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. Author manuscript; available in PMC: 2016 Nov 25.
Published in final edited form as: Cell Rep. 2016 Oct 18;17(4):966–976. doi: 10.1016/j.celrep.2016.09.064

Androgen Receptor Tumor Suppressor Function Mediated by Recruitment of Retinoblastoma Protein

Shuai Gao 1,2, Yanfei Gao 2,3, Housheng Hansen He 4, Dong Han 1, Wanting Han 1, Amy Avery 1, Jill A Macoska 1, Xiaming Liu 5, Sen Chen 2, Fen Ma 2, Shaoyong Chen 2, Steven P Balk 2, Changmeng Cai 1,2
PMCID: PMC5123835  NIHMSID: NIHMS822122  PMID: 27760327

SUMMARY

Although well characterized as a transcriptional activator that drives prostate cancer (PCa) growth, androgen receptor (AR) can function as a transcriptional repressor, and high-level androgens can suppress PCa proliferation. The molecular basis for this repression activity remains to be determined. Genes required for DNA replication are highly enriched amongst androgen-repressed genes, and AR is recruited to the majority of these genes, where it rapidly represses their transcription. This activity is enhanced in PCa cells expressing high AR levels and is mediated by recruitment of hypophosphorylated retinoblastoma protein (Rb). Significantly, AR also indirectly increases expression of DNA replication genes through stimulatory effects on other metabolic genes, with subsequent CDK activation and Rb hyperphosphorylation. In castration-resistant PCa cells, which are dependent on high-level AR expression, this anti-proliferative repression function might be exploited through treatment with androgen in combination with agents that suppress AR driven metabolic functions or cell cycle progression.

Keywords: prostate cancer, androgen receptor, AR, androgen deprivation therapy, transcriptional repression, DNA replication, DNA damage repair, metabolism, Retinoblastoma protein, Rb, E2F1

Graphical abstract

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INTRODUCTION

Androgen receptor (AR) is essential for normal prostate differentiation, but in prostate cancer (PCa) plays a pivotal role in tumor growth. Most patients with metastatic PCa respond to androgen deprivation therapy (ADT), but they generally relapse within several years despite castrate androgen levels (castration-resistant prostate cancer, CRPC) (Green et al., 2012; Yuan et al., 2014). Residual androgens contribute to persistent AR activity in these relapsed tumors, and many patients with CRPC will respond to therapies that further suppress androgen synthesis or to new AR antagonists (de Bono et al., 2011; Scher et al., 2012). However, these patients generally relapse within 1–2 years and AR activity is again restored in many tumors. Therefore, there continues to be a pressing need for new strategies directed at AR in PCa.

AR has been extensively characterized as a transcriptional activator, but it can also function as a transcriptional repressor (Cai et al., 2011; Grosse et al., 2012; Zhao et al., 2012). Directly AR repressed genes include AR and AKR1C3 (an androgen synthetic gene), revealing a negative feedback mechanism for regulating AR signaling (Cai et al., 2011; Stanbrough et al., 2006). This repression is dependent on LSD1, which can demethylate enhancer-associated mono- or di-methylated H3K4 (H3K4me1,2) (Shi et al., 2004). Significantly, high levels of androgens can suppress proliferation in some models, including VCaP cells (a CRPC cell line with AR gene amplification). Our previous pathway analyses of androgen-repressed genes in VCaP showed marked enrichment for genes involved in proliferation, while AR stimulated genes were associated with cellular metabolism (Cai et al., 2011). Overall these findings are consistent with the role of AR in normal prostate epithelium being to suppress growth and drive differentiation, and suggest that these growth inhibitory functions of AR are overcome during PCa development. However, the molecular mechanisms that contribute to androgen-mediated repression of proliferation remain to be established.

