A genome-wide RNA interference screen identifies new regulators of androgen receptor function in prostate cancer cells

Abstract

The androgen receptor (AR) is a mediator of both androgen-dependent and castration-resistant prostate cancers. Identification of cellular factors affecting AR transcriptional activity could in principle yield new targets that reduce AR activity and combat prostate cancer, yet a comprehensive analysis of the genes required for AR-dependent transcriptional activity has not been determined. Using an unbiased genetic approach that takes advantage of the evolutionary conservation of AR signaling, we have conducted a genome-wide RNAi screen in Drosophila cells for genes required for AR transcriptional activity and applied the results to human prostate cancer cells. We identified 45 AR-regulators, which include known pathway components and genes with functions not previously linked to AR regulation, such as HIPK2 (a protein kinase) and MED19 (a subunit of the Mediator complex). Depletion of HIPK2 and MED19 in human prostate cancer cells decreased AR target gene expression and, importantly, reduced the proliferation of androgen-dependent and castration-resistant prostate cancer cells. We also systematically analyzed additional Mediator subunits and uncovered a small subset of Mediator subunits that interpret AR signaling and affect AR-dependent transcription and prostate cancer cell proliferation. Importantly, targeting of HIPK2 by an FDA-approved kinase inhibitor phenocopied the effect of depletion by RNAi and reduced the growth of AR-positive, but not AR-negative, treatment-resistant prostate cancer cells. Thus, our screen has yielded new AR regulators including drugable targets that reduce the proliferation of castration-resistant prostate cancer cells.

Figure 1.

Effect of the AR regulators on AR-dependent transcriptional activity and cell proliferation in human prostate cancer cells. (A) LNCaP cells stably expressing an AR-responsive probasin-luciferase reporter gene were transfected with siRNAs against the indicated factors or a scrambled, nonsilencing siRNA (control), and after 48 h, were treated with 10 nM R1881 for 24 h. Luciferase activity was measured, normalized to protein, and presented as relative luminescence units (RLUs). (B) The efficiency of knockdown of each factor was determined at the mRNA level relative to RPL19 and shown as relative mRNA expression. (White bars) Nonsilencing siRNA; (black bars) the indicated siRNAs. (C) LNCaP-abl cells were transfected with siRNAs against the indicated factor or a scrambled siRNA for the control cells, and cell proliferation was measured after 7 d. Data are represented as relative fluorescence units (RFUs). (D) The extent of knockdown was determined and represented as in B, and relative mRNA expression is shown. The experiment was performed in triplicate with error bars representing the standard deviation.

Figure 2.

MED19 depletion affects AR target genes expression. MED19 knockdown decreases the expression of AR target genes in LNCaP (A) and LNCaP-abl (B) cells. Cells were transfected with control (siControl) or MED19 siRNA (siMED19), and either androgen deprived for 48 h and then treated with 10 nM R1881 for 24 h (LNCaP) or cultured in media with 10% charcoal-stripped FBS for 48 h (LNCaP-abl). Relative mRNA levels of the indicated genes were analyzed by qPCR and normalized to RPL19. Each assay was performed in duplicate, with error bars representing the range of the mean. (C–F) MED19 selectively regulates AR transcriptional activity. LNCaP-abl cells were transfected with control siRNA (siControl) or siRNA against MED19 (siMED19) together with (C) a plasmid expressing the Gal4 DNA-binding domain fused to the VP16 activation domain, along with a luciferase reporter gene driven by five Gal4-binding sites upstream of the E1b promoter. (D) A plasmid containing the human GR and a GR-responsive luciferase reporter, treated with 100 nM dexamethasone. (E) An AR-responsive luciferase reporter treated with 10 nM R1881. Luciferase activity was measured, normalized to protein, and presented as RLU. (F) The efficiency of MED19 knockdown was monitored at the mRNA level. Each assay was performed in triplicate, with error bars representing the standard deviation. The experiment was repeated twice with similar results.

Figure 3.

