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• W2080748037 abstract "•GR induction is a common feature of enzalutamide-resistant prostate cancer•GR expression and activity promote resistance to enzalutamide•GR binds and regulates a subset of AR targets in a enzalutamide-insensitive manner•AR inhibition induces high levels of GR in primed prostate cells The treatment of advanced prostate cancer has been transformed by novel antiandrogen therapies such as enzalutamide. Here, we identify induction of glucocorticoid receptor (GR) expression as a common feature of drug-resistant tumors in a credentialed preclinical model, a finding also confirmed in patient samples. GR substituted for the androgen receptor (AR) to activate a similar but distinguishable set of target genes and was necessary for maintenance of the resistant phenotype. The GR agonist dexamethasone was sufficient to confer enzalutamide resistance, whereas a GR antagonist restored sensitivity. Acute AR inhibition resulted in GR upregulation in a subset of prostate cancer cells due to relief of AR-mediated feedback repression of GR expression. These findings establish a mechanism of escape from AR blockade through expansion of cells primed to drive AR target genes via an alternative nuclear receptor upon drug exposure. The treatment of advanced prostate cancer has been transformed by novel antiandrogen therapies such as enzalutamide. Here, we identify induction of glucocorticoid receptor (GR) expression as a common feature of drug-resistant tumors in a credentialed preclinical model, a finding also confirmed in patient samples. GR substituted for the androgen receptor (AR) to activate a similar but distinguishable set of target genes and was necessary for maintenance of the resistant phenotype. The GR agonist dexamethasone was sufficient to confer enzalutamide resistance, whereas a GR antagonist restored sensitivity. Acute AR inhibition resulted in GR upregulation in a subset of prostate cancer cells due to relief of AR-mediated feedback repression of GR expression. These findings establish a mechanism of escape from AR blockade through expansion of cells primed to drive AR target genes via an alternative nuclear receptor upon drug exposure. Recently approved drugs that target androgen receptor (AR) signaling such as abiraterone and enzalutamide have rapidly become standard therapies for advanced-stage prostate cancer (Scher et al., 2012Scher H.I. Fizazi K. Saad F. Taplin M.E. Sternberg C.N. Miller K. de Wit R. Mulders P. Chi K.N. Shore N.D. et al.AFFIRM InvestigatorsIncreased survival with enzalutamide in prostate cancer after chemotherapy.N. Engl. J. Med. 2012; 367: 1187-1197Crossref PubMed Scopus (3395) Google Scholar, de Bono et al., 2011de Bono J.S. Logothetis C.J. Molina A. Fizazi K. North S. Chu L. Chi K.N. Jones R.J. Goodman Jr., O.B. Saad F. et al.COU-AA-301 InvestigatorsAbiraterone and increased survival in metastatic prostate cancer.N. Engl. J. Med. 2011; 364: 1995-2005Crossref PubMed Scopus (3391) Google Scholar). Despite their success, sustained response with these agents is limited by acquired resistance, which typically develops within ∼6–12 months. Clinical success of kinase inhibitors in other tumors such as melanoma, lung cancer, leukemia, and sarcoma is similarly transient (Sawyers et al., 2002Sawyers C.L. Hochhaus A. Feldman E. Goldman J.M. Miller C.B. Ottmann O.G. Schiffer C.A. Talpaz M. Guilhot F. Deininger M.W. et al.Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study.Blood. 2002; 99: 3530-3539Crossref PubMed Scopus (1061) Google Scholar, Chapman et al., 2011Chapman P.B. Hauschild A. Robert C. Haanen J.B. Ascierto P. Larkin J. Dummer R. Garbe C. Testori A. Maio M. et al.BRIM-3 Study GroupImproved survival with vemurafenib in melanoma with BRAF V600E mutation.N. Engl. J. Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6115) Google Scholar, Demetri et al., 2002Demetri G.D. von Mehren M. Blanke C.D. Van den Abbeele A.D. Eisenberg B. Roberts P.J. Heinrich M.C. Tuveson D.A. Singer S. Janicek M. et al.Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors.N. Engl. J. Med. 2002; 347: 472-480Crossref PubMed Scopus (3724) Google Scholar, Maemondo et al., 2010Maemondo M. Inoue A. Kobayashi K. Sugawara S. Oizumi S. Isobe H. Gemma A. Harada M. Yoshizawa H. Kinoshita I. et al.North-East Japan Study GroupGefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR.N. Engl. J. Med. 2010; 362: 2380-2388Crossref PubMed Scopus (4628) Google Scholar), resulting in numerous efforts to define mechanisms of acquired resistance. One strategy that has proven particularly useful is prolonged treatment of drug-sensitive preclinical models to derive drug-resistant sublines, followed by genome-wide profiling studies to ascertain differences that may play a causal role in conferring drug resistance. A common mechanism that has emerged from these kinase inhibitor studies is reactivation of the signaling pathway targeted by the drug, directly by mutation of the kinase target or indirectly by bypassing pathway inhibitor blockade through amplification of an alternative kinase (Glickman and Sawyers, 2012Glickman M.S. Sawyers C.L. Converting cancer therapies into cures: lessons from infectious diseases.Cell. 2012; 148: 1089-1098Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Both scenarios have been validated in clinical specimens and are guiding efforts to discover next-generation inhibitors and to develop rational drug combinations. Clinically relevant mechanisms of resistance to hormone therapy in prostate cancer have also been elucidated using preclinical models. Hormone therapy, through the use of drugs that lower serum testosterone or competitively block the binding of androgens to AR, has been the mainstay of treatment for metastatic prostate cancer for decades but is not curative. The late stage of disease, which is refractory to hormone therapy, is termed castration-resistant prostate cancer (CRPC). We previously examined the molecular basis of progression to CRPC in mouse models and discovered that increased AR expression was the primary mechanism (Chen et al., 2004Chen C.D. Welsbie D.S. Tran C. Baek S.H. Chen R. Vessella R. Rosenfeld M.G. Sawyers C.L. Molecular determinants of resistance to antiandrogen therapy.Nat. Med. 2004; 10: 33-39Crossref PubMed Scopus (1955) Google Scholar). We then used this observation to screen for antiandrogens that restore AR inhibition in the setting of increased AR levels. These efforts yielded three second-generation antiandrogens: enzalutamide, ARN-509, and RD162 (Tran et al., 2009Tran C. Ouk S. Clegg N.J. Chen Y. Watson P.A. Arora V. Wongvipat J. Smith-Jones P.M. Yoo D. Kwon A. et al.Development of a second-generation antiandrogen for treatment of advanced prostate cancer.Science. 2009; 324: 787-790Crossref PubMed Scopus (1710) Google Scholar, Clegg et al., 2012Clegg N.J. Wongvipat J. Joseph J.D. Tran C. Ouk S. Dilhas A. Chen Y. Grillot K. Bischoff E.D. Cai L. et al.ARN-509: a novel antiandrogen for prostate cancer treatment.Cancer Res. 2012; 72: 1494-1503Crossref PubMed Scopus (504) Google Scholar). Enzalutamide and ARN-509 were further developed for clinical use, culminating in US Food and Drug Administration (FDA) approval of enzalutamide in 2012 based on increased survival (Scher et al., 2012Scher H.I. Fizazi K. Saad F. Taplin M.E. Sternberg C.N. Miller K. de Wit R. Mulders P. Chi K.N. Shore N.D. et al.AFFIRM InvestigatorsIncreased survival with enzalutamide in prostate cancer after chemotherapy.N. Engl. J. Med. 2012; 367: 1187-1197Crossref PubMed Scopus (3395) Google Scholar). Now, with widespread use, resistance to enzalutamide is a major clinical problem. We and others have recently identified an AR point mutation as one resistance mechanism by derivation of drug-resistant sublines following prolonged exposure to enzalutamide or ARN-509 (Balbas et al., 2013Balbas M.D. Evans M.J. Hosfield D.J. Wongvipat J. Arora V.K. Watson P.A. Chen Y. Greene G.L. Shen Y. Sawyers C.L. Overcoming mutation-based resistance to antiandrogens with rational drug design.eLife. 2013; 2: e00499Crossref Scopus (303) Google Scholar, Joseph et al., 2013Joseph J.D. Lu N. Qian J. Sensintaffar J. Shao G. Brigham D. Moon M. Chow Maneval E. Chen I. Darimont B. et al.A clinically relevant androgen receptor mutation confers resistance to 2nd generation anti-androgens enzalutamide and ARN-509.Cancer Discov. 