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Edited by Owen N. Witte, University of California, Los Angeles, CA, and approved April 6, 2018 (received for review February 8, 2018) Significance

Histone demethylase JMJD1A promotes alternative splicing of AR variant 7 (AR-V7) in prostate cancer cells Lingling Fan a,b,1, Fengbo Zhang a,b,c,1, Songhui Xu a,b, Xiaolu Cui a,b, Arif Hussain b,d, Ladan
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Histone demethylase JMJD1A promotes alternative splicing of AR variant 7 (AR-V7) in prostate cancer cells Lingling Fan a,b,1, Fengbo Zhang a,b,c,1, Songhui Xu a,b, Xiaolu Cui a,b, Arif Hussain b,d, Ladan Fazli e, Martin Gleave e, Xuesen Dong e, and Jianfei Qi a,b,2 a Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201; b Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, MD 21201; c Department of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing , China; d Baltimore VA Medical Center, Baltimore, MD 21201; and e Vancouver Prostate Centre, University of British Columbia, Vancouver, BC V6H 3Z6, Canada Edited by Owen N. Witte, University of California, Los Angeles, CA, and approved April 6, 2018 (received for review February 8, 2018) Formation of the androgen receptor splicing variant 7 (AR-V7) is one of the major mechanisms by which resistance of prostate cancer to androgen deprivation therapy occurs. The histone demethylase JMJD1A (Jumonji domain containing 1A) functions as a key coactivator for AR by epigenetic regulation of H3K9 methylation marks. Here, we describe a role for JMJD1A in AR-V7 expression. While JMJD1A knockdown had no effect on full-length AR (AR-FL), it reduced AR- V7 levels in prostate cancer cells. Reexpression of AR-V7 in the JMJD1A-knockdown cells elevated expression of select AR targets and partially rescued prostate cancer cell growth in vitro and in vivo. The AR-V7 protein level correlated positively with JMJD1A in a subset of human prostate cancer specimens. Mechanistically, we found that JMJD1A promoted alternative splicing of AR-V7 through heterogeneous nuclear ribonucleoprotein F (HNRNPF), a splicing factor known to regulate exon inclusion. Knockdown of JMJD1A or HNRNPF inhibited splicing of AR-V7, but not AR-FL, in a minigene reporter assay. JMJD1A was found to interact with and promote the recruitment of HNRNPF to a cryptic exon 3b on AR pre-mrna for the generation of AR-V7. Taken together, the role of JMJD1A in AR-FL coactivation and AR-V7 alternative splicing highlights JMJD1A as a potentially promising target for prostate cancer therapy. histone demethylase JMJD1A androgen receptor RNA splicing prostate cancer Androgen deprivation therapy (ADT), which blocks androgen receptor (AR) activity, is the primary treatment for metastatic prostate cancer. Despite an initially beneficial response to ADT, a majority of patients with prostate cancer eventually become resistant to such therapies and progress to castrationresistant prostate cancer (CRPC), which is an incurable and ultimately lethal disease state. Activation of AR signaling despite low androgen levels is believed to underlie the development of CRPC in most cases (1, 2). Restoration of AR transcriptional activity in CRPC can occur through a variety of mechanisms, among which is the formation of AR splicing variants (AR-Vs) (3 5). Full-length AR (AR-FL) consists of an N-terminal transactivation domain (N-TAD), a DNA-binding domain (DBD), a hinge region, and a C-terminal ligand-binding domain (LBD). Although AR-Vs lack an LBD, they drive the AR transcriptional program in a constitutively active manner. AR-V7 (also called AR3) is one of most widely studied AR variants. Expression of AR-V7 is associated with resistance to AR signaling inhibitors and poor prognosis of prostate cancer (6, 7). The AR gene consists of eight exons that encode different regions: exon 1 encodes N-TAD, exons 2 and 3 encode the DBD, and exons 4 8 encode the hinge region and LBD. AR-FL and AR-V7 are derived from the same AR premrna through alternative splicing. In contrast to