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- 2019 American Association for Cancer Research. mide, which are newly developed inhibitors of androgen receptor
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(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 5 106 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. Cancer cells mental procedures. (2 106 cells) in a PBS/Matrigel mixture were injected sub- cutaneously into these castrated NOD/SCID mice at 8 week of Tandem affinity purification and mass spectrometry age under deep anesthesia, and the mice were maintained in a The SF3B2-TAP–expressing prostate cancer cell line was gen- temperature-controlled and pathogen-free room. All animals erated from 22Rv1 cells. The SF3B2–TAP complex was purified were handled according to approved protocols and the guide- from nuclear extracts by TAP technology as described previous- lines of the Animal Committee of Osaka University (Osaka, ly (15). The purified proteins were precipitated with trichloroa- Japan). The tumor size was measured weekly, and the tumor cetic acid and separated on SDS-PAGE gels. Protein bands stained volume was calculated according to the following formula: with silver were excised from the gel, in-gel digested with trypsin, tumor volume (mm3) ¼ length (width)2/2. When the tumor and analyzed by LC/MS-MS.
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Bioinformatics and AR-V7–positive prostate cancers (Supplementary Fig. S2E Deep-sequencing data are available under DRA accession and S2F) and AR-V7-GFP negative cells were increased by U2AF2 DRA006189, which includes the following: 3 PAR-CLIP libraries gRNA transfection (Supplementary Fig. S2B), the correlation and 8 RNA-seq libraries. The detailed methods are described in between U2AF2 expression and AR-V7 expression in the RNA-seq extended experimental procedures. data of CRPCs was not consistent compared with SF3B2 (Sup- plementary Fig. S2F). These results support the conclusion that SF3B2 is a strong candidate splicing factor involved in the regu- Results lation of AR-V7 expression. SF3B2 is identified as a splicing factor associated with AR-V7 Increased SF3B2 expression was detected in CRPCs and pros- expression by in silico analysis of transcriptome data and by tate cancers with high AR-V7 expression (Fig. 1F). SF3B2 expres- disruption of candidate genes by CRISPR/Cas9 in prostate sion was also increased in prostate cancers with high Gleason cancer scores (8–10), a pathologic indicator of prostate cancer malig- Because increased AR-V7 expression has been detected in nancy, consistent with a similar pattern of AR-V7 expression CRPCs (16), we first sought to identify splicing factor– (Fig. 1G). In contrast, SF3B2 expression did not correlate with encoding genes with increased expression in CRPCs and AR- full-length AR expression (Supplementary Fig. S2G). Notably, V7–positive prostate cancers (Fig. 1A and B). Using previously mutations in SF3B2 did not affect AR-V7 expression or the published microarray-based gene expression data in patients with Gleason score (Supplementary Fig. S2H). Higher SF3B2 expres- benign, localized prostate cancers and CRPCs (17), we compared sion was associated with poor progression-free survival in pros- the expression level of 309 genes encoding splicing factors and tate cancer (P ¼ 0.0042 by log-rank test; Fig. 1H). Interestingly, RNA-binding proteins, selected by gene ontology (GO) terms in high SF3B2 expression was also associated with poor overall AmiGO (Fig. 1A; ref. 18). This set of genes differentiated the three survival in bladder cancer (P ¼ 0.0237), acute myeloid leukemia sample groups by cluster analysis, with a general trend toward an (AML; P ¼ 0.0316), lung adenocarcinoma (P ¼ 0.0109), head and upregulation in CRPCs compared with localized prostate cancers neck squamous cell carcinoma (P ¼ 0.0491), and breast cancer and benign prostate samples (Fig. 1A). We further examined this (P ¼ 0.0441; Supplementary Fig. S3). Thus, high SF3B2 expres- subset of genes according to AR-V7 expression levels in the The sion was associated with aggressive phenotypes of various human Cancer Genome Atlas (TCGA) prostate cancer dataset (19). Using cancers, including prostate cancer, and SF3B2 may regulate the this in silico approach, we identified 21 genes encoding splicing expression of AR-V7 as well as other genes involved in cancer factors that were upregulated in both CRPC and TCGA prostate progression. tumors with high AR-V7 expression (Fig. 1B). To determine the functional effect of the candidate splicing Depletion of SF3B2 decreases the expression of AR-V7 and factors on AR-V7 expression, we generated CWR22Rv1 (hereafter androgen-responsive genes in human prostate cancer cells called 22Rv1) prostate cancer cells with stable knock-in of GFP To examine whether SF3B2 regulates AR-V7 in cells with intact immediately before the AR-V7 stop codon in AR cryptic exon 3 AR-V7 sequences, AR-V7 protein expression in SF3B2 gRNA– (AR-V7-GFP cells) using CRISPR/Cas9 (Fig. 1C; Supplementary transfected prostate cancer cells was analyzed in 22Rv1 and Fig. S1). The GFP cassette does not include polyA sequence to LNCaP95 cells, another prostate cancer cell line expressing endog- avoid affecting the use of the cryptic exon 3 polyA site (20). 22Rv1 enous AR-V7 (23). Transfection of SF3B2 gRNA decreased AR-V7 cells have a tandem duplication of exon 3 and cryptic exon 3 in AR protein expression in both cells, concomitantly correlating with gene (Fig. 1C; ref. 21). To avoid unexpected effect on further reduced levels of SF3B2 (Fig. 2A and B). analysis, we selected a clone that lost the tandem duplication To further confirm the role of SF3B2 in the regulation of AR-V7, (Fig. 1C; Supplementary Fig. S1A–S1C). Fusion of GFP with AR- we used the AR-V7-GFP signal as a marker for isolating SF3B2- V7 was confirmed by Western blotting (Supplementary Fig. S1A depleted cells in SF3B2 gRNA–transfected AR-V7-GFP cells, since and S1B) and FACS analysis of AR-V7 gRNA–transfected AR-V7- SF3B2 knockout cells could not be established and siRNAs against GFP cells (Supplementary Fig. S1D). Full-length AR (AR-FL) was SF3B2 could not efficiently decrease SF3B2 to examine its func- consistently expressed in AR-V7-GFP cells, compared with wild- tion. SF3B2 protein was significantly depleted in AR-V7-GFP– type 22Rv1 cells, while AR-V7 expression was increased in AR-V7- negative cells in SF3B2 gRNA–transfected AR-V7-GFP cells (top), GFP cells (Supplementary Fig. S1B and S1E). It may be due to while AR-V7-GFP–positive cells (top) had comparable amounts increasing stability of AR-V7 RNA, because AR-FL expression was of SF3B2 relative to control cells (Fig. 2C and D). Thus, we defined consistent (Supplementary Fig. S1B and S1E). We could not detect the isolated AR-V7-GFP–negative cells from SF3B2 gRNA– AR-V7-GFP protein by AR-V7–specific antibody (Supplementary transfected AR-V7-GFP cells as SF3B2-depleted cells. Indeed, Fig. S1B), although we could detect the protein by GFP antibody, SF3B2-depleted cells lost not only SF3B2 but also AR-V7 protein FACS analysis, or qPCR (Supplementary Fig. S1A, S1D, and S1E). expression, while a weak decrease in AR-FL protein was detected An addition of GFP at the end of AR-V7 may prevent the AR-V7– (Fig. 2E). In contrast, AR-V7 depletion did not diminish specific antibody from recognizing the C-terminus region of AR- the amounts of SF3B2 protein, suggesting that AR-V7 was V7. This cell line allowed for the examination of endogenous AR- not involved in the upregulation of SF3B2 expression (Fig. 2F). V7 expression through the evaluation of GFP. Transfection of Collectively, these results support a key role for SF3B2 in