In this study we used AR ChIP-seq and transcriptome profiling to identify genes that are directly repressed by AR. In VCaP cells, the androgen-repressed genes with linked AR binding sites were highly enriched for DNA replication. Mechanistically, we found this activity was mediated by androgen-stimulated recruitment of hypophosphorylated retinoblastoma protein (Rb) to AR binding sites, and was enhanced in PCa cells expressing higher level of AR. As androgens at intermediate levels can stimulate rather than impair cell cycle in the LNCaP PCa cell line, we further explored AR regulation of these genes in LNCaP. Although the expression of DNA replication genes was increased after 24h (hours) of androgen stimulation, the initial and direct effect of AR was similarly to suppress their transcription. The later increased transcription of these genes in LNCaP cells was associated with AR activation of metabolic genes and subsequent cyclin-dependent kinase (CDK) activation and Rb hyperphosphorylation. These findings demonstrate that AR has an Rb-dependent anti-proliferative function, and suggest this function may be therapeutically enhanced by combination treatments that stimulate AR while repressing its growth-promoting functions.

RESULTS

Global assessment of genes directly regulated by AR

We and others reported previously that treatment with androgen (dihydrotestosterone, DHT) could directly repress the expression of genes including AR, AKR1C3, and OPRK1 (a neuroendocrine-related gene) in PCa cells (Cai et al., 2011; Zhao et al., 2012). To further identify genes directly regulated by AR, we carried out AR ChIP-seq in VCaP cells treated with 10nM DHT for 4h. This yielded 12,210 high confidence binding peaks using a stringent threshold (p-value<10−15) (Figure S1A). We next identified genes with AR binding sites within 20 or 50 Kb of their transcriptional start sites (TSS), or within gene bodies, whose mRNA levels were altered by 24h DHT treatment (2-fold cutoff) in VCaP cells. By each criterion we found enrichment for AR binding sites and binding motifs in association with androgen-stimulated genes (Figure S1B and Table S1). There was less enrichment for AR binding sites in association with androgen-repressed genes. This may reflect weaker AR binding to a subset of androgen-repressed genes, as the average peak intensity in those gene loci was generally weaker than in androgen-stimulated genes (although the difference is not statistically significant) (Figure S1C). This lower enrichment also could reflect indirect AR binding to some of these sites or repression by other indirect mechanisms (Curtin et al., 2001; Shah et al., 2003).

Genes directly repressed by AR regulate DNA replication

Gene Ontology (GO) analysis of androgen-stimulated genes with associated AR binding sites in VCaP cells showed enrichment for lipid/sterol metabolism pathways, consistent with previous findings (Figure 1A and S1D) (Cai et al., 2011; Massie et al., 2011; Xu et al., 2006). In contrast, analysis of androgen-repressed genes with AR binding sites revealed enrichment for genes involved in DNA replication (Figure 1B and S1E). Since average intensity of AR binding was generally weaker at AR-repressed genes (see Figure S1C), we reanalyzed the data using less stringent thresholds (p-value<10−5 for peak calling and 1.5-fold cutoff for gene expression) to identify androgen-repressed genes that have AR binding sites within 20 Kb upstream of TSS or gene bodies. The GO analysis showed similar results as using more stringent parameters (not shown), and this analysis with less stringent criteria identified 89 androgen-repressed genes involved in DNA replication. The majority had linked AR binding sites (77/89), consistent with direct repression by AR (see Table S1).

Figure 1. Characterization of AR transcriptional repression activity.

Figure 1

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(A and B) VCaP cells in androgen-deprived medium were treated with 10nM DHT for 4h, followed by AR ChIP-Seq. Peaks were identified using MACS (p-Value<10−15). GO analyses of (A) AR-activated versus (B) -repressed genes (based on binding within 20 Kb of TSS). (C) qRT-PCR of indicated genes in VCaP cells treated with 10nM DHT for 0–24h. (D) ChIP analyses for AR, p-Pol2, and H3K4me2 at the indicated loci in VCaP cells treated with 10nM DHT for 4h. (E) VCaP xenografts at pre-castration (AD) or 4 days post-castration were analyzed by AR ChIP-qPCR at the indicated loci. (F) Scid mice (n=5) bearing recurrent VCaP xenografts were treated with 1mg of testosterone per animal (daily i.p. injection) for 5 days. mRNA extracted from tumor biopsies was analyzed by qRT-PCR, with post-treatment levels normalized to pre-treatment. See also Figure S1.

