Published OnlineFirst March 16, 2016; DOI: 10.1158/0008-5472.CAN-15-2116 Cancer Molecular and Cellular Pathobiology Research An Epigenetic Reprogramming Strategy to Resensitize Radioresistant Prostate Cancer Cells Claudia Peitzsch1,MonicaCojoc1, Linda Hein1,InaKurth1,KatrinMabert€ 1, Franziska Trautmann1, Barbara Klink2,3,4,EvelinSchrock€ 2,3,4, Manfred P. Wirth5, Mechthild Krause1,3,4,6,7, Eduard A. Stakhovsky8,GennadyD.Telegeev9,VladimirNovotny5, Marieta Toma10, Michael Muders10, Gustavo B. Baretton10, Fiona M. Frame11, Norman J. Maitland11, Michael Baumann1,3,4,6,7, and Anna Dubrovska1,3,4,7 Abstract Radiotherapy is a mainstay of curative prostate cancer treat- switch occurred during a course of radiotherapy that was ment, but risks of recurrence after treatment remain significant associated with stable genetic and epigenetic changes. Specif- in locally advanced disease. Given that tumor relapse can be ically, we found that irradiation triggered histone H3 meth- attributed to a population of cancer stem cells (CSC) that ylation at the promoter of the CSC marker aldehyde dehydro- survives radiotherapy, analysis of this cell population might genase 1A1 (ALDH1A1), stimulating its gene transcription. illuminate tactics to personalize treatment. However, this Inhibiting this methylation event triggered apoptosis, promot- direction remains challenging given the plastic nature of pros- ed radiosensitization, and hindered tumorigenicity of radio- tate cancers following treatment. We show here that irradiating resistant prostate cancer cells. Overall, our results suggest that prostate cancer cells stimulates a durable upregulation of stem epigenetic therapies may restore the cytotoxic effects of irra- cell markers that epigenetically reprogram these cells. In both diation in radioresistant CSC populations. Cancer Res; 76(9); tumorigenic and radioresistant cell populations, a phenotypic 2637–51. Ó2016 AACR. Introduction treated with surgery and radiotherapy alone or in combination with androgen ablation. Tumor radioresistance can be a limiting Prostate cancer is the most common cancer among men and the factor of the efficacy of radiotherapy for some patients with a high- third leading cause of cancer-related deaths in men worldwide risk prostate cancer (2, 3). Depending on the stage of disease, up to (1). A significant proportion of prostate cancer patients are 45% of prostate cancers can relapse after radiotherapy (4–6). diagnosed with potentially curable localized tumor that can be Tumor relapse after radiotherapy has been attributed to the population of cancer stem cells (CSC) or tumor-initiating cells (7–9). The CSC hypothesis argues that cancer cells are hierar- 1OncoRay-National Center for Radiation Research in Oncology, Fac- ulty of Medicine and University Hospital Carl Gustav Carus,Technische chically organized according to their tumorigenic potential, and Universitat€ Dresden and Helmholtz-Zentrum Dresden-Rossendorf, tumors are initiated and maintained by the populations of CSCs Dresden, Germany. 2Institute of Clinical Genetics, Faculty of Medicine (10). Decades of radiobiologic research have demonstrated that and University Hospital Carl Gustav Carus, Technische Universitat€ Dresden, Dresden, Germany. 3German Cancer Consortium (DKTK), the frequency of tumor cells with CSC characteristics and their Dresden, Germany. 4German Cancer Research Center (DKFZ), Heidel- intrinsic radiosensitivity varies between tumors (7, 11, 12). berg, Germany. 5Department of Urology, Faculty of Medicine and Radiobiologic studies provided evidence for the importance of University Hospital Carl Gustav Carus,TechnischeUniversitat€ Dresden, fi Dresden, Germany. 6Department of Radiation Oncology, Faculty of these cells for local tumor control and suggested that ef cient Medicine and University Hospital Carl Gustav Carus, Technische Uni- tumor treatment by irradiation might require eradication of the versitat€ Dresden, Dresden, Germany. 7Helmholtz-Zentrum Dresden- entire CSC population. The role of CSCs in tumor development Rossendorf, Institute of Radiation Oncology, Dresden, Germany. and recurrence has recently motivated investigations of CSC- 8National Cancer Institute, Kyiv, Ukraine. 9Institute of Molecular Biol- ogy and Genetics NAS of Ukraine, Kyiv, Ukraine. 