41903–41913. activation and fibrogenesis. J Pathol 2013; 229:264–273. 22 Ruiz-Ortega M, Rodriguez-Vita J, Sanchez-Lopez E, Carvajal G, Egido J. TGF-beta 49 Bechtel W, McGoohan S, Zeisberg EM, Muller GA, Kalbacher H, Salant DJ et al. signaling in vascular fibrosis. Cardiovasc Res 2007; 74:196–206. Methylation determines fibroblast activation and fibrogenesis in the kidney. 23 Fujii H, Kawada N. Inflammation and fibrogenesis in steatohepatitis. Nat Med 2010; 16: 544–550. J Gastroenterol 2012; 47:215–225. 50 Jones PA, Liang G. Rethinking how DNA methylation patterns are maintained. 24 Li C, Wilson PB, Levine E, Barber J, Stewart AL, Kumar S. TGF-beta1 levels in Nat Rev Genet 2009; 10: 805–811. pre-treatment plasma identify breast cancer patients at risk of developing post- 51 Wang Y, Fan PS, Kahaleh B. Association between enhanced type I collagen radiotherapy fibrosis. Int J Cancer 1999; 84:155–159. expression and epigenetic repression of the FLI1 gene in scleroderma fibro- 25 Westbury CB, Yarnold JR. Radiation fibrosis—current clinical and therapeutic blasts. Arthritis Rheum 2006; 54: 2271–2279. perspectives. Clin Oncol (R Coll Radiol) 2012; 24: 657–672. 52 Sun CY, Chang SC, Wu MS. Suppression of Klotho expression by protein-bound 26 Haydont V, Riser BL, Aigueperse J, Vozenin-Brotons MC. Specific signals involved uremic toxins is associated with increased DNA methyltransferase expression in the long-term maintenance of radiation-induced fibrogenic differentiation: a and DNA hypermethylation. Kidney Int 2012; 81:640–650. role for CCN2 and low concentration of TGF-beta1. Am J Physiol Cell Physiol 2008; 53 Bian EB, Huang C, Ma TT, Tao H, Zhang H, Cheng C et al. DNMT1-mediated PTEN 294: C1332–C1341. hypermethylation confers hepatic stellate cell activation and liver fibrogenesis 27 Hu S, Chen Y, Li L, Chen J, Wu B, Zhou X et al. Effects of adenovirus-mediated in rats. Toxicol Appl Pharmacol 2012; 264:13–22. delivery of the human hepatocyte growth factor gene in experimental radiation- 54 Hu B, Gharaee-Kermani M, Wu Z, Phan SH. Epigenetic regulation of myofibro- induced heart disease. Int J Radiat Oncol Biol Phys 2009; 75: 1537–1544. blast differentiation by DNA methylation. Am J Pathol 2010; 177:21–28. 28 Gottlober P, Steinert M, Bahren W, Weber L, Gerngross H, Peter RU. Interferon- 55 Weichenhan D, Plass C. The evolving epigenome. Hum Mol Genet 2013; 22: gamma in 5 patients with cutaneous radiation syndrome after radiation therapy. R1–R6. Int J Radiat Oncol Biol Phys 2001; 50:159–166. 56 Huang N, Tan L, Xue Z, Cang J, Wang H. Reduction of DNA hydroxymethylation 29 Puthawala K, Hadjiangelis N, Jacoby SC, Bayongan E, Zhao Z, Yang Z et al. in the mouse kidney insulted by ischemia reperfusion. Biochem Biophys Res Inhibition of integrin alpha(v)beta6, an activator of latent transforming growth Commun 2012; 422:697–702. factor-beta, prevents radiation-induced lung fibrosis. Am J Respir Crit Care Med 57 Tampe B, Tampe D, Muller CA, Sugimoto H, Lebleu V, Xu X et al. Tet3-mediated 2008; 177:82–90. hydroxymethylation of epigenetically silenced genes contributes to bone mor- 30 Daugherity EK, Balmus G, Al Saei A, Moore ES, Abi Abdallah D, Rogers AB et al. phogenic protein 7-induced reversal of kidney fibrosis. J Am Soc Nephrol 2014; The DNA damage checkpoint protein ATM promotes hepatocellular apoptosis 25: 905–912. and fibrosis in a mouse model of non-alcoholic fatty liver disease. Cell Cycle 2012; 58 Bian EB, Huang C, Wang H, Chen XX, Tao H, Zhang L et al. The role of methyl-CpG 11: 1918–1928. binding protein 2 in liver fibrosis. Toxicology 2013; 309:9–14. 31 Haase MG, Klawitter A, Bierhaus A, Yokoyama KK, Kasper M, Geyer P et al. 59 Mann J, Chu DC, Maxwell A, Oakley F, Zhu NL, Tsukamoto H et al. MeCP2 controls Inactivation of AP1 proteins by a nuclear serine protease precedes the onset of an epigenetic pathway that promotes myofibroblast transdifferentiation and radiation-induced fibrosing alveolitis. Radiat Res 2008; 169:531–542. fibrosis. Gastroenterology 2010; 138:705–714 714 e701-704. 