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Β8 Integrin Mediates Pancreatic Cancer Cell Radiochemoresistance 2 3 Sha Jin1 †, Wei-Chun Lee1 †, Daniela Aust2-4, Christian Pilarsky5, Nils Cordes1, 4,6-8 *

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Β8 Integrin Mediates Pancreatic Cancer Cell Radiochemoresistance 2 3 Sha Jin1 †, Wei-Chun Lee1 †, Daniela Aust2-4, Christian Pilarsky5, Nils Cordes1, 4,6-8 * Author Manuscript Published OnlineFirst on July 23, 2019; DOI: 10.1158/1541-7786.MCR-18-1352 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Jin and Lee et al., page 1 1 β8 integrin mediates pancreatic cancer cell radiochemoresistance 2 3 Sha Jin1 †, Wei-Chun Lee1 †, Daniela Aust2-4, Christian Pilarsky5, Nils Cordes1, 4,6-8 * 4 5 1OncoRay – National Center for Radiation Research in Oncology, Faculty of 6 Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, 7 Helmholtz-Zentrum Dresden - Rossendorf, 01307 Dresden, Germany 8 2Institute for Pathology, University Hospital Carl Gustav Carus, Technische 9 Universität Dresden, Dresden 01307, Germany 10 3NCT Biobank Dresden, University Hospital Carl Gustav Carus, Technische 11 Universität Dresden, Dresden 01307, Germany 12 4German Cancer Consortium (DKTK), Partner Site Dresden, Heidelberg 69120, 13 Germany 14 5Department of Surgery, Universitätsklinikum Erlangen, Friedrich-Alexander 15 Universität Erlangen, 91054 Erlangen, Germany 16 6Department of Radiotherapy and Radiation Oncology, Faculty of Medicine and 17 University Hospital Carl Gustav Carus, Technische Universität Dresden, 01307 18 Dresden, Germany 19 7Institute of Radiooncology - OncoRay, Helmholtz-Zentrum Dresden - Rossendorf, 20 Dresden, 01328 Dresden, Germany 21 8German Cancer Research Center (DKFZ) – Partner Site Dresden, 69192 Heidelberg, 22 Germany 23 24 Running Title: β8 integrin in pancreatic cancer cells 25 26 Conflict of interest: The authors declare no potential conflicts of interest. 27 28 Word count: 4926; Total number of Figures: 7; References: 41 Downloaded from mcr.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 23, 2019; DOI: 10.1158/1541-7786.MCR-18-1352 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Jin and Lee et al., page 2 29 30 Keyword: Pancreatic cancer; β8 integrin; radiochemoresistance; cell survival; 31 intracellular vesicle trafficking 32 33 Financial support: The RADIATE project has received funding from the European 34 Union’s Horizon 2020 research and innovation programme under the Marie 35 Sklodowska-Curie grant agreement No. 642623 36 37 Author Notes: 38 † S. Jin and W.-C. Lee contributed equally to this paper. 39 40 41 * Corresponding author: 42 Prof. Dr. Nils Cordes 43 OncoRay – National Center for Radiation Research in Oncology, Technische 44 Universität Dresden, Fetscherstrasse 74 / PF 41, 01307 Dresden, Germany 45 Phone: +49 (0)351–458–7401. 46 Fax: +49 (0)351–458–7311. 47 E-mail: [email protected] 48 49 Downloaded from mcr.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 23, 2019; DOI: 10.1158/1541-7786.MCR-18-1352 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Jin and Lee et al., page 3 50 Abstract 51 Pancreatic ductal adenocarcinoma (PDAC) stroma, composed of extracellular matrix 52 (ECM) proteins, promotes therapy resistance and poor survival rate. Integrin- 53 mediated cell/ECM interactions are well known to controls cancer cell survival, 54 proliferation and therapy resistance. Here, we identified β8 integrin in a high- 55 throughput knockdown screen in three-dimensional (3D), ECM-based cell cultures for 56 novel focal adhesion protein targets as critical determinant of PDAC cell 57 radiochemoresistance. Intriguingly, β8 integrin localizes with the golgi apparatus 58 perinuclearly in PDAC cells and resection specimen from PDAC patients. Upon 59 radiogenic genotoxic injury, β8 integrin shows a microtubule-dependent perinuclear- 60 to-cytoplasmic shift as well as strong changes in its proteomic interactome regarding 61 the cell functions transport, catalysis and binding. Parts of this interactome link β8 62 integrin to autophagy, which is diminished in the absence of β8 integrin. Collectively, 63 our data reveal β8 integrin to critically co-regulate PDAC cell radiochemoresistance, 64 intracellular vesicle trafficking and autophagy upon irradiation. 65 66 Implications 67 This study identified β8 integrin as essential determinant of PDAC cell 68 radiochemosensitivity and as novel potential cancer target. 69 Downloaded from mcr.