Author Manuscript Published OnlineFirst on August 20, 2019; DOI: 10.1158/0008-5472.CAN-18-3965 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 SF3B2-mediated RNA splicing drives human prostate cancer progression 2 3 Norihiko Kawamuraa,b,1, Keisuke Nimuraa,1,2, Kotaro Sagaa, Airi Ishibashia, Koji 4 Kitamuraa,c, Hiromichi Naganoa, Yusuke Yoshikawad, Kyoso Ishidaa,e, Norio 5 Nonomurab, Mitsuhiro Arisawad, Jun Luof & Yasufumi Kanedaa,2 6 7 aDivision of Gene Therapy Science, Osaka University Graduate School of Medicine, 8 Suita, Osaka 565-0871, Japan 9 bDepartment of Urology, Osaka University Graduate School of Medicine, Suita, 10 Osaka 565-0871, Japan 11 cDepartment of Otorhinolaryngology-Head and Neck surgery, Osaka University 12 Graduate School of Medicine, Suita, Osaka 565-0871, Japan 13 dGraduate School of Pharmaceutical Sciences, Osaka University 14 Yamada-oka 1-6, Suita, Osaka 565-0871, Japan 15 eDepartment of Gynecology, Osaka University Graduate School of Medicine, Suita, 16 Osaka 565-0871, Japan 17 fJames Buchanan Brady Urological Institute and Department of Urology, Johns 18 Hopkins University School of Medicine, 600 N. Wolfe St., Baltimore, MD 21287, 19 USA 20 1Co-first 21 2Corresponding author 22 23 Running title: SF3B2-mediated RNA splicing in prostate cancer progression 24 25 Keywords: SF3B2, RNA splicing, SF3b, AR-V7, prostate cancer 26 27 Significance: 28 RNA splicing factor SF3B2 is essential for the generation of an androgen receptor 29 (AR) variant that renders prostate cancer cells resistant to AR-targeting therapy. 30 31 Additional information: 32 This work was supported by Platform Project for Supporting Drug Discovery and 33 Life Science Research (Basis for Supporting Innovative Drug Discovery and Life 1 Downloaded from cancerres.aacrjournals.org on October 7, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 20, 2019; DOI: 10.1158/0008-5472.CAN-18-3965 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 34 Science Research (BINDS)) from AMED under Grant Number JP19am0101084 and 35 DAICEL, Inc. to K. Nimura. 36 37 Corresponding authors: 38 Keisuke Nimura, Division of Gene Therapy Science, Osaka University Graduate 39 School of Medicine, Suita, Osaka 565-0871, Japan 40 tel:+81-6-6879-3901; fax:+81-6-6879-3909 41 [email protected] 42 Yasufumi Kaneda, Division of Gene Therapy Science, Osaka University Graduate 43 School of Medicine, Suita, Osaka 565-0871, Japan 44 tel:+81-6-6879-3901; fax:+81-6-6879-3909 45 [email protected] 46 47 N.K., K.N., and Y.K. made patents application with DAICEL, Inc.. 48 49 The manuscript includes 4,980 words of text including introduction, methods, results, 50 discussion, and 7 figures. Supplemental Information includes Extended 51 Experimental Procedures, 8 supplementary figures, and 3 supplementary tables. 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 2 Downloaded from cancerres.aacrjournals.org on October 7, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 20, 2019; DOI: 10.1158/0008-5472.CAN-18-3965 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 67 68 Abstract 69 Androgen receptor splice variant-7 (AR-V7) is a constitutively active AR variant 70 implicated in castration-resistant prostate cancers. Here, we show that the RNA 71 splicing factor SF3B2, identified by in silico and CRISPR/Cas9 analyses, is a critical 72 determinant of AR-V7 expression and is correlated with aggressive cancer 73 phenotypes. Transcriptome and PAR-CLIP analyses revealed that SF3B2 controls 74 the splicing of target genes, including AR, to drive aggressive phenotypes. 75 SF3B2-mediated aggressive phenotypes in vivo were reversed by AR-V7 knockout. 76 Pladienolide B, an inhibitor of a splicing modulator of the SF3b complex, 77 suppressed the growth of tumors addicted to high SF3B2 expression. These 78 findings support the idea that alteration of the splicing pattern by high SF3B2 79 expression is one mechanism underlying prostate cancer progression and 80 therapeutic resistance. This study also provides evidence supporting SF3B2 as a 81 candidate therapeutic target for treating cancer patients. 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 3 Downloaded from cancerres.aacrjournals.org on October 7, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 20, 2019; DOI: 10.1158/0008-5472.CAN-18-3965 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 100 101 INTRODUCTION 102 Splicing of precursor mRNA (pre-mRNA) is a critical process involving the 103 alternative use of exons/introns leading to diverse mature mRNAs from a limited 104 numbers of genes (1). Recent genome-wide analyses of cancer transcriptomes 105 have revealed globally aberrant splicing profiles including exon skipping and intron 106 retention in mature mRNAs (2,3). Although the molecular mechanisms underlying 107 aberrant splicing in cancer are largely unknown, one of the causes of aberrant 108 splicing in cancers may be mutations in splicing factors including SF3B1, U2AF1, 109 SRSF2 and ZRSR2, as well as altered expression of splicing factors, such as 110 RBFOX2, MBNL1/2, and QKI (2,4). 