AR-FL mrna that includes all eight exons, AR-V7 is generated via use of a cryptic exon 3b located in the third intron of AR, which results in the formation of AR-V7 mrna that only includes exons 1 3 and 3b (4, 5). The various AR-Vs can form homodimers among themselves or heterodimers with AR-FL to transcribe target genes (8 10). The target genes regulated by AR-FL and AR-V7 overlap but are not identical (4, 11, 12). The role of AR-V7 in transcriptional regulation and CRPC progression has been extensively studied. However, the mechanisms controlling the alternative splicing of AR pre-mrna to generate AR- V7 remain poorly explored. JMJD1A (Jumonji domain containing 1A; i.e., KDM3A) is a histone demethylase that removes the repressive H3K9 methylation marks (H3K9me1 or H3K9me2) to regulate gene expression (13, 14). We previously reported that JMJD1A played a key role in the proliferation and survival of prostate cancer cells, in part, through regulation of the AR transcriptional program and elevation of c-myc levels (15, 16). JMJD1A interacts with AR and functions as a key AR coactivator through its histone demethylase activity (13, 15). This study reveals a role for JMJD1A in the alternative splicing of AR pre-mrna to generate AR-V7. Splicing of mrna is performed by the spliceosome, a large complex of proteins and snrnas that recognizes specific sequence elements within an intron (e.g., 5 splice site, 3 splice site), removes the intron, and ligates the adjacent exons. The splicing factors, which bind to specific sequence elements on premrnas and promote exon inclusion or skipping, regulate the efficiency of splice site recognition by the spliceosome. Heterogeneous nuclear ribonucleoprotein F (HNRNPF) and highly related HNRNPH are splicing factors that bind to the guanosine (G)-rich sequence in exons or introns, and thus function as either enhancers or inhibitors of alternatively spliced exons (17 21). In Significance Formation of androgen receptor splicing variant 7 (AR-V7), a constitutively active form of AR, plays a key role in the resistance of prostate cancer to hormone therapy. However, the mechanisms that regulate AR-V7 generation are poorly understood. Here, we identified a new role for histone demethylase JMJD1A (Jumonji domain containing 1A) in the formation of AR-V7 in prostate cancer cells. We found that JMJD1A facilitated recruitment of a splicing factor, heterogeneous nuclear ribonucleoprotein F, for alternative splicing and generation of AR-V7. The findings suggest that targeting JMJD1A may provide new therapeutic opportunity for prostate cancer. Author contributions: J.Q. designed research; L. Fan, F.Z., S.X., X.C., and L. Fazli performed research; A.H., M.G., and X.D. contributed new reagents/analytic tools; L. Fazli and J.Q. analyzed data; and J.Q. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Published under the PNAS license. 1 L. Fan and F.Z. contributed equally to this work. 2 To whom correspondence should be addressed. This article contains supporting information online at /pnas /-/DCSupplemental. Published online April 30, E4584 E4593 PNAS vol. 115 no. 20 this study, we found that JMJD1A interacts with HNRNPF and promotes the recruitment of HNRNPF to exon 3b of AR premrna, resulting in its splicing to create the AR-V7 variant. Results JMJD1A Knockdown Reduces the Level of AR-V7 in Prostate Cancer Cells. JMJD1A is a key AR coactivator in prostate cancer cells (15, 16). To examine the effect of androgens on JMJD1A and AR, we switched CWR22Rv1 prostate cancer cells (hereafter called Rv1 cells) to growth media containing 5% charcoalstripped FBS (which has low androgen levels) and supplemented the media with either 5 μm antiandrogen enzalutamide (to maximally block androgenic action) or 1 nm synthetic androgen R1881 (to restore androgens to physiological levels). The former mimics androgen deprivation conditions, while the latter reflects normal androgen conditions. We found that protein levels of JMJD1A and AR-FL in Rv1 cells were not affected irrespective of the androgen