Modulators of Prostate Cancer Cell Proliferation and Viability Identified by Short-Hairpin RNA Library Screening
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ABSTRACT: Recovery of hairpins targeting a known prostate cancer pathway testing the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets. Two cell lines assayed for 4 time points with a control and 2 experimental conditions. Two to four replicates of each instance are provided.
Project description:Prostate cancer is the most common cancer in men and AR downstream signalings promote prostate cancer cell proliferation. To investigate the AR signaling, we performed RNA sequence analysis in AR positive prostate cancer cell line, LNCaP. In addition, we used hormone-refractory prostate cancer model cells, Bicalutamide-resistant (BicR) to explore the differences of androgen signaling in prostate cancer progression. Short RNA sequence analysis of androgen-regulated miRNAs in two prostate cancer cells
Project description:Castrate-resistant prostate cancer (CRPC) is poorly characterized and heterogeneous and while the androgen receptor (AR) is of singular importance in early prostate cancer, other factors such as c-Myc and the E2F family also play a role in later stage disease. Hes6 is a transcription co-factor that has been associated with neurogenesis during gastrulation, a neuroendocrine phenotype in the prostate and metastasis in breast cancer but its role in prostate cancer remains uncertain. Here we show that Hes6 is controlled by c-Myc and AR and drives castration resistance in prostate cancer. Hes6 activates a cell-cycle enhancing transcriptional network that maintains tumour growth and nuclear AR localization in castrate conditions. We show aphysical interaction between E2F1 and both Hes6 and AR, and suggest a co-dependency of these transcription factors in castration-resistance. In the clinical setting, we have uncovered a Hes6-associated signature that predicts poor outcome in prostate cancer, which can be pharmacologically targeted. We have therefore shown for the first time the critical role of Hes6 in the development of CRPC and identified its potential in patient specific therapeutic strategies. This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
Project description:Study aimed at evaluating differences in Du145, PC3 and LNCaP human prostate cancer cells treated with ATX-101 and docetaxel for 24h.
Project description:Androgen Receptor (AR) is essential for the growth and progression of prostate cancer in both hormone-sensitive and hormone-refractory disease. We have designed a sequence-specific DNA binding polyamide (1) that targets the consensus androgen response element (ARE). This polyamide binds the PSA promoter ARE, inhibits androgen-induced expression of PSA and several other AR-regulated genes in cultured prostate cancer cells, and reduces AR occupancy at the PSA promoter and enhancer. Down-regulation of PSA by this polyamide was comparable to that produced by the synthetic anti-androgen bicalutamide (Casodex) at the same concentration. Genome-wide expression analysis reveals that a similar number of transcripts are affected by treatment with the polyamide and with bicalutamide. Direct inhibition of AR-DNA binding by sequence-specific DNA binding small molecules could offer an alternative approach to antagonizing AR activity. A polyamide (2) that targets a different DNA sequence is included as a control. Experiment Overall Design: DHT (dihydrotestosterone)-stimulated LNCaP cells that were treatment with polyamide 1, polyamide 2, bicalutamide were compared to control cells that were also DHT-stimulated. Cells not stimulated with DHT were also compared to the DHT-stimulated controls. Three biological replicates were included for each treatment/condition except the no-DHT induced controls, which were in biological duplicate.
