Development of a small molecule for treatment of castration resistant prostate cancer via androgen receptor and IL6/STAT3 pathways
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Anne Margrete Oyan
PROVIDER: E-MTAB-5102 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Combining different clinical agents to target multiple pathways in prostate cancer cells, including androgen receptor (AR) signaling, is potentially an effective strategy to improve outcomes for men with metastatic disease. We have previously demonstrated that sub-effective concentrations of an AR antagonist, bicalutamide, a histone deacetylase inhibitor, vorinostat (SAHA), and a hsp90 inhibitor, 17-AAG, act synergistically when combined to cause death of AR-dependent prostate cancer cells. In this study, expression profiling of human prostate cancer cells treated with bicalutamide, vorinostat (SAHA) or 17-AAG, alone or in paired combination, was employed to determine the molecular mechanisms underlying these synergistic interactions. We used microarray analysis to determine the global molecular profile contributing to the synergistic cell death in LNCaP human prostate cancer cells caused by combinations of bicalutamide, vorinostat (SAHA), or 17-AAG. LNCaP human prostate cancer cells were treated for 6 hours with drug treatments as follows: vehicle control, 5 uM bicalutamide, 1 uM vorinostat (SAHA), 40 nM 17-AAG, 5 uM bicalutamide + 40 nM 17-AAG, 40 nM 17-AAG + 1 uM vorinostat (SAHA), or 5 uM bicalutamide + 1 uM vorinostat (SAHA). Each treatment was performed in sextuplicate.
2014-03-27 | E-GEOD-56188 | biostudies-arrayexpress
Project description:Genome wide expression changes following 50uM Casodex treatment were investigated to determine regulatory targets commonly overlooked in gene function specific microarrays and for comparison of the effects of the mutant androgen receptor (AR) LNCaP cell line against the wild-type AR expressing PC-346C cells. LNCaP Prostate Cancer cells were treated for a period of 48h with or without 50uM Casodex following a 24h seeding period. At the selected time point, total RNA was harvested from the cells for hybridization and analysis by Nimblgen Systems Inc using the homo sapiens gene expression array.
2007-09-15 | E-GEOD-8533 | biostudies-arrayexpress
Project description:The androgen receptor (AR) mediates the action of androgens by binding to androgen-responsive elements (AREs) and subsequently regulating target genes involved in prostate carcinogenesis. The precise locations, true nature, and functional roles of AREs in human prostate cancer are still unknown. Here we redefine AREs by motif-resolution AR chromatin immunoprecipitation-exonuclease (ChIP-exo) assay in human prostate cancer cells and tumors. Surprisingly, we find that, in addition to canonical full-length AREs and half-site-like AREs, a significant portion of the four redefined ARE categories comprises non-canonical full-length AREs. The redefined AREs in enhanced AR binding regions in prostate tumors versus paired non-malignant adjacent tissues regulate a prostate cancer-relevant gene network not only centered on AR, but more interestingly, on novel AR target genes mTOR, BIRC5 and BCL2L1 involved in prostate cancer cell growth and survival. The precise redefinition of AREs has important implications for understanding how AR contributes to prostate carcinogenesis. To examine the differential AR binding in LNCaP cells before and after androgen stimulation, ChIP-Seq of androgen receptor is performed in LNCaP cells under the two conditions. To profile histone modification status in control LNCaP cells, MNase-Seq is performed with five different antibodies specific to certain histone marks. Each experiment includes two replicates.
2015-01-01 | E-GEOD-43720 | biostudies-arrayexpress
Project description:We introduce a family of multivalent peptidomimetic conjugates that modulate the activity of the androgen receptor (AR). Bioactive ethisterone ligands were conjugated to a set of sequence-specific peptoid oligomers. Certain multivalent peptoid conjugates enhance AR-mediated transcriptional activation. We identify a linear and a cyclic conjugate that exhibit potent anti-proliferative activity in LNCaP-abl cells, a model of therapy-resistant prostate cancer. The linear conjugate blocks AR action by competing for ligand binding. In contrast, the cyclic conjugate is active despite its inability to compete against endogenous ligand for binding to AR in vitro, suggesting a non-competitive mode of action. These results establish a versatile platform to design competitive and non-competitive AR modulators with potential therapeutic significance. We use microarray analysis to further elucidate the mechanism of AR antagonism and show that the compounds (cyc and n=8) are distinct. We used microarray analysis to see genone-wide effects on LNCaP-abl cells with moduators of AR activity LNCaP-abl cells were treated with compound and RNA was extracted and hybridized on Affymetrix microarrays Treatments: Vehicle is just Ethanol treated LNCaP-abl cells that were used as a control. n=8 and cyc are peptoid conjugates that modulate AR activity. n=8 is linear and cyc is a cyclic compounds. We wanted to look at gene expression when LNCaP-abl cells were treated with these compounds to show that they are distinct. C3 is a small molecule that inhibits the interaction between AR and beta-catenin (co-regulator protein). Initial results with this compound show potent anti-proliferative activity in LNCaP-abl cells and we wanted to further investigate the mechanism of action of this compound.
