Published OnlineFirst August 20, 2019; DOI: 10.1158/0008-5472.CAN-18-3965 Cancer Molecular Cell Biology Research SF3B2-Mediated RNA Splicing Drives Human Prostate Cancer Progression Norihiko Kawamura1,2, Keisuke Nimura1, Kotaro Saga1, Airi Ishibashi1, Koji Kitamura1,3, Hiromichi Nagano1, Yusuke Yoshikawa4, Kyoso Ishida1,5, Norio Nonomura2, Mitsuhiro Arisawa4, Jun Luo6, and Yasufumi Kaneda1 Abstract Androgen receptor splice variant-7 (AR-V7) is a General RNA splicing SF3B2 complex-mediated alternative RNA splicing constitutively active AR variant implicated in U2 castration-resistant prostate cancers. Here, we show U2 snRNA that the RNA splicing factor SF3B2, identified by 3’ 3’ in silico and CRISPR/Cas9 analyses, is a critical 5’ 3’ splice site 5’ SF3B7 AR-V7 5’ A U2AF2 AGA Exon ? determinant of expression and is correlated SF3B6(p14) SF3B4 SF3B1 SF3B4 SF3B1 with aggressive cancer phenotypes. Transcriptome SF3B5 SF3B2 SF3B3 SF3B2 SF3B3 and PAR-CLIP analyses revealed that SF3B2 con- SF3A3 SF3B2 complex SF3A3 SF3A1 SF3A1 SF3b complex trols the splicing of target genes, including AR, to AR pre-mRNA drive aggressive phenotypes. SF3B2-mediated CE3 aggressive phenotypes in vivo were reversed by AR-V7 mRNA AR mRNA AR-V7 knockout. Pladienolide B, an inhibitor of CE3 a splicing modulator of the SF3b complex, sup- Drive malignancy pressed the growth of tumors addicted to high While the SF3b complex is critical for general RNA splicing, SF3B2 promotes inclusion of the target exon through recognizing a specific RNA motif. SF3B2 expression. These findings support the idea © 2019 American Association for Cancer Research that alteration of the splicing pattern by high SF3B2 expression is one mechanism underlying prostate cancer progression and therapeutic resistance. This study also provides evidence supporting SF3B2 as a candidate therapeutic target for treating patients with cancer. Significance: RNA splicing factor SF3B2 is essential for the generation of an androgen receptor (AR) variant that renders prostate cancer cells resistant to AR-targeting therapy. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5204/F1.large.jpg. Introduction Splicing of precursor mRNA (pre-mRNA) is a critical process involving the alternative use of exons/introns leading to diverse mature mRNAs from a limited numbers of genes (1). Recent 1Division of Gene Therapy Science, Osaka University Graduate School of Med- icine, Suita, Osaka, Japan. 2Department of Urology, Osaka University Graduate genome-wide analyses of cancer transcriptomes have revealed School of Medicine, Suita, Osaka, Japan. 3Department of Otorhinolaryngology- globally aberrant splicing profiles including exon skipping and Head and Neck Surgery, Osaka University Graduate School of Medicine, Suita, intron retention in mature mRNAs (2, 3). Although the molecular Osaka, Japan. 4Graduate School of Pharmaceutical Sciences, Osaka University, mechanisms underlying aberrant splicing in cancer are largely 5 Suita, Osaka, Japan. Department of Gynecology, Osaka University Graduate unknown, one of the causes of aberrant splicing in cancers may be School of Medicine, Suita, Osaka, Japan. 6James Buchanan Brady Urological mutations in splicing factors including SF3B1, U2AF1, SRSF2 and Institute and Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland. ZRSR2, as well as altered expression of splicing factors, such as RBFOX2, MBNL1/2, and QKI (2, 4). Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Prostate cancer is one of most frequently detected cancers in men. Although the majority of localized prostate cancers are fi N. Kawamura and K. Nimura are the co- rst authors of this article. curable, progressive and metastatic prostate cancer contributes Corresponding Authors: Keisuke Nimura, Osaka University Graduate School of to approximately 307,000 cancer-related deaths each year world- Medicine, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3901; Fax: 81-6-6879- wide (5). Androgen deprivation therapy (ADT) is the first-line 3909; E-mail: [email protected]; and Yasufumi Kaneda, therapy for men with metastatic prostate cancer, but almost all [email protected] men develop castration-resistant prostate cancer (CRPC) after – Cancer Res 2019;79:5204 17 first-line ADT. Patients with CRPC may be treated with additional doi: 10.1158/0008-5472.CAN-18-3965 hormonal therapies, including abiraterone acetate and enzaluta- Ó2019 American Association for Cancer Research. mide, which are newly developed inhibitors of androgen receptor 5204 Cancer Res; 79(20) October 15, 2019 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst August 20, 2019; DOI: 10.1158/0008-5472.CAN-18-3965 SF3B2-Mediated RNA Splicing in Prostate Cancer Progression (AR) signaling with proven survival benefit in patients with volume reached approximately 100–300 mm3 for 22Rv1 or CRPC (6–9). A significant proportion of men with CRPC are approximately 200–400 mm3 for LNCaP95, the pladienolide B resistant to abiraterone acetate and enzalutamide, and nearly all derivative (5 mg/kg) or