2013; 3: 1020-1029Crossref PubMed Scopus (454) Google Scholar, Korpal et al., 2013Korpal M. Korn J.M. Gao X. Rakiec D.P. Ruddy D.A. Doshi S. Yuan J. Kovats S.G. Kim S. Cooke V.G. et al.An F876L Mutation in Androgen Receptor Confers Genetic and Phenotypic Resistance to MDV3100 (Enzalutamide).Cancer Discov. 2013; 3: 1030-1043Crossref PubMed Scopus (417) Google Scholar). This AR mutation has also been recovered from patients with resistance to ARN-509 but only in a minority of cases (Joseph et al., 2013Joseph J.D. Lu N. Qian J. Sensintaffar J. Shao G. Brigham D. Moon M. Chow Maneval E. Chen I. Darimont B. et al.A clinically relevant androgen receptor mutation confers resistance to 2nd generation anti-androgens enzalutamide and ARN-509.Cancer Discov. 2013; 3: 1020-1029Crossref PubMed Scopus (454) Google Scholar). Here, we define a potentially more prevalent mechanism of resistance by which tumors bypass AR blockade through upregulation of the glucocorticoid receptor (GR). We previously showed that LNCaP/AR xenograft tumors regress during the first 28 days of treatment with ARN-509 (Clegg et al., 2012Clegg N.J. Wongvipat J. Joseph J.D. Tran C. Ouk S. Dilhas A. Chen Y. Grillot K. Bischoff E.D. Cai L. et al.ARN-509: a novel antiandrogen for prostate cancer treatment.Cancer Res. 2012; 72: 1494-1503Crossref PubMed Scopus (504) Google Scholar), enzalutamide, or RD162 (Tran et al., 2009Tran C. Ouk S. Clegg N.J. Chen Y. Watson P.A. Arora V. Wongvipat J. Smith-Jones P.M. Yoo D. Kwon A. et al.Development of a second-generation antiandrogen for treatment of advanced prostate cancer.Science. 2009; 324: 787-790Crossref PubMed Scopus (1710) Google Scholar). In a pilot study to explore mechanisms of acquired resistance to these drugs, we treated mice continually and harvested tumors after progression (mean 163 days, Table S1A available online). Tissue from 15 resistant tumors obtained from long-term antiandrogen-treated mice (n = 6 ARN-509, n = 9 RD162) and from 3 control tumors from vehicle-treated mice were analyzed by expression array. Aggregated data from resistant and control tumors in this pilot cohort were compared to identify expression changes commonly associated with resistance (Figure 1A). Among the most upregulated genes in the resistant tumors was the GR (gene symbol NR3C1), which shares overlapping target specificity with AR (Mangelsdorf et al., 1995Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. The nuclear receptor superfamily: the second decade.Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6064) Google Scholar). Of note, several of the most differentially expressed genes were known androgen-regulated genes (confirmed by transcriptome analysis of short-term dihydrotestosterone (DHT)-treated LnCaP/AR cells in vitro [Table S1B]), but they were altered in directions that did not reflect restored AR signaling. On the one hand, SGK1 (Serum Glucocorticoid Induced Kinase 1), a known AR- and GR-induced target gene, was among the most upregulated genes, but several other androgen-induced genes (PMEPA1, SNAI2, KCNN2, LONRF1, and SPOCK1) were among the most repressed. Conversely, several androgen-repressed genes (UGT2B15, PMP22, CAMK2N1, and UGT2B17) were among the most upregulated (Figure 1A). These findings indicated that resistance in this model system is unlikely to be mediated by simple restoration of AR activity and raised the possibility that GR may play a role. To explore this question further, we generated an independent set of drug-resistant tumors (the validation cohort), focusing on the two second-generation antiandrogens in clinical use, enzalutamide and ARN-509 (Figure 1B). GR messenger RNA (mRNA) levels in resistant tumors were substantially higher compared to control (median 26.9-fold increase) or 4 day treated tumors (Figure 1C). Of the tissues analyzed by RT-qPCR, most were also analyzed for GR expression by western blot, based on availability of protein lysates (control, n = 6; 4 day, n = 5; resistant, n = 13). No GR was detected in control samples, minimal expression was noted in 4 day treated samples, and substantial expression was found in most resistant tumors in a pattern that tended to correlate with GR mRNA levels (Figure 1D). There was no correlation between GR expression and the specific antiandrogen treatment used (Table S1C). In contrast to GR, AR RNA