32 Zhao W, Robbins ME. Inflammation and chronic oxidative stress in radiation- 60 Zhu NL, Asahina K, Wang J, Ueno A, Lazaro R, Miyaoka Y et al. Hepatic stellate induced late normal tissue injury: therapeutic implications. Curr Med Chem 2009; cell-derived delta-like homolog 1 (DLK1) protein in liver regeneration. J Biol 16: 130–143. Chem 2012; 287: 10355–10367. 33 Veldwijk MR, Herskind C, Sellner L, Radujkovic A, Laufs S, Fruehauf S et al. 61 Tao H, Huang C, Yang JJ, Ma TT, Bian EB, Zhang L et al. MeCP2 controls the Normal-tissue radioprotection by overexpression of the copper-zinc and expression of RASAL1 in the hepatic fibrosis in rats. Toxicology 2011; 290: manganese superoxide dismutase genes. Strahlenther Onkol 2009; 185:517–523. 327–333. 34 Moding EJ, Kastan MB, Kirsch DG. Strategies for optimizing the response of 62 Suzuki M, Yamada T, Kihara-Negishi F, Sakurai T, Oikawa T. Direct association cancer and normal tissues to radiation. Nat Rev Drug Discov 2013; 12:526–542. between PU.1 and MeCP2 that recruits mSin3A-HDAC complex for 35 Freitinger Skalicka Z, Zolzer F, Beranek L, Racek J. Indicators of oxidative PU.1-mediated transcriptional repression. Oncogene 2003; 22: 8688–8698. stress after ionizing and/or non-ionizing radiation: superoxid dismutase and 63 Hu B, Gharaee-Kermani M, Wu Z, Phan SH. Essential role of MeCP2 in the malondialdehyde. J Photochem Photobiol B 2012; 117:111–114. regulation of myofibroblast differentiation during pulmonary fibrosis. Am J 36 Pandita TK, Richardson C. Chromatin remodeling finds its place in the DNA Pathol 2011; 178: 1500–1508. double-strand break response. Nucleic Acids Res 2009; 37: 1363–1377. 64 Maunakea AK, Chepelev I, Cui K, Zhao K. Intragenic DNA methylation modulates 37 Rossetto D, Truman AW, Kron SJ, Cote J. Epigenetic modifications in double- alternative splicing by recruiting MeCP2 to promote exon recognition. Cell Res strand break DNA damage signaling and repair. Clin Cancer Res 2010; 16: 2013; 23:1256–1269. 4543–4552. 65 Asuthkar S, Velpula KK, Chetty C, Gorantla B, Rao JS. Epigenetic regulation of 38 Koturbash I, Baker M, Loree J, Kutanzi K, Hudson D, Pogribny I et al. Epigenetic miRNA-211 by MMP-9 governs glioma cell apoptosis, chemosensitivity and dysregulation underlies radiation-induced transgenerational genome instability radiosensitivity. Oncotarget 2012; 3: 1439–1454. in vivo. Int J Radiat Oncol Biol Phys 2006; 66:327–330. 66 Rivera AL, Pelloski CE, Gilbert MR, Colman H, De La Cruz C, Sulman EP et al. 39 Koturbash I, Rugo RE, Hendricks CA, Loree J, Thibault B, Kutanzi K et al. Irradiation MGMT promoter methylation is predictive of response to radiotherapy and induces DNA damage and modulates epigenetic effectors in distant bystander prognostic in the absence of adjuvant alkylating chemotherapy for glioblastoma. tissue in vivo. Oncogene 2006; 25: 4267–4275. Neuro Oncol 2010; 12: 116–121. 40 Dickey JS, Baird BJ, Redon CE, Sokolov MV, Sedelnikova OA, Bonner WM. Inter- 67 Kim EH, Park AK, Dong SM, Ahn JH, Park WY. Global analysis of CpG methylation cellular communication of cellular stress monitored by gamma-H2AX induction. reveals epigenetic control of the radiosensitivity in lung cancer cell lines. Carcinogenesis 2009; 30: 1686–1695. Oncogene 2010; 29:4725–4731. 41 Coppes RP, van der Goot A, Lombaert IM. Stem cell therapy to reduce radiation- 68 Hofstetter B, Niemierko A, Forrer C, Benhattar J, Albertini V, Pruschy M et al. induced normal tissue damage. Semin Radiat Oncol 2009; 19:112–121. Impact of genomic methylation on radiation sensitivity of colorectal carcinoma. 