Molecular insights into DDX3X-androgen receptor mRNA regulation via non-canonical G-quadruplex in castration-resistant prostate cancer.

We report a new mechanism of androgen receptor (AR) mRNA regulation and cytoprotection in response to AR pathway inhibition (ARPI) stress in prostate cancer (PCA). AR mRNA translation is coordinately regulated by RNA binding proteins, YTHDF3 and G3BP1. Under ambient conditions m6A-modified AR mRNA is bound by YTHDF3 and translationally stimulated, while m6A-unmodified AR mRNA is bound by G3BP1 and translationally repressed. When AR-regulated PCA cell lines are subjected to ARPI stress, m6A-modified AR mRNA is recruited from actively translating polysomes (PSs) to RNA-protein stress granules (SGs), leading to reduced AR mRNA translation. After ARPI stress, m6A-modified AR mRNA liquid–liquid phase separated with YTHDF3, while m6A-unmodified AR mRNA phase separated with G3BP1. Accordingly, these AR mRNA messages form two distinct YTHDF3-enriched or G3BP1-enriched clusters in SGs. ARPI-induced SG formation is cell-protective, which when blocked by YTHDF3 or G3BP1 silencing increases PCA cell death in response to ARPI stress. Interestingly, AR mRNA silencing also delays ARPI stress-induced SG formation, highlighting its supportive role in triggering this stress response. Our results define a new mechanism for stress adaptive cell survival after ARPI stress involving SG-regulated translation of AR mRNA, mediated by m6A RNA modification and their respective regulatory proteins.

Prostate cancer (CaP) driven by androgen receptor (AR) is treated with androgen deprivation; however, therapy failure results in lethal castration-resistant prostate cancer (CRPC). AR-low/negative (ARL/-) CRPC subtypes have recently been characterized and cannot be targeted by hormonal therapies, resulting in poor prognosis. RNA-binding protein (RBP)/helicase DDX3 (DEAD-box helicase 3 X-linked) is a key component of stress granules (SG) and is postulated to affect protein translation. Here, we investigated DDX3-mediated posttranscriptional regulation of AR mRNA (messenger RNA) in CRPC. Using patient samples and preclinical models, we objectively quantified DDX3 and AR expression in ARL/- CRPC. We utilized CRPC models to identify DDX3:AR mRNA complexes by RNA immunoprecipitation, assess the effects of DDX3 gain/loss-of-function on AR expression and signaling, and address clinical implications of targeting DDX3 by assessing sensitivity to AR-signaling inhibitors (ARSI) in CRPC xenografts in vivo. ARL/- CRPC expressed abundant AR mRNA despite diminished levels of AR protein. DDX3 protein was highly expressed in ARL/- CRPC, where it bound to AR mRNA. Consistent with a repressive regulatory role, DDX3 localized to cytoplasmic puncta with SG marker PABP1 in CRPC. While induction of DDX3-nucleated SGs resulted in decreased AR protein expression, inhibiting DDX3 was sufficient to restore 1) AR protein expression, 2) AR signaling, and 3) sensitivity to ARSI in vitro and in vivo. Our findings implicate the RBP protein DDX3 as a mechanism of posttranscriptional regulation for AR in CRPC. Clinically, DDX3 may be targetable for sensitizing ARL/- CRPC to AR-directed therapies.

