A comprehensive analysis of coregulator recruitment, androgen receptor function and gene expression in prostate cancer

(A) Overview of genes included in custom Agilent oligoarray Rows, categories of genes included on 8 × 15K custom Agilent oligoarray. Columns, Number of genes identified for inclusion on the array, and number of genes for which Agilent catalogue probes were available for inclusion. (B) Overview of 452 AR target gene signature Gene name, HUGO gene symbol; FC, fold change (C) Overview of coregulators considered, prioritized and withheld for analysis A PudMed search for papers that contain the terms ‘AR’ and ‘CaP’ in their title and/or abstract was performed. Abstracts fulfilling these criteria were screened for reference to coregulator function, and if so, full-length papers were reviewed individually to verify description of a bona fide AR-associated coregulator. Left to right: Column 1: 181 coregulators for which literature search was done. Column 2: 51 coregulators for which differential protein expression has been reported in CaP when compared to benign prostate (yes entries). Column 3: 22 coregulators for which differential expression in CaP correlated with aggressive disease, and were analyzed in Figures 4–6 (yes entries). Column 4: 18 coregulators for which siRNA-mediated silencing did not affect AR expression, CaP cell morphology or CaP cell survival and were included in final analyses (yes entries).

(A) Androgen regulation of AR target gene expression in VCaP cells VCaP cells were seeded in medium supplemented with charcoal-stripped FBS (CSS). 2 days later, medium was changed and cells were treated with 5 nm R1881 or ethanol vehicle for 48 hr. Cells were harvested and AR target gene expression was evaluated using real-time RT-PCR. Target gene mRNA levels were normalized with the values obtained from GAPDH expression and are expressed as relative expression values, taking the value obtained from one of the vehicle-treated samples as 1. Columns, means of values obtained from three independent biological replicates; bars, sem. Note the consistency of androgen regulation of majority (7/8) of AR target genes obtained from LNCaP cells also in VCaP cells. (B) Kinetics of androgen regulation of AR target gene expression LNCaP cells were seeded in medium supplemented with charcoal-stripped FBS (CSS). 2 days later, medium was changed and cells were with 5 nm R1881 or ethanol for 4 or 8 hr. Timepoints were chosen as androgen regulation of androgen-induced AR target genes is detectable at 4 hr, and androgen regulation of androgen-repressed AR target genes becomes apparent at 8 hr. Target gene mRNA levels were normalized with the values obtained from GAPDH expression and are expressed as relative expression values, taking the value obtained from one of the vehicle-treated samples at the 4 hr time point as 1. Columns, means of values obtained from three independent biological replicates; bars, sem values. Note the consistency in kinetics of androgen regulation of newly recognized AR target genes (bottom 2 rows) with that of the well-known ARE-driven genes PSA, SCAP, FN1 and SERPINB5 (top row). (C) Representative real-time RT-PCR validation of Agilent oligoarray data Side-by-side comparison of Agilent oligoarray data and real-time RT-PCR data using same RNA samples. Expression data shown are derived from genes for which Agilent oligoarray expression was low (Y axis values, top row), and thus likely less reliable.

(A) Real-time RT-PCR validation of siRNA-mediated coregulator knock-out efficiency LNCaP cells were transfected with On Target siRNA SmartPools directed against 22 coregulators, against AR, or with non-targeting On Target Plus control SmartPool. At 96 hr after transfection, cells were harvested and RNA was extracted for real-time RT-PCR analysis. Target gene mRNA levels were normalized to GAPDH expression and are expressed as relative expression, taking the value obtained from one of the control siRNA-transfected conditions as 1. Columns, means of values obtained from three independent biological replicates; bars, sem. Gray bars, control siRNA-transfected cells; Black bars, specific siRNA-transfected cells (B) Morphology of LNCaP cells following coregulator silencing LNCaP cells were transfected with individual On Target siRNA SmartPools directed against each of the 22 coregulators, against AR, or a non-targeting On Target Plus control SmartPool. At 96 hr after transfection, cells were imaged using Infinity Analyze software. (C) Effects of coregulator silencing on AR protein expression levels LNCaP cells were transfected with individual On Target siRNA SmartPools directed against each of the 22 coregulators or against AR, or with non-targeting On Target Plus control SmartPools (c). At 96 hr after transfection, cells were harvested and total protein extracts were subjected to western blot analysis using an antibody directed against AR. To control for potential loading differences, blots were stripped and reprobed with an antibody targeted at β-actin.