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 23, 2019; DOI: 10.1158/1541-7786.MCR-18-1352 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Jin and Lee et al., page 4 70 Introduction 71 Pancreatic ductal adenocarcinoma (PDAC) is one of the five most lethal 72 malignancies in the world. While the 5-year overall survival rate is about 15-20% for 73 resectable patients (1,2) most patients presenting at late stage with a treatment- 74 refractory disease survive significantly less. Neoadjuvant chemotherapy (e.g. 75 gemcitabine, nab-paclitaxel, FOLFIRINOX (folinic acid, 5-fluorouracil, irinotecan, and 76 oxaliplatin) is administered to patients, whose tumors seem irresectable or borderline 77 resectable. Alternatives such as radiation therapy and biologicals were neither 78 systematically evaluated in large clinical trials nor showed great advantages over 79 standard of care yet (2,3). 80 One putative cancer target in PDAC are PDAC cell/extracellular matrix (ECM) 81 interactions as PDACs are stroma-rich tumors (4). This stroma consists of numerous 82 ECM components such collagens, laminins, fibronectin, and hyaluronic acid (5). Cells 83 interact with ECM components via cell adhesion receptors that coalesce with 84 receptor tyrosine kinases, adapter and signaling molecules to form focal adhesion 85 complexes. These membranous multiprotein complexes are essentially co-regulating 86 key cell functions like survival, cell death, proliferation, metastasis and therapeutic 87 resistance (6,7). In previous work, we and others have documented the 88 radiochemosensitizing potential of integrin and focal adhesion protein targeting, as 89 the major family facilitating cell/ECM interactions. Examples for preclinically identified 90 novel targets are β1 integrin (8), αvβ3 integrin (9), αvβ6 integrin (10), FHL2 (11), 91 APPL1 and 2 (12), Caveolin 1 (8,13), as well as small integrin binding ligand N-linked 92 glycoproteins (called SIBLINGs) or secreted protein acidic and rich in cysteine (called 93 SPARC) families (14). Particularly the anti-integrin approaches were exploited for 94 molecular imaging (15), while biologicals inhibiting focal adhesion proteins (FAPs) 95 have not found their way into the clinic. Owing to the critical function of focal Downloaded from mcr.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 23, 2019; DOI: 10.1158/1541-7786.MCR-18-1352 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Jin and Lee et al., page 5 96 adhesions for prosurvival signaling in normal and cancer cells, we established a high- 97 throughput esiRNA-based screening for more physiological three-dimensional (3D) 98 cell cultures to more systematically characterize the role of FAPs for PDAC 99 radiochemoresistance. Intriguingly, we identified β8 integrin as top druggable target 100 eliciting radiosensitization of PDAC cells. 101 β8 integrin, a 769-amino acids containing type I transmembrane protein, 102 consists of a large extracellular domain, a VWFA domain, four cysteine-rich repeats 103 and a short cytosolic domain (16,17). Recent studies demonstrated that, unlike other 104 β integrins, the cytosolic tail of β8 integrin shares no apparent homology and does 105 not directly influence cell adhesion. This suggests β8 integrin signaling to be distinct 106 from other β integrins (16). A connection between β8 and αV integrin as well as the 107 TGFβ signaling cascade has been reported (18). Others exhibited β8 integrin 108 involvement in liver cancer resistance to gefitinib (19), interactions with EphB4 109 receptor (20), as well as dependency of differentiation and radiosensitivity on β8 110 integrin in glioma cells (21). 111 Based on the fact that PDAC is highly therapy-refractory, there appears an 112 imbalance in survival and death mechanisms per se and under treatment. Partly, the 113 therapy resistance found in PDAC arises from autophagy (22). Autophagy is a highly 114 conserved catabolic process involving the formation of double-membraned vesicles 115 known as autophagosomes that engulf cellular proteins and organelles for delivery to 116 the lysosome (23). The impact of autophagy seems tissues and cancer type 117 dependent. In general, autophagy has two opposing function. One is cytoprotective 118 eliciting therapeutic resistance; the other one is cytotoxic inducing autophagic cell 119 death. Recent studies have shown that autophagy as a prosurvival and resistance 120 mechanism against chemotherapy treatment in PDAC (24,25). Downloaded from mcr.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 23, 2019; DOI: 10.1158/1541-7786.MCR-18-1352 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Jin and Lee et al., page 6 121 In the present study, we explored the function of β8 integrin in PDAC therapy 122
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