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Targeting chromatin binding regulation of constitutively active AR variants to overcome prostate cancer resistance to endocrine-based therapies.

Chan SC, Selth LA, Li Y, Nyquist MD, Miao L, Bradner JE, Raj GV, Tilley WD, Dehm SM - Nucleic Acids Res. (2015)

Bottom Line: Dimerization was an absolute requirement for constitutive AR-V DNA binding and transcriptional activation.Treatment with the bromodomain and extraterminal (BET) inhibitor JQ1 resulted in inhibition of AR-V chromatin binding and impaired AR-V driven PCa cell growth in vitro and in vivo.Importantly, this was associated with a novel JQ1 action of down-regulating AR-V transcript and protein expression.

View Article: PubMed Central - PubMed

Affiliation: Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55905, USA.

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BET inhibitors repress expression of AR and ARv567es in prostate cancer cells. (A) Binding of full-length AR or ARv567es to FASN ARBS1 was tested in R1-AD1 or R1-D567 cells, respectively, treated with vehicle (DMSO) or JQ1 (0.5 mM) in the presence or absence of 1 nM dihydrotestosterone (DHT) as indicated. (B) Western blots with antibodies specific for the AR NTD, AR-V7 or ERK-2 (loading control) with lysates from indicated PCa cell lines treated 24 h with vehicle (DMSO) or JQ1 (doses: 0.1, 0.5, 1, 5, 10 μM) in medium containing 10% CSS. (C) Quantitative RT-PCR analysis of total AR mRNA levels in PCa cell lines treated as in (B). (D) Western blots with antibodies specific for the AR NTD or β-actin (loading control) with lysates from LNCaP cells treated 24 h with vehicle (DMSO) or active S(+) or inactive R(–) JQ1 stereoisomers as indicated. (E) Gene track view of BRD2, BRD3, BRD4 and H3K27Ac ChIP-seq data at the AR locus. Data were obtained from NCBI Gene Expression Omnibus (GEO), representing VCaP cells treated with 0.5 μM JQ1 or vehicle control (DMSO) (GSE27823, (28)) or DHT-treated LNCaP cells (GSE27823, (29)). The FDR-adjusted P value signifying JQ1-mediated loss of BRD2 binding at this site in VCaP cells was derived using diffReps (34). (F) Binding of BRD2 to the AR 5′utr region was tested in R1-AD1 or R1-D567 cells treated with vehicle (DMSO) or JQ1 (0.5 μM) as indicated.
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Figure 8: BET inhibitors repress expression of AR and ARv567es in prostate cancer cells. (A) Binding of full-length AR or ARv567es to FASN ARBS1 was tested in R1-AD1 or R1-D567 cells, respectively, treated with vehicle (DMSO) or JQ1 (0.5 mM) in the presence or absence of 1 nM dihydrotestosterone (DHT) as indicated. (B) Western blots with antibodies specific for the AR NTD, AR-V7 or ERK-2 (loading control) with lysates from indicated PCa cell lines treated 24 h with vehicle (DMSO) or JQ1 (doses: 0.1, 0.5, 1, 5, 10 μM) in medium containing 10% CSS. (C) Quantitative RT-PCR analysis of total AR mRNA levels in PCa cell lines treated as in (B). (D) Western blots with antibodies specific for the AR NTD or β-actin (loading control) with lysates from LNCaP cells treated 24 h with vehicle (DMSO) or active S(+) or inactive R(–) JQ1 stereoisomers as indicated. (E) Gene track view of BRD2, BRD3, BRD4 and H3K27Ac ChIP-seq data at the AR locus. Data were obtained from NCBI Gene Expression Omnibus (GEO), representing VCaP cells treated with 0.5 μM JQ1 or vehicle control (DMSO) (GSE27823, (28)) or DHT-treated LNCaP cells (GSE27823, (29)). The FDR-adjusted P value signifying JQ1-mediated loss of BRD2 binding at this site in VCaP cells was derived using diffReps (34). (F) Binding of BRD2 to the AR 5′utr region was tested in R1-AD1 or R1-D567 cells treated with vehicle (DMSO) or JQ1 (0.5 μM) as indicated.

Mentions: Based on these mechanistic findings, we hypothesized that disruption of AR-V:ARE interactions may represent a viable strategy for overcoming AR-V-mediated therapeutic resistance in CRPC. To develop preclinical support for this chromatin targeting concept, we evaluated JQ1, an inhibitor of the BET family of chromatin readers (49) shown to impair genome-wide chromatin binding and transcriptional activation of androgen-activated full-length AR (28). BET family chromatin readers BRD2, 3 and 4 engage with acetylated lysine residues, including H3K27Ac, which constitute chromatin marks enriched at active enhancers and associated transcription start-sites of transcriptionally active genes (50). As expected, JQ1 reduced androgen-mediated AR engagement with FASN ARBSI in R1-AD1 cells (Figure 8A). More strikingly, JQ1 completely inhibited constitutive AR-V engagement with this site in R1-D567 cells (Figure 8A). Unexpectedly, we noted loss of AR and ARv567es protein when R1-AD1 and R1-D567 cells were treated with therapeutically-relevant doses of JQ1 (28,49–51), which corresponded with down-regulated AR mRNA expression (Figure 8B and C). The CRPC C4-2, 22Rv1 and VCaP cell lines also displayed dose-dependent reductions in AR and/or AR-V expression in response to JQ1 treatment (Figure 8B and C). In the LNCaP cell line, down-regulated AR expression was observed for (+)-JQ1 but not (−)-JQ1, indicating specificity for the active stereoisomer (Figure 8D) (49).