This project is to study the molecular basis of LSD1-dependent AR suppression in prostate cancer cells. Androgen receptor (AR) is highly expressed in prostate cancer (PCa) and plays a pivotal role in tumor growth. Patients generally respond to androgen deprivation therapy (ADT). However, the tumors invariably relapse, and these relapsed tumors (called castration-resistant prostate cancer, CRPC) are generally more aggressive and relatively resistant to current AR antagonist treatments. Significantly, these relapsed tumors express increased levels of AR mRNA and express multiple AR regulated genes, indicating that AR transcriptional activity has been restored. Despite the critical role AR plays in PCa development and progression to CRPC, the mechanisms that contribute to the consistent substantial increases in AR mRNA in CRPC are poorly understood. Using a VCaP xenograft model, we initially detected a rapid and substantial upregulation of AR mRNA upon castration, suggesting a negative feedback loop regulating AR mRNA levels, which may make a significant contribution to increasing AR mRNA in CRPC (Cancer Research 2009, 69:6027). In a recent published study (Cancer Cell 2011, 20:457), we reported that the agonist liganded AR binds to an enhancer in the second intron of the AR gene, and that increased AR gene expression and progression to CRPC is associated with increased H3K4 methylation and increased recruitment of other transcription factors to this site. Significantly, in contrast to AR function as a transcriptional activator on previously studied androgen responsive elements (AREs), we found that the agonist liganded AR functions as a direct transcriptional repressor at this site. The repression is due to AR recruitment of an H3K4 demethylase, LSD1. Interestingly, AR binding to this site and repression of AR gene expression occurs at slightly higher androgen levels than those required for classical androgen regulated genes, consistent with a negative feedback loop to regulate AR signaling. Pathway analyses of additional AR repressed genes showed marked enrichment for genes mediating DNA synthesis and cell cycle progression, while AR stimulated genes were associated with lipid/protein synthesis and cellular metabolism. Significantly, a set of these androgen repressed genes that are associated with proliferation are overexpressed in CRPC. Taken together, these results indicate that the agonist liganded AR functions as a transcriptional repressor on a subset of genes that enhance proliferation. We propose that ADT is effective because it initially suppresses all AR functions, but that the partial restoration of androgen levels and AR activity in CRPC cells may provide a strong growth advantage by stimulating cellular metabolism without downregulation of AR repressed genes that enhance cellular proliferation. Combining the expression array data and ChIP-seq analysis on AR in VCaP and VCaP-derived castration-resistant VCS2 cells, we have identified gene subsets with AR direct binding on the gene locus. Interestingly, while AR binding is highly enriched (∼2fold) in AR-activated gene subset, the binding is less enriched (1.4fold) in AR-suppressed gene subset. The lower enrichment could mean that fewer genes in the AR –repressed group are directly regulated by AR, but could also be in part technical and reflect somewhat weaker binding of AR to AR-repressed genes. To further elucidate the molecular basis for AR suppression function, we focused on the role of LSD1 in the global AR transcriptional repression as previous results showed that expressions of a number of AR-suppressed genes are dependent on LSD1 activity. LSD1 was initially identified in corepressor complexes and shown to function by demethylating mono- and dimethylated H3K4. However, it was subsequently shown to also function as a coactivator through demethylation of repressive mono- and dimethylated H3K9 when associated with AR. Our data indicate that the association with AR does not determine the coactivator versus corepressor function of LSD1, and that it is instead determined by properties of the element to which it is being recruited. Through genome wide ChIP-seq analysis of LSD1 in VCaP and LNCaP cells, we found that LSD1 binding is significantly enriched for both AR-activated and –repressed (both ∼1.5fold) genes, suggesting that both AR activation and repression are dependent on LSD1 activity. Furthermore, we searched the transcription factor-binding motif on LSD1 binding sites. While E2F1 is highly enriched for LSD1 binding in both activation and repression loci, the ZBTB (zinc finger and BTB domain containing) transcription repressor binding motif was only significantly enriched in repression loci. Among this family, PLZF and LRF are highly expressed in prostate cancer cells. Therefore, the current work has focused on these two factors and we propose that LSD1 mediates AR gene suppression through interaction with ZBTB transcription repressors. Citation Format: Changmeng Cai, Housheng He, Sen Chen, X. Shirley Liu, Myles Brown, Steven P. Balk. LSD1 mediates global AR transcription suppression in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B12.