42 Mothersill C, Seymour C. Are epigenetic mechanisms involved in radiation- Int J Radiat Oncol Biol Phys 2010; 76:1512–1519. induced bystander effects? Front Genet 2012; 3:74. 69 De Schutter H, Kimpe M, Isebaert S, Nuyts S. A systematic assessment of 43 Kuhmann C, Weichenhan D, Rehli M, Plass C, Schmezer P, Popanda O. DNA radiation dose enhancement by 5-Aza-2'-deoxycytidine and histone deacetylase methylation changes in cells regrowing after fractioned ionizing radiation. inhibitors in head-and-neck squamous cell carcinoma. Int J Radiat Oncol Biol Phys Radiother Oncol 2011; 101:116–121. 2009; 73:904–912. 44 Sanders YY, Ambalavanan N, Halloran B, Zhang X, Liu H, Crossman DK et al. 70 Kumar A, Rai PS, Upadhya R, Vishwanatha, Prasada KS, Rao BS et al. Gamma- Altered DNA methylation profile in idiopathic pulmonary fibrosis. Am J Respir Crit radiation induces cellular sensitivity and aberrant methylation in human tumor Care Med 2012; 186:525–535. cell lines. Int J Radiat Biol 2011; 87: 1086–1096. 45 Rabinovich EI, Kapetanaki MG, Steinfeld I, Gibson KF, Pandit KV, Yu G et al. 71 Antwih DA, Gabbara KM, Lancaster WD, Ruden DM, Zielske SP. Radiation- Global methylation patterns in idiopathic pulmonary fibrosis. PLoS ONE 2012; 7: induced epigenetic DNA methylation modification of radiation-response e33770. pathways. Epigenetics 2013; 8: 839–848.

Oncogene (2015) 2145 – 2155 © 2015 Macmillan Publishers Limited Epigenetics in radiation-induced fibrosis C Weigel et al 2153 72 Kaup S, Grandjean V, Mukherjee R, Kapoor A, Keyes E, Seymour CB et al. 101 Nawroth I, Alsner J, Deleuran BW, Dagnaes-Hansen F, Yang C, Horsman MR et al. Radiation-induced genomic instability is associated with DNA methylation Peritoneal macrophages mediated delivery of chitosan/siRNA nanoparticle to changes in cultured human keratinocytes. Mutat Res 2006; 597:87–97. the lesion site in a murine radiation-induced fibrosis model. Acta Oncol 2013; 52: 73 Lahtz C, Bates SE, Jiang Y, Li AX, Wu X, Hahn MA et al. Gamma irradiation does 1730–1738. not induce detectable changes in DNA methylation directly following exposure 102 Stone HB. Leg contracture in mice: an assay of normal tissue response. Int J of human cells. PLoS ONE 2012; 7: e44858. Radiat Oncol Biol Phys 1984; 10:1053–1061. 74 Chaudhry MA, Omaruddin RA. Differential DNA methylation alterations in 103 Chung YL, Wang AJ, Yao LF. Antitumor histone deacetylase inhibitors suppress radiation-sensitive and -resistant cells. DNA Cell Biol 2012; 31:908–916. cutaneous radiation syndrome: implications for increasing therapeutic gain in 75 Koturbash I, Boyko A, Rodriguez-Juarez R, McDonald RJ, Tryndyak VP, Kovalchuk I cancer radiotherapy. Mol Cancer Ther 2004; 3:317–325. et al. Role of epigenetic effectors in maintenance of the long-term persistent 104 Purrucker JC, Fricke A, Ong MF, Rube C, Rube CE, Mahlknecht U. HDAC inhibition bystander effect in spleen in vivo. Carcinogenesis 2007; 28: 1831–1838. radiosensitizes human normal tissue cells and reduces DNA double-strand break 76 Prunotto M, Budd DC, Gabbiani G, Meier M, Formentini I, Hartmann G et al. repair capacity. Oncol Rep 2010; 23:263–269. Epithelial-mesenchymal crosstalk alteration in kidney fibrosis. J Pathol 2012; 105 Kramer M, Dees C, Huang J, Schlottmann I, Palumbo-Zerr K, Zerr P et al. 228: 131–147. Inhibition of H3K27 histone trimethylation activates fibroblasts and induces 77 Pogribny I, Koturbash I, Tryndyak V, Hudson D, Stevenson SM, Sedelnikova O fibrosis. Ann Rheum Dis 2013; 72:614–620. et al. Fractionated low-dose radiation exposure leads to accumulation of DNA 106 Perugorria MJ, Wilson CL, Zeybel M, Walsh M, Amin S, Robinson S et al. Histone damage and profound alterations in DNA and histone methylation in the methyltransferase ASH1 orchestrates fibrogenic gene transcription during murine thymus. Mol Cancer Res 2005; 3:553–561. myofibroblast transdifferentiation. Hepatology 2012; 56: 1129–1139. 