These genes mediate a variety of DNA replication related functions including DNA synthesis, modification, metabolism, and repair. Significantly, 50/77 of these genes were overexpressed in a previously reported group of metastatic CRPC versus primary PCa samples (Stanbrough et al., 2006), indicting the importance of these DNA replication genes in driving PCa progression (see Table S1). The DHT-stimulated downregulation of a subset of these genes was confirmed by qRT-PCR (Figure 1C).

We then selected a minichromosome maintenance complex gene (MCM7) and a Fanconi anemia complementation group gene (FANCI) for further validation (also including AKR1C3 and OPRK1). ChIP-qPCR confirmed AR binding at the sites identified by ChIP-seq for each gene, and showed decreased Ser5 phosphorylated RNA pol II (p-Pol2, mark of transcriptional activation) and decreased H3K4me2 upon DHT stimulation (Figure 1D), consistent with transcriptional repression.

To determine if AR was binding to these sites in vivo, we examined the effects of ADT on AR occupancy in VCaP xenografts. Androgen deprivation led to marked decreases in AR binding at all tested loci in vitro and in vivo (Figure S1F and Figure 1E). Finally, to determine if androgens could repress DNA replication genes in vivo, we examined castration-resistant VCaP xenografts before or after treatment with high-dose testosterone (which causes a rapid reduction in tumor volume in this model) (Cai et al., 2011). As shown in Figure 1F, except for LMNB1, the expression of each gene examined was decreased by testosterone. Finally, we examined if AR binds to those repressive sites in normal prostate and PCa cells from human patient tissues using a public ChIP-seq database (Pomerantz et al., 2015). In those samples, over 50% of the DNA replication genes (as well as other AR-repressed genes) consistently harbor nearby AR binding sites at the same location as in DHT treated VCaP cells (Figure S1G–H). Together these findings support the conclusion that AR functions as a direct transcriptional repressor at these sites both in vitro and in vivo.

AR repression of DNA replication genes is mediated by recruitment of Rb

Many of these DNA replication related genes are targets of E2F and Rb (Dick and Rubin, 2013). Therefore, we hypothesized that AR may increase the recruitment of Rb to these genes and thereby enhance their Rb-mediated repression. Indeed, DHT rapidly increased Rb binding at the major AR binding sites (S) in MCM7 and BLM (which overlap with the promoters) as well as in FANCI and LMNB1 (within the gene bodies), and this Rb recruitment persisted at 24h (Figure 2A). DHT also stimulated Rb binding at the promoter regions of FANCI and LMNB1 (P). This AR binding and recruitment of Rb could be decreased by the AR antagonist enzalutamide (Figure S2A and Figure 2B). To further validate this Rb recruitment in vivo, we carried out Rb ChIP in tissue samples from castration-resistant VCaP xenografts with short-term testosterone treatment. As seen in Figure 2C, Rb binding was increased upon testosterone treatment. Finally, we assessed for histone deacetylases (HDACs), which are recruited by Rb as mediators of gene repression. Binding of HDAC1 and 2, was DHT-stimulated and this recruitment led to deacetylation of H3K27 (Figure S2B).

Figure 2. AR repression of DNA replication genes is mediated by recruitment of Rb.

Figure 2

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(A) ChIP-qPCR for Rb recruitment by 10nM DHT (4h or 24h) at the indicated loci in VCaP cells. (B) ChIP-qPCR for Rb recruitment by DHT alone (4h) versus DHT plus 5μM enzalutamide (pretreat 4h) in VCaP cells. (C) Scid mice bearing VCaP CRPC xenografts were treated with vehicle (n=4) or 1mg of testosterone (n=4) for 1 week. Biopsies of tumor tissue were fixed and subjected to ChIP-qPCR of Rb at indicated loci. (D) AR ChIP was performed first in VCaP cells treated with DHT for 4h, followed by Re-ChIP of Rb. (E) Plasmids expressing Rb or AR fragments (HA tagged, FL: 1-919aa; N: 1-539aa; ND: 1-628aa; DL: 538-919aa; L: 662-919aa) were cotransfected in 293 cells. AR protein fragments were immunoprecipitated by anti-HA beads followed by Rb immunoblotting. (F and G) VCaP cells were transfected with non-target control (siNTC) or RB siRNA (siRB) for 2d in hormone-depleted medium and then treated with DHT for 24h, followed by (F) immunoblotting for Rb and (G) by qRT-PCR for indicated DNA replication genes. (H) qRT-PCR for expression of indicated genes in C4-2 cells treated with 10 nM DHT for 0–24h. (I) ChIP-qPCR for Rb recruitment by 10nM DHT (4h) at the indicated loci in C4-2 cells. (J and K) C4-2 cells were transfected with siNTC or siRB for 2d in hormone-reduced medium (2% FBS/8% hormone-depleted FBS) and then treated with 10nM DHT for 24h, followed by (J) immunoblotting for Rb and by (K) flow cytometry cell cycle analysis. See also Figure S2.