10Department of specific biomarkers for the analysis of CSC populations in tumor Pathology, Faculty of Medicine and University Hospital Carl Gustav pretreatment biopsies for the prediction of clinical outcome and € 11 Carus, Technische Universitat Dresden, Dresden, Germany. YCR Can- selection of the optimal treatment strategy (13, 14). It is also cer Research Unit, Department of Biology, University of York, York, United Kingdom. assumed that the combination of radiotherapy with agents that target or radiosensitize the CSC population might be beneficial for Note: Supplementary data for this article are available at Cancer Research fi Online (http://cancerres.aacrjournals.org/). the treatment re nement (14). However, compelling evidence suggests a high diversity of CSCs and plasticity of their features M. Cojoc and L. Hein contributed equally to this article. imposed by tumor treatment, which could challenge the devel- Corresponding Author: Anna Dubrovska, Technische Universitat€ Dresden, opment of CSC-related predictive biomarkers and CSC-targeted Fetscherstr. 74, Dresden 01307, Germany. Phone: 4935-1458-7150; Fax: therapy. Furthermore, CSCs, which are selected or induced fol- 4903-5145-87311; E-mail: [email protected] lowing chemo- or radiotherapy, are very difficult to detect before doi: 10.1158/0008-5472.CAN-15-2116 treatment and might acquire treatment resistance, resulting in Ó2016 American Association for Cancer Research. tumor relapse (15). www.aacrjournals.org 2637 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst March 16, 2016; DOI: 10.1158/0008-5472.CAN-15-2116 Peitzsch et al. In this study, we show that irradiation induces genetic and pituitary extract (Life Technologies), 2 ng/mL leukemia inhibitory epigenetic alterations that affect prostate cancer cell tumorigenic- factor (Peprotech) 2 ng/mL stem cell factor (Peprotech), 100 ng/mL ity and radioresistance. The epigenetic changes driven by irradi- cholera toxin (Sigma-Aldrich), and 1 ng/mL granulocyte macro- ation are mediated by histone methylation, which induces the phage colony-stimulating factor (GM-CSF; Miltenyi Biotec; ref. 17). expression of ALDH1A1 gene regulating the maintenance of the Cells were cultured in the presence of irradiated (60 Gy) STO prostate CSCs and their radioresistance. Targeting of the histone 3 (mouse embryonic fibroblast) cells. After expansion, the primary methylation with DZNep, an inhibitor of lysine methyltransferase epithelial cells were plated at density of 8 Â 104 cells/mL in collagen enhancer of zeste homologue 2 (EZH2), leads to the downregula- I–coated chamber slides (Corning) without feeder cells. Cells were tion of ALDH1A1 expression and tumor cell radiosensitization. pretreated with DMSO (control cells) or DZNep at concentration of Furthermore, irradiation causes long-term alterations in the 1 mmol/L for 24 hours, irradiated with 4 Gy or left nonirradiated expression of stem cell markers and induces tumor cell repro- and fixed with4% formaldehyde 24hours after irradiation. Primary gramming. To our knowledge, this is the first study demonstrating prostate cells were used for the experiments at the early passages (up that radioresistant cell populations within prostate cancer cells to 6 passages for the cells from patient 312/13 and up to 5 passages undergo a phenotypic switch during the course of irradiation. We for the cells from patient 311/13). found that in contrast to nonirradiated cells, ALDH activity is not indicative of a radioresistant cell subset in the cells surviving the Human tissue samples course of fractionated irradiation. Moreover, our study demon- Clinical material was collected at the Department of Urology, strates that radioresistant prostate cancer cells are more sensitive University Hospital Carl Gustav Carus, Technische Universit€at to the DZNep treatment, which induces apoptosis and abrogates Dresden and Kyiv National Cancer Institute with informed con- tumorigenicity in combination with X-ray irradiation. sent and approval from the local ethics committee (Institutional Our findings suggest that radioresistant properties of cancer Review Board of the Faculty of Medicine, Technische Universit€at cells are dynamic in nature and that a combination of radiother- Dresden, EK49022015 and Ethics Committee of Kyiv National apy with drugs that prevent tumor cell reprogramming may be Cancer Institute, protocol no. 44, respectively). The tumor spec- beneficial for eradication of tumor-initiating and radioresistant imen PT1 was taken from a lymph node of a 69-year-old patient cell populations. Future research must extend this work by using (pT3bpN1L1V0R0G3a, Gleason score 4 þ 3 ¼ 7, preoperative PSA additional experiments on primary cultures and biopsies, and by level 6.58 ng/mL) with newly diagnosed prostate cancer, who combination of DZNep treatment with radiotherapy in xenograft underwent primary surgery (bilateral lymphadenectomy and pros- mouse models of human prostate cancer. tate vesiculectomy). The tumor specimen PT2 is a radical prosta- tectomy tissue taken from a 64-year-old patient (pT2c N0 M0, Gleason score 3 þ 4 ¼ 7, preoperative PSA level 19.8 ng/mL). The Materials and Methods