bioRxiv preprint doi: https://doi.org/10.1101/597112; this version posted April 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Title page 2 3 Inhibiting TG2 sensitizes lung cancer to radiotherapy through interfering 4 TOPOIIα-mediated DNA repair 5 6 Xiao Lei#, Zhe Liu#, Kun Cao#, Yuanyuan Chen#, Jianming Cai, Fu Gao*, Yanyong 7 Yang* 8 9 #Authors contributed equally to this work. 10 11 Department of Radiation Medicine, Faculty of Naval Medicine, Second Military 12 Medical University, 800, Xiangyin Road, 200433, Shanghai, P.R. China; 13 14 *Corresponding author: Yanyong Yang, Fu Gao and Jianming Cai. 15 Address: Department of Radiation Medicine, Faculty of Naval Medicine, Second 16 Military Medical University; 800, Xiangyin Road, 200433, Shanghai, P.R. China. Fax: 17 +86-21-81871148. E-mail: [email protected], [email protected], 18 [email protected]; 19 20 Running title: Targeting TG2 sensitizes lung cancer to radiotherapy 21 22 Keywords: TG2, Radiosensitization, TOPOIIα, NSCLC, DNA repair 23 24 Conflicts of interest 25 The authors have no conflicts of interest to disclose. 26 1 bioRxiv preprint doi: https://doi.org/10.1101/597112; this version posted April 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 27 Abstract 28 Radiotherapy is an indispensable strategy for lung cancer, however, treatment failure 29 or reoccurrence is often found in patients due to the developing radioresistance. Novel 30 approaches are required for radiosensitizing to improve the therapeutic efficacy. In 31 present study, we found that transglutaminase 2 (TG2) confers radioresistance in 32 non-small cell lung cancer (NSCLC) cells through regulating TOPOIIα and promoting 33 DNA repair. Our data showed that TG2 inhibitor or knockdown increased NSCLC 34 radiosensitivity in vivo and in vitro. We found that TG2 translocated into nucleus and 35 located to DSB sites, surprisingly, knockdown TG2 or glucosamine inhibited the 36 phosphorylation of ATM, ATR and DNA-Pkcs. Through IP-MS assay and functional 37 experiments, we identified that TOPOIIα as an downstream factor of TG2. Moreover, 38 we found that TGase domain account for the interaction with TOPOIIα. Finally, we 39 found that TG2 expression was correlated with poor survival in lung adenocarcinoma 40 instead of squamous cell carcinoma. In conclusion, we demonstrated that inhibiting 41 TG2 sensitize NSCLC to IR through interfere TOPOIIα mediated DNA repair, 42 suggesting TG2 as a potential radiosensitizing target in NSCLC. 43 2 bioRxiv preprint doi: https://doi.org/10.1101/597112; this version posted April 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 44 Introduction 45 Radiotherapy is an indispensable strategy in treating lung cancer, of which 80% is 46 non-small cell lung cancer (NSCLC) with poor outcomes (1, 2). Despite the advance 47 in physical techniques, novel approaches in radiosensitizing from biological aspect 48 are required to overcome the growing radioresistance during radiotherapy. The current 49 research involving radiosensitization mainly falls in the following fields: DNA 50 damage repair, poly (adenosine diphosphate–ribose) polymerase inhibitors, histone 51 deacetylase inhibitors, tumor hypoxia and redox conditions, antiangiogenic drugs etc 52 (3). However, most of these drugs are in research process or clinical trials, efficacy as 53 well as normal tissue toxicity limits their application. 54 Transglutaminase 2 (TG2), a member of Transglutaminases family, exerts 55 multiple physiological functions and is associated with cancer cell survival, metastatic 56 behavior and chemoresistance (4-7). It has been proved that TG2 was related to 57 multiple drug resistance including cisplatin, histone deacetylase inhibitor, EGFR-TKI 58 etc (5, 8, 9). Recently, the prognostic value of elevated TG2 for patient survival has 59 been illustrated in NSCLC and are attracting more and more attention (10, 11). These 60 studies indicated that TG2 might be critical for radiation resistance in NSCLC. When 61 we are preparing this manuscript, Sheng et al. reported that TG2 inhibitor KCC009 62 induces radiosensitization in lung adenocarcinoma cells(12). However, the detailed 63 role of TG2 in NSCLC radioresistance and the underlying mechanism remains 64 unclear. 65 Previous studies indicated that TG2 was related to DNA damage repair, which is 66 aberrant active in cancer cells (13-15). Previous study had showed that ATM inhibitor 67 KU55933 abrogated the constitutively activation of TG2 induced by genotoxic drug 68 MNNG (13). ATM mediated NF-kB activation increased the level of TG2. TG2 was 69 also proved as a target of p53 and involved in DNA damage repair, and knockdown of 70 p53 reduced the level of TG2 (14, 15). But the response of TG2 to ionizing radiation 71 and the exact role of TG2 in DNA repair remains to be uncovered. 