Hormonal regulation of androgen receptor messenger RNA in the medial preoptic area of the male rat.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract In patients with castration-resistant prostate cancer (CRPC), clinical resistances such as androgen receptor (AR) mutation, AR overexpression, and AR splice variants (ARVs) limit the effectiveness of second-generation antiandrogens (SGAs). Several strategies have been implemented to develop novel antiandrogens to circumvent the occurring resistance. Here, we found and identified a bifunctional small molecule Z15, which is both an effective AR antagonist and a selective AR degrader. Z15 could directly interact with the ligand-binding domain (LBD) and activation function-1 region of AR, and promote AR degradation through the proteasome pathway. In vitro and in vivo studies showed that Z15 efficiently suppressed AR, AR mutants and ARVs transcription activity, downregulated mRNA and protein levels of AR downstream target genes, thereby overcoming AR LBD mutations, AR amplification, and ARVs-induced SGAs resistance in CRPC. In conclusion, our data illustrate the synergistic importance of AR antagonism and degradation in advanced prostate cancer treatment. Editor's evaluation The present study reports the discovery and preclinical evaluation of a novel therapeutic agent for the treatment of castration-resistance prostate cancer through inducing degradation of androgen receptor. The major strength of this study is the identification of a novel lead compound and its interesting in vitro and in vivo activities in prostate cancer models. https://doi.org/10.7554/eLife.70700.sa0 Decision letter eLife's review process Introduction Prostate cancer (PCa) is one of the most common cancers and the second leading cause of cancer-related death for men in western countries (Siegel et al., 2022; Sung et al., 2021). Advanced PCa initially responds to androgen deprivation therapy (ADT), but invariably fails and recurs as lethal castration-resistant prostate cancer (CRPC) (Harris et al., 2009; Desai et al., 2021). Androgen receptor (AR) signaling plays a crucial role in the progress and survival of CRPC (Dai et al., 2017). Second-generation antiandrogens (SGAs), such as enzalutamide (ENZa), abiraterone, apalutamide, and darolutamide, improve the overall survival time and decline prostate-specific antigen (PSA) levels in patients with CRPC (Sternberg et al., 2020; Armstrong et al., 2019; Smith et al., 2021; Smith et al., 2022; Ryan et al., 2015; de Bono et al., 2011). Despite the initial benefit of these agents, their success in treating CRPC has been eliminated by the emergence of drug resistance. Multiple possible mechanisms for the development of drug resistance have thus far been identified, including mutations in the AR LBD, amplification of AR, expression of AR splice variants (ARVs), and intra-tumoral de novo androgen synthesis (Buttigliero et al., 2015; Robinson et al., 2015; Karantanos et al., 2015). Therefore, more effective therapies are urgently required to conquer the SGAs drug resistance. Several strategies have been implemented to develop novel antiandrogens to circumvent the occurring resistance. The first strategy is to develop new competitive antiandrogens targeting the AR hormone-binding pocket (HBP) site, such as darolutamide (Smith et al., 2022). Another strategy is to target the AR signaling axis beyond the HBP site, which includes activation function-1 (AF1), activation function-2, binding function 3, and the DNA binding site through active compounds, such as EPI-001, VPC-14449 (Caboni and Lloyd, 2013). Recently, down-regulating both AR protein and AR mRNA levels has attracted attention due to their potential in the discovery and development of new antiandrogens. The most exciting progress is AR degradation based on the proteolysis targeting chimeras (PROTACs) concept, with various of these AR PROTACs developed with a DC50 (drug concentration that results in 50% protein degradation) potency up to 1 nM. However, low cell permeability, poor pharmacokinetic properties, and complex chemical structures may restrict the clinical application of PROTAC drugs (He et al., 2020). What’s more, LBD-targeted AR PROTACs cannot degrade