Author Manuscript Published OnlineFirst on April 27, 2020; DOI: 10.1158/0008-5472.CAN-19-2549 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Loss of a negative feedback loop between IRF8 and AR promotes 2 prostate cancer growth and enzalutamide resistance 3 4 Hongxi Wu1, †, Linjun You1, †, Yan Li1, †, Zhili Zhao1, †, Guangjiang Shi1, Zhen 5 Chen1, Zhuo Wang1, Xianjing Li1, Shijia Du1, Wanli Ye1, Xiaofang Gao1, Jingjing 6 Duan1, Yan Cheng1, Weiyan Tao1, Jinsong Bian4, Jin-Rong Zhou3, Qingyi Zhu2, * & 7 Yong Yang1, * 8 9 1State Key Laboratory of Natural Medicines, China Pharmaceutical University, 10 Nanjing 211198, China 11 2Department of Urology, Jiangsu Province Hospital of Traditional Chinese Medicine, 12 Nanjing 210029, China 13 3Nutrition/Metabolism Laboratory, Department of Surgery/General Surgery, Harvard 14 Medical School, Boston, Massachusetts 15 4Department of Pharmacology, Yong Loo Lin School of Medicine, National University 16 of Singapore, 117597, Singapore 17 18 Footnotes: † They contributed equally to this paper. 19 20 Running title: IRF8 inhibits prostate cancer progress. 21 22 Keywords: Prostate cancer; Castration-resistant prostate cancer; Interferon regulatory 23 factor 8; Enzalutamide resistance; Androgen receptor 24 25 Disclosures of Potential Conflicts of Interest: We declare no conflict of interest. 26 27 Corresponding Author: 28 Prof. Yong Yang, State Key Laboratory of Natural Medicines, China Pharmaceutical 29 University, Nanjing 211198, China. Phone and Fax: 86-025-86185622; E-mail: 30 [email protected] 31 Dr. Qingyi Zhu, Department of Urology, Jiangsu Province Hospital of Traditional 32 Chinese Medicine, Nanjing 210029. Phone and Fax: 86-025-86617141-90506; E-mail: 33 [email protected]. 34 35 36 37 38 39 1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 27, 2020; DOI: 10.1158/0008-5472.CAN-19-2549 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 40 Abstract 41 In incurable castration-resistant prostate cancer (CRPC), resistance to the novel 42 androgen receptor (AR) antagonist enzalutamide (ENZ) is driven mainly by AR 43 overexpression. Here we report that the expression of interferon regulatory factor 8 44 (IRF8) is increased in primary prostate cancer (PCa) but decreased in CRPC 45 compared to normal prostate tissue. Decreased expression of IRF8 positively 46 associated with CRPC progression and ENZ resistance. IRF8 interacted with AR and 47 promoted its degradation via activation of the ubiquitin/proteasome systems. 48 Epigenetic knockdown of IRF8 promoted AR-mediated PCa progression and ENZ 49 resistance in vitro and in vivo. Furthermore, IFNα increased expression of IRF8 and 50 improved the efficacy of ENZ in CRPC by targeting the IRF8-AR axis. We also 51 provide preliminary evidence for the efficacy of IFNα with hormonotherapy in a 52 clinical study. Collectively, this study identifies IRF8 both as a tumor suppressor in 53 PCa pathogenesis and a potential alternative therapeutic option to overcome ENZ 54 resistance. 55 56 The statement of significance 57 Findings identify IRF8-mediated AR degradation as a mechanism of resistance to AR 58 targeted therapy, highlighting the therapeutic potential of IFNα in targeting IRF8-AR 59 axis in CRPC. 60 2 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 27, 2020; DOI: 10.1158/0008-5472.CAN-19-2549 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 61 Introduction 62 Androgen and androgen receptor (AR) signaling pathway play a critical role in the 63 carcinogenesis and progression of prostate cancer (PCa), which is the second leading 64 cause of cancer-related deaths in North America (1). Consequently, androgen 65 depletion therapy (ADT) has been the first-line therapy for primary PCa for decades. 66 Despite the initial efficacy of ADT, regeneration of tumour will occur eventually in 67 almost every patient, leading to alleged castration-resistant prostate cancer (CRPC). 68 AR-targeted therapy is a gold standard therapy for CRPC (2-4). Enzalutamide 69 (ENZ) is approved for the treatment of CRPC patients, based on its ability to block 70 androgen binding to AR in a competitive manner, inhibiting AR nuclear translocation 71 and DNA fixation (5-7). Despite the success of ENZ in improving the overall survival 72 of CRPC patients, inherent or acquired ENZ resistance (ENZR) remains a major 73 clinical challenge (8-10). AR deregulation, including overexpression of AR 74 full-length (ARfl) and AR variants (ARvs), has been identified as a unique factor 75 consistently associated with the progression of PCa to CRPC and ENZR (9); therefore, 76 studying the mechanisms of AR deregulation is critical to improve the efficacy of 77 current treatments. AR undergoes degradation mainly by the ubiquitin/proteasome 78 system, as well as modification of protein stability triggered by ubiquitin-like 79 signaling pathways, such as ISGylation (Interferon stimulated gene) (11,12). AR is a 80 type I interferon (IFN) regulated protein and disruption of interferon system genes 81 plays a novel function in malignant transformation of PCa (12-15). However, the 3 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 27, 2020; DOI: 10.1158/0008-5472.CAN-19-2549 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 82 mechanism for AR maintaining its stability under the influence of interferon system 83 remains unknown. 