2012-12-21 | E-GEOD-37796 | biostudies-arrayexpress
Project description:Androgen receptor (AR) is a transcription factor that plays a central role in the growth and development of the normal prostate and its malignant transformation. More recently, a majority of prostate cancers have been shown to harbor recurrent gene fusions of the androgen-regulated gene, TMPRSS2, to the oncogenic ETS transcription factor ERG. Here we employed chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-Seq) to explore the genome-wide localization of these transcription factors in human prostate cancer cell lines as well as tissues. Unexpectedly, transcriptional networks emanating from AR and ERG were found to be highly overlapping. Furthermore, AR was found to regulate known 5’ fusion partners in prostate cancer including TMPRSS2, as well as negatively regulating its own expression. While induced by androgen through fusion to TMPRSS2, ERG itself was shown to inhibit AR expression and positively regulate the genomic locus of wild-type ERG, thus revealing multiple levels of molecular cross-talk between AR and ERG. Importantly, androgen-sensitive prostate cancer cells in which ERG is overexpressed are able to proliferate and invade in the absence of androgen. Thus, we dissected the intertwined genomic landscape of two master transcriptional regulators of prostate cancer and suggest a role for ERG in maintaining transcriptional networks necessary for androgen-independent prostate cancer growth. These studies may suggest that future therapies against prostate cancer should target both AR and ERG, rather than AR alone, in order to achieve maximum effectiveness. ChIP_Seq examination of histone modifications and key transcription factors in LNCaP and VCaP prostate cancer cell lines in un-treated, vehicle treated or 10nM R1881 treated conditions. LNCaP ChIP-Seq experiments include samples GSM353609-GSM353618, GSM353625-GSM353628, GSM353633-GSM353635, GSM353641-GSM353644, and GSM353648. VCaP ChIP-Seq experiments include samples GSM353601-GSM353608, GSM353619-GSM353624, GSM353629-GSM353632, and GSM353645-GSM353647. In addition, we performed re-ChIP of AR and ERG in VCaP cells (GSM356767), and examined the effect of ERG knockdown on AR and ERG binding (samples GSM353636-GSM353639). To study ectopic ERG binding we performed ERG ChIP-Seq in stable RWPE+ERG or control cells (samples GSM353649-GSM353650). AR ChIP-Seq was also done in the AR-positive but ETS fusion-negative 22RV1 cells (GSM353640). To further study transcription factor binding and chromatin state we performed ChIP-Seq of AR, ERG, H3K4me3, H3K9me3, H3K27me3 and RNA Pol II in a metastatic prostate tumor tissue (samples GSM353651-GSM353656). To couple the ChIP-Seq experiments with gene expression, we have also done Illumian SAGE-tag profiling in LNCaP cells following androgen treatment for 0, 24 and 48hrs. These DGE experiments correspond to samples GSM353657-GSM353659.
2010-05-17 | E-GEOD-14092 | biostudies-arrayexpress
Project description:The Polycomb group (PcG) protein enhancer of zeste homolog 2 (EZH2) plays an essential role in histone methylation-mediated epigenetic gene silencing. We demonstrate that under physiological conditions, cyclin-dependent kinase 1 (CDK1) and 2 (CDK2) bind to and phosphorylate EZH2 at threonine 350 (T350) in an evolutionally conserved motif. T350 phosphorylation is required for EZH2 function in silencing of genes that promote differentiation and inhibit proliferation and migration. Blockage of T350 phosphorylation by generating a T350A mutation largely diminishes the global effect of EZH2 on gene silencing. Seven experiments have been designed. LNCaP cells were transfected with si-Control, si-EZH2, si-EZH2 plus RNAi resistant EZH2 plasmid, or si-EZH2 plus RNAi resistant T350A mutated EZH2 plsamid for 60 hours, or treated with DMSO, 5 uM TSA, or 10 uM Roscovitine for 24 hours. Two replicates per experiment.
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Project description:We performed androgen receptor (AR) ChIP-seq after GFP control or FOXA1 over-expression in two AR driven cancer models; LNCaP prostate cancer cell line and MDA-MB-453 molecular apocrine breast cancer cell line.