ARVs which were associated with unfavorable clinical outcomes in patients with CRPC (Fettke et al., 2020). The selective estrogen receptor degrader fulvestrant approved by the FDA in 2002 expanded treatment choices for advanced breast cancer (Bross et al., 2003), which gave rise to next-generation novel degraders with promising antitumor activity in recent years (Nardone et al., 2019). Bradbury et al., 2011 suggested that similar specific downregulation or degradation of AR might be proved beneficial in the treatment of CRPC. Therefore, selective AR degraders (SARDs) which could synthetically degrade and antagonize AR may be an efficient strategy to overcome the drug resistance in the antiandrogen therapy of CRPC. Based on structural modification of the AR antagonists and the tissue-selective AR agonist enobosarm, Miller et al. designed a series of SARDs, namely UT-155, UT-69, and UT-34, which could induce AR ubiquitin-proteasome degradation via binding to AF-1 of the AR to reduce its stability (Ponnusamy et al., 2017; Ponnusamy et al., 2019; Hwang et al., 2019). Notably, the degradation potency of these compounds for ARVs is quite limited. In the present study, we determined that Z15 screened by rational drug design as an AR antagonist and degrader via direct binding to the AR LBD and AR AF1, could overcome AR LBD mutations, AR amplification, and ARVs-induced SGAs resistance of CRPC in vitro and in vivo. Results Identifying Z15 as an AR inhibitor To develop novel AR inhibitors and overcome antiandrogen resistance, we previously constructed a common molecular characteristic pharmacophore model, and screened ~7.5 million compounds from the ZINC lead-like database and ChemDiv database. About 47,202 compounds matched more than four features of the filtering model. Next, these compounds were docked into the HBP of the antagonistic AR. Then, compounds with the top 1000 docking scores were chosen for ADMET prediction by Discovery Studio v3.5. Finally, 80 hits with high drug-likeness were selected and purchased for further bioactivity evaluation (Figure 1—figure supplement 1 and Supplementary file 1a). To preliminarily evaluate the influences for AR transcriptional activity of these 80 candidates, human prostate cancer cells PC-3 co-transfected with wild-type AR (wt-AR) and PSA-luc were incubated with 5α-dihydrotestosterone (DHT) and 10 μM candidate compounds for 24 h. The cell lysates were collected and AR transcriptional activity was detected by dual-luciferase reporter assay. We identified 19 compounds that showed more than 25% AR transcription inhibition activity, among which compound Z15 (structure shown in Figure 1A) exhibited the most potent AR inhibition activity (Figure 1—figure supplement 2A). Nevertheless, the glucocorticoid receptor (GR) transcription inhibition activity of Z15 was quite feeble (Figure 1—figure supplement 2B–C). Figure 1 with 4 supplements see all Download asset Open asset Z15 specifically inhibits the transcription activity of AR and AR mutants. (A) Chemical structure of Z15. (B) Dual-luciferase reporter assay to measure PSA-luc reporter luciferase activities in PC-3 cells co-transfected with Renilla, AR, and PSA promoter expression vector plasmids, stimulated by 5 nM DHT, and treated with different concentrations of Z15 for 24 hr. (C) LNCaP, (D) VCaP, (E) and 22Rv1 cells co-transfected with Renilla and PSA promoter expression vector plasmids, stimulated by 5 nM DHT, and treated with different concentrations of Z15 for 24 h. (F) Dual-luciferase reporter assay to measure PSA-luc reporter luciferase activities in LNCaP cells stimulated by 5 nM DHT, and treated with different concentrations of Z15 or ENZa for 24 hr. (G) Dual-luciferase reporter assays to measure MMTV-luc reporter luciferase activities in PC-3 cells co-transfected with Renilla and MMTV promoter expression vector plasmids stimulated by 100 nM Dex, and treated with different concentrations of Z15 for 24 hr. (H) Dual-luciferase reporter assays to measure PSA-luc reporter luciferase activities in PC-3 cells co-transfected with Renilla, AR_T877A mutation, and PSA promoter expression vector plasmids stimulated by 5 nM DHT treated with different concentrations of Z15 for 24 hr. (I) PC-3 cells