78 Rugo RE, Mutamba JT, Mohan KN, Yee T, Chaillet JR, Greenberger JS et al. 107 Sun G, Reddy MA, Yuan H, Lanting L, Kato M, Natarajan R. Epigenetic histone Methyltransferases mediate cell memory of a genotoxic insult. Oncogene 2011; methylation modulates fibrotic gene expression. J Am Soc Nephrol 2010; 21: 30: 751–756. 2069–2080. 79 Armstrong CA, Jones GD, Anderson R, Iyer P, Narayanan D, Sandhu J et al. 108 Zager RA, Johnson AC. Renal ischemia-reperfusion injury upregulates histone- DNMTs are required for delayed genome instability caused by radiation. modifying enzyme systems and alters histone expression at proinflammatory/ Epigenetics 2012; 7:892–902. profibrotic genes. Am J Physiol Renal Physiol 2009; 296: F1032–F1041. 80 Bauersachs J. Regulation of myocardial fibrosis by MicroRNAs. J Cardiovasc 109 Abrass CK, Hansen K, Popov V, Denisenko O. Alterations in chromatin are Pharmacol 2010; 56:454–459. associated with increases in collagen III expression in aging nephropathy. Am J 81 Jiang X, Tsitsiou E, Herrick SE, Lindsay MA. MicroRNAs and the regulation of Physiol Renal Physiol 2011; 300: F531–F539. fibrosis. FEBS J 2010; 277: 2015–2021. 110 Zeybel M, Hardy T, Wong YK, Mathers JC, Fox CR, Gackowska A et al. Multi- 82 Dai Y, Khaidakov M, Wang X, Ding Z, Su W, Price E et al. MicroRNAs involved in generational epigenetic adaptation of the hepatic wound-healing response. Nat the regulation of postischemic cardiac fibrosis. Hypertension 2013; 61:751–756. Med 2012; 18: 1369–1377. 83 Pandit KV, Milosevic J, Kaminski N. MicroRNAs in idiopathic pulmonary fibrosis. 111 Tang X, Peng R, Ren Y, Apparsundaram S, Deguzman J, Bauer CM et al. BET Transl Res 2011; 157:191–199. bromodomain proteins mediate downstream signaling events following growth 84 Patel V, Noureddine L. MicroRNAs and fibrosis. Curr Opin Nephrol Hypertens 2012; factor stimulation in human lung fibroblasts and are involved in bleomycin- 21: 410–416. induced pulmonary fibrosis. Mol Pharmacol 2013; 83:283–293. 85 Srivastava SP, Koya D, Kanasaki K. MicroRNAs in kidney fibrosis and diabetic 112 Ramadoss S, Chen X, Wang CY. Histone demethylase KDM6B promotes nephropathy: roles on EMT and EndMT. Biomed Res Int 2013; 2013: 125469. epithelial-mesenchymal transition. J Biol Chem 2012; 287: 44508–44517. 86 He Y, Huang C, Zhang SP, Sun X, Long XR, Li J. The potential of microRNAs in 113 Ghosh AK, Varga J. The transcriptional coactivator and acetyltransferase p300 in liver fibrosis. Cell Signal 2012; 24: 2268–2272. fibroblast biology and fibrosis. J Cell Physiol 2007; 213:663–671. 87 Bowen T, Jenkins RH, Fraser DJ. MicroRNAs, transforming growth factor beta-1, 114 Ghosh AK, Bhattacharyya S, Varga J. The tumor suppressor p53 abrogates and tissue fibrosis. J Pathol 2013; 229:274–285. Smad-dependent collagen gene induction in mesenchymal cells. J Biol Chem 88 Metheetrairut C, Slack FJ. MicroRNAs in the ionizing radiation response and in 2004; 279: 47455–47463. radiotherapy. Curr Opin Genet Dev 2013; 23:12–19. 115 Bhattacharyya S, Ghosh AK, Pannu J, Mori Y, Takagawa S, Chen G et al. Fibroblast 89 Dickey JS, Zemp FJ, Martin OA, Kovalchuk O. The role of miRNA in the direct and expression of the coactivator p300 governs the intensity of profibrotic response indirect effects of ionizing radiation. Radiat Environ Biophys 2011; 50: 491–499. to transforming growth factor beta. Arthritis Rheum 2005; 52:1248–1258. 90 Zhao L, Lu X, Cao Y. MicroRNA and signal transduction pathways in tumor 116 Oliva J, Dedes J, Li J, French SW, Bardag-Gorce F. Epigenetics of proteasome radiation response. Cell Signal 2013; 25: 1625–1634. inhibition in the liver of rats fed ethanol chronically. World J Gastroenterol 2009; 91 Xiao F, Zuo Z, Cai G, Kang S, Gao X, Li T. miRecords: an integrated resource for 15:705–712. microRNA-target interactions. Nucleic Acids Res 2009; 37: D105–D110. 