vehicle was administered intraperito- men develop acquired resistance to these agents over a period of neally to each mouse on days 0, 2, 4, and 6, as described 1–2 years. Because both abiraterone acetate and enzalutamide previously (12) with minor modification. The relative tumor exhibit anticancer effects through inhibiting of the ligand-binding volume was calculated as the ratio between the tumor volume domain at the C-terminus of the AR, they may not suppress AR at time t and the tumor volume at the start of treatment. signaling mediated by truncated AR splice variants (AR-V) lacking the ligand-binding domain (10). Among the many AR-Vs that Flow cytometry analysis and cell sorting have been characterized (11), AR-V7 is most compatible with AR-V7-GFP cells were cultured in medium containing detection due to its high frequency and abundance relative to 0.5 mg/mL puromycin for selection after transfection with guide other AR-Vs. Detection of AR-Vs has been associated with aggres- RNA (gRNA) targeting to genes of interest or control gRNA for sive prostate cancers and CRPC progression (10). These findings 5 days. FACSAria II (BD Biosciences) was used for flow cytometry. suggest a critical role of splicing in modulating the activity of AR, a At least 50,000 events were collected per sample. After negative key prostate cancer drug target associated with CRPC progression. selection for doublet cells, the remaining gated cells were ana- However, key splicing factors that are critical for AR-V7 generation lyzed and sorted on the basis of GFP expression levels. Dead cells have not been definitively identified and characterized. were stained by propidium iodide (Dojindo) and Annexin V In this study, we identified SF3B2 (also known as SF3b145 or (BioLegend). The data were analyzed using FlowJo software SAP145) as a positive splicing regulatory factor for AR-V7 by (Version 10). in silico analyses of RNA-binding factors associated with AR-V7 expression using transcriptome data from prostate cancer patients Quantitative RT-PCR followed by examination of AR-V7 expression after the candidate RNA was isolated using the Isogen RNA extraction kit (Wako splicing factors were knocked out by the CRISPR/Cas9 system. Pure Chemicals). cDNA was synthesized from total RNA using the By genome-wide RNA splicing analysis and photoactivatable High-Capacity cDNA Reverse Transcription Kit (Applied Biosys- ribonucleoside-enhanced cross-linking immunoprecipitation tems). qPCR was performed using the SYBR Green PCR Master (PAR-CLIP), we show that SF3B2 controls the inclusion of Mix (Applied Biosystems) and the CFX384 Real-Time System SF3B2-bound exons and an exclusion of SF3B2-bound introns. (Bio-Rad). The primers used in this study are listed in Supple- Moreover, pladienolide B, an inhibitor of a splicing modulator mentary Table S1. Genomic DNA contamination was examined SF3B complex, repressed SF3B2-addicted tumor growth under by evaluating reverse transcription reaction samples lacking castration conditions in vivo. Collectively, these results indicate reverse transcriptase. that SF3B2 is a critical determinant of RNA splicing and gene RNA-seq expression patterns and controls the expression of key genes Sequencing libraries from at least two biological replicate associated with CRPC progression, such as AR-V7. RNA samples were prepared using NEBNext Ultra RNA Library Prep Kits for Illumina (NEB) following the manufacturer's Materials and Methods instructions. Cell culture and transfection RNA precipitation using Halo-tag The 22Rv1 prostate cancer cell line was purchased from the The Halo-tag was knocked-in immediately before the stop ATCC. LNCaP95 was a gift from J. Luo (Johns Hopkins University codon of the SF3B2 gene in 22Rv1 cells. The total cell extracts School of Medicine, Baltimore, MD). Cell line authentication was of 5 Â 106 SF3B2-Halo knocked-in cells were incubated with fi Mycoplasma not performed. All cells were con rmed to be negative 50 mL of Halo-Link Resin (Promega) overnight at 4 C to precip- before inoculation of cells into mice (TaKaRa). The length of time itate SF3B2–RNA complexes. Contaminated genomic DNA was between thawing and use is less than 3 months. The detailed removed from the extracted RNA using 1 ml of TURBO DNase method is described in extended experimental procedures. (Thermo Fisher Scientific). Enrichment of RNAs was determined by quantitative RT-PCR. Xenograft prostate cancer model All experiments using mice was approved by Osaka Univer- PAR-CLIP sity Animal Experiments committee and was performed fol- PAR-CLIP was performed according to the original proto- lowing the guidelines. The in vivo tumor growth of human col (13) and another protocol with some modifications (14). prostate cancer cells was determined using a subcutaneous The antibodies used in this study are listed in Supplementary transplant xenograft model. Male NOD/SCID mice (Charles Table S2. The detailed method is described in extended experi- River) were castrated surgically at 7 weeks of age.