(A) Nomenclature used to describe the effect of loss of coregulator on androgen regulation of AR target gene expression co+, androgen regulation is more pronounced (+) after loss of coregulator expression and the direction of androgen regulation remains consistent (co); co-, androgen regulation is less pronounced (-) after loss of coregulator expression and the direction of androgen regulation remains consistent (co); op+, androgen regulation is more pronounced (+) after loss of coregulator expression and the direction of androgen regulation is opposite (op); op-, as androgen regulation is less pronounced (-) after loss of coregulator expression and direction of androgen regulation is opposite (op). Androgen-induced, gene which expression is increased in control siRNA (c)-transfected cells after androgen stimulation; androgen-repressed gene which expression is decreased in control siRNA (c)-transfected cells after androgen stimulation. Spec, specific for one coregulator (B) Overview of coregulators that affect androgen regulation of the genes encoding RAB27A and GNB4 (C) Experimental validation of recruitment of 6 representative coregulators to the genes encoding RAB27A and GNB4 ChIP studies verifying recruitment of 6 coregulators at AREs within the genes encoding RAB27A and GNB4. ChIP was done on LNCaP cells that had been treated with 5nM R1881 or vehicle for 16 hr. Coregulators analyzed via ChIP were chosen based on availability of suitable antibodies. (D) Time course of androgen-dependent recruitment of AR (left), NCOA3 (middle) and WDR77 (right) to AREs in the genes encoding RAB27A and GNB4 ChIP was done on LNCaP cells that had been treated with 5nM R1881 or vehicle for 1, 4, 16 or 48 hr. (E) Impact of knock-down of WDR77 or NCOA3 on kinetics of androgen-regulation of RAB27A and GNB4 LNCaP cells were transfected with On Target siRNA SmartPools directed against WDR77 or NCOA3, or with non-targeting On Target Plus control SmartPool (c). At 42 hr after transfection, cells were treated with 5nM R1881 (+) or vehicle (-). After 1, 4, 16 or 48 hr, cells were harvested and RNA was extracted for real-time RT-PCR analysis. Target gene mRNA levels were normalized to GAPDH expression and are expressed as relative expression, taking the value obtained from one of the control siRNA-transfected conditions for each siRNA group at each time point as 1. Columns, means of values obtained from three independent biological replicates; bars, sem. Gray bars, control siRNA-transfected cells; Black bars, specific siRNA-transfected cells. (Top left) Effects of WDR77 (top) and NCOA3 (bottom) silencing on GNB4 mRNA expression; (Top right) Effects of WDR77 (top) and NCOA3 (bottom) silencing on RAB27A mRNA expression: (Bottom panels) verification of WDR77 siRNA-mediated silencing (left), verification of NCOA3 siRNA-mediated silencing (right).

(A) Co-immunoprecipitation assay shows interaction between IRF1 and STAT3 in LNCaP cells. (B) Overlap in genome-wide IRF1- and STAT3-dependent androgen-responsive gene expression. Results reflect the effect of 48 hr treatment of LNCaP cells with 5nM R1881. R1881 or vehicle treatment was administered 42 hr after siRNA transfection. Numbers, number of genes. (C) Overlap in IPA functional annotation between the androgen-responsive genes that depend on WDR77 and p53 (from Figure 3, panel F) and the androgen-responsive genes that depend on IRF1 and STAT3 (from panel B of this figure) (p<2.2.E-16). Genes subjected to IPA are those listed in the center of Venn diagrams shown in panel F of Figure 3 and panel B of this figure. Numbers, number of functional annotations.

n = number of CaP clinical specimens for which DNA sequencing or copy number variation data is available. Numbers in columns indicate the percentage of CaP cases in each study for which somatic mutations and/or copy number variations that affect corresponding coregulator have been reported.