DHT-dependent AR activity in LNCaP cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE60721 | GEO | 2016/03/15
SECONDARY ACCESSION(S): PRJNA259364
REPOSITORIES: GEO
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Project description:We previously encountered regulatory processes where dihydrotestosterone (DHT) exerted its inhibitory effect on parathyroid hormone-related protein (PTHrP) gene repression through the estrogen receptor (ER)M-NM-1, but not the androgen receptor (AR) in breast cancer MCF-7 cells. Here, we investigated whether such an aberrant ligand-nuclear receptor (NR) interaction is present in prostate cancer LNCaP cells. First, we confirmed that LNCaP cells expressed a functional AR and at negligible levels of ERM-NM-1, and progesterone receptors. Both suppression of PTHrP and activation of the PSA genes were observed after treatment of E2, DHT and R5020. Consistent with the previous notion that the AR in LNCaP cells lost the ligand specificity due to a mutation AR (Thr-Ala877), our study using siRNA targeting each NR revealed that the AR, but not the other NRs, monopolized the role as the mediator of shared hormone-dependent regulation. These results were invariably associated with nuclear translocation of this mutant AR. Microarray of the genes regulated by either DHT, E2 or R5020 downstream of the AR (Thr-Ala877) revealed that more than half genes overlapped in LNCaP cells. Noticeably, AR (wild-type, wt) and AR (Thr-Ala877) were equally responsible for the E2-AR interactions. Fluorescent microscopic experiments demonstrated that both EGFP-AR (wt) and EGFP-AR (Thr-Ala877) were exclusively localized within the nucleus after E2 or DHT treatment. Further, a promoter assay revealed that breast cancer MCF-7 and Rv22 cells also exhibited such an aberrant E2-AR (wt) signaling. We postulate entangled interactions between the AR (wt) and E2 in a certain hormone-sensitive cancer cells. Total RNAs from the LNCaP cells transfected with control siRNA (siCT) or siRNA for AR (siAR) transfected LNCaP cells before 24 hr followed by exposed to 10-7M of DHT, E2 or R5020 exposure for another 24 h, respectively, were used.
2014-06-19 | E-GEOD-58615 | biostudies-arrayexpress
Project description:We previously encountered regulatory processes where dihydrotestosterone (DHT) exerted its inhibitory effect on parathyroid hormone-related protein (PTHrP) gene repression through the estrogen receptor (ER)α, but not the androgen receptor (AR) in breast cancer MCF-7 cells. Here, we investigated whether such an aberrant ligand-nuclear receptor (NR) interaction is present in prostate cancer LNCaP cells. First, we confirmed that LNCaP cells expressed a functional AR and at negligible levels of ERα, and progesterone receptors. Both suppression of PTHrP and activation of the PSA genes were observed after treatment of E2, DHT and R5020. Consistent with the previous notion that the AR in LNCaP cells lost the ligand specificity due to a mutation AR (Thr-Ala877), our study using siRNA targeting each NR revealed that the AR, but not the other NRs, monopolized the role as the mediator of shared hormone-dependent regulation. These results were invariably associated with nuclear translocation of this mutant AR. Microarray of the genes regulated by either DHT, E2 or R5020 downstream of the AR (Thr-Ala877) revealed that more than half genes overlapped in LNCaP cells. Noticeably, AR (wild-type, wt) and AR (Thr-Ala877) were equally responsible for the E2-AR interactions. Fluorescent microscopic experiments demonstrated that both EGFP-AR (wt) and EGFP-AR (Thr-Ala877) were exclusively localized within the nucleus after E2 or DHT treatment. Further, a promoter assay revealed that breast cancer MCF-7 and Rv22 cells also exhibited such an aberrant E2-AR (wt) signaling. We postulate entangled interactions between the AR (wt) and E2 in a certain hormone-sensitive cancer cells.