co-transfected with Renilla, AR_F876L mutation, and PSA promoter expression vector plasmids, treated with different concentrations of Z15 for 24 hr. All experiments were performed in triplicate. Results are shown as mean ± sd. *p<0.05, **p<0.01, ***p<0.001 vs DHT or Dex group. ENZa, enzalutamide; DHT, dihydrotestosterone; Dex, dexamethasone; Mif, mifepristone. Z15 selectively suppresses AR and AR mutant transcriptional activity To further investigate the AR inhibition potency of Z15, we optimized the synthesis route and prepared a sufficient amount of Z15 (Figure 1—figure supplement 3). Next, we performed a dual-luciferase reporter assay in several human PCa cell lines including wt-AR-transfected PC-3 and LNCaP cells. The results indicated that Z15 could inhibit DHT-induced transcriptional activities of both exogenous and endogenous AR in a dose-dependent manner (Figure 1B–C). Unexpectedly, Z15 showed potent AR transcription inhibition activity in AR overexpression and ENZa-insensitive VCaP cells (Figure 1D). In another ENZa resistance 22Rv1 cells which naturally express AR and ARV7, Z15 also inhibited DHT-activated AR transcriptional activity (Figure 1E). Moreover, the AR transcription inhibition IC50 (half-maximal inhibitory concentration) of Z15 in LNCaP cells was ~0.22 μM, which was comparable to ENZa (Figure 1F). We further detected the selectivity of Z15 in GR-positive PC-3 cells, the results indicated that Z15 hardly inhibited dexamethasone activated GR transcriptional activity compared to the GR antagonist mifepristone (Figure 1G). Then, we compared AR, GR, estrogen receptor (ER), and progesterone receptor (PR) transcription inhibition activities of Z15 by dual-luciferase reporter assay. The transcription inhibition IC50 of Z15 was 0.41 μM for AR (Figure 1—figure supplement 4A), over 20 μM for GR and ER (Figure 1—figure supplement 4B–C), and 9.29 μM for PR (Figure 1—figure supplement 4D), which suggests that Z15 is a highly selective AR inhibitor. AR LBD point mutations such as AR T877A (a flutamide-resistant mutation) and AR F876L (ENZa- and apalutamide-resistant mutation), are key causes leading to antiandrogen resistance. Dual-luciferase reporter assay results indicated Z15 could efficiently inhibit DHT-induced both AR T877A and AR F876L transcriptional activities (Figure 1H–I). Taken together, these data illustrate Z15 as a potent selective AR inhibitor both for wild-type and mutated ARs. Z15 inhibits the AR pathway Next, we assessed the influence of Z15 on LNCaP cells transcriptome by RNA-sequencing analysis. Obviously, Z15 dose-dependently inhibited a series of DHT-activated AR downstream genes (Figure 2A). Then, we detected three canonical AR downstream-regulated genes (PSA, PMEPA1, and TMPRSS2) by quantitative real-time PCR (qRT-PCR) assay. The results revealed that Z15 significantly inhibited the mRNA expression levels of these genes (Figure 2B), consistent with the findings of RNA-sequencing. Furthermore, Z15 also decreased DHT-induced PSA mRNA levels in the antiandrogen resistance 22Rv1 and VCaP cells (Figure 2C). Figure 2 with 3 supplements see all Download asset Open asset Z15 downregulates AR target genes and ARlevels. (A) LNCaP cells treated with vehicle, 0.5, or 5 μM Z15 in the presence of 5 nM DHT for 24 hr before performing RNA-sequencing. Heatmap shows the expression levels of AR target genes. (B) The mRNA levels of PSA, PMEPA1, and TMPRSS2 measured by quantitative-PCR and normalized to GAPDH in LNCaP cells treated with vehicle or different concentrations of Z15 in the presence of 5 nM DHT for 24 hr. (C) The mRNA levels of PSA measured by quantitative-PCR and normalized to GAPDH in 22Rv1 and VCaP cells treated with vehicle or different concentrations of Z15 in the presence of 5 nM DHT for 24 hr. (D) Western blot analysis of LNCaP cells treated with indicated concentrations of Z15 in the presence of 5 nM DHT for 24 hr, before cell lysing and determining PSA and AR protein levels. (E) Western blot analysis performed in 22Rv1 cells. (F) Western blot analysis performed in VCaP cells. (G) Western blot analysis of LNCaP cells treated with indicated concentrations of Z15 in the absence of DHT for 24 hr, before cell lysing, and determining AR protein