or proteins levels were not consistently different across the treatment groups (Figures 1C and 1D). To explore AR and GR signaling in more detail, we established cells lines from control and drug-resistant tumors by adaptation to growth in vitro. LREX′ (LnCaP/AR Resistant to Enzalutamide Xenograft derived) was derived from an enzalutamide-resistant tumor with high GR expression, and CS1 was derived from a vehicle-treated tumor. We also developed a flow cytometry-based assay to measure GR expression on a cell-by-cell basis. In both LNCaP/AR and CS1, most cells showed no evidence of GR expression, with the exception of a small subpopulation (black arrow, discussed later) (Figure 1E). In contrast, essentially all LREX′ cells expressed GR. Intracellular AR staining confirmed that AR levels in LREX′ did not notably differ from control cells (Figure S1A). Having established the LREX′ model as representative of high GR expression, we next confirmed that these cells maintain a resistant phenotype in vivo. LREX′ or control cells were injected into castrated mice that were then immediately initiated on antiandrogen treatment. LREX′ showed robust growth, whereas LNCaP/AR or CS1 lines were unable to establish tumors in the presence of antiandrogen (Figures 2A and 2B ). Strong expression of GR was confirmed in multiple LREX′ xenograft tumors by western blot and by IHC (Figures S2 and S1B). As expected, untreated LNCaP/AR tumors were negative for GR expression with the exception of rare GR-positive cells (Figure 2C). Although many of these GR-positive cells had morphologic features of stromal or endothelial cells (blue arrows), some appeared epithelial (black arrow), which is consistent with the flow cytometry analysis (Figure 1E, black arrows). To determine whether GR expression is required to maintain the drug-resistant phenotype, LREX′ cells were infected with a small hairpin RNA (shRNA)-targeting GR (shGR), and stable knockdown of GR protein was confirmed (Figure 2F). Tumor growth of shGR-infected LREX′ cells was significantly delayed relative to nontargeted (shNT)-infected cells in castrated mice treated with enzalutamide (Figure 2D). In contrast, shGR had no impact on the growth of GR-negative CS1 xenografts, diminishing the possibility of an off-target effect (Figure 2E). Of note, shGR LREX′ xenografts harvested on day 49 showed decreased GR protein knockdown compared to the preimplantation levels, which is indicative of selective pressure against GR silencing in the setting of enzalutamide treatment (Figure 2F). These findings provide direct evidence that GR drives enzalutamide resistance in vivo. To determine whether GR expression is a feature of clinical antiandrogen resistance, we evaluated GR expression in bone metastases from patients receiving enzalutamide. Bone marrow samples were obtained prior to enzalutamide treatment (baseline) and again after 8 weeks of treatment, as previously reported in a cohort of abiraterone-treated patients (Efstathiou et al., 2012Efstathiou E. Titus M. Tsavachidou D. Tzelepi V. Wen S. Hoang A. Molina A. Chieffo N. Smith L.A. Karlou M. et al.Effects of abiraterone acetate on androgen signaling in castrate-resistant prostate cancer in bone.J. Clin. Oncol. 2012; 30: 637-643Crossref PubMed Scopus (155) Google Scholar). Using a GR immunohistochemistry (IHC) assay optimized for use in bone marrow samples, we quantified the percentage of GR-positive tumor cells and dichotomized the data based on clinical response. Patients who continued to benefit from therapy for greater than 6 months were defined as good responders, whereas those in whom therapy was discontinued earlier than 6 months due to a lack of clinical benefit were classified as poor responders (Figure 3A). Consistent with the designation of good versus poor clinical response based on treatment status at 6 months, 11 of 13 good responders but only 1 of 14 poor responders had a maximal PSA decline greater than 50% (Figure 3B). Akin to the findings in the preclinical model, GR positivity at baseline was low—3% of tumor cells in good responders and 8% in poor responders. Of note, 3 of 22 tumors had evidence of high GR expression at baseline (≥20% of tumor cells), and all three had a poor clinical response (Figures 