the gRNAs against the 21 candidate genes revealed that a distinct AR- regulation of AR-V7 expression. V7-GFP–negative population that was the most striking in We next performed genome-wide mRNA expression analysis to SF3B2 gRNA–transfected cells without cell death (Fig. 1D and determine whether SF3B2 depletion led to altered expression of E; Supplementary Fig. S2A–S2D). U2AF2 (also known as androgen-responsive genes (Fig. 2G and H; Supplementary U2AF65) is known to be correlated with AR-V7 expression (22). Fig. S4A). SF3B2 depletion resulted in the decreased expression Although an increase in U2AF2 expression was detected in CRPCs of 67 out of 100 androgen-responsive genes belonging to the gene
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A B Genes encoding splicing factors and RNA-binding proteins (309 genes) 468 2 −4−20 Microarray data set (n = 122) TCGA data set (RNA-seq n = 498) Grasso et al. (17) AR-V7 low PC vs highly positive PC Localized prostate cancer vs mCRPC Genes whose expression are increased in AR-V7 Genes whose expression are increased in CRPC highly positive PCa
41 Genes 21 Genes 12 Genes
C AR-FL AR-V7 1 234567 8 1 23 Cryptic exon 3
UGA AUG UGA AUG AR in 22Rv1 Cells Cryptic exon 3 Cryptic exon 3 Exon 1 2343 5678 5’ 3’ 309 Genes encoding splicing factors and RNA-binding proteins Tandem duplication CRPC Localized PC Stop codon Benign Stop codon 5’ CE3 3’ 5’ CE3 3’
5’ Homology arm 3’ Homology arm Cryptic exon 3 Knock-in Exon 1 2 3 4 5 6 7 8 5’ 3’ GFP T2A Neo R 3’ GFP-2A-Neo R Stop codon
D Control gRNA E F P < 0.0001 15,000 SF3B2 gRNA 40 P = 0.0083 P < 0.0001 100 35 13 P = 0.6047 30 12,000 80 25 12 9,000 60 20 15
Count 40 10 11 6,000 SF3B2 Expression 20 5 3,000 Percentage of AR-V7– negative population 0 10 0 0 1 2 3 10 10 10 10 Benign CRPC
AR-V7-GFP Localized PC Control gRNA AR-V7 NegativeAR-V7 Positive SF3B2 gRNASF3B2 #1 gRNASF3B2 #2 gRNA #3 AR-V7 High Positive
G H Prostate cancer (TCGA) P < 0.0001 3,000 P = 0.9856 P < 0.0001 1.0 SF3B2 High (n = 242) P = 0.0069 P = 0.0718 P = 0.9208 16,000 7.5 n P = 0.0044 0.8 SF3B2 Low ( = 242) 2,000 12,000 5.0 0.6 P = 0.9844 1,000 8,000 2.5 0.4 P = 0.0042 AR-V7 Expression AR-FL Expression SF3B2 Expression 0 4,000 0 0.2 Progression-free survival 678−10 678−10 678−10 0.0 Gleason score Gleason score Gleason score 0 20 40 60 80 100 120 140 160 180 Postoperative months
Figure 1. Identification of SF3B2 as a splicing modulating factor associated with AR-V7 expression. A, Heatmap of 309 genes encoding splicing factors or RNA-binding proteins. Published microarray data for prostate cancers were reanalyzed (17). Specimen types were color-coded and included CRPC (n ¼ 35), localized prostate cancer (PC; n ¼ 59), and benign prostate tissue samples (n ¼ 28). B, Schematic of the screening for candidate genes associated with the splicing for AR-V7. C, Schematic illustration of AR-FL and AR-V7 splicing and AR-V7-GFP knock-in in CWR22Rv1 prostate cancer cells. D, FACS analysis of AR-V7-GFP expression in control or SF3B2 gRNA-transfected AR-V7-GFP cells. Light blue, SF3B2 gRNA; red, control gRNA; dark blue, overlap area of SF3B2 gRNA and control gRNA. E, Percentages of AR-V7-GFP–negative cells following transfection with control gRNA and SF3B2 gRNA (n ¼ 3). F, Correlation between SF3B2 expression and CRPC in published microarray data (17) and correlation between SF3B2 and AR-V7 expression in the TCGA data (19). AR-V7 negative [n ¼ 437, reads per kilobase million (RPKM) < 1], AR-V7 positive (n ¼ 49, RPKM >1), and AR-V7 high positive (n ¼ 12, RPKM > 3). G, Correlation between SF3B2/AR-FL (full length)/AR-V7 expression and Gleason scores in TCGA data (19). Gleason score 6 (n ¼ 45), 7 (n ¼ 247), and 8–10 (n ¼ 205). H, Kaplan–Meier curves of progression-free survival in TCGA prostate cancer patients with SF3B2 high or low expression divided by the median. log, log-rank test.
