Limits...
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.

Show MeSH

Related in: MedlinePlus

The role of RUNX1 in androgen-dependent and –independent prostate cancer cells(A) Knockdown of RUNX1 showed positive effects on androgen-dependent cell proliferation in prostate cancer. LNCaP or VCaP cells were transfected with siRUNX1 or siControl and then treated with vehicle or 10 nM DHT. Cell growth was evaluated at day 3 by MTS assay. Data represent mean + s.d., n = 4. ** P <0.01. (B) Knockdown of RUNX1 in both LNCaP and DU145 cells. Both cells were transfected with siControl or siRUNX1 #1 for 48 h. Western blot analysis of RUNX1 was performed. β-actin was used as a loading control. (C) LNCaP cells were transfected with siRUNX1 #1 or siControl and then the cell proliferation was assessed by cell counting on day 3. Data represent mean + s.d., n = 4. ** P <0.01. (D) DU145 cells were transfected with siRUNX1 #1 or siControl and then the cell proliferation was assessed by cell counting on day 3. Data represent mean + s.d., n = 4. * P <0.05. (E) Effect of RUNX1 overexpression in cell proliferation of DU145 cells. DU145 cells were transfected with RUNX1 expression vector or control. Western blot analysis of RUNX1 was performed. β-actin was used as a loading control. The cell proliferation was assessed by cell counting on day 3. Data represent mean + s.d., n = 4. ** P <0.01. (F) Knockdown of RUNX1 showed negative effects on androgen-independent cell proliferation in prostate cancer. DU145 cells were transfected with siRUNX1 or siControl. Western blot analysis of RUNX1 was performed. β-actin was used as a loading control. Cell growth was evaluated by MTS assay on day 3. Data represent mean + s.d., n = 4. ** P <0.01. (G) Knockdown of RUNX1 and EZH2. DU145 cells were transfected with siEZH2, siRUNX1 or siControl. Western blot analysis of RUNX1 and EZH2 was performed. β-actin was used as a loading control. (H) DU145 cells were transfected with siEZH2, siRUNX1 or siControl. Cell growth was evaluated by MTS assay on day 3. Data represent mean + s.d., n = 4. * P <0.05; ** P <0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4385850&req=5

Figure 5: The role of RUNX1 in androgen-dependent and –independent prostate cancer cells(A) Knockdown of RUNX1 showed positive effects on androgen-dependent cell proliferation in prostate cancer. LNCaP or VCaP cells were transfected with siRUNX1 or siControl and then treated with vehicle or 10 nM DHT. Cell growth was evaluated at day 3 by MTS assay. Data represent mean + s.d., n = 4. ** P <0.01. (B) Knockdown of RUNX1 in both LNCaP and DU145 cells. Both cells were transfected with siControl or siRUNX1 #1 for 48 h. Western blot analysis of RUNX1 was performed. β-actin was used as a loading control. (C) LNCaP cells were transfected with siRUNX1 #1 or siControl and then the cell proliferation was assessed by cell counting on day 3. Data represent mean + s.d., n = 4. ** P <0.01. (D) DU145 cells were transfected with siRUNX1 #1 or siControl and then the cell proliferation was assessed by cell counting on day 3. Data represent mean + s.d., n = 4. * P <0.05. (E) Effect of RUNX1 overexpression in cell proliferation of DU145 cells. DU145 cells were transfected with RUNX1 expression vector or control. Western blot analysis of RUNX1 was performed. β-actin was used as a loading control. The cell proliferation was assessed by cell counting on day 3. Data represent mean + s.d., n = 4. ** P <0.01. (F) Knockdown of RUNX1 showed negative effects on androgen-independent cell proliferation in prostate cancer. DU145 cells were transfected with siRUNX1 or siControl. Western blot analysis of RUNX1 was performed. β-actin was used as a loading control. Cell growth was evaluated by MTS assay on day 3. Data represent mean + s.d., n = 4. ** P <0.01. (G) Knockdown of RUNX1 and EZH2. DU145 cells were transfected with siEZH2, siRUNX1 or siControl. Western blot analysis of RUNX1 and EZH2 was performed. β-actin was used as a loading control. (H) DU145 cells were transfected with siEZH2, siRUNX1 or siControl. Cell growth was evaluated by MTS assay on day 3. Data represent mean + s.d., n = 4. * P <0.05; ** P <0.01.

Mentions: We explored the role of RUNX1 in prostate cancer cell proliferation. In LNCaP cells, we observed that androgen-dependent cell proliferation is inhibited by siRUNX1 transfection (Fig.5A). This effect of RUNX1 knockdown was also confirmed in VCaP cells. Next, we analyzed the role of RUNX1 in AR-negative prostate cancer cells. In DU145 cells, which represent androgen-independent cell proliferation, RUNX1 is expressed and effectively knocked down by siRUNX1 treatment (Fig.5B). By cell counting assays, we showed that proliferation of DU145 cells was promoted by RUNX1 knockdown in contrast to LNCaP cells, where proliferation is repressed (Fig.5C, D). The negative effect of RUNX1 on proliferation is also confirmed by overexpression of RUNX1 in DU145 cells (Fig.5E). We further analyzed whether this growth inhibitory effect of RUNX1 in androgen-independent cells was mediated by the function of EZH2 or not. We showed that RUNX1 knockdown by two different siRNAs promotes cell proliferation (Fig.5F). Surprisingly, the siEZH2-mediated growth inhibition was relieved by RUNX1 knockdown, suggesting EZH2 growth stimulatory effect is mediated partially at least by RUNX1 repression (Fig.5G, H). These results suggested that RUNX1 negatively regulates androgen-independent signaling for cell proliferation despite a positive effect on AR-mediated cell growth.