3C and 3D). At 8 weeks, the mean percentage of GR-positive cells was higher than baseline levels in both response groups but was more significantly elevated in poor responders (29% versus 8%, p = 0.009). In addition, the percentage of GR-positive cells at 8 weeks was significantly higher in poor compared to good responders (29% versus 10%, p = 0.02) (Figures 3C and 3D), and similar results were obtained when the analysis was limited to patients from whom matched baseline and 8 week samples were available for analysis (Figure 3E). Furthermore, when GR IHC data were dichotomized based on PSA decline instead of clinical response, GR induction was also associated with a limited PSA decline (Figure S2). These findings establish a correlation between GR expression and clinical response to enzalutamide and raise the possibility that AR inhibition may induce GR expression in some patients. The fact that poor PSA decline also correlates with GR expression raises the question of whether transcriptional regulation of a canonical AR target gene may be regulated by GR.Figure S2GR Induction Dichotomized Based on PSA Response, Related to Figure 3Show full captionGR IHC scores in matched baseline and 8 week samples (same as in Figure 3E) dichotomized based on maximal PSA response ± SEM. Comparisons are by Mann-Whitney test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) GR IHC scores in matched baseline and 8 week samples (same as in Figure 3E) dichotomized based on maximal PSA response ± SEM. Comparisons are by Mann-Whitney test. Having implicated GR as a potential mediator of antiandrogen resistance, we next asked whether restored AR pathway activity also plays a role by comparing the mRNA transcript levels of 74 direct AR target genes in control, 4 day, and resistant tumors from the validation cohort (Figure S3), as well as eight LREX′ tumors (Figure 4A) (see Experimental Procedures and Table S2 for details on gene selection).Figure 4Variable Expression of AR Target Genes in LREX′ In Vivo and after Glucocorticoid Treatment In VitroShow full caption(A) Normalized expression array signal (Illumina HT-12) of a suite of 74 AR target genes in control (n = 10), 4 day (n = 8), and LREX′ (n = 8, right) xenograft tumors. Genes are ranked by degree of restoration of expression in resistant tissue ([Res-4 day]/[Control-4 day]). All resistant tissues were continued on antiandrogen treatment through time of harvest.(B) Fractional restoration values of each of the 74 AR targets in LREX′ xenografts (n = 8) or resistant tissues from the validation cohort (n = 12, see also Figure S3).(C) GR mRNA in resistant tissues used in (B).(D) Relative Expression ± SEM of AR target genes in the LREX′ cell line in steroid depleted media after 8 hr of treatment with the indicated agonists in vitro. Enzalutamide, 10 μM; V, Vehicle.See also Figures S3 and S4.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Normalized expression array signal (Illumina HT-12) of a suite of 74 AR target genes in control (n = 10), 4 day (n = 8), and LREX′ (n = 8, right) xenograft tumors. Genes are ranked by degree of restoration of expression in resistant tissue ([Res-4 day]/[Control-4 day]). All resistant tissues were continued on antiandrogen treatment through time of harvest. (B) Fractional restoration values of each of the 74 AR targets in LREX′ xenografts (n = 8) or resistant tissues from the validation cohort (n = 12, see also Figure S3). (C) GR mRNA in resistant tissues used in (B). (D) Relative Expression ± SEM of AR target genes in the LREX′ cell line in steroid depleted media after 8 hr of treatment with the indicated agonists in vitro. Enzalutamide, 10 μM; V, Vehicle. See also Figures S3 and S4. Consistent with the data generated in the pilot cohort (Figure 1A), some AR target genes in resistant tissues showed elevated levels relative to control (SGK1 and STK39), whereas other genes (NDRG1, TIPARP, and PMEPA1) showed no evidence of restored expression. To examine restoration of AR signaling across the entire set of 74 target genes, we calculated a fractional restoration value using log 2 transformed expression values and the equation (resistant – 4 day)/(control – 4 day). With this approach, a gene whose expression in resistant tissue