levels. (H) Western blot analysis of 22Rv1 cells treated with indicated concentrations of Z15 in the absence of DHT for 24 hr, before cell lysing, and determining AR protein levels. Experiments were performed in triplicate. Results are shown as mean ± sd. *p<0.05, **p<0.01, ***p<0.001 vs DHT group. We further detected the influence of Z15 on AR and PSA protein levels in LNCaP cells. As demonstrated in Figure 2D, Z15 reduced DHT-activated PSA protein levels significantly, which was in line with the qRT-PCR analysis. Surprisingly, AR protein levels were also downregulated by Z15, quite different from the effects of ENZa (Figure 2—figure supplement 1A–B). Notably, Z15 potently inhibited PSA and AR protein levels in ENZa resistance 22Rv1 and VCaP cells (Figure 2E–F and Figure 2—figure supplement 1C–G). Then, we evaluated the AR DC50 of Z15 in LNCaP and 22Rv1 cells. The AR DC50 of Z15 in LNCaP cells was 1.05 μM (Figure 2G and Figure 2—figure supplement 1H), while in 22Rv1 cells it was 1.16 μM and the ARV7 DC50 was 2.24 μM (Figure 2H and Figure 2—figure supplement 1I). In addition, we performed a 4D-label free proteomics study to analyze the effect of Z15 on global protein levels in LNCaP cells. Among 5334 quantifiable proteins, AR LBD-targeted PROTAC molecule ARV-110 significantly reduced 34 proteins and Z15 downregulated 69 proteins compared to the DHT group (Figure 2—figure supplement 2 file 1d-e). Both Z15 and ARV-110 reduced AR, KLK3, and TMPRSS2 protein levels significantly (Figure 2—figure supplement 2A–B). KEGG analysis also proved that these two compounds had a similar influence on the functional pathways (Figure 2—figure supplement 2C–D). Additionally, to verify the specificity of Z15 downregulated AR protein levels, we chose 3 AR pathway related but independent proteins GR, HSP90 (AR chaperonin), and cyclin-dependent kinases 7 (CDK7) as controls. Western blot analysis indicated that Z15 has no influence on GR, HSP90, and CDK7 protein levels in 22Rv1 cells (Figure 2—figure supplement 3). Collectively, these data suggest that Z15 is a novel specific AR pathway inhibitor, which may play a role as an AR antagonist as well as an AR and ARV7 degrader. Z15 inhibits DHT-induced AR nuclear translocation Androgen-binding initiates AR activation, induces its conformational change, and reveals the nuclear localization signal of AR. The hormone-bound AR dimerizes and translocates to the nucleus, where it binds to DNA and interacts with a series of transcriptional coregulators to regulate target gene expression. Accordingly, we investigated whether Z15 disturbed androgen-induced AR nuclear translocation. As shown in Figure 3A–B, the DHT treatment could promote the importing of AR into the nuclear compared to untreated group, while both ENZa and Z15 blocked DHT-induced AR nuclear translocation. This result proves that Z15 can inhibit DHT-induced AR nuclear translocation. Figure 3 Download asset Open asset Z15 inhibits AR nuclear localization. (A) Nuclear localization of AR in LNCaP cells treated with vehicle or 5 μM compounds in the presence of 5 nM DHT for 4 h. (B) Quantitative analysis of AR nuclear localization.Experiments were performed in triplicate. Z15 binds directly to AR LBD and AR AF1 Since the chemical structure of Z15 is remarkably different from that of previously reported AR antagonists, we next evaluated whether Z15 directly binds to AR in a similar manner as ENZa. The AR competitive binding assay was performed to demonstrate the direct interaction between Z15 and AR, whereby compounds in competition with the radioligand [3H] DHT in cytosolic lysates from LNCaP cells were measured. Synthetic androgen R1881 displayed strong binding potency to AR with an IC50 value of 0.45 nM, which indicated the feasibility of this assay system. The binding affinity between ENZa and AR was 121.2 nM. Interestingly, Z15 showed a comparable binding affinity to ENZa, with an IC50 value of 63.3 nM (Figure 4A). In addition, our fluorescence polarization assay demonstrated Z15 could compete with androgen binding to AR LBD (Figure 4—figure supplement 1). Besides, the biolayer interferometry (BLI) measurement also revealed that both ENZa and Z15 possess AR LBD binding ability (Figure 4B, Figure 