Project description:Following androgen ablation treatment for advanced prostate cancer, almost all men relapse after a period of initial response to therapy, which eventually is life threatening. We have previously found that purine-rich element binding protein, PUR alpha, was significantly repressed in androgen-independent prostate cancer cell lines in comparison to an androgen-dependent line. Moreover, over-expressing PURa in androgen-independent prostate cancer cells attenuated their cell proliferation. The aim of the studies described here was to uncover some of the mechanisms by which over-expression of PURa attenuates cell proliferation. A set of common genes induced by over-expressing PURa both in PC3 and LNCaP cells was analyzed by DNA microarray. The results were then validated utilizing quantitative reverse transcription-PCR. Using a 5.3-kb region of the PSA promoter containing androgen response elements, the participation of PURa in androgen regulated gene expression was determined. Genes involved in stress response and cell differentiation were induced in cells over-expressing PURa. Some of the genes that are targets of androgen regulation are also induced. Most strikingly, ectopic expression of PURa induced transcriptional activity of the 5.3-kb PSA promoter containing androgen response elements, without androgen stimulation. Based upon the consideration that some of the genes involved in cell stress and differentiation are also regulated by androgens our data suggest that PURa shares some common pathway regulated by the androgen receptor. These findings suggest that regulation of PURa expression in prostate cancer cells may serve as a therapeutic target for hormone refractory prostate cancer. Experiment Overall Design: PC3 cells (low in PURa expression) was stably over-expressed PURa. Total RNA of the cells was collected simultaneously with that of PC3 transfected with mock vector. Experiment Overall Design: LNCaP cells (Express PURa more than PC3 cells) was PURa-over-expressed with retrovirus vector (MSCV-PIG) and selected with puromycin for 72hr. Total RNA of the cells was collected simultaneously with that of LNCaP cells infected with mock vector. Experiment Overall Design: One set of RNA derived from PC3 cells and two independent RNA derived from LNCaP cells retrovirus infection were used for DNA microarray. Quality and concentration of total RNA was verified using Agilent Bioanalyzer and Nanodrop Spectrophotometer, respectively. T7 oligo(dT) primer (Sigma-Proligo, Boulder, CO) and 5ug total RNA are combined for first strand cDNA synthesis. Following second strand cDNA synthesis and cDNA cleanup (Phase Lock Gel Light, Eppendorf, Hamburg, Germany) an in vitro transcription reaction was performed overnight using T7 RNA transcript labeling kit provided by Enzo Life Sciences, Inc. (Farmingdale, NY). IVT reactions were cleaned using RNeasy Mini Kit (Qiagen) and concentration determined by Nanodrop Spectrophotometer. 15ug cRNA was fragmented using 5X fragmentation buffer (made in-house). Hybridization cocktail was made in accordance with Affymetrix eukaryotic expression array protocol (Affymetrix, Santa Clara, CA) and combined with fragmented cRNA. 10ug cRNA was loaded onto Human U133Plus 2.0 genome arrays (Affymetrix, Santa Clara, CA) and hybridized overnight for 16 hours. After hybridization, staining and washing of the arrays was performed following the Affymetrix eukaryotic expression array protocol, including staining with streptavidin-phycoerythrin, antibody stain, and a second streptavidin-phycoerythrin stain. After washing and staining, all arrays were scanned with the Affymetrix GeneChip Scanner and data collected with GeneChip Operating System 1.4 (GCOS, Affymetrix, Santa Clara, CA). Experiment Overall Design: The quality of the microarray experiments was assessed with affyPLM and Affy, two Bioconductor packages for statistical analysis of microarray data. To estimate the gene expression signals, data analysis was conducted on the chipsâ CEL file probe signal values at the Affymetrix probe pair (perfect match (PM) probe and mismatch (MM) probe) level, using the statistical algorithm Robust Multiarray Analysis (RMA) expression measure (Irizarry RA et al. 2003) with Affy. This probe level data processing includes a normalization procedure utilizing the quantile normalization method (Bolstad BM et al. 2003) to reduce the obscuring variation between microarrays, which might be introduced during the processes of sample preparation, manufacture, fluorescence labeling, hybridization and/or scanning. Exploratory data analysis (EDA) was performed with the preprocessed data above. Between-treatment and between-replicate variations were examined with the pair-wise MvA plots, in which the base 2 log ratios (M) between two samples are plotted against their averaged base 2 log signals (A). With the signal estimates, Principal Component analysis (PCA) was also performed to assess sample variability. Experiment Overall Design: With the signal intensities obtained above, an empirical Bayes method with the Gamma-Gamma modeling, as implemented in the bioconductor package EBarrays, was used to estimate the posterior probabilities of the differential expression of genes between PURA and Vector only sample conditions (Newton MA et al. 2001, Kendziorski Cm et al. 2003, Newton MA et al. 2004). The criterion of the posterior probability > 0.5, which means the posterior odds favoring change, was used to produce the differentially expressed gene list. Experiment Overall Design: All Bioconductor packages are available at http://www.bioconductor.org and all computation was performed under R environment (http://www.r-project.org).