equals the expression in control tumors calculates as 1, whereas a gene whose expression in resistance equals its expression after 4 days of antiandrogen treatment equals 0. (Values greater than one indicate hyperrestoration in resistance relative to control, and values below zero suggest further inhibition as compared to acute treatment.) These data confirmed that the pattern of restoration varied gene by gene, but this pattern was consistent in LREX′ xenografts and in the validation cohort tumors (Pearson r 0.64, p = 7.54 × 10−10; Figure 4B). This finding is most consistent with a model in which AR remains inhibited in drug-resistant tumors, but expression of certain AR target genes is restored by an alternative transcription factor, possibly GR. The fact that restoration values were somewhat higher in the LREX′ analysis correlates with higher GR expression in these tumors (Figure 4C). To determine whether GR can drive expression of this subset of AR target genes, we compared, in vitro, DHT-induced (AR) and dexamethasone (Dex)-induced (GR) expression of seven AR targets that represent the spectrum of restoration noted in the in vivo analysis, as well as PSA (Figure 4D). All eight genes were regulated by DHT as expected, and this regulation was blocked by enzalutamide. Thus, AR signaling remains intact and can be inhibited by antiandrogens in these drug-resistant cells, making an AR-dependent mechanism of drug resistance less likely. In contrast to DHT, the effect of Dex on these same target genes was variable but closely matched the pattern observed in drug-resistant xenografts. For example, Dex strongly induced SGK1 and STK39 but did not induce TIPARP, NDRG1, and PMEPA1. Of note, KLK3 (PSA) was comparably induced by either DHT or Dex, providing evidence that persistent PSA expression in patients responding poorly to enzalutamide could be driven by GR. As expected, enzalutamide did not notably affect Dex activity. To confirm that this pattern of GR-dependent gene expression is not unique to LREX′ cells, we introduced a GR-expressing retrovirus into parental LNCaP/AR cells and observed a similar pattern of DHT- versus Dex-induced gene expression (Figures S4A and S4B). To be sure that the effects of Dex in these models are mediated through GR, we cotreated cells with a previously described competitive GR antagonist that lacks AR binding called compound 15 (Wang et al., 2006Wang J.C. Shah N. Pantoja C. Meijsing S.H. Ho J.D. Scanlan T.S. Yamamoto K.R. Novel arylpyrazole compounds selectively modulate glucocorticoid receptor regulatory activity.Genes Dev. 2006; 20: 689-699Crossref PubMed Scopus (78) Google Scholar). Compound 15 significantly decreased expression of Dex-induced genes, confirming that Dex activity in the LREX′ model is GR dependent (Figure S4C). Lastly, small interfering RNA (siRNA) experiments targeting AR confirmed that AR is not necessary for Dex-mediated gene activation (Figure S4D). Collectively, these experiments demonstrate that GR is able to drive expression of certain AR target genes independent of AR. To explore AR and GR transcriptomes in an unbiased fashion, we performed expression profiling after short-term treatment of LREX′ cells with DHT or Dex in the presence or absence of enzalutamide. AR and GR signatures were respectively defined as all genes with absolute expression change greater than 1.6-fold (FDR < 0.05) after 1 nM DHT or 100 nM Dex treatment (Table S3). Of the 105 AR signature genes and 121 GR signature genes, 52 were common to both lists (Figure 5A). An even larger proportion of AR or GR signature genes (>80%) showed evidence of regulation by the reciprocal receptor using different thresholds for expression differences (Table S3). Heatmap analysis of these genes confirmed significant overlap in DHT- versus Dex-induced gene expression and showed that Dex-induced gene expression is not impacted by enzalutamide treatment (Figure 5B). These findings support the hypothesis that GR activity can bypass enzalutamide-mediated AR inhibition by regulating a distinct but significantly overlapping transcriptome. We next addressed the question of whether transcriptomes of enzalutamide-resistant tumors are more likely to be explained by AR- or GR-driven gene expression