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Targeting Actin Inhibits Repair of Doxorubicin-Induced DNA Damage

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Targeting Actin Inhibits Repair of Doxorubicin-Induced DNA Damage Pfitzer et al. Cell Death and Disease (2019) 10:302 https://doi.org/10.1038/s41419-019-1546-9 Cell Death & Disease ARTICLE Open Access Targeting actin inhibits repair of doxorubicin-induced DNA damage: a novel therapeutic approach for combination therapy Lisa Pfitzer1,ChristinaMoser1, Florian Gegenfurtner1,AnjaArner1,FlorianFoerster1, Carina Atzberger1, Themistoklis Zisis1, Rebekka Kubisch-Dohmen2, Johanna Busse2, Rebecca Smith3,4, Gyula Timinszky3,5, Olga V. Kalinina6, Rolf Müller 7,ErnstWagner2, Angelika M. Vollmar1 and Stefan Zahler 1 Abstract Severe side effects often restrict clinical application of the widely used chemotherapeutic drug doxorubicin. In order to decrease required substance concentrations, new concepts for successful combination therapy are needed. Since doxorubicin causes DNA damage, combination with compounds that modulate DNA repair could be a promising strategy. Very recently, a role of nuclear actin for DNA damage repair has been proposed, making actin a potential target for cancer therapy in combination with DNA-damaging therapeutics. This is of special interest, since actin- binding compounds have not yet found their way into clinics. We find that low-dose combination treatment of doxorubicin with the actin polymerizer chondramide B (ChB) synergistically inhibits tumor growth in vivo. On the cellular level we demonstrate that actin binders inhibit distinctive double strand break (DSB) repair pathways. Actin 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; manipulation impairs the recruitment of replication factor A (RPA) to the site of damage, a process crucial for homologous recombination. In addition, actin binders reduce autophosphorylation of DNA-dependent protein kinase (DNA-PK) during nonhomologous end joining. Our findings substantiate a direct involvement of actin in nuclear DSB repair pathways, and propose actin as a therapeutic target for combination therapy with DNA-damaging agents such as doxorubicin. Introduction increase therapeutic effectiveness while reducing unwan- The anthracycline doxorubicin (Doxo) is widely used for ted adverse effects, combination therapy concepts have to therapy of a broad range of cancer types. However, like be developed. any other chemotherapy, treatment with Doxo induces OnemodeofactionofDoxoistostabilizethe unwanted side effects (e.g. cardiovascular morbidity due topoisomerase II complex during DNA replication, to both acute and delayed cardiotoxicity1,2). Nevertheless, which induces DNA double strand breaks (DSB) and Doxo is still an indispensable part of cancer therapy. To promotes apoptosis3,4. Cells respond to such DNA damage by starting various repair mechanisms. Target- ing specificproteinsinvolvedintheseDNArepair Correspondence: Stefan Zahler ([email protected]) pathways (repair factors) has been suggested as a pro- 1Department of Pharmacy, Pharmaceutical Biology, Ludwig Maximilian University Munich, Munich, Germany mising approach for cancer therapy. DSBs can be 2Department of Pharmacy, Pharmaceutical Biology and Biotechnology— repaired by different repair pathways, depending on the Biotechnology and Nanomedicine, Ludwig Maximilian University Munich, functional context of the cell. Nonhomologous end Munich, Germany Full list of author information is available at the end of the article. joining (NHEJ) works independently of sequence Edited by M. Daugaard © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Pfitzer et al. Cell Death and Disease (2019) 10:302 Page 2 of 14 homology and cell cycle. During NHEJ, DNA-dependent inhibition by application of actin binders as a novel protein kinase (DNA-PK) is formed by binding of its strategy for combination therapy with the DNA-damaging catalytic subunit (DNA-PKcs) to a DNA-bound Ku70/80 agent doxorubicin. heterodimer, leading to its activation. Activated DNA- PK phosphorylates itself and other targets involved in Materials and methods – NHEJ5 7. The other DSB repair pathways depend on Cell culture DNA end resection, i.e. the generation of single stranded MDA-MB-231 cells were purchased from CLS (Eppel- DNA (ssDNA). Alternative end joining (alt-EJ) utilizes heim, Germany), 4T1-luc2 (mouse) from Caliper- PARP-1-mediated annealing at short homologous DNA PerkinElmer (Alameda, CA, USA), T24 and HeLa from sequences (microhomologies)8,9. Homology directed DSMZ (Braunschweig, Germany). U2OS reporter cells26 repair (HDR) and single strand annealing (SSA), how- were kindly provided by Jeremy M. Stark (Beckman ever, both depend on extensive end resection, as repli- Research Institute of the City of Hope, USA). MDA-MB- cation factor A (RPA)—a crucial initiation factor for 231 and HeLa were cultured in DMEM (Dulbecco´s both pathways—can only bind to ssDNA. During HDR, Modified Eagle Medium), T24 and U2OS in McCoy’s5A RPA mediates Rad51-dependent strand invasion of medium, 4T1 cells in RPMI. Media were purchased from intact homologous regions on the sister chromatid PAA Laboratories (Pasching, Austria). All cells were cul- – during S-phase for accurate repair10 12.SSAanneals tivated with 10% FCS (fetal calf serum) and 1% penicillin/ homologous repeat sequences that flank the DSB, which streptomycin (Sigma-Aldrich) and were grown at 37 °C, is facilitated by Rad52 bound to the ssDNA-RPA com- 5% CO2. HeLa cells were tested for mycoplasma every – plexes12 14. fourth month. Cells were not used for more than 15 Surprisingly, sufficient nuclear levels of actin and its passages after thawing. ability to polymerize have been suggested to be required for efficient repair of irradiation or UV-induced DNA Reagents damage15,16. It seems that DSBs have to be actively clus- Jasplakinolide (Jaspla) was purchased from R&D Sys- tered in an actin-dependent way during the G1 phase of tems (Bio-Techne GmbH, Wiesbaden, Germany) and the cell cycle before being repaired in the G2 phase17. Latrunculin B (LB) from Sigma-Aldrich (Taufkirchen, Very recently, nuclear Arp2/3 complex18,19 and myosins18 Germany). Chondramide B (ChB) was isolated as descri- have been identified as crucial factors for actin-dependent bed previously27. Doxorubicin hydrochloride (Doxo) was mobility of DSBs for efficient repair by HDR. To date, purchased from Sigma-Aldrich (Taufkirchen, Germany). these mechanisms have been investigated mainly in model Antibodies are listed in Supplementary information. systems that can be tightly controlled to allow for max- imum mechanistic insight. However, the role of actin in In vivo mouse models the DNA damage repair processes in a complex and more All animal experiments were approved by the Govern- clinically relevant setting is far from being understood. As ment of Upper Bavaria in accordance with the German the obvious requirement of actin during DSB repair sug- Animal Welfare and Institutional guidelines. gests actin as an attractive target for combination therapy with DNA-damaging substances, we used two classes of Tumor growth model actin-binding compounds: polymerizers, such as jaspla- 5×106 MDA-MB-231 cells were injected sub- kinolide (Jaspla)20 or chondramide B (ChB)21, and a cutaneously into the flank of SCID mice (C.B-17/Icr- depolymerizer, like latrunculin B (LB)22 in combination HanHsd-Prkdcscid, Harlan Laboratories, USA). Forty with the clinically relevant drug Doxo. Despite the fact mice were divided equally into four treatment groups: that actin binders alone have shown antitumor char- solvent (DMSO), two single groups treated intravenously – acteristics in different experimental setups23 25, actin is either with Doxo 1.5 mg/kg body weight (once a week) or not directly targeted yet for clinical application due to ChB 0.6 mg/kg body weight (thrice a week) and a com- toxicity issues. bination treatment group. In this study we show that combination of subtoxic doses of actin-binding substances with classical che- In vivo comet motherapy is possible and leads to synergistic effects on 1×106 4T1-luc cells were injected subcutaneously into tumor growth in vivo. We demonstrate the failure of DSB BALB/cOlaHsd mice (Envigo, Netherlands) and tumors repair due to actin manipulation after Doxo-induced grown for 7–9 days. Mice were then treated with 3 mg/kg DNA damage, both in vitro and in vivo. On a molecular Doxo i.v. or in combination with 0.1 mg/kg LB i.p. for 24 level, we describe inhibition of RPA recruitment during h. Tumors were harvested, single-cell suspensions pre- HDR and SSA, as well as decreased autophosphorylation pared and cells seeded accordingly for alkaline comet of DNA-PK during NHEJ. We propose DNA repair assay. Official journal of the Cell Death Differentiation Association Pfitzer et al. Cell Death and Disease (2019) 10:302
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