117 Kalash R, Berhane H, Au J, Rhieu BH, Epperly MW, Goff J et al. Differences in 92 Lakner AM, Steuerwald NM, Walling TL, Ghosh S, Li T, McKillop IH et al. Inhibitory irradiated lung gene transcription between fibrosis-prone C57BL/6NHsd and effects of microRNA 19b in hepatic stellate cell-mediated fibrogenesis. fibrosis-resistant C3H/HeNHsd mice. In Vivo 2014; 28:147–171. Hepatology 2012; 56: 300–310. 118 Qin L, Han YP. Epigenetic repression of matrix metalloproteinases in myofibro- 93 Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G et al. The miR-200 blastic hepatic stellate cells through histone deacetylases 4: implication in tissue family and miR-205 regulate epithelial to mesenchymal transition by targeting fibrosis. Am J Pathol 2010; 177: 1915–1928. ZEB1 and SIP1. Nat Cell Biol 2008; 10: 593–601. 119 Vakhrusheva O, Smolka C, Gajawada P, Kostin S, Boettger T, Kubin T et al. Sirt7 94 Thum T, Gross C, Fiedler J, Fischer T, Kissler S, Bussen M et al. MicroRNA-21 increases stress resistance of cardiomyocytes and prevents apoptosis and contributes to myocardial disease by stimulating MAP kinase signalling in inflammatory cardiomyopathy in mice. Circ Res 2008; 102:703–710. fibroblasts. Nature 2008; 456: 980–984. 120 Hromas R, Williamson EA, Fnu S, Lee YJ, Park SJ, Beck BD et al. Chk1 95 Roderburg C, Urban GW, Bettermann K, Vucur M, Zimmermann H, Schmidt S phosphorylation of Metnase enhances DNA repair but inhibits replication fork et al. Micro-RNA profiling reveals a role for miR-29 in human and murine liver restart. Oncogene 2012; 31: 4245–4254. fibrosis. Hepatology 2011; 53:209–218. 121 Nguyen AT, Xiao B, Neppl RL, Kallin EM, Li J, Chen T et al. DOT1L regulates 96 Bernardo BC, Gao XM, Winbanks CE, Boey EJ, Tham YK, Kiriazis H et al. dystrophin expression and is critical for cardiac function. Genes Dev 2011; 25: Therapeutic inhibition of the miR-34 family attenuates pathological cardiac 263–274. remodeling and improves heart function. Proc Natl Acad Sci USA 2012; 109: 122 Dong Q, Oh JE, Chen W, Kim R, Kim RH, Shin KH et al. Radioprotective effects of 17615–17620. Bmi-1 involve epigenetic silencing of oxidase genes and enhanced DNA repair in 97 Volkmann I, Kumarswamy R, Pfaff N, Fiedler J, Dangwal S, Holzmann A et al. normal human keratinocytes. J Invest Dermatol 2011; 131: 1216–1225. MicroRNA-mediated epigenetic silencing of sirtuin1 contributes to impaired 123 Li X, Corsa CA, Pan PW, Wu L, Ferguson D, Yu X et al. MOF and H4 K16 acetylation angiogenic responses. Circ Res 2013; 113:997–1003. play important roles in DNA damage repair by modulating recruitment of DNA 98 Mayr C, Hemann MT, Bartel DP. Disrupting the pairing between let-7 and Hmga2 damage repair protein Mdc1. Mol Cell Biol 2010; 30: 5335–5347. enhances oncogenic transformation. Science 2007; 315: 1576–1579. 124 Tjeertes JV, Miller KM, Jackson SP. Screen for DNA-damage-responsive histone 99 Pandit KV, Corcoran D, Yousef H, Yarlagadda M, Tzouvelekis A, Gibson KF et al. modifications identifies H3K9Ac and H3K56Ac in human cells. EMBO J 2009; 28: Inhibition and role of let-7d in idiopathic pulmonary fibrosis. Am J Respir Crit Care 1878–1889. Med 2010; 182:220–229. 125 Price BD, D'Andrea AD. Chromatin remodeling at DNA double-strand breaks. 100 Sequeira-Lopez ML, Weatherford ET, Borges GR, Monteagudo MC, Pentz ES, Cell 2013; 152:1344–1354. Harfe BD et al. The microRNA-processing enzyme dicer maintains 126 FitzGerald J, Moureau S, Drogaris P, O'Connell E, Abshiru N, Verreault A et al. juxtaglomerular cells. J Am Soc Nephrol 2010; 21:460–467. Regulation of the DNA damage response and gene expression by the Dot1L