4—figure supplement 2A). These data suggested that Z15 could antagonize AR by directly targeting the LBD region. AR LBD targeted compound ARV-110 has been shown as an efficient AR degrader in preclinical research, however, it could not induce ARV7 degradation in 22Rv1 cells (Figure 4—figure supplement 3A–C). Since Z15 could degrade both AR and ARV7, we wondered if Z15 could also bind to other regions of AR to induce ARV7 degradation. Hence, we investigated the binding affinity between Z15 and AR AF1, as AF1 is an important drug target region of AR. The surface plasmon resonance assay indicated that Z15 could directly bind to AR AF1 with a KD value of 0.93 μM (Figure 4C). Z15 was also detected to potently bind to AR AF1 with a comparable binding affinity to AR AF1 inhibitor UT-34 by BLI assay (Figure 4—figure supplement 2B–C). Unexpectedly, UT-34 could not induce ARV7 degradation in 22Rv1 cells from western blot analysis (Figure 4—figure supplement 3D–F). As a control, we did not find any binding potency between AR AF1 and ENZa even at 200 μM (Figure 4—figure supplement 2D). These data illustrate that Z15 potently inhibits ARV7 by directly binding to AR AF1. Figure 4 with 3 supplements see all Download asset Open asset Z15 directly binds to AR. (A) Competitive binding assay to detect binding affinity of R1881, ENZa, and Z15 to AR LBD, 1 nM radioligand [3H] DHT and LNCaP cytosol were used. (B) Biolayer interferometry measurements of Z15 binding to AR LBD. (C) Sensorgram and steady state fitted results of surface plasmon resonance assay to detect binding affinity between Z15 and AF1. Experiments were performed in triplicate. Z15 promotes AR degradation through the proteasome pathway We have shown that Z15 could reduce AR and ARV7 protein levels and conjectured that it is an AR degrader. To confirm this hypothesis, we detected the influence of Z15 on AR protein and mRNA levels in LNCaP cells without DHT treatment. Certainly, Z15 reduced AR protein levels in a dose-dependent manner without influencing the AR mRNA levels (Figure 5A and Figure 5—figure supplement 1A). Moreover, we observed similar effects of Z15 on AR protein and mRNA levels in ENZa resistance cell lines 22Rv1 (Figure 5B and Figure 5—figure supplement 1B–C) and VCaP cells (Figure 5C and Figure 5—figure supplement 1D). Western blot analysis for AR in LNCaP cells treated with protein synthesis inhibitor cycloheximide, showed that Z15 accelerated AR degradation (Figure 5D and Figure 5—figure supplement 1E). These data indicate that Z15 is indeed an AR degrader. Figure 5 with 1 supplement see all Download asset Open asset Z15 promotes AR degradation in proteasome pathway-dependent manner. A-C Western blot analysis of AR protein levels, and quantitative-PCR normalized to GAPDH of AR mRNA levels in LNCaP (A), 22Rv1 (B), and VCaP (C) cells treated with indicated concentrations of Z15 in the absence of DHT for 24 hr. (D) Western blot analysis of AR in LNCaP cells treated with 100 μg/mL CHX in the presence or absence of 5 μM Z15 for indicated time points. (E) Western blot analysis of AR protein levels in LNCaP and VCaP cells treated with 5 μM Z15 or/and 5 μM MG 132 for 8 hr. (F) Immunoprecipitation done using anti-AR and immunoblotting with anti-Myc antibody in 22Rv1 cells co-transfected with Myc-tag CW7-UB plasmids treated with or without 5 μM Z15 in the presence of 5 μM Mg132 for 12 hr. Input: immunoblot of lysates probed with AR antibody. Experiments were performed in triplicate. All results are shown as mean ± sd. CHX, cycloheximide. The ubiquitin-proteasome pathway (UPP) is the main participant that regulates intracellular protein degradation. To explore whether Z15 promoted AR degradation through UPP, LNCaP cells were treated with Z15 in the presence or absence of proteasome inhibitor MG132. Indeed, Z15 reduced the AR protein levels after 8 hr treatment, while AR protein levels reduction was counteracted by MG132. Similarly, Z15 induced AR protein decline was also counteracted by MG132 in VCaP cells (Figure 5E and Figure 5—figure supplement 1F–G). Furthermore, Z15 treatment strikingly induced ubiquitination of AR (Figure 5F). Together, these results indicate that Z15 degrades AR through the UPP. Z15 inhibits