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And EZH2-Regulated Gene, Has Differential Roles in AR-Dependent and -Independent Prostate Cancer www.impactjournals.com/oncotarget/ Oncotarget, Vol. 6, No.4 RUNX1, an androgen- and EZH2-regulated gene, has differential roles in AR-dependent and -independent prostate cancer Ken-ichi Takayama1,2, Takashi Suzuki3, Shuichi Tsutsumi4, Tetsuya Fujimura5, Tomohiko Urano1,2, Satoru Takahashi6, Yukio Homma5, Hiroyuki Aburatani4 and Satoshi Inoue1,2,7 1 Department of Anti-Aging Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan 2 Department of Geriatric Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan 3 Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan 4 Genome Science Division, Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, Meguro-ku, Tokyo, Japan 5 Department of Urology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan 6 Department of Urology, Nihon University School of Medicine, Itabashi-ku, Tokyo, Japan 7 Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, Japan Correspondence to: Satoshi Inoue, email: [email protected] Keywords: RUNX1, androgen receptor, EZH2, prostate cancer Received: October 02, 2014 Accepted: December 09, 2014 Published: December 10, 2014 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Androgen receptor (AR) signaling is essential for the development of prostate cancer. Here, we report that runt-related transcription factor (RUNX1) could be a key molecule for the androgen-dependence of prostate cancer. We found RUNX1 is a target of AR and regulated positively by androgen. Our RUNX1 ChIP-seq analysis indicated that RUNX1 is recruited to AR binding sites by interacting with AR. In androgen-dependent cancer, loss of RUNX1 impairs AR-dependent transcription and cell growth. The RUNX1 promoter is bound by enhancer of zeste homolog 2 (EZH2) and is negatively regulated by histone H3 lysine 27 (K27) trimethylation. Repression of RUNX1 is important for the growth promotion ability of EZH2 in AR-independent cells. In clinical prostate cancer samples, the RUNX1 expression level is negatively associated with EZH2 and that RUNX1 loss correlated with poor prognosis. These results indicated the significance of RUNX1 for androgen-dependency and that loss of RUNX1 could be a key step for the progression of prostate cancer. INTRODUCTION treatment for prostate cancer and efficiently inhibits the growth of androgen-dependent tumors. Unfortunately, the Prostate cancer is the most frequently diagnosed majority of these cancers become refractory to hormone cancer in men [1]. Androgen and its cognate receptor, deprivation therapy and emerges as castration-resistant. the androgen receptor (AR), are involved in prostate Such castration-resistant prostate cancer (CRPC) is a oncogenesis through the transcriptional regulation of significant clinical challenge, and investigation into the target gene networks [2]. To determine the genomic biologic mechanisms that contribute to tumor regrowth distribution of AR binding sites, it is important to consider is of critical importance [1]. Mechanisms of castration- binding sites for AR collaborating transcription factors resistance can be grouped into two categories. The first such as FOXA1 [3, 4], NKX3-1 [5], GATA2, OCT1 [3] is enhancement of AR signaling caused by mutation, and members of the ETS family of transcription factors amplification of the AR gene or other mechanisms that [6, 7]. allow for the activation of AR even with castration levels Androgen-deprivation therapy is a standard of serum testosterone [1, 8-10]. The other group uses www.impactjournals.com/oncotarget 2263 Oncotarget a mechanism to bypass AR signaling pathways [11], analysis (Fig.1A). Two robust AR binding sites were which allows cancer cells to survive in the absence of identified around the RUNX1 locus, one (ARBS #1) androgen-dependent or -independent AR activation. For is 50 kb downstream and the other (ARBS #2) is 200 example, cells that lack AR expression or had repressed kb upstream (Fig.1B, Supplementary Fig.1A) by AR AR signaling have been reported in large numbers of Chromatin immunoprecipitation and sequencing (ChIP- metastatic tumors derived from prostate cancer patients seq). In addition, RUNX1 upregulation by DHT treatment [12]. was also observed and this induction was inhibited by The runt-related transcription factor (RUNX) family short interference RNA (siRNA) targeting AR-treatment consists of transcription factors such as RUNX1, RUNX2, (Supplementary Fig.1B, C). We also confirmed RUNX1 and RUNX3. Each is capable of forming heterodimers with induction by androgen in other AR positive prostate cancer the common core-binding