Using ChIP-Re-ChIP in VCaP cells we next determined that AR and Rb are associated in a complex at these repressive sites (Figure 2D). Experiments in cells overexpressing AR and Rb were then carried out to determine if a specific AR domain mediates the interaction. This indicated that Rb preferentially interacts with a N-terminal region of AR (Figure 2E), consistent with a previous study (Lu and Danielsen, 1998). However, we cannot exclude the possibility that AR interaction with Rb is indirect and mediated or enhanced by another protein, or that binding of the endogenous proteins is transient unless stabilized by chromatin binding.

We next treated VCaP cells with RB siRNA to determine if Rb contributes to the AR-mediated repression of these genes. As expected, RB knockdown (Figure 2F) increased the expression of each gene in the absence of DHT (Figure 2G). Moreover, the repression in response to DHT was decreased in the Rb knockdown cells. Finally, AR-mediated repression of OPRK1 (no detectable Rb binding site, see Figure S3D) was not affected by RB siRNA.

We next examined C4-2 cells, a cell line derived from a CRPC LNCaP xenograft that has increased AR expression and sensitivity to low levels of androgen, and whose proliferation can be suppressed at high levels of androgen (Gregory et al., 2001). C4-2 cells also exhibited androgen-repressed expression of DNA replication genes (Figure 2H), and DHT stimulation of Rb binding was similarly observed (Figure 2I). Therefore, we speculated that impairing AR recruitment of Rb may decrease the ability of DHT to suppress C4-2 cell proliferation. As seen in Figure 2J and K, DHT treatment significantly decreased the S phase fraction, but did not suppress that in cells transfected with RB siRNA.

Rb binding globally associates with AR transcriptional repression

To globally assess the overlap between AR and Rb binding, we carried out Rb ChIP-seq in 10nM DHT treated (4h) VCaP cells and identified 9,797 binding sites (Figure 3A). As expected, these Rb binding sites were markedly enriched for the E2F binding motif (Figure S3A) and analysis of the top 1000 genes showed enrichment for genes mediating DNA replication (Figure S3B). Significantly, a large portion of Rb binding sites (6,056/9,797, ~70%) overlapped with AR binding sites (Figure 3A) and these sites were similarly enriched for E2F binding motifs (Figure 3B). Moreover, higher intensity of AR binding correlated with stronger Rb binding at these sites within AR-repressed genes (Figure 3C). Among the 77 AR-repressed DNA replication gene subset, 74 genes (~95%) had Rb binding sites and 66 genes (~85%) had AR and Rb overlapping binding sites (Figure S3C–D).

Figure 3. Rb binding broadly associates with AR-mediated transcriptional repression.

Figure 3

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(A) Rb ChIP-seq in VCaP cells treated with 10nM DHT for 4h. Peak calling was done using MACS (p-Value<10−5). Overlapping Rb and AR sites in DHT treated VCaP cells were shown. (B) Motif enrichment analysis for AR and Rb overlapping sites using Cistrome tool (lowest p-value is set to 1×10−30). (C) Heat maps for AR and Rb binding intensity within the AR-repressed gene loci (ranking from high to low based on AR binding intensity). (D) BETA analyses for the association of AR and Rb binding sites with expressions of AR-activated versus -repressed genes (detail in Supplemental Experimental Procedures). See also Figure S3.