© 2015 Macmillan Publishers Limited Oncogene (2015) 2145 – 2155 Epigenetics in radiation-induced fibrosis C Weigel et al 2154 histone methyltransferase and the 53Bp1 tumour suppressor. PLoS ONE 2011; 152 Wynn TA, Ramalingam TR. Mechanisms of fibrosis: therapeutic translation for 6: e14714. fibrotic disease. Nat Med 2012; 18: 1028–1040. 127 Fnu S, Williamson EA, De Haro LP, Brenneman M, Wray J, Shaheen M et al. 153 Ko YA, Mohtat D, Suzuki M, Park AS, Izquierdo MC, Han SY et al. Cytosine Methylation of histone H3 lysine 36 enhances DNA repair by nonhomologous methylation changes in enhancer regions of core pro-fibrotic genes characterize end-joining. Proc Natl Acad Sci USA 2011; 108:540–545. kidney fibrosis development. Genome Biol 2013; 14: R108. 128 Goodarzi AA, Kurka T, Jeggo PA. KAP-1 phosphorylation regulates CHD3 154 Sandoval J, Heyn H, Moran S, Serra-Musach J, Pujana MA, Bibikova M et al. nucleosome remodeling during the DNA double-strand break response. Nat Validation of a DNA methylation microarray for 450000 CpG sites in the Struct Mol Biol 2011; 18:831–839. human genome. Epigenetics 2011; 6:692–702. 129 Guo CY, Mizzen C, Wang Y, Larner JM. Histone H1 and H3 dephosphorylation are 155 Park PJ. ChIP-seq: advantages and challenges of a maturing technology. Nat Rev differentially regulated by radiation-induced signal transduction pathways. Genet 2009; 10: 669–680. Cancer Res 2000; 60: 5667–5672. 156 Mancuso M, Pazzaglia S, Tanori M, Hahn H, Merola P, Rebessi S et al. Basal cell 130 Gupta A, Sharma GG, Young CS, Agarwal M, Smith ER, Paull TT et al. Involvement carcinoma and its development: insights from radiation-induced tumors in of human MOF in ATM function. Mol Cell Biol 2005; 25: 5292–5305. Ptch1-deficient mice. Cancer Res 2004; 64:934–941. 131 Huen MS, Grant R, Manke I, Minn K, Yu X, Yaffe MB et al. RNF8 transduces the 157 Mancuso M, Pasquali E, Leonardi S, Tanori M, Rebessi S, Di Majo V et al. DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Oncogenic bystander radiation effects in Patched heterozygous mouse Cell 2007; 131: 901–914. cerebellum. Proc Natl Acad Sci USA 2008; 105: 12445–12450. 132 Li DQ, Ohshiro K, Reddy SD, Pakala SB, Lee MH, Zhang Y et al. E3 ubiquitin ligase 158 Wick W, Furnari FB, Naumann U, Cavenee WK, Weller M. PTEN gene transfer in COP1 regulates the stability and functions of MTA1. Proc Natl Acad Sci USA 2009; human malignant glioma: sensitization to irradiation and CD95L-induced 106: 17493–17498. apoptosis. Oncogene 1999; 18: 3936–3943. 133 Ogiwara H, Ui A, Otsuka A, Satoh H, Yokomi I, Nakajima S et al. Histone 159 Zhang Y, Chen LH, Wang L, Wang HM, Zhang YW, Shi YS. Radiation-inducible acetylation by CBP and p300 at double-strand break sites facilitates SWI/SNF PTEN expression radiosensitises hepatocellular carcinoma cells. Int J Radiat Biol chromatin remodeling and the recruitment of non-homologous end joining 2010; 86: 964–974. factors. Oncogene 2011; 30: 2135–2146. 160 Sparfel L, Pinel-Marie ML, Boize M, Koscielny S, Desmots S, Pery A et al. 134 Penicud K, Behrens A. DMAP1 is an essential regulator of ATM activity and Transcriptional signature of human macrophages exposed to the environmental function. Oncogene 2013; 33:525–531. contaminant benzo(a)pyrene. Toxicol Sci 2010; 114:247–259. 135 Chailleux C, Tyteca S, Papin C, Boudsocq F, Puget N, Courilleau C et al. Physical 161 Dees C, Schlottmann I, Funke R, Distler A, Palumbo-Zerr K, Zerr P et al. The Wnt interaction between the histone acetyl transferase Tip60 and the DNA double- antagonists DKK1 and SFRP1 are downregulated by promoter hypermethylation strand breaks sensor MRN complex. Biochem J 2010; 426:365–371. in systemic sclerosis. Ann Rheum Dis 2014; 73:1232–1239. 