Oncogene (2015) 34, 2145–2155 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc 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 transgenera- tional 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