Targeting chromatin binding regulation of constitutively active AR variants to overcome prostate cancer resistance to endocrine-based therapies.

Chan SC, Selth LA, Li Y, Nyquist MD, Miao L, Bradner JE, Raj GV, Tilley WD, Dehm SM - Nucleic Acids Res. (2015)

BET inhibitors repress expression of AR and ARv567es in prostate cancer cells. (A) Binding of full-length AR or ARv567es to FASN ARBS1 was tested in R1-AD1 or R1-D567 cells, respectively, treated with vehicle (DMSO) or JQ1 (0.5 mM) in the presence or absence of 1 nM dihydrotestosterone (DHT) as indicated. (B) Western blots with antibodies specific for the AR NTD, AR-V7 or ERK-2 (loading control) with lysates from indicated PCa cell lines treated 24 h with vehicle (DMSO) or JQ1 (doses: 0.1, 0.5, 1, 5, 10 μM) in medium containing 10% CSS. (C) Quantitative RT-PCR analysis of total AR mRNA levels in PCa cell lines treated as in (B). (D) Western blots with antibodies specific for the AR NTD or β-actin (loading control) with lysates from LNCaP cells treated 24 h with vehicle (DMSO) or active S(+) or inactive R(–) JQ1 stereoisomers as indicated. (E) Gene track view of BRD2, BRD3, BRD4 and H3K27Ac ChIP-seq data at the AR locus. Data were obtained from NCBI Gene Expression Omnibus (GEO), representing VCaP cells treated with 0.5 μM JQ1 or vehicle control (DMSO) (GSE27823, (28)) or DHT-treated LNCaP cells (GSE27823, (29)). The FDR-adjusted P value signifying JQ1-mediated loss of BRD2 binding at this site in VCaP cells was derived using diffReps (34). (F) Binding of BRD2 to the AR 5′utr region was tested in R1-AD1 or R1-D567 cells treated with vehicle (DMSO) or JQ1 (0.5 μM) as indicated.
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Figure 8: BET inhibitors repress expression of AR and ARv567es in prostate cancer cells. (A) Binding of full-length AR or ARv567es to FASN ARBS1 was tested in R1-AD1 or R1-D567 cells, respectively, treated with vehicle (DMSO) or JQ1 (0.5 mM) in the presence or absence of 1 nM dihydrotestosterone (DHT) as indicated. (B) Western blots with antibodies specific for the AR NTD, AR-V7 or ERK-2 (loading control) with lysates from indicated PCa cell lines treated 24 h with vehicle (DMSO) or JQ1 (doses: 0.1, 0.5, 1, 5, 10 μM) in medium containing 10% CSS. (C) Quantitative RT-PCR analysis of total AR mRNA levels in PCa cell lines treated as in (B). (D) Western blots with antibodies specific for the AR NTD or β-actin (loading control) with lysates from LNCaP cells treated 24 h with vehicle (DMSO) or active S(+) or inactive R(–) JQ1 stereoisomers as indicated. (E) Gene track view of BRD2, BRD3, BRD4 and H3K27Ac ChIP-seq data at the AR locus. Data were obtained from NCBI Gene Expression Omnibus (GEO), representing VCaP cells treated with 0.5 μM JQ1 or vehicle control (DMSO) (GSE27823, (28)) or DHT-treated LNCaP cells (GSE27823, (29)). The FDR-adjusted P value signifying JQ1-mediated loss of BRD2 binding at this site in VCaP cells was derived using diffReps (34). (F) Binding of BRD2 to the AR 5′utr region was tested in R1-AD1 or R1-D567 cells treated with vehicle (DMSO) or JQ1 (0.5 μM) as indicated.
Mentions: Based on these mechanistic findings, we hypothesized that disruption of AR-V:ARE interactions may represent a viable strategy for overcoming AR-V-mediated therapeutic resistance in CRPC. To develop preclinical support for this chromatin targeting concept, we evaluated JQ1, an inhibitor of the BET family of chromatin readers (49) shown to impair genome-wide chromatin binding and transcriptional activation of androgen-activated full-length AR (28). BET family chromatin readers BRD2, 3 and 4 engage with acetylated lysine residues, including H3K27Ac, which constitute chromatin marks enriched at active enhancers and associated transcription start-sites of transcriptionally active genes (50). As expected, JQ1 reduced androgen-mediated AR engagement with FASN ARBSI in R1-AD1 cells (Figure 8A). More strikingly, JQ1 completely inhibited constitutive AR-V engagement with this site in R1-D567 cells (Figure 8A). Unexpectedly, we noted loss of AR and ARv567es protein when R1-AD1 and R1-D567 cells were treated with therapeutically-relevant doses of JQ1 (28,49–51), which corresponded with down-regulated AR mRNA expression (Figure 8B and C). The CRPC C4-2, 22Rv1 and VCaP cell lines also displayed dose-dependent reductions in AR and/or AR-V expression in response to JQ1 treatment (Figure 8B and C). In the LNCaP cell line, down-regulated AR expression was observed for (+)-JQ1 but not (−)-JQ1, indicating specificity for the active stereoisomer (Figure 8D) (49).

Bottom Line: Dimerization was an absolute requirement for constitutive AR-V DNA binding and transcriptional activation.Treatment with the bromodomain and extraterminal (BET) inhibitor JQ1 resulted in inhibition of AR-V chromatin binding and impaired AR-V driven PCa cell growth in vitro and in vivo.Importantly, this was associated with a novel JQ1 action of down-regulating AR-V transcript and protein expression.

View Article: PubMed Central - PubMed

Affiliation: Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55905, USA.

Show MeSH
Related in: MedlinePlus