136 Sun Y, Jiang X, Chen S, Fernandes N, Price BD. A role for the Tip60 histone 162 Kim IG, Kim SY, Kim HA, Kim JY, Lee JH, Choi SI et al. Disturbance of DKK1 acetyltransferase in the acetylation and activation of ATM. Proc Natl Acad Sci USA level is partly involved in survival of lung cancer cells via regulation of ROMO1 2005; 102: 13182–13187. and gamma-radiation sensitivity. Biochem Biophys Res Commun 2013; 443: 137 Chernikova SB, Dorth JA, Razorenova OV, Game JC, Brown JM. Deficiency in Bre1 49–55. impairs homologous recombination repair and cell cycle checkpoint response to 163 Shou J, Ali-Osman F, Multani AS, Pathak S, Fedi P, Srivenugopal KS. Human radiation damage in mammalian cells. Radiat Res 2010; 174:558–565. Dkk-1, a gene encoding a Wnt antagonist, responds to DNA damage and its 138 Wu J, Chen Y, Lu LY, Wu Y, Paulsen MT, Ljungman M et al. Chfr and RNF8 overexpression sensitizes brain tumor cells to apoptosis following alkylation synergistically regulate ATM activation. Nat Struct Mol Biol 2011; 18: 761–768. damage of DNA. Oncogene 2002; 21:878–889. 139 Niemantsverdriet M, de Jong E, Langendijk JA, Kampinga HH, Coppes RP. 164 Cisneros J, Hagood J, Checa M, Ortiz-Quintero B, Negreros M, Herrera I et al. Synergistic induction of profibrotic PAI-1 by TGF-beta and radiation depends on Hypermethylation-mediated silencing of p14(ARF) in fibroblasts from p53. Radiother Oncol 2010; 97:33–35. idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 2012; 303: 140 Zhu F, Li Y, Zhang J, Piao C, Liu T, Li HH et al. Senescent cardiac fibroblast L295–L303. is critical for cardiac fibrosis after myocardial infarction. PLoS ONE 2013; 8: 165 Khan S, Guevara C, Fujii G, Parry D. p14ARF is a component of the p53 response e74535. following ionizing irradiation of normal human fibroblasts. Oncogene 2004; 23: 141 Ruzankina Y, Pinzon-Guzman C, Asare A, Ong T, Pontano L, Cotsarelis G et al. 6040–6046. Deletion of the developmentally essential gene ATR in adult mice leads to 166 Simon M, Voss D, Park-Simon TW, Mahlberg R, Koster G. Role of p16 and p14ARF age-related phenotypes and stem cell loss. Cell Stem Cell 2007; 1:113–126. in radio- and chemosensitivity of malignant gliomas. Oncol Rep 2006; 16: 142 Shan B, Xu J, Zhuo Y, Morris CA, Morris GF. Induction of p53-dependent 127–132. activation of the human proliferating cell nuclear antigen gene in chromatin by 167 Sanders YY, Pardo A, Selman M, Nuovo GJ, Tollefsbol TO, Siegal GP et al. Thy-1 ionizing radiation. J Biol Chem 2003; 278: 44009–44017. promoter hypermethylation: a novel epigenetic pathogenic mechanism in 143 Sharma GG, So S, Gupta A, Kumar R, Cayrou C, Avvakumov N et al. MOF and pulmonary fibrosis. Am J Respir Cell Mol Biol 2008; 39:610–618. histone H4 acetylation at lysine 16 are critical for DNA damage response and 168 Zhang J, Yang Y, Wang Y, Zhang J, Wang Z, Yin M et al. Identification of hub double-strand break repair. Mol Cell Biol 2010; 30:3582–3595. genes related to the recovery phase of irradiation injury by microarray and 144 Linard C, Gremy O, Benderitter M. Reduction of peroxisome proliferation- integrated gene network analysis. PLoS ONE 2011; 6: e24680. activated receptor gamma expression by gamma-irradiation as a mechanism 169 Wu CH, Tang SC, Wang PH, Lee H, Ko JL. Nickel-induced epithelial-mesenchymal contributing to inflammatory response in rat colon: modulation by the transition by reactive oxygen species generation and E-cadherin promoter 5-aminosalicylic acid agonist. J Pharmacol Exp Ther 2008; 324: 911–920. hypermethylation. J Biol Chem 2012; 287: 25292–25302. 145 Arora H, Qureshi R, Park AK, Park WY. Coordinated regulation of ATF2 by 170 Almeida C, Nagarajan D, Tian J, Leal SW, Wheeler K, Munley M et al. The role miR-26b in gamma-irradiated lung cancer cells. PLoS ONE 2011; 6: e23802. of alveolar epithelium in radiation-induced lung injury. PLoS ONE 2013; 8: 146 Imaeda M, Ishikawa H, Yoshida Y, Takahashi T, Ohkubo Y, Musha A et al. e53628. Long-term pathological and immunohistochemical features in the liver after 171 Shintani S, Hamakawa H, Nakashiro K, Shirota T, Hatori M, Tanaka M et al. Friend intraoperative whole-liver irradiation in rats. J Radiat Res (e-pub ahead of print leukaemia insertion (Fli)-1 is a prediction marker candidate for radiotherapy 23 February 2014; doi:10.1093/jrr/rru005). resistant oral squamous cell carcinoma. Int J Oral Maxillofac Surg 2010; 39: 147 Das C, Lucia MS, Hansen KC, Tyler JK. CBP/p300-mediated acetylation of histone 1115–1119. H3 on lysine 56. Nature 2009; 459: 113–117. 