Mediator subunits differentially affect AR transcriptional activity and prostate cancer cell proliferation. (A) LNCaP cells stably expressing an AR-responsive probasin-luciferase reporter gene were transfected with siRNAs against the indicated Mediator complex subunits or a scrambled, nonsilencing siRNA (control) and, after 48 h, were treated with 10 nM R1881 for 24 h. Luciferase activity was measured and normalized to protein and presented as relative luminescence units (RLUs). (B) The efficiency of knockdown of each factor was determined at the mRNA level relative to RPL19 and shown as relative mRNA expression. (White bars) Nonsilencing siRNA; (black bars) the indicated siRNAs. (C) LNCaP-abl cells were transfected with siRNAs against the indicated Mediator complex subunits or a scrambled, nonsilencing siRNA (control), and cell proliferation was measured after 7 d as in Figure 1. Data are shown as relative fluorescence units (RFUs). (D) Efficiency of knockdown at the mRNA level is shown relative to RPL19. (White bars) Nonsilencing siRNA; (black bars) the indicated siRNAs. Each assay was performed in triplicate, with error bars representing the standard deviation.

Figure 4.

HIPK2 depletion affects AR target gene expression. HIPK2 knockdown decreases the expression of AR target genes in LNCaP (A) and LNCaP-abl (B) cells. Cells were transfected with control (siControl) or HIPK2 siRNA (siHIPK2) and either androgen deprived for 48 h and then treated with 10 nM R1881 for 24 h (LNCaP) or cultured in media with 10% charcoal-stripped FBS for 48 h (LNCaP-abl). Relative mRNA levels of the indicated genes were analyzed by qPCR and normalized to RPL19. Each assay was performed in duplicate, with error bars representing the range of the mean.

Figure 5.

Effects of MED19 and HIPK2 depletion on gene expression in LNCaP-abl cells. (A) Depiction of the procedure used to examine MED19 and HIPK2-responsive genes. (B) Number of genes up-regulated and down-regulated by >1.5-fold upon MED19 and HIPK2 depletion in LNCaP-abl cells. (C) Numbers of genes in MED19 and HIPK2 siRNA-depleted LNCaP-abl cells that overlap with siRNA knockdown of AR in LNCaP-abl cells. (D) Microarray heat map of changes in gene expression in LNCaP-abl cells depleted of HIPK2, MED19, AR, and parental LNCaP cells depleted of AR siRNA. (E) Gene Ontology analysis of the genes affected by siRNA depletion of the indicated factor in LNCaP-abl cells. (*) The data sets for LNCaP siAR, LNCaP-abl siAR were taken from Gene Expression Omnibus accession numbers GSE7868 and GSE11428 (Wang et al. 2009).

Figure 6.

HIPK2 and MED19 depletion selectively affects the proliferation of AR-expressing prostate cancer cells. AR-positive prostate cancer cell lines (A) LNCaP and (B) LNCaP-abl and AR-negative (C) HEK293 and (D) PC3 cells were transfected with control siRNA (siControl), HIPK2 siRNA (siHIPK2), or MED19 siRNA (siMED19), and cell proliferation was measured at the indicated days and shown as relative fluorescence units (RFUs). (E–H) The efficiency of knockdown was monitored at the mRNA level for each factor in the four cell lines relative to RLP19. Each experiment was repeated at least two times. A representative experiment is shown.

Figure 7.

A kinase inhibitor to HIPK2 recapitulates the effects of HIPK2 depletion on prostate cancer cell proliferation and AR target genes expression. (A) LNCaP-abl, LNCaP, PC3, and HEK293 cells were treated with 5 μM BAY 43-9006, and cell proliferation was measured after 4 d. Data are shown as relative fluorescence units (RFUs). (B) LNCaP-abl cells were treated with 5 μM BAY 43-9006 or vehicle (DMSO) in the presence (siControl) or absence of HIPK2 (siHIPK2), and the mRNA levels of AR target genes FKBP5, CDK1, and UBEC2 were measured by qPCR relative to RPL19. (C) Impact of BAY 43-9006 treatment on LNCaP-abl cell proliferation as a function of HIPK2. LNCaP-abl cells were depleted of HIPK2 as in B, and cell proliferation was measured after 7 d in the absence (DMSO) and presence of 5 μM BAY 43-9006. Each experiment was performed at least two times with similar results.