factor (CBF) β subunit [13]. cells (Supplementary Fig.1D). Next, we investigated RUNX-CBFβ heterodimers bind to their consensus target histone modification patterns in the RUNX1 locus by sequence, TGT/CGGT, and either activate or repress the ChIP-seq (Fig.1B, Supplementary Fig.1E). RUNX1 has transcription of target genes. Interestingly, each RUNX two distinct transcriptional start sites (TSSs: Promoter 1 protein has differential function. RUNX1 is essential for and 2), and we found that promoter 2 is occupied with hematopoiesis [14], RUNX2 for osteogenesis [15] and highly acetylated histone H3 by ChIP-seq analysis. RUNX3 for neurogenesis [16]. In addition, RUNX genes Although both ARBSs are modified with monomethylation are associated with different types of malignancy, and they of histone H3K4, an active enhancer mark, we showed that function as both oncogenes and tumor suppressors in a ARBS #1 has a highly androgen-dependent transcriptional tissue-specific manner [13]. activity by luciferase assay (Fig.1C). Highly acetylated In the present study, we demonstrated the molecular histone modification was observed only in the promoter mechanism and clinical significance of RUNX1 2 of RUNX1 (and not in that of CLIC6) around the expression in prostate cancer. By genome-wide analysis ARBS#1, implicating that this region acts as an enhancer of AR-binding sites (ARBSs), we identified RUNX1 as for RUNX1 induction (Fig.1B). These results suggest that an AR target gene that is induced by androgens. RUNX1 RUNX1 is directly regulated by AR binding. expression is necessary for AR signaling and androgen- Next, we analyzed the role of RUNX1 in prostate dependent prostate cancer cell proliferation. Furthermore, cancer cells and its association with AR. To examine we showed that RUNX1 expression is correlated with a whether AR interacts with RUNX1, we transfected 293T good prognosis for prostate cancer patients. Enhancer of cells with expression vectors of Flag-tagged AR and Myc- zeste homolog 2 (EZH2), which is a major contributor to tagged RUNX1 and its deletion mutants (Fig.1D, upper). androgen-independent signaling in prostate cancer [17], We then performed immunoprecipitation by anti-Myc represses RUNX1 transcription, and the expression of antibody and immunoblotted with anti-Flag antibody. RUNX1 is negatively associated with EZH2 in clinical We observed ligand dependent interaction of AR with samples. We also observed that repression of RUNX1 by RUNX1. When the N-terminal region of RUNX1 was EZH2 enhanced androgen-independent prostate cancer deleted, this interaction was diminished, suggesting the cell growth. Taken together, our results showed that N-terminal domain is responsible for AR interaction RUNX1 plays a critical role in androgen-dependent and (Fig.1D, lower). We also showed the interaction of –independent prostate cancer cell growth. Our findings endogenous AR and RUNX1 in LNCaP cells (Fig.1E). Our also highlighted the significance of RUNX1 loss in the results indicate that androgen-regulated RUNX1 interacts progression of prostate cancer. with AR that regulates gene transcription. RESULTS Genome-wide analysis of RUNX1 binding sites reveals androgen-dependent recruitment of RUNX1 to ARBSs RUNX1 is a direct target of AR and interacts with AR We investigated the genome-wide binding of RUNX1 in androgen-dependent transcriptional regulation We previously reported the global analysis of by ChIP-seq. LNCaP cells were treated with vehicle androgen signaling by mapping ARBSs and androgen- or DHT for 24 h and then ChIP was performed using a regulated transcripts [18, 19, 20]. Using quantitative RUNX1 specific antibody. Enriched DNA was sequenced reverse transcriptase-PCR (qRT-PCR) analysis, we using a next generation sequencer and sequenced tags found that RUNX1 is highly induced by R1881 10 nM were mapped to the human genome (Fig.2A). We obtained treatment in LNCaP cells (more than 50-fold following 1125 significant RUNX1 binding sites in vehicle-treated 48 h of treatment) (Fig.1A). RUNX1 protein levels cells and 2040 in DHT-treated cells using MACS (ref. 21, were also increased as demonstrated by western blot P-value < 1.0E-5, Fig.2B). We have previously reported www.impactjournals.com/oncotarget 2264 Oncotarget Figure 1: RUNX1, a direct target of AR in prostate cancer cells, interacts with AR androgen-dependently. (A) RUNX1 is induced by androgen. LNCaP cells were treated with 10 nM R1881 or vehicle. Expression level of RUNX1 mRNA was measured by qRT-PCR. Data represent mean + s.d., n = 3. Western blot analysis of RUNX1 was performed. β-actin was used as a loading control.
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