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ABC22Rv1 LNCaP95 Control gRNA SF3B2 gRNA 250K
200K
150K
Control-gRNA SF3B2-gRNA FSC Control-gRNA SF3B2-gRNA (bulk) (bulk) (bulk) (bulk) 100K SF3B2 SF3B2 10.2 10.8 50K Control Lower Upper AR-FL AR-FL 11.1 11 0 AR-V7 AR-V7 101 102 103 104 101 102 103 104 1 0.06 10.7 ACTB ACTB AR-V7-GFP 11 11
DEF
Control SF3B2-Depletion Control AR-V7 KO AR-FL SF3B2
Control-gRNA SF3B2-gRNA (upper) SF3B2-gRNA (lower) SF3B2 AR-V7 ACTB SF3B2 ACTB ACTB
GH0.20 0.15 0-100 0.10 0.05 0.00 Control 0-100
0-100
RNA-seq SF3B2 dep 0-100 Log10 FPKM + 1 2 TMPRSS2 TMPRSS2 1 KLK3 0-300
Control 0-300 NKX3-1 KLK4 0-300
RNA-seq SF3B2 dep 0-300
Control KLK3
SF3B2 Depletion
Figure 2. SF3B2 depletion decreases the expression of AR-V7 and androgen-response genes. A and B, Western blots of the indicated proteins in SF3B2 gRNA-transfected CWR22Rv1 cells (A) and LNCaP95 cells (B). The intensity of each band was normalized to that of ACTB. C, FACS analysis of SF3B2 gRNA-transfected AR-V7-GFP cells showing the "upper" and "lower" cell populations. D, Western blots of lower SF3B2 protein expression in AR-V7-GFP cells with "lower" GFP expression compared with those with "upper" GFP expression. E, Western blots of the indicated proteins in SF3B2-depleted AR-V7-GFP cells. F, Western blots of SF3B2 in AR-V7–depleted AR-V7-GFP cells. G, Heatmap of expression data showing decreased expression of the AR-target genes in SF3B2-depleted or nontarget gRNA-transfected AR-V7-GFP cells. H, TMPRSS2 and KLK3 mRNA expression in SF3B2-depleted AR-V7-GFP cells.