72 Here, we report that TG2 confers to radioresistance in NSCLC and enhanced 3 bioRxiv preprint doi: https://doi.org/10.1101/597112; this version posted April 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 73 DNA repair capacity through directly interacting with DNA topoisomerase IIα 74 (TOPOIIα). We found that ionizing radiation (IR) resulted in a rapid nuclear 75 translocation of TG2 and knockdown TG2 significantly inhibited DNA repair. TG2 76 was found to bind and activate TOPOIIα in nucleus to initial DNA damage repair 77 processes, such as phosphorylation of ATM, ATR and DNA-PKcs. Moreover, we used 78 a clinically used TG2 inhibitor, glucosamine, and found it significantly sensitized lung 79 cancer to IR in vivo and in vitro. Finally, we found TG2 was significantly correlated 80 with the survival in lung adenocarcinoma instead of squamous cell carcinoma patients, 81 which suggest possible prognostic value of TG2 in lung adenocarcinoma. These data 82 provide the possibility of clinical translation of TG2 inhibitor in the radiosensitization 83 of lung cancer. 84 4 bioRxiv preprint doi: https://doi.org/10.1101/597112; this version posted April 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 85 Results 86 Inhibition of TG2 sensitizes lung cancer cells to ionizing radiation 87 It has been proved that TG2 high expression was related to chemoresistance of 88 multiple cancers (16-18). To determine whether TG2 participates in radioresistance in 89 NSCLC, firstly we used a TG2 inhibitor, glucosamine, which was already used in 90 clinics as an anti-inflammatory drug. We found that glucosamine effectively reduced 91 cell viability in A549 cells, while showed little influence on normal lung BEAS-2B 92 cells (Fig. 1A). Besides, glucosamine effectively inhibited TG2 level at the 93 concentration of 5mM in A549 cells (Fig. 1B). Compared with normal lung BEAS-2B 94 cells, TG2 expression was also found to be elevated in lung adenocarcinoma cell lines 95 including A549, H1975 and H358 cells. (Fig. 1C, S1A). By using colony formation 96 assay, we found that glucosamine or TG2 knockdown significantly sensitized A549, 97 H1299, H460 cells to IR, while glucosamine showed no further sensitizing effects on 98 TG2 knockdown cells (Fig. 1D-F). This data was also confirmed in CRISPR Cas9 99 mediated TG2 knockout cells (Fig. 1G). Alternatively, we used apoptosis assay to 100 determine cellular damage in TG2 inhibited cells. It was found that glucosamine 101 treatment resulted in more apoptotic cells in response to IR, while glucosamine 102 showed no sensitizing effects on BEAS-2B cells (Fig. 1H, Fig. S1B, C). 103 Radiation induces TG2 nuclear translocation and initiates DNA damage response 104 To figure out how TG2 confers to radioresistance, we investigated its subcellular 105 location and the relationship with DNA damage repair, the main effects of radiation 106 response. By using Immunofluorescence staining and nuclear protein western blot 107 assay, we found that radiation rapidly induced TG2 nuclear translocation, which could 108 be inhibited by glucosamine (Fig. 2A, B, S2B). Based on distinct functions of TG2, 109 we used calcium inhibitor perillyl alcohol (POH), TG2 activity inhibitor cystamine, 110 and NF-kB inhibitor QNZ, our data showed that QNZ inhibited radiation-induced 111 nuclear translocation of TG2 (Fig. S2A). Moreover, we found that glucosamine 112 treatment significantly inhibited the phosphorylation of DNA-PKcs, ATM and ATR, 113 which are critical for initiating DNA damage repair (Fig. 2C). Further, we used a 5 bioRxiv preprint doi: https://doi.org/10.1101/597112; this version posted April 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 114 siRNA of TG2 to investigate its role in DDR, and found the same effects on 115 DNA-PKcs, ATM and ATR inhibition. However, TG2 siRNA combined with 116 glucosamine treatment didn’t showed any additive effects (Fig. 2D). To investigate 117 the influence of TG2 inhibitor on DNA damage, we examined γH2AX foci and found 118 that TG2 knockout significantly impaired DNA repair in response to IR (Fig. 2E, F). 119 By using a comet assay, we confirmed that more DNA damage remains unrepaired in 120 cells treated with glucosamine (Fig. 2G, H, I). 121 TG2 interacts with TOPOⅡα and participates in DNA repair 122 To identify the specific target of TG2, we conducted an Immunoprecipitation–Mass 123 Spectrometry (IP-MS) assay in A549 cells. Through bioinformatics analysis, we 124 found that 134 proteins bind to TG2 in radiation group compared with normal group 125 (Fig.