An integrated network of androgen receptor, polycomb, and TMPRSS2-ERG gene fusions in prostate cancer progression

Abstract

Chromosomal rearrangements fusing the androgen-regulated gene TMPRSS2 to the oncogenic ETS transcription factor ERG occur in approximately 50% of prostate cancers, but how the fusion products regulate prostate cancer remains unclear. Using chromatin immunoprecipitation coupled with massively parallel sequencing, we found that ERG disrupts androgen receptor (AR) signaling by inhibiting AR expression, binding to and inhibiting AR activity at gene-specific loci, and inducing repressive epigenetic programs via direct activation of the H3K27 methyltransferase EZH2, a Polycomb group protein. These findings provide a working model in which TMPRSS2-ERG plays a critical role in cancer progression by disrupting lineage-specific differentiation of the prostate and potentiating the EZH2-mediated dedifferentiation program.

Figure 1. Genome-wide location analysis of AR in prostate cancer

(A) Venn diagram showing overlap of the ChIP-Seq AR binding sites in the LNCaP and VCaP prostate cancer cells. (B) The consensus motif identified in the AR binding sites. De novo motif search was performed using the MEME program (Bailey and Elkan, 1994). (C) An enrichment network linking AR, TMPRSS2-ERG pathways and the polycomb-mediated de-differentiation program. AR-bound genes (purple node with black ring) were derived by ChIP-Seq in the VCaP cells and analyzed by the Molecular Concept Map in Oncomine^TM. Each node represents one molecular concept or gene set with node size proportional to the number of genes. Each edge represents a statistically significant (P < 1 × 10⁻¹⁰) overlap of genes in the two linked nodes. Molecular concepts were grouped into 5 major clusters indicated by oval rings of distinct color. See also Figure S1 and Tables S1-4.

Figure 2. ChIP-Seq analyses of ERG binding sites in prostate cancer

(A) ChIP-Seq analysis of the VCaP cells detected ERG binding to previously reported target genes (Tomlins et al., 2008). (B) Venn diagram showing overlap between the ERG binding sites identified in the VCaP cells and the RWPE+ERG cells. The RWPE cells were infected with lentivirus overexpressing ERG or lacZ and selected for stable clones. ChIP-Seq of ERG was performed in the stable RWPE+ERG cells using the RWPE+lacZ as control. (C) Distribution of AR or ERG binding sites relative to the transcriptional start sites (TSS) of all RefSeq genes. The Y-axis on the left and right represents the density of ERG (ERG BS) and AR binding sites (ARBS), respectively. See also Figure S2. (D) The consensus sequence motif identified in the ERG binding sites by the MEME program.

Figure 3. ERG and AR co-occupancy of target genes in prostate cancer

(A) Overlap between the binding sites of different regulators. Significance of overlap was measured relative to their respective overlap with the NRSF binding sites. *P<0.05, ** P<0.01, and ***P<0.001 by hypergeometric test. (B) ERG binds to a majority of the AR-bound genes. AR or ERG ChIP-Seq binding sites were each assigned to the nearest gene on the genome to derive the list of bound genes. (C) Overlap between the ERG-bound genes and the genes that are bound by both AR and ERG as determined by re-ChIP-Seq using an anti-ERG and an anti-AR antibody. (D) Representative genes co-occupied by AR and ERG. On the Y-axes are the number of reads of AR (left, in blue) and ERG (right, in red) binding sites. Above the plot are the TSS and direction denoted by arrows, exons by black box, and introns by horizontal lines, respectively. The genomic coordinates are indicated below the plot. (E) ChIP-PCR confirms AR and ERG co-occupancy on selected genes. The Y-axis on the left represents AR ChIP enrichment in VCaP cells treated with R1881 normalized to the ethanol (Ethl) treated cells. The Y-axis on the right represents ChIP enrichment (in log scale) by an anti-ERG antibody normalized to IgG. Error bars: n=3, mean ± SEM. The 3′ intronic region of the KIAA0066 gene was used as a negative control. (F) AR and ERG co-occupancy in a prostate cancer tissue. A prostate tumor tissue that expresses both AR and TMPRSS2-ERG were analyzed by ChIP-Seq. (G) Physical interaction between the AR and ERG proteins. VCaP cells were immunoprecipitated by various antibodies and immunoblotted for AR. To deplete DNA, VCaP cell lysates were pre-incubated with ethidium bromide for 30 min before immunoprecipitation. Representative experiment of 4 independent co-immunoprecipitation assessments is shown. (H) Interaction of ERG with AR in vitro via the ETS domain. HaloTag-ERG protein and mutants were generated by in vitro transcription/translation in wheat germ extracts. Recombinant GST-AR was incubated with the in vitro translated protein products and glutathione beads used in pull down assays. The interaction between AR and various domains of ERG are summarized and indicated as + or -. See also Figure S3 and Table S5.

Figure 4. ERG negatively regulates the AR gene

(A) ChIP-Seq showing AR and ERG co-binding to the regulatory regions of the AR gene. The Y-axes are as denoted as in Figure 3D. (B) ChIP-PCR confirms ERG binding to the AR promoter. (C) Ectopic ERG overexpression represses the AR transcript. VCaP and LNCaP prostate cancer cells and RWPE benign prostate epithelial cells were infected by LacZ or ERG adenovirus. QRT-PCR was used to assay the ERG and AR transcript normalized to GAPDH. Error bars: n=3, mean ± SEM. (D) ERG represses the AR protein. VCaP (in the presence or absence of androgen R1881), LNCaP, RWPE and 22RV1 cells were infected with ERG for 48hr before immunoblotting. (E) ERG knockdown de-represses the AR protein. RNA interference of ERG was done in VCaP cells in the presence or absence of androgen. See also Figure S4.

