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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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EZH2-dependent H3K27 methylation repressed RUNX1 expression(A) ChIP-seq analysis of the RUNX1 promoter region. LNCaP cells were treated with vehicle or 10 nM R1881 for 24 h. ChIP-seq analysis by K27me3 and AcH3 were performed. Signal distribution at the RUNX1 promoter is shown. (B) Effect of EZH2 knockdown. LNCaP cells were treated with siEZH2 #1 and #2 (10 nM). Western blot analysis of AR and RUNX1 was performed. β-actin was used as a loading control. (C) Analysis of K27me3 at the 5′-upstream region of RUNX1. LNCaP cells were treated with siEZH2 #1 or siControl. After 48 h incubation, cells were treated with vehicle or 10 nM DHT for 24 h. ChIP analysis was performed by using anti-K27me3 antibody. Enrichment of the 5′ upstream region of RUNX1 was quantified using qPCR. Data represent mean + s.d., n = 3. * P <0.05; ** P <0.01. (D) Analysis of EZH2 recruitment to 5′-upstream region of RUNX1. LNCaP cells were treated with vehicle or DHT. ChIP analysis was performed by using anti-EZH2 antibody. Enrichment of the promoter, 5′-upstream and ARBS regions of RUNX1 was quantified using qPCR. The SLIT2 promoter was used as a positive control for EZH2 recruitment. Data represent mean + s.d., n = 3. (E) ChIP-seq analysis of EZH2 binding at the RUNX1 promoter. LNCaP cells were treated with DHT for 24 h. Distribution of AR and EZH2 binding in RUNX1 regions is shown.
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Figure 4: EZH2-dependent H3K27 methylation repressed RUNX1 expression(A) ChIP-seq analysis of the RUNX1 promoter region. LNCaP cells were treated with vehicle or 10 nM R1881 for 24 h. ChIP-seq analysis by K27me3 and AcH3 were performed. Signal distribution at the RUNX1 promoter is shown. (B) Effect of EZH2 knockdown. LNCaP cells were treated with siEZH2 #1 and #2 (10 nM). Western blot analysis of AR and RUNX1 was performed. β-actin was used as a loading control. (C) Analysis of K27me3 at the 5′-upstream region of RUNX1. LNCaP cells were treated with siEZH2 #1 or siControl. After 48 h incubation, cells were treated with vehicle or 10 nM DHT for 24 h. ChIP analysis was performed by using anti-K27me3 antibody. Enrichment of the 5′ upstream region of RUNX1 was quantified using qPCR. Data represent mean + s.d., n = 3. * P <0.05; ** P <0.01. (D) Analysis of EZH2 recruitment to 5′-upstream region of RUNX1. LNCaP cells were treated with vehicle or DHT. ChIP analysis was performed by using anti-EZH2 antibody. Enrichment of the promoter, 5′-upstream and ARBS regions of RUNX1 was quantified using qPCR. The SLIT2 promoter was used as a positive control for EZH2 recruitment. Data represent mean + s.d., n = 3. (E) ChIP-seq analysis of EZH2 binding at the RUNX1 promoter. LNCaP cells were treated with DHT for 24 h. Distribution of AR and EZH2 binding in RUNX1 regions is shown.

Mentions: We analyzed whether RUNX1 is involved in androgen-independent prostate cancer development. By ChIP-seq analysis of histone modifications, we investigated the modification patterns of the RUNX1 promoter (histoneH3 K4me3, K27me3, and AcH3). We found that the RUNX1 promoter is occupied with modified histones not only the markers of activation, but also the repressive marker, H3K27me3 (Fig.4A). In prostate cancer cells, EZH2 overexpression is known to promote cancer progression [17]. EZH2 is a part of the polycomb complex that induces gene repression through histone H3K27 methylation. We observed that knockdown of EZH2 enhances the expression of RUNX1 (Fig.4B) and represses H3K27 methylation in the RUNX1 promoter (Fig.4C). By ChIP assay we further investigated whether the RUNX1 promoter is associated with EZH2 or not. SLIT2 is a representative target of EZH2 in prostate cancer and its promoter is occupied with EZH2 [23]. In LNCaP cells, we observed EZH2 recruitment, comparable with that of the SLIT2 promoter, to the 5′-upstream region and the promoter of RUNX1 (Fig.4D). By ChIP-seq, we further analyzed the regions occupied by EZH2 and identified multiple EZH2 binding sites in 5′-upstream region of the RUNX1 gene (Fig.4E). Similar results were also observed in VCaP and DU145 cells (Supplementary Fig.4). These results suggest the regulation of RUNX1 by EZH2 dependent H3K27 methylation.