conditions (Fig. 1A). Interestingly, although knockdown of JMJD1A in Rv1 cells had little effect on the protein levels of AR-FL, we found reduced levels of a protein band at 80 kda, which is consistent with the size of AR-Vs (Fig. 1A). Using an AR-V7 specific antibody, Western blot analysis demonstrated that JMJD1A knockdown indeed reduced the protein levels of AR-V7 in Rv1 cells (Fig. 1A). Furthermore, qrt-pcr analysis showed that JMJD1A knockdown in Rv1 cells reduced mrna levels of AR-V7, but not AR-FL, under either androgen-deprived or normal conditions (Fig. 1B). LN95 and VCaP are two other prostate cancer cell lines known to express AR-V7, albeit at lower levels than Rv1 cells (Fig. S1A). Similar to our findings with Rv1 cells, JMJD1A knockdown in LN95 cells reduced mrna levels of AR-V7, but not AR-FL, under both normal and androgen-deprived conditions (Fig. 1C). Unlike Rv1 or LN95 cells, mrna levels of both AR-FL and AR-V7 were up-regulated in VCaP cells upon androgen deprivation (Fig. 1D), likely due to the transcriptional up-regulation of AR pre-mrna in this cell line (22, 23). Nonetheless, JMJD1A knockdown in VCaP cells reduced mrna levels of AR-V7, but not AR-FL, under either androgen deprivation or normal conditions (Fig. 1D). Taken together, these results indicate that JMJD1A promotes the expression of AR-V7, but not AR-FL, in prostate cancer cells known to express AR-V7. To confirm the relevance of our findings to human prostate cancer, we evaluated the expression of JMJD1A and AR-V7 in a BIOCHEMISTRY PNAS PLUS Fig. 1. (A) Rv1 cells were transduced with plko.1 control or JMJD1A shrnas for 24 h. Then, cells were maintained in growth media that contained 5% charcoal-stripped-fbs supplemented with either enzalutamide (Enza; 5 μm) or R1881 (1 nm). After 24 h, cell lysates were collected for Western blot analyses using the indicated antibodies. (B) Rv1 cells described in A were analyzed by qrt-pcr for transcripts of JMJD1A, AR-V7, or AR-FL. (C) LN95 cells (plko.1 or JMJD1A-knockdown) were treated and analyzed by qrt-pcr as described in A and B. (D) VCaP cells (plko.1 or JMJD1A-knockdown) were treated and analyzed by qrt-pcr as described in A and B. In B D, the comparison with statistical significance (plko.1 vs. shjmjd1a) is indicated with an asterisk (**P 0.01, *P 0.05; ANOVA). (E) Immunohistochemistry staining of JMJD1A or AR-V7 was performed on prostate cancer TMA. The staining was developed with DAB (brown) and counterstained with hematoxylin (blue). Shown are representative images of high (Upper) and low (Lower) levels of JMJD1A or AR- V7 staining. (F) Quantification of JMJD1A or AR-V7 staining of prostate cancer TMA (n = 40). The staining of JMJD1A or AR-V7 was scored as 3 (strong), 2 (moderate), 1 (weak), or 0 (none). Scores of 3 or 2 were defined as high, and scores of 1 or 0 were defined as low. The Mann Whitney U test was used for statistical analysis (P 0.01). Fan et al. PNAS vol. 115 no. 20 E4585 human prostate cancer tissue microarray (TMA) containing tumor samples of Gleason grades 3 5. JMJD1A and AR-V7 staining revealed primarily nuclear localization, but varying degrees of cytoplasmic staining of both proteins were also observed in some samples (Fig. 1E). Based on the relative staining intensity, we defined AR-V7 staining as either high (strong or moderate) or low (weak or none). JMJD1A staining was likewise defined as either high or low. Approximately 69.2% of JMJD1Ahigh tumors exhibited high staining of AR-V7, whereas only 16.7% of JMJD1A-low tumors exhibited high staining of AR-V7, with the majority (83.3%) stained at low intensity (Fig. 1F). These results indicate that JMJD1A may also promote the expression of AR-V7 in a subset of human prostate cancer tissues. We previously reported that JMJD1A knockdown reduced levels of c-myc in prostate cancer cells (15). To evaluate the possible involvement of c-myc in modulating AR-V7 expression, we knocked down c-myc in Rv1, LN95, and VCaP cells but found no change in AR-V7 mrna levels (Fig. S1B). This indicates that c-myc does