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SF3B2-Mediated RNA Splicing in Prostate Cancer Progression
set of NELSON_RESPONSE_TO_ANDROGEN_UP (24), includ- CLIP signals of SF3B2 were detected across the meta-exon or ing KLK3 [also known as prostate-specific antigen (PSA)] and -intron regions (Fig. 4D and E). The SF3B2 signals were stronger transmembrane protease serine 2 (TMPRSS2), but not ACTB in exons than in introns, possibly reflecting that the removed (Fig. 2G and H; Supplementary Fig. S4C). Thus, SF3B2 depletion introns were degraded. Notably, transcripts with SF3B2-bound in prostate cancer cells led to significant decreases in the expres- exons were significantly decreased by SF3B2 depletion (P ¼ sion of AR-V7 and the majority of androgen-responsive genes. 1 10 11, Fig. 4F), while transcripts with SF3B2-bound introns demonstrated increased intron retention (P ¼ 2 10 15, PAR-CLIP analysis identifies global SF3B2-binding sites Fig. 4G). Collectively, these results support a critical role for To investigate the genome-wide molecular functions of SF3B2, SF3B2 in the inclusion/exclusion of exons and introns. we determined the binding sites of SF3B2 by PAR-CLIP analy- sis (13). A single band at the expected size was detected in PAR- The SF3B2-recognizing motif in cryptic exon 3 is critical for the CLIP of tandem affinity purification-tagged SF3B2 (SF3B2-TAP) generation of the AR-V7 transcript from whole-cell extracts of 22Rv1 cells with stable SF3B2-TAP To further demonstrate the critical role of SF3B2 binding to expression (Fig. 3A). SF3B2-bound RNAs from three independent cryptic exon 3 (as shown in Fig. 3) in the splicing of AR-V7, we experiments were analyzed by deep sequencing, yielding approx- designed two gRNAs for the SF3B2-recognizing motif to disrupt imately 460 M of reads in total. SF3B2-binding sites were found the SF3B2 binding sequence (Fig. 5A). While control gRNA not only in coding regions, including exons and introns, but also recognizing the sequence between the cryptic exon 3 and the intergenic regions including promoters (Fig. 3B; Supplementary SF3B2-binding site did not increase the GFP-negative population Fig. S4D and S4E). in AR-V7-GFP cells compared with the control gRNA that does not To discover the SF3B2-recognizing RNA motif, the reads with a contain the matched sequence in the human genome (Figs. 1E T-to-C conversion by the PAR-CLIP reaction were analyzed by and 5B and C), both gRNA #1 and #2 targeting the SF3B2- PARalyzer (25) and cERMIT (26). cERMIT identified the CNNGU recognizing motif significantly increased in the AR-V7-GFP– RNA sequence as the SF3B2-recognizing RNA motif (P < 1 10 8, negative population (P ¼ 0.0001 or P < 0.0001; Fig. 5C). The compared with 1,000 random scores), following analysis of 3,154 data indicate that the SF3B2-binding site is necessary for inclusion clusters identified by PARalyzer and the exclusion of clusters of cryptic exon 3 into AR mRNA, that is, the generation of the assigned to repeats and rRNA (Fig. 3C and D). mature AR-V7 transcript. Notably, SF3B2 bound to AR exon 1 and cryptic exon 3, the We next examined splicing between exon 3 and cryptic exon inclusion of which would lead to the AR-V7 transcript (Fig. 3E). 3 or between exon3 and exon4 in AR by quantitative PCR SF3B2 depletion significantly decreased transcription from these (qPCR) in the AR-V7-GFP–negative population isolated from exons (Fig. 3E), although SF3B2 depletion did not significantly the SF3B2 core motif in cryptic exon 3–targeting gRNA- affect the expression of AR-FL protein (Fig. 2E). This difference transfected AR-V7-GFP cells (Fig. 5D–F). Splicing between exon might be caused by the stability of AR-FL protein. To examine 3 and cryptic exon 3 was significantly decreased in the isolated whether endogenous SF3B2 binds to the SF3B2-binding sites in AR-V7-GFP–negative population, while splicing between exon AR, we established 22Rv1 cells carrying a Halo-tag knock-in 3 and exon 4 was not affected by disruption of the SF3B2- immediately before the stop codon of SF3B2 (Fig. 3F). RNA binding site in cryptic exon 3 (Fig. 5E). Conversely, SF3B2 precipitation analysis of SF3B2-Halo revealed an enrichment of overexpression increased splicing between exon 3 and cryptic RNAs in AR exon1 and cryptic exon 3 (Fig. 3F). Thus, PAR-CLIP exon 3 without having a significant effect on splicing between analysis of SF3B2 revealed the genome wide SF3B2-binding sites, exon 3 and exon 4 (Fig. 5F). Thus, SF3B2 binding to AR cryptic and confirmed SF3B2 binding to AR pre-mRNA including cryptic exon 3 of the AR plays a key role in the inclusion of this target exon3, a critical exon for AR-V7. exon into AR-V7 mRNA.