Figure 5. ERG attenuates AR transcriptional activity

(A) ChIP-Seq showing AR and ERG co-binding to the regulatory regions of representative AR target genes. The Y-axes are as denoted in Figure 3D. (B) Androgen-induced genes are significantly enriched for repression by ERG. Androgen-induced genes were obtained from microarray analysis of time-course androgen treatment of LNCaP cells. ERG-repressed genes are down-regulated in the ERG+ (n=35) relative to the ETS- (n=31) prostate tumors based on cancer gene expression microarray profiling. (C) ERG overexpression represses AR target genes. LNCaP cells were infected with ERG or lacZ for 48 hrs prior to qRT-PCR analysis. ERG, PLAT and PLAU were used as positive controls for ERG overexpression. (D) ERG knockdown de-represses androgen–induced genes. VCaP cells were hormone starved for 2 days and treated with either ethanol, synthetic androgen R1881, or R1881 with concurrent RNA interference targeting ERG for 48hrs before qRT-PCR analysis. The level of ERG knockdown is shown in Figure S4B. (E) Suppression of the KLK3 and TMPRSS2 promoters by ERG. LNCaP cells were co-transfected with various promoter reporter constructs along with pRL-TK (the internal control) at 24hrs post-infection of ERG or LacZ, incubated for another 24hrs and then monitored for luciferase activity. Error bars: n=3, mean ± SEM. See also Figure S5.

Figure 6. ERG regulates the neoplastic properties of prostate cancer cells independent of androgen signaling

(A) Ectopic ERG overexpression induces prostate cancer cell invasion in the absence of androgen. The VCaP and LNCaP prostate cancer were hormone-starved for 1 day, infected with ERG or control for another 2 days in the absence of androgen. Error bars: n=3, mean ± SEM. (B) Ectopic ERG overexpression induces prostate cancer cell invasion independent of AR. VCaP and LNCaP cells were subjected to RNA interference against AR or control for 1 day before adenovirus infection of ERG or LacZ. Error bars: n=3, mean ± SEM. (C) Ectopic ERG overexpression induces VCaP cell growth independent of androgen. VCaP cells were hormone-deprived for 48 hrs before infection with LacZ or ERG adenovirus in the presence or absence of androgen. Error bars: n=3, mean ± SEM. (D–E) Ectopic ERG overexpression induces prostate cancer cell growth independent of AR expression. VCaP and LNCaP cells were subjected to RNA interference targeting AR or control for 1 day before adenovirus infection by ERG or LacZ. Cell proliferation was assayed at 48hrs or 96hrs after infection. (F) Ectopic ERG overexpression in prostate cancer cells increases cell growth. VCaP cells were infected with lentivirus expressing ERG or GUS (control). Stable clones expressing ERG (VCaP+ERG) or control (VCaP+GUS) were plated equally, and assayed for cell proliferation in regular medium. (G) Ectopic ERG overexpression confers cell growth in the absence of AR signaling. Equal numbers of stable VCaP+ERG and control cells were hormone-deprived for 48 hrs and assayed for cell proliferation in hormone-deprived medium.

Figure 7. ERG induces EZH2–mediated epigenetic silencing

(A–B) ChIP-Seq and ChIP-PCR shows ERG binding to the EZH2 promoter in VCaP cells. (C) ERG knockdown in VCaP cells decreases EZH2 protein. (D) ERG overexpression increases EZH2 protein. LNCaP and RWPE cells were infected with ERG or LacZ adenovirus for 48hrs before immunoblot analysis. (E) EZH2 is expressed at significantly (P<0.001) higher levels in the ERG+ (n=35) than the ETS- (n=31) prostate tumors. (F) ERG overexpression in the LNCaP cells activates EZH2 and represses known EZH2 target genes. LNCaP cells were infected with LacZ or ERG adenovirus for 48 hrs and analyzed by qRT-PCR. Error bars: n=3, mean ± SEM. See also Figure S6. (G) Polycomb-related signatures and androgen-induced genes predict the status of TMPRSS2-ERG gene fusion in localized prostate tumors. The target genes of PcG proteins in embryonic stem cells were derived from a previous study (Ben-Porath et al., 2008), while the control gene signatures were taken from the ChIP-Seq experiments. Androgen-induced or–repressed genes were from microarray profiling of the LNCaP cells following androgen treatment. A random signature not related to ERG (by removing ERG-regulated genes) was used as an absolute negative control (rand_nonERG). The Polycomb signatures and the androgen-induced genes have prediction scores significantly (P<0.001) better than the other gene sets. (H) A model of TMPRSS2-ERG in prostate cancer by disrupting prostate-specific differentiation and potentiating a stem cell-like de-differentiation program. (i) androgen signaling leads to normal prostate differentiation. (ii) formation of the TMPRSS2-ERG somatic mutation. (iii) inhibition of AR expression and direct interaction with AR by the TMPRSS2-ERG fusion product. (iv) ERG binding to AR target gene loci for negative regulation. (v,viii) activation of epigenetic silencing, stem cell like state and oncogenesis. (vi) induction of EZH2. (vii) induction of H3K27 marks and epigenetic silencing of pro-differentiation genes.