not affect AR-V7 expression in these prostate cancer cell lines. JMJD1A Promotes the Expression of Select AR Targets Through AR-V7. JMJD1A is known to interact with and coactivate AR-FL in prostate cancer cells (13, 15). To assess if JMJD1A also interacts with AR-V7, we coexpressed myc-jmjd1a with either Flagtagged AR-FL or AR-V7 in 293T cells and performed immunoprecipitation studies using anti-flag M2 beads. Myc-JMJD1A coprecipitated with Flag-AR-FL, but not with Flag-AR-V7 (Fig. 2A), indicating that JMJD1A does not directly interact with AR- V7, and thus may not function as its coactivator. Of note, AR-FL and AR-V7 were shown to regulate the expression of both common and unique AR targets (4, 11, 12). If this were true, we reasoned that restoration of AR-V7 expression in JMJD1Aknockdown Rv1 cells should be able to rescue expression of AR targets that depend on AR-V7. To determine the dependency of AR targets on AR-FL or AR-V7, we knocked down AR-FL using shrna that targeted the AR LBD domain and Fig. 2. (A) Flag-tagged AR-V7 or AR-FL was cotransfected with myc-jmjd1a in 293T cells for 24 h. Cell lysates were then subjected to immunoprecipitation (IP) with anti-flag M2 beads. Bound proteins were analyzed by Western blotting with myc or Flag antibodies. (B) Rv1 cells were transduced with plko.1 control, AR-V7 shrna, or AR-FL shrna. After 48 h, cell lysates were analyzed by Western blotting with the AR antibody (N-20) that recognizes the AR N-terminal region. (C) Rv1 cells described in B were maintained in growth media that contained 5% charcoal-stripped FBS supplemented with enzalutamide (Enza; 5 μm) or R1881 (1 nm). After 24 h, RNAs were collected and analyzed by qrt-pcr for transcripts of representative AR target genes that include NKX3.1, FKBP5, and SREBF1. (D) AR-V7 was reexpressed in JMJD1A-knockdown Rv1 cells via lentiviral transduction (shjmjd1a+ar-v7). Cell lysates were analyzed by Western blotting with antibodies against AR-V7 or JMJD1A. (E) Rv1 cells (plko.1, shjmjd1a, or shjmjd1a+ar-v7) were treated and analyzed by qrt-pcr as described in C. InC and E, the comparison with statistical significance is indicated with an asterisk (**P 0.01, *P 0.05; ANOVA). E Fan et al. AR-V7 using shrna that targeted the AR-V7 3 -UTR. Western blot and qrt-pcr analysis showed that levels of AR-FL or AR- V7 in Rv1 cells were efficiently and specifically knocked down by the respective shrnas (Fig. 2B and Fig. S2A). As another control, AR-FL or AR-V7 shrnas showed no effect on the mrna level of AR-V1, another form of AR variant (Fig. S2A). Of note, knockdown of AR-V7 significantly reduced the level of AR-Vs (Fig. 2B), indicating that AR-V7 is the major form of AR-Vs in Rv1 cells. We next performed qrt-pcr analysis on a panel of well-characterized AR targets that include KLK3, NKX3.1, SLC45A3, FKBP5, NDRG1, SREBF1, STEAP1, and STEAP2. In Rv1 cells, the sample AR targets can be categorized into three groups (Fig. 2C and Fig. S2B): (i) AR targets, such as NKX3.1, KLK3, SLC45A3, or STEAP2, depend on AR-FL in normal androgen conditions but become dependent on AR- V7 under androgen-deprived conditions; (ii) AR targets, such as FKBP5, NDRG1, or STEAP1, depend on both AR-FL and AR- V7 in normal androgen conditions but depend mainly on AR- V7 under androgen-deprived conditions; and (iii) AR targets, such as SREBF1, are only dependent on AR-V7 under either normal or androgen-deprived conditions. Therefore, we chose NKX3.1, FKBP5, and SREBF1 as representative examples of each of the three AR target groups regulated by AR-FL and/or AR-V7 in Rv1 cells. We restored AR-V7 expression in JMJD1Aknockdown Rv1 cells to the control levels (Fig. 2D) and examined expression of representative AR targets by qrt-pcr (Fig. 2E). Under androgen deprivation conditions, reexpression of AR-V7 in JMJD1A-knockdown Rv1 cells rescued expression of NKX3.1, FKBP5, and SREBF1 (Fig. 2E), consistent with the dependency of these AR targets on AR-V7 in the absence of androgen (Fig. 2C). In contrast, under normal androgen conditions, reexpression