2014-06-19 | GSE58615 | GEO
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Project description:The discovery that enhancers are regulated transcription units, encoding eRNAs, has raised new questions about the mechanisms of their activation. Here, we report an unexpected molecular mechanism that underlies ligand-dependent enhancer activation, based on DNA nicking to relieve torsional stress from eRNA synthesis. Using dihydrotestosterone (DHT)-induced binding of androgen receptor (AR) to prostate cancer cell enhancers as a model, we show rapid recruitment, within minutes, of DNA topoisomerase I (TOP1) to a large cohort of AR-regulated enhancers. Furthermore, we show that the DNA nicking activity of TOP1 is a prerequisite for robust eRNA synthesis and enhancer activation and is kinetically accompanied by the recruitment of ATR and the MRN complex, followed by additional components of DNA damage repair machinery to the AR-regulated enhancers. Together, our studies reveal a linkage between eRNA synthesis and ligand-dependent TOP1-mediated nicking - a strategy exerting quantitative effects on eRNA expression in regulating AR-bound enhancer-dependent transcriptional programs. Genome-wide binding analysis of AR, TOP1, MRE11 in prostate cancer cell line LNCaP with or without 5alpha-dihydrotestosterone (DHT) treatment. Nascent RNA analysis by global nuclear run-on (GRO-seq) in LNCaP cells transfected with siRNA with or without DHT treatment. Distribution of transcriptionally engaged RNA Pol II in LNCaP cells with or without DHT treatment by precision nuclear run-on and sequencing (PRO-seq).
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Project description:Enzalutamide (formerly MDV3100 and available commercially as Xtandi), a novel androgen receptor (AR) signaling inhibitor, blocks the growth of castration-resistant prostate cancer (CRPC) in cellular model systems and was shown in a clinical study to increase survival in patients with metastatic CRPC. Enzalutamide inhibits multiple steps of AR signaling: (1) binding of androgens to AR, (2) AR nuclear translocation, and (3) association of AR with DNA. Here we used Affymetrix human genome microarray technology to investigate the global programme of gene expression of LNCaP cells in response to enzalutamide alone and in the context of DHT-stimulated androgen receptor gene expression. LNCaP cells were grown in RPMI 1640 supplemented with 5% hormone depleted FBS and treated with vehicle (control sample) , DHT (100 nM), enzalutamide (1 or 10 M-BM-5M) or DHT (100 nM) plus enzalutamide (1 or 10 M-BM-5M)for 16 hours for RNA extraction and hybridization. Each condition was done in triplicate.
2013-04-01 | E-GEOD-44905 | biostudies-arrayexpress
Project description:We previously encountered regulatory processes where dihydrotestosterone (DHT) exerted its inhibitory effect on parathyroid hormone-related protein (PTHrP) gene repression through the estrogen receptor (ER)α, but not the androgen receptor (AR) in breast cancer MCF-7 cells. Here, we investigated whether such an aberrant ligand-nuclear receptor (NR) interaction is present in prostate cancer LNCaP cells. First, we confirmed that LNCaP cells expressed a functional AR and at negligible levels of ERα, and progesterone receptors. Both suppression of PTHrP and activation of the PSA genes were observed after treatment of E2, DHT and R5020. Consistent with the previous notion that the AR in LNCaP cells lost the ligand specificity due to a mutation AR (Thr-Ala877), our study using siRNA targeting each NR revealed that the AR, but not the other NRs, monopolized the role as the mediator of shared hormone-dependent regulation. These results were invariably associated with nuclear translocation of this mutant AR. Microarray of the genes regulated by either DHT, E2 or R5020 downstream of the AR (Thr-Ala877) revealed that more than half genes overlapped in LNCaP cells. Noticeably, AR (wild-type, wt) and AR (Thr-Ala877) were equally responsible for the E2-AR interactions. Fluorescent microscopic experiments demonstrated that both EGFP-AR (wt) and EGFP-AR (Thr-Ala877) were exclusively localized within the nucleus after E2 or DHT treatment. Further, a promoter assay revealed that breast cancer MCF-7 and Rv22 cells also exhibited such an aberrant E2-AR (wt) signaling. We postulate entangled interactions between the AR (wt) and E2 in a certain hormone-sensitive cancer cells. Total RNAs from the LNCaP cells transfected with control siRNA (siCT) or siRNA for AR (siAR) transfected LNCaP cells before 24 hr followed by exposed to 10-7M of DHT, E2 or R5020 exposure for another 24 h, respectively, were used.
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Project description:The major pioneer factor activity of FOXA1 in PCa is to facilitate AR recruitment to androgen-regulated enhancers. Therefore, we hypothesized that the decreased FOXA1 binding and enhancer availability by LSD1 inhibition may result in the impairment of subsequent AR recruitment to enhancers. To globally test this hypothesis, we performed AR ChIP-seq in LNCaP cells treated with an LSD1 inhibitors. Consistent with previous reports, DHT treatment can dramatically induce AR binding to chromatin. Significantly, LSD1 inhibitor treatment in presence of DHT stimulation markedly decreased the total number of AR binding peaks and their intensity. We further assessed the impact of LSD1 inhibition on overall AR transcriptional output using RNA-seq data.
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