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RUNX1, an androgen- and EZH2-regulated gene, has differential roles in AR-dependent and -independent prostate cancer.

Takayama K, Suzuki T, Tsutsumi S, Fujimura T, Urano T, Takahashi S, Homma Y, Aburatani H, Inoue S - Oncotarget (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anti-Aging Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

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.

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Knockdown of RUNX1 decreased the androgen-responsive transcriptional program(A) Knockdown of RUNX1 by siRNA transfection. LNCaP cells were transfected with siControl, siRUNX1 #1 and #2 (10 nM). Cells were transfected with vehicle or 10 nM DHT for 24 h. Western blot analysis of AR and RUNX1 was performed. β-actin was used as a loading control. (B) ChIP-seq analysis of AR binding with depleting RUNX1 expression. LNCaP cells were treated with siControl or siRUNX1 for 48 h. AR ChIP was performed after DHT treatment for 24h. ARBSs (Fold > 10, P < 10−5) were determined by MACS. (C) ChIP analysis of AR binding with depleting RUNX1 expression. LNCaP cells were treated with vehicle or 10 nM DHT for 24 h. ChIP analysis was performed using an AR-specific antibody. Enrichment of the ARBS was quantified using qPCR. Data represent mean + s.d., n = 3. (D) Global analysis of RUNX1 effects on androgen regulation of AR-binding genes. LNCaP cells were treated with siControl and siRUNX1 #1. After 48 h incubation, cells were treated with vehicle or 10 nM DHT for 24 h. Microarray analysis was performed and two clusters (Cluster I and II) were identified as genes positively regulated genes by RUNX1. (E) RUNX1 effects on androgen regulation of AR-binding genes. LNCaP cells were transfected with siControl or siRUNX1 #1 and #2 and then treated with 10 nM DHT or vehicle. Expression level of mRNA of genes in cluster I was measured by qRT-PCR. Data represent mean + s.d., n = 3. * P <0.01. (F) Effects of RUNX1 on transcriptional activity of AR. LNCaP cells were transfected with siControl or siRUNX1 #1 and #2. Luciferase vectors including PSA and TACC2-ARBSs were used. Cells were treated with 10 nM DHT or vehicle for 24 h. Data represent mean + s.d., n = 3. * P <0.01.
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Figure 3: Knockdown of RUNX1 decreased the androgen-responsive transcriptional program(A) Knockdown of RUNX1 by siRNA transfection. LNCaP cells were transfected with siControl, siRUNX1 #1 and #2 (10 nM). Cells were transfected with vehicle or 10 nM DHT for 24 h. Western blot analysis of AR and RUNX1 was performed. β-actin was used as a loading control. (B) ChIP-seq analysis of AR binding with depleting RUNX1 expression. LNCaP cells were treated with siControl or siRUNX1 for 48 h. AR ChIP was performed after DHT treatment for 24h. ARBSs (Fold > 10, P < 10−5) were determined by MACS. (C) ChIP analysis of AR binding with depleting RUNX1 expression. LNCaP cells were treated with vehicle or 10 nM DHT for 24 h. ChIP analysis was performed using an AR-specific antibody. Enrichment of the ARBS was quantified using qPCR. Data represent mean + s.d., n = 3. (D) Global analysis of RUNX1 effects on androgen regulation of AR-binding genes. LNCaP cells were treated with siControl and siRUNX1 #1. After 48 h incubation, cells were treated with vehicle or 10 nM DHT for 24 h. Microarray analysis was performed and two clusters (Cluster I and II) were identified as genes positively regulated genes by RUNX1. (E) RUNX1 effects on androgen regulation of AR-binding genes. LNCaP cells were transfected with siControl or siRUNX1 #1 and #2 and then treated with 10 nM DHT or vehicle. Expression level of mRNA of genes in cluster I was measured by qRT-PCR. Data represent mean + s.d., n = 3. * P <0.01. (F) Effects of RUNX1 on transcriptional activity of AR. LNCaP cells were transfected with siControl or siRUNX1 #1 and #2. Luciferase vectors including PSA and TACC2-ARBSs were used. Cells were treated with 10 nM DHT or vehicle for 24 h. Data represent mean + s.d., n = 3. * P <0.01.

Mentions: We next analyzed the effect of RUNX1 on AR binding by ChIP-seq. We treated LNCaP cells with short interference RNA (siRNA) targeting RUNX1 or control RNA (siControl) (Fig.3A). We performed AR ChIP after treating cells with DHT. In our ChIP and ChIP-seq study, the number of significant ARBSs obtained was decreased and enrichments of AR binding are inhibited by RUNX1 knockdown, suggesting a positive role of RUNX1 for AR binding (Fig.3B, C). The overlap of ARBSs with RUNX1 binding, and the reduction of ARBSs by RUNX1 knockdown were also observed in VCaP cells (Supplementary Fig.2B). These results indicate a significant role for RUNX1 recruitment to ARBS in AR binding.


