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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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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. (B) ChIP-seq analysis of AR, AcH3 and K4me1 in the RUNX1 locus. Two ARBSs were identified (ARBS #1 at 60 kb 3′-downstream region, ARBS #2: at 250 kb 5′ -upstream region). Two promoter loci and the direction of transcription were indicated by arrows. (C) Luciferase assay was performed in LNCaP cells. Luciferase vectors including ARBS #1 and #2 were used. Cells were treated with 10 nM DHT or vehicle for 24 h. Data represent mean + s.d., n = 3. (D) (Upper) Three Myc-tagged expression vectors of RUNX1 and deleted RUNX1 variants were constructed. (Lower) Androgen dependent interaction of RUNX1 with AR. 293T cells were transiently transfected with Flag-AR and Myc-RUNX1 and after 24 h incubation cells were treated with vehicle or 10 nM DHT for 24 h. Immunoprecipitation by Myc-antibody and western blot analysis of Flag and Myc was performed. (E) Endogenous interaction of AR with RUNX1. LNCaP cells were treated with vehicle or 10 nM DHT for 24 h. Cell lysates were immunoprecipitated by RUNX1 antibody. Western blot analysis of RUNX1 and AR was performed.
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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. (B) ChIP-seq analysis of AR, AcH3 and K4me1 in the RUNX1 locus. Two ARBSs were identified (ARBS #1 at 60 kb 3′-downstream region, ARBS #2: at 250 kb 5′ -upstream region). Two promoter loci and the direction of transcription were indicated by arrows. (C) Luciferase assay was performed in LNCaP cells. Luciferase vectors including ARBS #1 and #2 were used. Cells were treated with 10 nM DHT or vehicle for 24 h. Data represent mean + s.d., n = 3. (D) (Upper) Three Myc-tagged expression vectors of RUNX1 and deleted RUNX1 variants were constructed. (Lower) Androgen dependent interaction of RUNX1 with AR. 293T cells were transiently transfected with Flag-AR and Myc-RUNX1 and after 24 h incubation cells were treated with vehicle or 10 nM DHT for 24 h. Immunoprecipitation by Myc-antibody and western blot analysis of Flag and Myc was performed. (E) Endogenous interaction of AR with RUNX1. LNCaP cells were treated with vehicle or 10 nM DHT for 24 h. Cell lysates were immunoprecipitated by RUNX1 antibody. Western blot analysis of RUNX1 and AR was performed.

Mentions: We previously reported the global analysis of androgen signaling by mapping ARBSs and androgen-regulated transcripts [18, 19, 20]. Using quantitative reverse transcriptase-PCR (qRT-PCR) analysis, we found that RUNX1 is highly induced by R1881 10 nM treatment in LNCaP cells (more than 50-fold following 48 h of treatment) (Fig.1A). RUNX1 protein levels were also increased as demonstrated by western blot analysis (Fig.1A). Two robust AR binding sites were identified around the RUNX1 locus, one (ARBS #1) is 50 kb downstream and the other (ARBS #2) is 200 kb upstream (Fig.1B, Supplementary Fig.1A) by AR Chromatin immunoprecipitation and sequencing (ChIP-seq). In addition, RUNX1 upregulation by DHT treatment was also observed and this induction was inhibited by short interference RNA (siRNA) targeting AR-treatment (Supplementary Fig.1B, C). We also confirmed RUNX1 induction by androgen in other AR positive prostate cancer cells (Supplementary Fig.1D). Next, we investigated histone modification patterns in the RUNX1 locus by ChIP-seq (Fig.1B, Supplementary Fig.1E). RUNX1 has two distinct transcriptional start sites (TSSs: Promoter 1 and 2), and we found that promoter 2 is occupied with highly acetylated histone H3 by ChIP-seq analysis. Although both ARBSs are modified with monomethylation of histone H3K4, an active enhancer mark, we showed that ARBS #1 has a highly androgen-dependent transcriptional activity by luciferase assay (Fig.1C). Highly acetylated histone modification was observed only in the promoter 2 of RUNX1 (and not in that of CLIC6) around the ARBS#1, implicating that this region acts as an enhancer for RUNX1 induction (Fig.1B). These results suggest that RUNX1 is directly regulated by 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)