172 Kis E, Szatmari T, Keszei M, Farkas R, Esik O, Lumniczky K et al. Microarray analysis 148 Alimova I, Birks DK, Harris PS, Knipstein JA, Venkataraman S, Marquez VE et al. of radiation response genes in primary human fibroblasts. Int J Radiat Oncol Biol Inhibition of EZH2 suppresses self-renewal and induces radiation sensitivity in Phys 2006; 66: 1506–1514. atypical rhabdoid teratoid tumor cells. Neuro Oncol 2013; 15:149–160. 173 Polistena A, Johnson LB, Rome A, Wittgren L, Back S, Osman N et al. Matrilysin 149 Xu B, Chen H, Xu W, Zhang W, Buckley S, Zheng SG et al. Molecular expression related to radiation and microflora changes in murine bowel. J Surg mechanisms of MMP9 overexpression and its role in emphysema pathogenesis Res 2011; 167:e137–e143. of Smad3-deficient mice. Am J Physiol Lung Cell Mol Physiol 2012; 303:L89–L96. 174 Nadlonek NA, Weyant MJ, Yu JA, Cleveland Jr. JC, Reece TB, Meng X et al. 150 Kao GD, McKenna WG, Guenther MG, Muschel RJ, Lazar MA, Yen TJ. Histone Radiation induces osteogenesis in human aortic valve interstitial cells. J Thorac deacetylase 4 interacts with 53BP1 to mediate the DNA damage response. J Cell Cardiovasc Surg 2012; 144: 1466–1470. Biol 2003; 160: 1017–1027. 175 Hamama S, Gilbert-Sirieix M, Vozenin MC, Delanian S. Radiation-induced 151 Massague J. TGFbeta signalling in context. Nat Rev Mol Cell Biol 2012; 13: enteropathy: molecular basis of pentoxifylline-vitamin E anti-fibrotic effect 616–630. involved TGF-beta1 cascade inhibition. Radiother Oncol 2012; 105:305–312.

Oncogene (2015) 2145 – 2155 © 2015 Macmillan Publishers Limited Epigenetics in radiation-induced fibrosis C Weigel et al 2155 176 Abderrahmani R, Francois A, Buard V, Benderitter M, Sabourin JC, Crandall DL 180 Ao X, Zhao L, Davis MA, Lubman DM, Lawrence TS, Kong FM. Radiation produces et al. Effects of pharmacological inhibition and genetic deficiency of plasmino- differential changes in cytokine profiles in radiation lung fibrosis sensitive and gen activator inhibitor-1 in radiation-induced intestinal injury. Int J Radiat Oncol resistant mice. J Hematol Oncol 2009; 2:6. Biol Phys 2009; 74: 942–948. 181 Moriconi F, Christiansen H, Raddatz D, Dudas J, Hermann RM, Rave-Frank M et al. 177 Rave-Frank M, Malik IA, Christiansen H, Naz N, Sultan S, Amanzada A et al. Rat Effect of radiation on gene expression of rat liver chemokines: in vivo and in vitro model of fractionated (2Gy/day) 60Gy irradiation of the liver: long-term effects. studies. Radiat Res 2008; 169:162–169. Radiat Environ Biophys 2013; 52:321–338. 182 Malik IA, Moriconi F, Sheikh N, Naz N, Khan S, Dudas J et al. Single-dose gamma- 178 Liu DG, Wang TM. Role of connective tissue growth factor in irradiation induces up-regulation of chemokine gene expression and recruit- experimental radiation nephropathy in rats. Chin Med J (Engl) 2008; 121: ment of granulocytes into the portal area but not into other regions of rat 1925–1931. hepatic tissue. Am J Pathol 2010; 176: 1801–1815. 179 Ostrau C, Hulsenbeck J, Herzog M, Schad A, Torzewski M, Lackner KJ et al. 183 Balli D, Ustiyan V, Zhang Y, Wang IC, Masino AJ, Ren X et al. Foxm1 transcription Lovastatin attenuates ionizing radiation-induced normal tissue damage in vivo. factor is required for lung fibrosis and epithelial-to-mesenchymal transition. Radiother Oncol 2009; 92: 492–499. EMBO J 2013; 32: 231–244.