Project description:Analysis of genes regulated by miR-23b/-27b overexpression in aggressive PC3-ML cells, confirmed by antagomiR inhibition of miR-23b and miR-27b in the relatively indolent cell line LNCaP. Genes that were downregulated in PC3-ML overexpression and upregulated with LNCaP inhibition were further explored as downstream targets of miR-23b/-27b. PC3-Ml cells were transduced with miR-23b/-27b or a scrambled miRNA control, and only cells expressing greater than 95% transduction efficiency were used for array. LNCaP cells were transfected with antagomiRs to miR-23b and miR-27b, or a non-coding control.
Project description:The epidemiologic association between statin use and decreased risk of advanced prostate cancer suggests that statins may inhibit prostate cancer development and/or progression. Studies were performed to determine the effects of a model statin, atorvastatin (ATO), on the proliferation and differentiation of prostate cancer cells, and to identify possible mechanisms of ATO action. ATO inhibited the in vitro proliferation of both LNCaP and PC3 human prostate cancer cells in dose-dependent fashion. The greater inhibitory activity of ATO in PC3 cells was associated with induction of autophagy in that cell line, as demonstrated by increased expression of LC3-II. miR-182 was consistently upregulated by ATO in PC3 cells, but not in LNCaP cells. ATO upregulation of miR-182 in PC3 cells was p53-independent and was reversed by geranylgeraniol. Transfection of miR-182 inhibitors decreased expression of miR-182 by >98% and attenuated the antiproliferative activity of ATO. miR-182 expression in PC3 cells was also increased in response to stress induced by serum withdrawal, suggesting that miR-182 upregulation can occur due to nutritional stress. Bcl2 and p21 were identified to be potential target genes of miR-182 in PC3 cells. Bcl2 was downregulated and p21 was upregulated in PC3 cells exposed to ATO. These data suggest that miR-182 may be a stress-responsive miRNA that mediates ATO action in prostate cancer cells. Gene and miRNA expression in two prostate cancer cell lines treated with Atorvastatin vs. untreated control.
Project description:The epidemiologic association between statin use and decreased risk of advanced prostate cancer suggests that statins may inhibit prostate cancer development and/or progression. Studies were performed to determine the effects of a model statin, atorvastatin (ATO), on the proliferation and differentiation of prostate cancer cells, and to identify possible mechanisms of ATO action. ATO inhibited the in vitro proliferation of both LNCaP and PC3 human prostate cancer cells in dose-dependent fashion. The greater inhibitory activity of ATO in PC3 cells was associated with induction of autophagy in that cell line, as demonstrated by increased expression of LC3-II. miR-182 was consistently upregulated by ATO in PC3 cells, but not in LNCaP cells. ATO upregulation of miR-182 in PC3 cells was p53-independent and was reversed by geranylgeraniol. Transfection of miR-182 inhibitors decreased expression of miR-182 by >98% and attenuated the antiproliferative activity of ATO. miR-182 expression in PC3 cells was also increased in response to stress induced by serum withdrawal, suggesting that miR-182 upregulation can occur due to nutritional stress. Bcl2 and p21 were identified to be potential target genes of miR-182 in PC3 cells. Bcl2 was downregulated and p21 was upregulated in PC3 cells exposed to ATO. These data suggest that miR-182 may be a stress-responsive miRNA that mediates ATO action in prostate cancer cells. Gene and miRNA expression in two prostate cancer cell lines treated with Atorvastatin vs. untreated control.