using gene set enrichment analysis (GSEA). To define gene sets that distinguish AR and GR activity, expression of AR and GR signature genes was first evaluated by GSEA in the DHT- and Dex-treated samples from which they were derived. As expected, GR signature genes were enriched in the Dex-treated samples, and AR signature genes were enriched with DHT treatment (Figure 5C). Because several of the genes did not distinguish AR and GR status due to their overlapping transcriptional activities, we refined the lists into AR selective genes (defined as the AR-induced signature genes that were also more highly expressed in DHT-treated samples relative to Dex treated samples, n = 39) and GR selective genes (defined as the converse, n = 67) (Table S3). GSEA analysis of these selective gene lists revealed that GR selective genes were strongly enriched in the enzalutamide-resistant LREX′ tumors, whereas AR selective genes were strongly enriched in the control tumors (Figure 5D). These data provide compelling, unbiased evidence that drug resistance is associated with a transition from AR- to GR-driven transcriptional activity. One prediction of this model is that GR should occupy a substantial portion of AR binding sites in drug-resistant cells. To address this question, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) experiments to define AR and GR DNA binding sites in LREX′ cells after DHT and Dex treatment, respectively. Of note, 52% of the AR binding sites identified after DHT treatment were bound by GR after Dex treatment (Figure 5E). We examined the remaining 48% of AR peaks more closely to be sure that these peaks were not scored as GR negative simply because they fell just below the threshold set by our peak calling parameters. When we plotted the average AR and GR signal as a measure of the relative strength of AR and GR peaks, we found little evidence of GR binding at the AR unique sites (Figure S5A), confirming that these peaks were indeed unique to AR. Next we conducted motif analysis to explore potential differences between AR/GR overlap versus AR unique sites. The core ARE/GRE consensus sequence was present in both groups (66% and 68% of peaks), but AR/GR overlap peaks were relatively enriched for the FoxA motif (64% versus 45% of peaks; p = 2.2 × 10−16) (Figure 5E). Similar analysis of the GR cistrome defined GR unique and AR/GR overlap peaks and revealed that a higher proportion of GR binding sites were unique to GR. Interestingly, GR unique peaks were highly enriched for the FoxA motif (Figure 5F), whereas the classic ARE/GRE was not reported by the motif discovery algorithm (MEME) and was found only 25% of the time. Although these cistrome studies provide evidence of substantial overlap between AR and GR binding sites in enzaluamide-resistant cells, several lines of evidence indicate that the transcriptional differences in DHT- versus Dex-induced gene expression cannot be explained solely by DNA binding. For example, ChIP RT-qPCR experiments showed significant AR and GR DNA binding at genes induced by both receptors (SGK1, FKBP5, PSA) but also at genes such as NDRG1 that are transcriptionally activated by DHT, but not Dex (Figure S5B). Integrative ChIP-seq and transcriptome analysis provided further evidence that DNA binding is not sufficient to determine transcriptional competence. Of the 56 AR signature genes found to have an AR binding peak, 49 showed at least some transcriptional regulation by GR (1.2-fold expression change, p < 0.05). 38 of these 49 GR regulated genes (78%) had an overlapping AR/GR binding peak, confirming substantial overlap at coregulated genes. But GR peaks were also found in three of the seven AR targets genes (43%) with no apparent GR transcriptional regulation (Figure S5C). Others have reported evide" @default.
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• W2080748037 date "2013-12-01" @default.
• W2080748037 modified "2023-10-17" @default.
• W2080748037 title "Glucocorticoid Receptor Confers Resistance to Antiandrogens by Bypassing Androgen Receptor Blockade" @default.
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• W2080748037 doi "https://doi.org/10.1016/j.cell.2013.11.012" @default.
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