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)

EZH2-dependent H3K27 methylation repressed RUNX1 expression(A) ChIP-seq analysis of the RUNX1 promoter region. LNCaP cells were treated with vehicle or 10 nM R1881 for 24 h. ChIP-seq analysis by K27me3 and AcH3 were performed. Signal distribution at the RUNX1 promoter is shown. (B) Effect of EZH2 knockdown. LNCaP cells were treated with siEZH2 #1 and #2 (10 nM). Western blot analysis of AR and RUNX1 was performed. β-actin was used as a loading control. (C) Analysis of K27me3 at the 5′-upstream region of RUNX1. LNCaP cells were treated with siEZH2 #1 or siControl. After 48 h incubation, cells were treated with vehicle or 10 nM DHT for 24 h. ChIP analysis was performed by using anti-K27me3 antibody. Enrichment of the 5′ upstream region of RUNX1 was quantified using qPCR. Data represent mean + s.d., n = 3. * P <0.05; ** P <0.01. (D) Analysis of EZH2 recruitment to 5′-upstream region of RUNX1. LNCaP cells were treated with vehicle or DHT. ChIP analysis was performed by using anti-EZH2 antibody. Enrichment of the promoter, 5′-upstream and ARBS regions of RUNX1 was quantified using qPCR. The SLIT2 promoter was used as a positive control for EZH2 recruitment. Data represent mean + s.d., n = 3. (E) ChIP-seq analysis of EZH2 binding at the RUNX1 promoter. LNCaP cells were treated with DHT for 24 h. Distribution of AR and EZH2 binding in RUNX1 regions is shown.
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Figure 4: EZH2-dependent H3K27 methylation repressed RUNX1 expression(A) ChIP-seq analysis of the RUNX1 promoter region. LNCaP cells were treated with vehicle or 10 nM R1881 for 24 h. ChIP-seq analysis by K27me3 and AcH3 were performed. Signal distribution at the RUNX1 promoter is shown. (B) Effect of EZH2 knockdown. LNCaP cells were treated with siEZH2 #1 and #2 (10 nM). Western blot analysis of AR and RUNX1 was performed. β-actin was used as a loading control. (C) Analysis of K27me3 at the 5′-upstream region of RUNX1. LNCaP cells were treated with siEZH2 #1 or siControl. After 48 h incubation, cells were treated with vehicle or 10 nM DHT for 24 h. ChIP analysis was performed by using anti-K27me3 antibody. Enrichment of the 5′ upstream region of RUNX1 was quantified using qPCR. Data represent mean + s.d., n = 3. * P <0.05; ** P <0.01. (D) Analysis of EZH2 recruitment to 5′-upstream region of RUNX1. LNCaP cells were treated with vehicle or DHT. ChIP analysis was performed by using anti-EZH2 antibody. Enrichment of the promoter, 5′-upstream and ARBS regions of RUNX1 was quantified using qPCR. The SLIT2 promoter was used as a positive control for EZH2 recruitment. Data represent mean + s.d., n = 3. (E) ChIP-seq analysis of EZH2 binding at the RUNX1 promoter. LNCaP cells were treated with DHT for 24 h. Distribution of AR and EZH2 binding in RUNX1 regions is shown.
Mentions: We analyzed whether RUNX1 is involved in androgen-independent prostate cancer development. By ChIP-seq analysis of histone modifications, we investigated the modification patterns of the RUNX1 promoter (histoneH3 K4me3, K27me3, and AcH3). We found that the RUNX1 promoter is occupied with modified histones not only the markers of activation, but also the repressive marker, H3K27me3 (Fig.4A). In prostate cancer cells, EZH2 overexpression is known to promote cancer progression [17]. EZH2 is a part of the polycomb complex that induces gene repression through histone H3K27 methylation. We observed that knockdown of EZH2 enhances the expression of RUNX1 (Fig.4B) and represses H3K27 methylation in the RUNX1 promoter (Fig.4C). By ChIP assay we further investigated whether the RUNX1 promoter is associated with EZH2 or not. SLIT2 is a representative target of EZH2 in prostate cancer and its promoter is occupied with EZH2 [23]. In LNCaP cells, we observed EZH2 recruitment, comparable with that of the SLIT2 promoter, to the 5′-upstream region and the promoter of RUNX1 (Fig.4D). By ChIP-seq, we further analyzed the regions occupied by EZH2 and identified multiple EZH2 binding sites in 5′-upstream region of the RUNX1 gene (Fig.4E). Similar results were also observed in VCaP and DU145 cells (Supplementary Fig.4). These results suggest the regulation of RUNX1 by EZH2 dependent H3K27 methylation.

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