We then performed Binding and Expression Target Analysis (BETA) (Wang et al., 2013) to globally determine the association of Rb binding with AR-regulation of gene expression. As seen in Figure 3D, Rb unique binding sites (no AR binding, left panel) were only weakly associated with AR-repressed genes. In contrast, AR-Rb overlapping sites (middle panel) were very highly associated with AR-repressed genes, while AR unique sites (no Rb binding, right panel) were most strongly associated with AR-activated genes. Collectively, these gene-specific and global analyses of Rb and AR binding sites strongly indicate that androgen-stimulated Rb binding is a mechanism mediating AR dependent transcriptional repression.

AR directly represses and indirectly enhances expression of genes regulating DNA replication in LNCaP cells

While DNA replication genes were repressed by 24h of DHT in VCaP and C4-2 cells, many were increased by treatment with 10nM DHT at 24h in the LNCaP cell line (Figure 4A). However, they were not increased at earlier time points that were sufficient for induction of typical AR regulated genes. We confirmed that DHT did not increase expression of DNA replication genes in LNCaP cells until 16–24h by qRT-PCR (Figure S4A). Protein expression was also differentially regulated by androgen in LNCaP versus VCaP cells (Figure S4B). As a control, OPRK1 was repressed similarly by androgens in both cell lines. This delayed stimulation suggested that these genes were not directly induced by AR. To further test this hypothesis, we pre-treated LNCaP cells with cycloheximide (CHX) to block new protein synthesis prior to androgen treatment. Significantly, while CHX did not prevent the DHT-stimulated increase in PLZF, it prevented the increase in each of the DNA replication genes examined (Figure S4C).

Figure 4. AR mediated repression of DNA replication genes is circumvented by the indirect stimulatory effect of androgens but is enhanced by AR overexpression in CRPC cells.

Figure 4

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(A) Expressions of 77 AR-repressed DNA replication gene subset and a group of AR-activated genes in LNCaP cells treated with 10nM DHT for 0–24h. (B) ChIP-qPCR at indicated AR-dependent gene loci for AR and p-Pol2 binding in LNCaP cells treated with 10nM DHT for 4–24h. (C) LNCaP or VCaP cells in hormone-depleted medium were treated with 10nM DHT for 24h, followed by cell cycle analysis. (D) Immunoblotting for phosphorylated-Rb (Ser780) in LNCaP or VCaP cells treated with DHT for 0–24h. (E) ChIP-qPCR for Rb recruitment by DHT stimulation (0–24h) at the indicated loci in LNCaP cells. (F) Immunoblotting for AR or FLAG in LN-Ctrl versus LN-AR. (G) qRT-PCR for the indicated genes in LN-Ctrl versus LN-AR cells treated with DHT for 0–24h. (H) ChIP-qPCR for DHT-stimulated (0–16h) AR binding in LN-Ctrl versus LN-AR cells. (I) ChIP-qPCR for DHT-stimulated Rb recruitment (0–24h) in LN-AR cells. (J and K) C4-2 cells were treated with palbociclib (0.1μM) or DHT (10nM) for (J) 1d, followed by cell cycle analysis, or (K) 0–4d, followed by cell counting. See also Figure S4.

We next assessed AR binding and transcriptional activation of these genes over a time course after DHT treatment. DHT stimulated AR binding within 4h in LNCaP cells (Figure 4B), and this binding could be prevented by enzalutamide (Figure S4D). Significantly, this AR binding was also associated with an initial decrease of p-Pol2 (4–16h), which was only reversed after 24h of treatment. As controls, AR binding at PSA versus OPRK1 gene loci led to progressive increases or decreases in recruitment of p-Pol2, respectively.

Significantly, while 10nM DHT stimulates proliferation of LNCaP cells cultured in steroid-depleted medium, it does not stimulate the proliferation of VCaP cells (Figure 4C). This may reflect a VCaP requirement for additional growth factors that are lost in steroid-depleted serum (Cai et al., 2011). As expected, the time course for DHT-stimulated induction of DNA replication genes in LNCaP cells is similar to the time course for the increase of S-phase cells (Figure S4E). Consistent with these findings, Rb phosphorylation is stimulated by 24h DHT treatment in LNCaP cells, but remained unchanged in VCaP cells (Figure 4D and S4F–G). Importantly, this observation indicates that decreased Rb phosphorylation is not a mechanism contributing to the androgen-stimulated increase in Rb binding to DNA replication genes in VCaP or LNCaP cells.