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. (B) ChIP-seq analysis of AR, AcH3 and K4me1 in the RUNX1 locus. Two ARBSs were identified (ARBS #1 at 60 kb 3′-downstream region, ARBS #2: at 250 kb 5′ -upstream region). Two promoter loci and the direction of transcription were indicated by arrows. (C) Luciferase assay was performed in LNCaP cells. Luciferase vectors including ARBS #1 and #2 were used. Cells were treated with 10 nM DHT or vehicle for 24 h. Data represent mean + s.d., n = 3. (D) (Upper) Three Myc-tagged expression vectors of RUNX1 and deleted RUNX1 variants were constructed. (Lower) Androgen dependent interaction of RUNX1 with AR. 293T cells were transiently transfected with Flag-AR and Myc-RUNX1 and after 24 h incubation cells were treated with vehicle or 10 nM DHT for 24 h. Immunoprecipitation by Myc-antibody and western blot analysis of Flag and Myc was performed. (E) Endogenous interaction of AR with RUNX1. LNCaP cells were treated with vehicle or 10 nM DHT for 24 h. Cell lysates were immunoprecipitated by RUNX1 antibody. Western blot analysis of RUNX1 and AR was performed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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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. (B) ChIP-seq analysis of AR, AcH3 and K4me1 in the RUNX1 locus. Two ARBSs were identified (ARBS #1 at 60 kb 3′-downstream region, ARBS #2: at 250 kb 5′ -upstream region). Two promoter loci and the direction of transcription were indicated by arrows. (C) Luciferase assay was performed in LNCaP cells. Luciferase vectors including ARBS #1 and #2 were used. Cells were treated with 10 nM DHT or vehicle for 24 h. Data represent mean + s.d., n = 3. (D) (Upper) Three Myc-tagged expression vectors of RUNX1 and deleted RUNX1 variants were constructed. (Lower) Androgen dependent interaction of RUNX1 with AR. 293T cells were transiently transfected with Flag-AR and Myc-RUNX1 and after 24 h incubation cells were treated with vehicle or 10 nM DHT for 24 h. Immunoprecipitation by Myc-antibody and western blot analysis of Flag and Myc was performed. (E) Endogenous interaction of AR with RUNX1. LNCaP cells were treated with vehicle or 10 nM DHT for 24 h. Cell lysates were immunoprecipitated by RUNX1 antibody. Western blot analysis of RUNX1 and AR was performed.
Mentions: We previously reported the global analysis of androgen signaling by mapping ARBSs and androgen-regulated transcripts [18, 19, 20]. Using quantitative reverse transcriptase-PCR (qRT-PCR) analysis, we found that RUNX1 is highly induced by R1881 10 nM treatment in LNCaP cells (more than 50-fold following 48 h of treatment) (Fig.1A). RUNX1 protein levels were also increased as demonstrated by western blot analysis (Fig.1A). Two robust AR binding sites were identified around the RUNX1 locus, one (ARBS #1) is 50 kb downstream and the other (ARBS #2) is 200 kb upstream (Fig.1B, Supplementary Fig.1A) by AR Chromatin immunoprecipitation and sequencing (ChIP-seq). In addition, RUNX1 upregulation by DHT treatment was also observed and this induction was inhibited by short interference RNA (siRNA) targeting AR-treatment (Supplementary Fig.1B, C). We also confirmed RUNX1 induction by androgen in other AR positive prostate cancer cells (Supplementary Fig.1D). Next, we investigated histone modification patterns in the RUNX1 locus by ChIP-seq (Fig.1B, Supplementary Fig.1E). RUNX1 has two distinct transcriptional start sites (TSSs: Promoter 1 and 2), and we found that promoter 2 is occupied with highly acetylated histone H3 by ChIP-seq analysis. Although both ARBSs are modified with monomethylation of histone H3K4, an active enhancer mark, we showed that ARBS #1 has a highly androgen-dependent transcriptional activity by luciferase assay (Fig.1C). Highly acetylated histone modification was observed only in the promoter 2 of RUNX1 (and not in that of CLIC6) around the ARBS#1, implicating that this region acts as an enhancer for RUNX1 induction (Fig.1B). These results suggest that RUNX1 is directly regulated by 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