84 Activity of the interferon system is mainly regulated by interferon regulatory 85 factor 8 (IRF8), a member of the IRF family (IRF1-9) (16). Loss of IRF8 in immune 86 cells leads to the occurrence of chronic myelogenous leukemia and aberrant 87 methylation of IRF8 gene in nonhemopoietic cells play an increasingly important role 88 in tumorigenesis (17-25). Among all the IRFs, only IRF8 facilitates a protein-protein 89 interactions model by interacts with proteins containing the PEST motif, a region that 90 plays an important role in protein degradation by the proteasome system, including 91 AR degradation (26,27). Emerging evidences suggest that IRF8 may function in the 92 cytosol ubiquitylation system (16,28), but the exact role and the regulation 93 mechanism of IRF8 in cancers, especially in PCa tissues, have not been explored. 94 More importantly, the relationship between IRF8 with AR in PCa and whether IRF8 95 interacts with AR (containing the PEST motif) and functions in the regulation of AR 96 stability are still unknown. 97 The present study provides evidences to support an advantaged role for IRF8 in 98 CRPC and ENZR, and this role of IRF8 is likely mediated through regulating AR 99 stability. We then evaluated the potential of IFNα targeting IRF8 to improve the 100 therapeutic efficacy of hormonotherapy in PCa, suggesting that IFNα combined with 101 ENZ is an attractive therapeutic strategy for CRPC and ENZR. 102 4 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 27, 2020; DOI: 10.1158/0008-5472.CAN-19-2549 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 103 Materials and Methods 104 Reagents 105 Fetal bovine serum (FBS) and charcoal-stripped, dextran-treated fetal bovine serum 106 (CSS, depleted androgen and any other steroid) were purchased from Biological 107 Industries (Israel). Enzalutamide (MedChemExpress, USA), R1881, 108 Dihydrotestosterone (DHT) (Meilunbio, China), EGF, IGF-1 were commercially 109 obtained (Peprotech, USA). Serial dilutions of all drugs were made using DMSO. 110 111 Cell lines and primary cultures 112 PC-3 cells were cultured in F12 Medium, HEK293T were cultured in DMEM 113 Medium, 22RV1 cells and LNCaP cells were cultured in RPMI-1640 Medium. PC-3, 114 22RV1, LNCaP and HEK293T cells were purchased from were purchased from Cell 115 Bank of the Chinese Academy of Sciences (Shanghai, China), two stable 116 LNCaP-sh-IRF8-Puro (LNCaP-shIRF8) and LNCaP-sh-negative control 117 (LNCaP-shNC)-Puro cells were purchased from GenePharma Technology Co. Ltd. 118 (GenePharma, Shanghai, China). All cells were authenticated by the Short Tandem 119 Repeat DNA profiling (Cobioer, Nanjing, China) and confirmed Mycoplasma free 120 using GMyc-PCR Mycoplasma Test Kit (YeSen; Shanghai, China, 40601ES10) after 121 last experiment, and used within 15 cell passages after thawing. All cell lines were 122 cultured in medium supplemented with 10% fetal bovine serum (Sigma), 1% 123 Penicillin-Streptomycin (Gibico), 5% CO2, 37 °C. 5 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 27, 2020; DOI: 10.1158/0008-5472.CAN-19-2549 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 124 Plasmids, siRNA and DNA transient transfections 125 Plasmids including pcDNA3 (Vector), pcDNA3-hIRF8 (IRF8), pcDNA3-hAR (AR), 126 containing the whole CDS domains were constructed by Genscript (Nanjing, China); 127 double luciferase reporter plasmid pEZX-FR03-hIRF8-luc, carrying the IRF8 128 promoter (-1106-+166bp), was constructed by FulenGen (Guangzhou, China); 129 3xFlag-Ub plasmid was generously provided by Dr. Guo-qiang Xu (Soochow 130 University, China). For transient transfections, cells were seeded into six-well plates 131 at 150,000 cells per well and transfected with IRF8 or AR expression vectors using 132 empty vector (pvector) as control. The total plasmid DNA was adjusted to the same 133 with empty vector. 134 siRNA and shRNA targeting IRF8 are as follows: siNC (shNC): TTC TCC GAA 135 CGT GTC ACG TTT C; siRNA1# (shIRF8A): GCA GTT CTA TAA CAG CCA GGG; 136 siRNA2# (shIRF8B): GGG AAG AGT TTC CGG ATA TGG.