We reported previously that AR stimulates G1/S cell cycle progression in LNCaP cells by increasing the transcription of multiple genes involved in metabolism, with a subsequent increase in TORC1 activity and translation of D cyclins (Xu et al., 2006). Therefore, we considered that the increased expression of DNA replication genes in LNCaP cells might be a downstream consequence, rather than an upstream driver, of CDK activation and Rb phosphorylation. To test this hypothesis we treated LNCaP with a CDK2 inhibitor, roscovitine, which effectively prevented the DHT-stimulated increase in Rb phosphorylation (Figure S4H). Significantly, this blocked the DHT-induced expression of DNA replication genes, but not PSA, indicating that the DHT-mediated stimulation of these DNA replication genes is a downstream consequence of increased G1/S-CDK activity (Figure S4I–J).

As phosphorylation of Rb disrupts its binding to E2F, we hypothesized that the initial DHT-stimulated recruitment of Rb, if mediated jointly by AR and E2F, would be diminished by 24h in LNCaP cells. Indeed, the Rb recruitment was lost at 24h (Figure 4E). This is in contrast with DHT-stimulated Rb binding in VCaP cells that persisted at 24h (see Figure 2A). Based on these results, we conclude that AR initially represses E2F regulated genes in LNCaP cells by enhancing the binding of hypophosphorylated Rb.

We further carried out Rb ChIP-seq in 10nM DHT treated (4h) LNCaP cells and identified 21,932 binding peaks (Figure S4K). The Rb binding profile in VCaP cells substantially overlapped that in LNCaP cells (6,871 peaks, ~70% of total Rb binding sites in VCaP). Amongst those overlapping Rb binding sites, ~70% (4,811/6,871) were linked with AR binding in VCaP cells, suggesting these sites may similarly be involved in mediating AR repression activity in LNCaP cells. Interestingly, a large subset of peaks (15,061) were unique, suggesting additional functions in LNCaP cells. Significantly, for the 77 DNA replication gene subset, all the AR and Rb overlapping sites identified in VCaP were also present in LNCaP cells (Figure S4L). Together these data indicate that a direct function of AR in LNCaP, similarly to VCaP cells, is to repress expression of genes mediating DNA replication. This repression may then be overcome by AR-stimulated secondary mechanisms that lead to Rb hyperphosphorylation.

AR overexpression enhances AR mediated repression of DNA replication genes

We next sought to further determine the basis for the differential regulation of DNA replication genes in LNCaP versus VCaP and C4-2 cells. One possible mechanism could be that repression is favored by higher level of liganded AR. To test this we examined if DNA replication genes would be repressed by higher concentrations of DHT in LNCaP cells. Indeed, we observed a biphasic response with maximum increases at 10nM DHT after 24h, but expression did not decline below baseline at up to 1μM DHT (Figure S4M).

Since both VCaP and C4-2 cells express higher levels of AR relative to LNCaP (Figure S4N), we next hypothesized that AR repression activity may be favored by overexpression of AR protein. To test this we stably overexpressed AR in LNCaP cells (LN-AR) (Figure 4F) and assessed the effect of DHT on DNA replication genes. Basal expression of each DNA replication gene examined was increased in LN-AR cells (Figure 4G), which is consistent with increased basal AR activity and decreased dependence on exogenous androgens for proliferation (Chen et al., 2004). However, in contrast to the LNCaP control cells (LN-Ctrl), these DNA replication genes were repressed by DHT in the LN-AR cells at 16–24h (Figure 4G).

We next assessed effects of AR overexpression on chromatin binding. DHT-stimulated AR binding to each of the genes examined was increased and more persistent in the LN-AR cells (Figure 4H), and p-Pol2 was decreased at 24h (Figure S4O). Significantly, in contrast to diminished Rb binding at 24h of DHT treatment in LNCaP cells (see Figure 4E), Rb binding persisted at 24h in LN-AR cells (Figure 4I). Taken together, these results indicate that higher level of AR in C4-2 and VCaP cells favor repression.