111 Prostate cancer is one of most frequently detected cancers in men. 112 Although the majority of localized prostate cancers are curable, progressive and 113 metastatic prostate cancer contributes to ~307,000 cancer deaths each year 114 worldwide (5). Androgen deprivation therapy (ADT) is the front line therapy for men 115 with metastatic prostate cancer, but almost all men develop castration-resistant 116 prostate cancer (CRPC) after first-line ADT. CRPC patients may be treated with 117 additional hormonal therapies, including abiraterone acetate and enzalutamide, 118 which are newly developed inhibitors of androgen-receptor (AR) signaling with 119 proven survival benefit in patients with CRPC (6–9). A significant proportion of men 120 with CRPC are resistant to abiraterone acetate and enzalutamide, and nearly all 121 men develop acquired resistance to these agents over a period of 1-2 years. 122 Because both abiraterone acetate and enzalutamide exhibit anti-cancer effects 123 through inhibiting of the ligand-binding domain at the C-terminus of the AR, they 124 may not suppress AR signaling mediated by truncated AR splice variants (AR-Vs) 125 lacking the ligand-binding domain (10). Among the many AR-Vs that have been 126 characterized (11), AR-V7 is most compatible with detection due to its high 127 frequency and abundance relative to other AR-Vs. Detection of AR-Vs has been 128 associated with aggressive prostate cancers and CRPC progression (10). These 129 findings suggest a critical role of splicing in modulating the activity of AR, a key 130 prostate cancer drug target associated with CRPC progression. However, key 131 splicing factors that are critical for AR-V7 generation have not been definitively 132 identified and characterized. 4 Downloaded from cancerres.aacrjournals.org on October 7, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 20, 2019; DOI: 10.1158/0008-5472.CAN-18-3965 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 133 In the present study, we identified SF3B2 (also known as SF3b145 or 134 SAP145) as a positive splicing regulatory factor for AR-V7 by in silico analyses of 135 RNA binding factors associated with AR-V7 expression using transcriptome data 136 from prostate cancer patients followed by examination of AR-V7 expression after 137 the candidate splicing factors were knocked out by the CRISPR/Cas9 system. By 138 genome-wide RNA splicing analysis and photoactivatable ribonucleoside-enhanced 139 cross-linking immunoprecipitation (PAR-CLIP), we show that SF3B2 controls the 140 inclusion of SF3B2-bound exons and an exclusion of SF3B2-bound introns. 141 Moreover, Pladienolide B, an inhibitor of a splicing modulator SF3B complex, 142 repressed SF3B2-addicted tumor growth under castration conditions in vivo. 143 Collectively, these results indicate that SF3B2 is a critical determinant of RNA 144 splicing and gene expression patterns and controls the expression of key genes 145 associated with CRPC progression, such as AR-V7. 146 147 148 Materials and Methods 149 1. Cell culture and transfection 150 The 22Rv1 prostate cancer cell line was purchased from the American Type Culture 151 Collection. LNCaP95 was a gift from Dr. Luo. Cell line authentication was not 152 performed. All cells were confirmed to be mycoplasma-negative before inoculation 153 of cells into mice (TaKaRa). The length of time between thawing and use is less than 154 3 months. The detailed method is described in extended experimental procedures. 155 156 2. Xenograft prostate cancer model 157 All experiments using mice was approved by Osaka University Animal Experiments 158 committee and was performed following the guidelines. The in vivo tumor growth of 159 human prostate cancer cells was determined using a subcutaneous transplant 160 xenograft model. Male NOD/SCID (non-obese diabetic/severe combined 161 immunodeficient) mice (Charles River) were castrated surgically at 7 weeks of age. 162 Cancer cells (2 x 106 cells) in a PBS / Matrigel mixture were injected subcutaneously 163 into these castrated NOD/SCID mice at 8 week of age under deep anesthesia, and 164 the mice were maintained in a temperature-controlled and pathogen-free room. All 165 animals were handled according to approved protocols and the guidelines of the 5 Downloaded from cancerres.aacrjournals.org on October 7, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 20, 2019; DOI: 10.1158/0008-5472.CAN-18-3965 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.