SF3B2 regulates the splicing and expression of target genes SF3B2 overexpression enhances tumor growth in vivo and SF3B2 is a component of the SF3B1 (also known as SF3b155 increases the expression of AR-V7 and its target genes or SAP155) complex, known as SF3b, which functions as a Because high SF3B2 expression is associated with aggressive component of U2-small nuclear ribonucleoprotein (snRNP) in cancer phenotypes in patients (Fig. 1; Supplementary Fig. S3), we pre-mRNA splicing (27–29). We next explored the genome- next examined whether SF3B2 overexpression could alter phe- wide functions of SF3B2 in the regulation of splicing and gene notypes in prostate cancer cells. Consistent with the results expression. SF3B2 depletion affected approximately one-fourth showing that SF3B2 increased AR-V7 splicing (Fig. 5), SF3B2 of the detected splicing events (Supplementary Fig. S5A and overexpression increased AR-V7 protein expression in two pros- S5B) and increased intron retention (IR) and exon skipping tate cancer cell lines, while the full-length AR protein was not (ES) (Supplementary Fig. S5C and S5D). The expression of significantly affected (Fig. 6A–C). Because AR-V7 promotes tumor SF3B2-target genes was decreased according to the location and growth in vivo, along with an increase in its target gene expres- number of SF3B2-binding sites (Fig. 4A and B), implicating sion (23, 30), we examined the effect of SF3B2 overexpression on that SF3B2 binding to an intron iscriticaltocontrolling gene tumor growth in vivo under an androgen-depleted conditions expression, whereas the peak height of the SF3B2 binding site (Fig. 6D and E). We demonstrated that SF3B2 overexpression (xRPM) was not associated with a decrease in expression of significantly promoted tumor growth in 22Rv1 and LNCaP95 SF3B2 target genes (Fig. 4C). cells under castration conditions (P < 0.05, Fig. 6D and E). To examine whether SF3B2 modulates both an inclusion of Notably, SF3B2-mediated aggressive cancer phenotype was exons and an exclusion of introns in the SF3B2-binding sites, we reversed by AR-V7 knockout (Fig. 6D and E; Supplementary performed meta-exon or -intron analysis of transcripts or PAR- Fig. S6), indicating that regulation of AR-V7 splicing is one of CLIP signals of SF3B2 at SF3B2-bound sites (Fig. 4D–G). PAR- the critical roles of SF3B2 in CRPC.
www.aacrjournals.org Cancer Res; 79(20) October 15, 2019 5209
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
Kawamura et al.