Blocking G1/S-CDK activity enhances androgen-repression of cell proliferation in CRPC cells

We next hypothesized that preventing Rb hyperphosphorylation by blocking G1/S-CDK activity may further enhance the growth suppressive activity of androgens in CRPC models. Therefore, we examined the combination effect of androgen and palbociclib, a CDK4/6 inhibitor (Finn et al., 2015), on expression of DNA replication genes and on cell proliferation. As seen in Figure S4P and Q, palbociclib in C4-2 cells inhibited Rb phosphorylation and thereby enhanced AR-mediated repression of DNA replication genes. Significantly, both cell cycle analysis and proliferation assay showed that palbociclib treatment effectively increased the growth repressive effect of DHT (Figure 4J–K, and S4R), suggesting a potential therapeutic strategy for treating CRPC patients.

DISCUSSION

AR is a transcriptional activator, but can also function as a transcriptional repressor by direct and indirect mechanisms (Cai et al., 2011; Zhao et al., 2012). Our previous analyses showed that DHT repressed genes in VCaP derived models were highly enriched in genes involved in DNA replication (Cai et al., 2011). In this study we examined AR ChIP-seq data and found closely linked AR binding sites in a large fraction of DHT-repressed DNA replication genes, supporting the conclusion that they are directly repressed by AR. Moreover, while most of these genes were increased by DHT in LNCaP cells, we confirmed that the rapid direct action of DHT was still to repress these genes, and that their subsequent increase was indirect and due to AR stimulation of other metabolic pathways. Overall, based on these results, we conclude that AR functions directly as a transcriptional repressor on DNA replication genes, and suggest that this reflects its normal differentiating function in prostate epithelium. However, this growth inhibitory function of AR is clearly circumvented in PCa cells, where stimulatory effects of AR on metabolic pathways may override its differentiating functions and drive tumor growth.

Significantly, many genes involved in DNA replication are regulated by Rb and E2F (Dick and Rubin, 2013). Rb had been found previously to interact with AR, but it appeared to act as an AR coactivator on androgen-stimulated genes in these studies (Lu and Danielsen, 1998; Yeh et al., 1998). The basis for this activity is not clear, but could possibly reflect sequestration of HDACs or other transcriptional corepressors. In other studies, androgen-mediated repression of EZH2 and SKP2 were found to be dependent on Rb or the related p107/p130, but these effects were mediated by unclear indirect mechanisms (Bohrer et al., 2010; Jiang et al., 2012). This report shows that AR enhances Rb binding to a series of E2F-regulated DNA replication genes, indicating that AR functions in concert with E2F to recruit Rb and suppress these genes. Importantly, Rb ChIP-seq shows substantial overlap between Rb and AR binding sites, and these overlapping sites correlated with AR transcriptional repression activity, suggesting a broad role of Rb in AR mediated transcriptional repression. An AR-Rb-E2F containing complex may assemble directly on promoters that contain AR binding sites or indirectly by chromatin looping in genes with AR binding sites that are distal to the promoter and E2F sites. Importantly, this recruitment of Rb is consistent with the ability of high-dose androgens to suppress CRPC expressing higher level of AR in model systems and in a subset of patients (Schweizer et al., 2015). Furthermore, our results suggest that high-dose androgen therapy may be less effective in tumors that have downregulated or lost Rb expression.

Amongst the genes directly repressed by AR were multiple MCM genes, which are required to assemble origins of DNA replication (Labib et al., 2000). Interestingly, AR has been reported previously to function as a licensing factor that must be degraded in early G1 phase in order to assemble these origins of replication, so that high levels of androgen that maintain AR activity may suppress DNA replication (D’Antonio et al., 2009). Although our findings differ mechanistically from this previous study, our results similarly indicate that AR, by suppressing expression of MCM genes, may impair origin of replication assembly. Many genes that are activated for DNA replication are also involved in DNA damage repair pathways, and a subset of these DNA damage repair genes were found in a recent study to be transcriptionally activated by AR in LNCaP cells (Polkinghorn et al., 2013). These results are not inconsistent with our findings, as the reported increases were after 24–48h, and the direct immediate effects of androgen were not assessed. However, mechanistically our data would indicate that many of these genes are not directly transcriptionally activated by AR, but are instead directly repressed, with their increased expression after 24h being through indirect mechanisms. In particular, based on our data we suggest that expression of at least a subset of these DNA repair genes is induced as a downstream consequence of G1/S-CDK activation (Xu et al., 2006).