of AR-V7 in the JMJD1A-knockdown Rv1 cells fully rescued SREBF1 expression, partly rescued FKBP5 expression, but had no effect on NKX3.1 expression (Fig. 2E). These data are consistent with the dependency of SREBF1 on AR-V7, partial dependency of FKBP5 on AR-V7, BIOCHEMISTRY PNAS PLUS Fig. 3. (A and B) Rv1 cells (plko.1, shjmjd1a, or shjmjd1a+ar-v7) were seeded at low density and maintained for 3 wk in 5% charcoal-stripped FBS media with or without 1 nm R1881 supplement. Colony number ( 100 μm in diameter) was scored after 3 wk. Representative images of colonies are shown in A,and the colony number per high-power field is shown in B. +R1881, P for any pairwise comparison (ANOVA); R1881, P for any pairwise comparison (ANOVA). (C) Rv1 cells (plko.1, shjmjd1a, or shjmjd1a+ar-v7) were injected s.c. into immune-deficient NSG mice (n = 20 per group). After 1 wk, half of the mice in each group were castrated, while the other half were sham-castrated. The xenograft tumors were collected, and tumor weight was measured 2 wk later. For sham conditions, P for any pairwise comparison (ANOVA); for castration conditions, P for any pairwise comparison (ANOVA). (D) Example staining of Ki67 (Upper) and active caspase-3 (Lower) on tumor sections derived from the indicated Rv1 cells. The staining was developed by DAB (brown) and counterstained by hematoxylin (blue). The image width is 0.5 mm. (E) Percentage of Ki67-positive cells per field on the staining described in D. P 0.01 for shjmjd1a vs. shjmjd1a+ar-v7 (ANOVA); P for other pairwise comparisons (ANOVA). (F) Number of positive cells for active caspase-3 per field on staining described in D. P for plko.1 vs. shjmjd1a (ANOVA); P 0.01 for other pairwise comparisons (ANOVA). Fan et al. PNAS vol. 115 no. 20 E4587 and dependency of NKX3.1 on AR-FL in the presence of androgens (Fig. 2C). Taken together, the above results demonstrate that JMJD1A-dependent expression of AR-V7 in Rv1 cells promotes expression of AR targets that rely on AR-V7. AR-V7 Is a Downstream Effector of JMJD1A in Rv1 Cells. To assess the effect of AR-V7 on JMJD1A-dependent cell growth, we evaluated colony formation by JMJD1A-knockdown Rv1 cells upon restoring the expression of AR-V7 in these cells (shjmjd1a+ AR-V7), as described in Fig. 2D. The Rv1 cells were allowed to form colonies in the presence or absence of androgen. Compared with the plko.1 control, JMJD1A-knockdown Rv1 cells showed about a fivefold reduction in colony formation (Fig. 3 A and B). Reexpression of AR-V7 in JMJD1A-knockdown Rv1 cells partially increased their ability to form colonies (Fig. 3 A and B). Comparable results were seen for Rv1 cells grown in the presence or absence of androgen, although the presence of androgen slightly increased colony formation (Fig. 3 A and B). To further test the role of AR-V7 in JMJD1A-dependent tumor growth in vivo, we used a xenograft prostate tumor model in which Rv1 cells were injected s.c. into immune-deficient NSG mice. Compared with control cells, JMJD1A-knockdown Rv1 cells showed an 13-fold reduction in tumor weights in the control mice, with no tumor formation in castrated mice (Fig. 3C). Reexpression of AR-V7 in JMJD1A-knockdown Rv1 cells partly rescued xenograft tumor formation in both control and castrated mice (Fig. 3C). We next performed immunohistochemistry staining for the proliferation marker Ki67 and the apoptosis marker active caspase-3 on xenograft tumor sections. JMJD1A knockdown reduced the percentage of Ki67-positive cells, concomitant with an increased number of cells positive for active caspase-3 (Fig. 3 D F). These results suggest that JMJD1A knockdown inhibits proliferation and promotes apoptosis in xenograft prostate tumors. In contrast, the patterns of Ki67 and active caspase-3 staining detected in JMJD1A-knockdown tumors were partially reversed after AR-V7 reexpression (Fig. 3 D F). Overall, these findings confirmed that AR-V7 could function as a downstream effector of JMJD1A to drive the growth and survival o
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