RUNX1, an androgen- and EZH2-regulated gene, has differential roles in AR-dependent and -independent prostate cancer.

Takayama K, Suzuki T, Tsutsumi S, Fujimura T, Urano T, Takahashi S, Homma Y, Aburatani H, Inoue S - Oncotarget (2015)

Knockdown of RUNX1 decreased the androgen-responsive transcriptional program(A) Knockdown of RUNX1 by siRNA transfection. LNCaP cells were transfected with siControl, siRUNX1 #1 and #2 (10 nM). Cells were transfected with vehicle or 10 nM DHT for 24 h. Western blot analysis of AR and RUNX1 was performed. β-actin was used as a loading control. (B) ChIP-seq analysis of AR binding with depleting RUNX1 expression. LNCaP cells were treated with siControl or siRUNX1 for 48 h. AR ChIP was performed after DHT treatment for 24h. ARBSs (Fold > 10, P < 10−5) were determined by MACS. (C) ChIP analysis of AR binding with depleting RUNX1 expression. LNCaP cells were treated with vehicle or 10 nM DHT for 24 h. ChIP analysis was performed using an AR-specific antibody. Enrichment of the ARBS was quantified using qPCR. Data represent mean + s.d., n = 3. (D) Global analysis of RUNX1 effects on androgen regulation of AR-binding genes. LNCaP cells were treated with siControl and siRUNX1 #1. After 48 h incubation, cells were treated with vehicle or 10 nM DHT for 24 h. Microarray analysis was performed and two clusters (Cluster I and II) were identified as genes positively regulated genes by RUNX1. (E) RUNX1 effects on androgen regulation of AR-binding genes. LNCaP cells were transfected with siControl or siRUNX1 #1 and #2 and then treated with 10 nM DHT or vehicle. Expression level of mRNA of genes in cluster I was measured by qRT-PCR. Data represent mean + s.d., n = 3. * P <0.01. (F) Effects of RUNX1 on transcriptional activity of AR. LNCaP cells were transfected with siControl or siRUNX1 #1 and #2. Luciferase vectors including PSA and TACC2-ARBSs were used. Cells were treated with 10 nM DHT or vehicle for 24 h. Data represent mean + s.d., n = 3. * P <0.01.
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Figure 3: Knockdown of RUNX1 decreased the androgen-responsive transcriptional program(A) Knockdown of RUNX1 by siRNA transfection. LNCaP cells were transfected with siControl, siRUNX1 #1 and #2 (10 nM). Cells were transfected with vehicle or 10 nM DHT for 24 h. Western blot analysis of AR and RUNX1 was performed. β-actin was used as a loading control. (B) ChIP-seq analysis of AR binding with depleting RUNX1 expression. LNCaP cells were treated with siControl or siRUNX1 for 48 h. AR ChIP was performed after DHT treatment for 24h. ARBSs (Fold > 10, P < 10−5) were determined by MACS. (C) ChIP analysis of AR binding with depleting RUNX1 expression. LNCaP cells were treated with vehicle or 10 nM DHT for 24 h. ChIP analysis was performed using an AR-specific antibody. Enrichment of the ARBS was quantified using qPCR. Data represent mean + s.d., n = 3. (D) Global analysis of RUNX1 effects on androgen regulation of AR-binding genes. LNCaP cells were treated with siControl and siRUNX1 #1. After 48 h incubation, cells were treated with vehicle or 10 nM DHT for 24 h. Microarray analysis was performed and two clusters (Cluster I and II) were identified as genes positively regulated genes by RUNX1. (E) RUNX1 effects on androgen regulation of AR-binding genes. LNCaP cells were transfected with siControl or siRUNX1 #1 and #2 and then treated with 10 nM DHT or vehicle. Expression level of mRNA of genes in cluster I was measured by qRT-PCR. Data represent mean + s.d., n = 3. * P <0.01. (F) Effects of RUNX1 on transcriptional activity of AR. LNCaP cells were transfected with siControl or siRUNX1 #1 and #2. Luciferase vectors including PSA and TACC2-ARBSs were used. Cells were treated with 10 nM DHT or vehicle for 24 h. Data represent mean + s.d., n = 3. * P <0.01.
Mentions: We next analyzed the effect of RUNX1 on AR binding by ChIP-seq. We treated LNCaP cells with short interference RNA (siRNA) targeting RUNX1 or control RNA (siControl) (Fig.3A). We performed AR ChIP after treating cells with DHT. In our ChIP and ChIP-seq study, the number of significant ARBSs obtained was decreased and enrichments of AR binding are inhibited by RUNX1 knockdown, suggesting a positive role of RUNX1 for AR binding (Fig.3B, C). The overlap of ARBSs with RUNX1 binding, and the reduction of ARBSs by RUNX1 knockdown were also observed in VCaP cells (Supplementary Fig.2B). These results indicate a significant role for RUNX1 recruitment to ARBS in AR binding.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anti-Aging Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

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.

Show MeSH
Related in: MedlinePlus