ADTs are initially effective in most patients, but by relieving repression of multiple genes mediating DNA replication, it may also be contributing to the eventual emergence of CRPC. Interestingly, a recent study in the PTEN deficient mouse PCa model found that androgen ablation increased the eventual emergence of invasive PCa (Jia et al., 2013). It is also intriguing that androgen levels decline with age (Kaufman and Vermeulen, 2005). While it has been suggested that a resulting increase in the ratio of estrogen to testosterone may stimulate the development of PCa, it is plausible that modest decreases in androgen levels by themselves may relieve repression of androgen-repressed genes and thereby contribute to PCa development. Finally, the beneficial effects of 5α-reductase inhibitors in PCa trials could possibly have been compromised by induction of AR-repressed genes (Kang and Chung, 2013). While it has been long recognized that AR has both growth promoting and differentiating function, we anticipate that further studies elucidating the precise molecular basis for these functions will lead to the development of more selective and effective AR targeted therapies.

METHODS

ChIP

VCaP cell lines were treated with 10nM DHT for 4h, followed by ChIP-seq analyses (He et al., 2010). GEO accession number for the published AR ChIP-Seq dataset is GSE32345. Antibodies and primers used for ChIP analyses are in Supplemental Experimental Procedures.

RT-PCR and immunoblotting

Gene expression was measured using Taqman one-step RT-PCR reagents and results were normalized to co-amplified GAPDH. For immunoblotting, cells were lysed with RIPA buffer with protease inhibitors. Gels shown are representative of at least 3 independent experiments. Primers/probes and primary antibodies are in Supplemental Experimental Procedures.

VCaP xenografts and LNCaP stable lines

Xenograft generation has been previously described (Cai et al., 2011). For LNCaP-AR cells, Flag tagged AR was transfected in LNCaP cells and stable lines were established by G418 selection.

Statistical analysis

Data in bar graphs represent mean±SD of at least 3 biological repeats. Statistical analysis was performed using Student’s t-test by comparing treatment versus vehicle or otherwise as indicated. p-Value<0.05 (*) was considered to be statistically significant.

Supplementary Material

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Acknowledgments

This work is supported by grants from NIH (K99 CA172948 to H.H.H., R00 CA166507 to C.C., R00 CA135592 to S.C., P01 CA163227, and Prostate Cancer SPORE P50 CA090381) and DOD (W81XWH-15-1-0554 to S.G., W81XWH-14-1-0245 to Y.G., W81XWH-10-1-0557 to H.H.H., W81XWH-15-1-0519 to C.C., W81XWH-11-1-0295 and W81XWH-13-1-0266 to S.P.B.).

Footnotes

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Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.

Additional experimental procedures and methods are listed in the Supplementary Experimental Procedures.

AUTHOR’S CONTRIBUTIONS

S.G., Y.G., D.H., W.H., X.L., S. C., F.M., A.A., and C.C. performed experiments; H.H.H., D.H., J.A.M., and C.C. were involved in data processing and bioinformatics analysis; S.P.B., S.C., and C.C. designed research, analyzed data and wrote the manuscript. All authors discussed the results and commented on the manuscript. As co-first authors, Y.G. discovered the differential activities of AR on DNA replication and S.G. determined the role of Rb in mediating the repression activity of AR. As co-corresponding authors, S.C. supported and supervised the studies by Y.G. and was involved in manuscript writing while S.P.B. and C.C. were responsible for overall research design, data analyzing and manuscript preparation.

ACCESSION NUMBERS

The GEO accession for Rb ChIP-seq data is GSE76141.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS822122-supplement-1.pdf (5.1MB, pdf)
2
NIHMS822122-supplement-2.pdf (51.2KB, pdf)

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