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Project description:While thousands of long non-coding RNAs (lncRNAs) are expressed in higher eukaryotes, the potential regulatory roles of lncRNAs in regulated gene transcription programs remain rather poorly understood. Here, we report that two lncRNAs highly overexpressed in aggressive prostate cancer, PRNCR1 and PCGEM1, bind successively to the androgen receptor (AR) and strongly enhance both ligand-dependent and ligand-independent AR-mediated gene activation programs and proliferation in prostate cancer cells. Binding of PRNCR1 to the C-terminally acetylated AR on enhancers and its association with DOT1L appear to be required for recruitment of the second lncRNA, PCGEM, to the N-terminally methylated AR. Unexpectedly, recognition of the H3K4me3 promoter mark by the PHD finger-domain of Pygopus2, recruited by PCGEM1, proves to enhance selective looping of AR-bound enhancers to target gene promoters in these cells, revealing a novel aspect of ligand-induced enhancer-promoter interactions. In “resistant” prostate cancer cells, these overexpressed lncRNAs can interact with, and are required for the robust activation of both truncated and full length AR, causing DHT-independent activation of the AR transcriptional program and cell proliferation. Conditionally-expressed short hairpin RNA (shRNA)-mediated targeting of these lncRNAs in these resistant cancer cell lines strongly suppressed xenograft growth in vivo. Together, these results suggest that these overexpressed lncRNAs can potentially serve as a required component of castration-resistance in prostatic tumors. Global Run On (GRO) assay followed by high throughput sequencing (GRO-seq); after knocking down PYGO2 LNCaP cells were grown to 30-50% confluence and siRNA/ASO transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Control samples were transfected with scramble ASO and control siRNA, respectively. On the following day of transfection, the cells were cultured in UltraCULTURE (Phenol red free) + 5% Charcoal Dextran Stripped (CDS) serum for 48 hours. For androgen induction, we treat cells with DHT from a 100 uM stock in 70% ethanol to a final concentration of 100 nM for 1 hour Control siRNA, -DHT Control siRNA, +DHT Pygo2 siRNA, -DHT Pygo2 siRNA, +DHT
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2011-11-16 | E-GEOD-28264 | biostudies-arrayexpress
Project description:While thousands of long non-coding RNAs (lncRNAs) are expressed in higher eukaryotes, the potential regulatory roles of lncRNAs in regulated gene transcription programs remain rather poorly understood. Here, we report that two lncRNAs highly overexpressed in aggressive prostate cancer, PRNCR1 and PCGEM1, bind successively to the androgen receptor (AR) and strongly enhance both ligand-dependent and ligand-independent AR-mediated gene activation programs and proliferation in prostate cancer cells. Binding of PRNCR1 to the C-terminally acetylated AR on enhancers and its association with DOT1L appear to be required for recruitment of the second lncRNA, PCGEM, to the N-terminally methylated AR. Unexpectedly, recognition of the H3K4me3 promoter mark by the PHD finger-domain of Pygopus2, recruited by PCGEM1, proves to enhance selective looping of AR-bound enhancers to target gene promoters in these cells, revealing a novel aspect of ligand-induced enhancer-promoter interactions. In M-bM-^@M-^\resistantM-bM-^@M-^] prostate cancer cells, these overexpressed lncRNAs can interact with, and are required for the robust activation of both truncated and full length AR, causing DHT-independent activation of the AR transcriptional program and cell proliferation. Conditionally-expressed short hairpin RNA (shRNA)-mediated targeting of these lncRNAs in these resistant cancer cell lines strongly suppressed xenograft growth in vivo. Together, these results suggest that these overexpressed lncRNAs can potentially serve as a required component of castration-resistance in prostatic tumors. Global Run On (GRO) assay followed by high throughput sequencing (GRO-seq); ChIRP-seq data for a lincRNA (PCGEM1). LNCaP cells were grown to 30-50% confluence and siRNA/ASO transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturerM-bM-^@M-^Ys instructions. Control samples were transfected with scramble ASO and control siRNA, respectively. On the following day of transfection, the cells were cultured in UltraCULTURE (Phenol red free) + 5% Charcoal Dextran Stripped (CDS) serum for 48 hours. For androgen induction, we treat cells with DHT from a 100 uM stock in 70% ethanol to a final concentration of 100 nM for 1 hour we have sequenced the DNA sequences that are associated with the presence of a RNA molecule on a genome wide scale. PCGEM1 probe, -DHT PCGEM1 probe, +DHT
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