A TAP B Other 2.1% Promoter 9.3% 5’UTR 0.75% Exon 9.5% GFP-TAP SF3B2-TAP Intergenic 39.5% 150 kDa Intron 31.8%
100 kDa TTS 3’UTR 3.4% 75 kDa 3.6%
50 kDa C 2.0 P < 1 × 10-8 37 kDa 1.5
Bits 1.0
25 kDa 0.5 0.0 SF3B2 1 2345 D Gene name Representative PAR-CLIP clusters xRPM FPKM AR ------AGCAACUGUGUCUGUCUGAGGUUCCUGUGGCCAUCUUU------9.0 100.9 WDR74 ------CCACUCCACGCAUCGACCUGGUAUUGCAGUACUUCC------139.0 25.1 NEMF --UUGCCUAAGGAGGGGUGAACCGGCCCAGGUCGGAAACGGAGCAGGUCAAAACUCCCGUGCUGAUCAGUAGUGGGAUUGCGCCUGUG- 157.0 8.0 TRAF4 ------AAGCUGGCAAUGGCACGGCAUGUGGAGGAGAGUGUGA------12.9 55.7 POLR3GL ------AGAUGUCAGGUCCGAUUGA------13.9 11.7 MALAT1 ------AGACAGGUGGGAGAUUAUGAUCA------22.9 332.5 NOTCH1 ------AGGAGGCCGCCGCCCGGGCGCAGAGGGCAGCCGGUGGGGAGGCA------79.2 18.6 EIF4G3 ------UUCUCGGGUUGGAAUUU------13.7 12.4 RAB3B ------CCUGGUUAGUACUUGGAUGGG------1.8 5.4 ZC3H3 UUGAUUCGGCUGAUCUGGCUGGCUAGGCGGGUGUCCCCUUCCUUCCUCACUGCUUCAUGUGCGUCCCUCCCGAAGCU------420.7 11.7 MATR3 ------UACACUGCUGGGAGAACAGCAGCCAAUAGCUGGUUGGCAUUCUGGCCCUGGUU------12.4 97.9 NAGLU ------AGGUGCUGGCUAGUGACAGCCGCUUCUUGCUGGGCA------55.6 23.3 SETD1B ------UCUAGAGCCUCGUGGGUAACGGCAUAGUGUGGCCAGGUGUGUAGGGAAUG------44.1 22.5 RNF187 ------AACUCAGGUCUUCAGGGAGAG------18.4 128.7 ATP5O ------GUUCAGGUAUACGGUAUUGAAGGUC------6.9 32.5 CCNK ------AGUAGGAACUGGUAUGGAUG------18.3 16.7 RIT1 ------GAGGGGUCACAGGUCAGUGCCGGAGCCUCCGCGAGUGAAGGAA 3.8 22.0 ANKS3 ------ACUCCCCAGCCCCCAGCCCUCCUCAUCCUUGUC------2.4 3.8 DHX34 ------CUGGCCCUGGCUGGUGCCACACACAGGCCU------3.0 14.6 MLL5 ------AAAGACAUCAAAUUAAUCUUGUGAAUGCAG------2.4 17.5
AR E F F1 F1 F1
0-3,000 0-3,000 Exon 1 2 3 4 5 6 7 8 9 Control 0-3,000 0-3,000 5’ 3’
0-3,000 0-3,000
RNA-seq SF3B2 R1 Cryptic exon 3 R1 R1 dep 0-3,000 0-3,000 * 0-100 0-2,000 2.5 SF3B2 PAR CLIP 0-100 0-2,000 2.0 AR 1.5 Exon 1 Exon 3 Cryptic exon 3 1.0 0.5
Relative SF3B2 binding 0 ex1 CE3 ex8 ex1 CE3 ex8 SF3B2-Halo + −
Figure 3. Identification of SF3B2-binding RNA regions. A, Top, autoradiograph of 32P-labeled RNA crosslinked with TAP-tagged SF3B2 (SF3B2-TAP) protein separated by SDS-PAGE. Bottom, Western blot analysis of SF3B2. B, Pie chart of approximately 63,000 SF3B2-binding sites with T-to-C mutations above threshold. C, The SF3B2-recognizing RNA motif. A total of 3,154 clusters excluding repeats and rRNA were analyzed. D, Sequence alignment of representative SF3B2-binding sites. Gray characters indicate the SF3B2-recognizing motif. E, mRNA expression and SF3B2 PAR-CLIP signals in AR. F, RNA precipitation analysis of endogenous SF3B2 with Halo-tag knock-in. Top, primer positions. Bottom, enrichment of RNAs by RNA precipitation of SF3B2-Halo. , P < 0.05; n ¼ 3. FPKM, fragments per kilobase of transcript per million mapped reads; xRPM, crosslinked-reads per million.