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)

The role of RUNX1 in androgen-dependent and –independent prostate cancer cells(A) Knockdown of RUNX1 showed positive effects on androgen-dependent cell proliferation in prostate cancer. LNCaP or VCaP cells were transfected with siRUNX1 or siControl and then treated with vehicle or 10 nM DHT. Cell growth was evaluated at day 3 by MTS assay. Data represent mean + s.d., n = 4. ** P <0.01. (B) Knockdown of RUNX1 in both LNCaP and DU145 cells. Both cells were transfected with siControl or siRUNX1 #1 for 48 h. Western blot analysis of RUNX1 was performed. β-actin was used as a loading control. (C) LNCaP cells were transfected with siRUNX1 #1 or siControl and then the cell proliferation was assessed by cell counting on day 3. Data represent mean + s.d., n = 4. ** P <0.01. (D) DU145 cells were transfected with siRUNX1 #1 or siControl and then the cell proliferation was assessed by cell counting on day 3. Data represent mean + s.d., n = 4. * P <0.05. (E) Effect of RUNX1 overexpression in cell proliferation of DU145 cells. DU145 cells were transfected with RUNX1 expression vector or control. Western blot analysis of RUNX1 was performed. β-actin was used as a loading control. The cell proliferation was assessed by cell counting on day 3. Data represent mean + s.d., n = 4. ** P <0.01. (F) Knockdown of RUNX1 showed negative effects on androgen-independent cell proliferation in prostate cancer. DU145 cells were transfected with siRUNX1 or siControl. Western blot analysis of RUNX1 was performed. β-actin was used as a loading control. Cell growth was evaluated by MTS assay on day 3. Data represent mean + s.d., n = 4. ** P <0.01. (G) Knockdown of RUNX1 and EZH2. DU145 cells were transfected with siEZH2, siRUNX1 or siControl. Western blot analysis of RUNX1 and EZH2 was performed. β-actin was used as a loading control. (H) DU145 cells were transfected with siEZH2, siRUNX1 or siControl. Cell growth was evaluated by MTS assay on day 3. Data represent mean + s.d., n = 4. * P <0.05; ** P <0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385850&req=5

Figure 5: The role of RUNX1 in androgen-dependent and –independent prostate cancer cells(A) Knockdown of RUNX1 showed positive effects on androgen-dependent cell proliferation in prostate cancer. LNCaP or VCaP cells were transfected with siRUNX1 or siControl and then treated with vehicle or 10 nM DHT. Cell growth was evaluated at day 3 by MTS assay. Data represent mean + s.d., n = 4. ** P <0.01. (B) Knockdown of RUNX1 in both LNCaP and DU145 cells. Both cells were transfected with siControl or siRUNX1 #1 for 48 h. Western blot analysis of RUNX1 was performed. β-actin was used as a loading control. (C) LNCaP cells were transfected with siRUNX1 #1 or siControl and then the cell proliferation was assessed by cell counting on day 3. Data represent mean + s.d., n = 4. ** P <0.01. (D) DU145 cells were transfected with siRUNX1 #1 or siControl and then the cell proliferation was assessed by cell counting on day 3. Data represent mean + s.d., n = 4. * P <0.05. (E) Effect of RUNX1 overexpression in cell proliferation of DU145 cells. DU145 cells were transfected with RUNX1 expression vector or control. Western blot analysis of RUNX1 was performed. β-actin was used as a loading control. The cell proliferation was assessed by cell counting on day 3. Data represent mean + s.d., n = 4. ** P <0.01. (F) Knockdown of RUNX1 showed negative effects on androgen-independent cell proliferation in prostate cancer. DU145 cells were transfected with siRUNX1 or siControl. Western blot analysis of RUNX1 was performed. β-actin was used as a loading control. Cell growth was evaluated by MTS assay on day 3. Data represent mean + s.d., n = 4. ** P <0.01. (G) Knockdown of RUNX1 and EZH2. DU145 cells were transfected with siEZH2, siRUNX1 or siControl. Western blot analysis of RUNX1 and EZH2 was performed. β-actin was used as a loading control. (H) DU145 cells were transfected with siEZH2, siRUNX1 or siControl. Cell growth was evaluated by MTS assay on day 3. Data represent mean + s.d., n = 4. * P <0.05; ** P <0.01.
Mentions: We explored the role of RUNX1 in prostate cancer cell proliferation. In LNCaP cells, we observed that androgen-dependent cell proliferation is inhibited by siRUNX1 transfection (Fig.5A). This effect of RUNX1 knockdown was also confirmed in VCaP cells. Next, we analyzed the role of RUNX1 in AR-negative prostate cancer cells. In DU145 cells, which represent androgen-independent cell proliferation, RUNX1 is expressed and effectively knocked down by siRUNX1 treatment (Fig.5B). By cell counting assays, we showed that proliferation of DU145 cells was promoted by RUNX1 knockdown in contrast to LNCaP cells, where proliferation is repressed (Fig.5C, D). The negative effect of RUNX1 on proliferation is also confirmed by overexpression of RUNX1 in DU145 cells (Fig.5E). We further analyzed whether this growth inhibitory effect of RUNX1 in androgen-independent cells was mediated by the function of EZH2 or not. We showed that RUNX1 knockdown by two different siRNAs promotes cell proliferation (Fig.5F). Surprisingly, the siEZH2-mediated growth inhibition was relieved by RUNX1 knockdown, suggesting EZH2 growth stimulatory effect is mediated partially at least by RUNX1 repression (Fig.5G, H). These results suggested that RUNX1 negatively regulates androgen-independent signaling for cell proliferation despite a positive effect on AR-mediated cell growth.

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