proliferation and induces in CRPC cell lines As Z15 exhibited AR and ARV7 inhibition and degradation we next investigated the effects of Z15 on cell proliferation activity in CRPC cell lines VCaP and 22Rv1 cells. In VCaP cells, Z15 showed comparable proliferation inhibition potency with ARV-110 However, in 22Rv1 cells, the proliferation inhibition activity of Z15 was than that of ARV-110 Both Z15 and ARV-110 displayed inhibition effects on the proliferation activity of PC-3 and cells (Figure To the effects of Z15 on CRPC cell activity, we 22Rv1 and PC-3 cells to 1 μM Z15 or ARV-110 for 2 As a Z15 significantly decreased the 22Rv1 cell compared to both and both Z15 and ARV-110 showed no influence on the PC-3 cell (Figure we proved that through and AR, Z15 could selectively inhibit the proliferation of AR and ARV7 CRPC cell Furthermore, based on PCa we PCa and treated the with 1 μM Z15 for 7 The results indicated that Z15 significantly inhibited PCa proliferation compared to the group (Figure What’s more, western blot analysis indicated that Z15 also promoted the of VCaP and 22Rv1 cells in a dose-dependent manner and while Z15 showed no influence on the of AR cells (Figure and Figure supplement Figure with 1 supplement see all Download asset Open asset Z15 selectively inhibits proliferation and induces of CRPC cells. (A) VCaP, and PC-3 cells treated with different concentrations of Z15 or ARV-110 for hr, cell proliferation detected by assay. (B) of PC-3 and 22Rv1 cells treated with or without 1 μM Z15 or ARV-110 for were (C) PCa treated with 1 μM Z15 or for 7 observed by (D) Western blot analysis of protein levels in VCaP cells treated with indicated concentrations of Z15 for 24 hr. (E) Western blot analysis of protein levels in 22Rv1 cells treated with indicated concentrations of Z15 for 24 hr. Experiments were performed in triplicate. Results are shown as mean ± sd. *p<0.05, **p<0.01, ***p<0.001 vs group. Z15 inhibits CRPC experiments proved that Z15 is a selective AR degrader and antagonist with activity in To evaluate the PCa inhibition activity of Z15 in we of 22Rv1 cells in the of an of were treated with vehicle control, 10 Z15, or 20 Z15 for were no effects observed in as assessed by the (Figure of with 10 and 20 Z15 both suppressed 22Rv1 and decreased the significantly (Figure In addition, western blot analysis indicated that AR, ARV7, and PSA protein levels in the were significantly in both 10 and 20 Z15 treatment (Figure Figure supplement analysis also revealed that Z15 reduced the and PSA protein levels in (Figure Taken together, our data indicate that Z15 could inhibit the of CRPC both in vitro and in vivo. Figure 7 with 1 supplement see all Download asset Open asset Z15 suppresses 22Rv1 progress in vivo. (A) from 22Rv1 cells treated with control, or 20 Z15 a for by (B) other (C) on the (D) Western blot analysis of AR, ARV7, and PSA protein levels in (E) analysis of proliferation and PSA levels in Results are shown as mean ± sd. *p<0.05, **p<0.01, ***p<0.001 vs group. Identifying Z15 as AR inhibitor Since Z15 showed CRPC inhibition we the and ZINC database to Z15 chemical structure compounds (Figure supplement Supplementary file with more than to Z15 were for further bioactivity Dual-luciferase reporter assay indicated that most of these compounds inhibited DHT-activated AR transcriptional activity at 1 μM for (Figure supplement that the group plays an role in the AR inhibitory activity of Z15 and its Western blot analysis revealed that these active also reduced AR and DHT-induced PSA protein levels (Figure supplement Furthermore, we detected the AR transcription inhibition IC50 of active Z15 Among these and exhibited the AR transcription inhibition while also showed comparable AR inhibition activity compared to Z15 (Figure Western blot analysis revealed that these active could reduce AR and PSA protein levels in a dose-dependent manner. Notably, of these compounds showed AR downregulation activity than Z15 (Figure and Figure supplement Together, these results indicate that through chemical structural modification to Z15, more and more selective AR degraders with AR inhibition activity might be found in the Figure 8 with 3 supplements see all Download asset Open asset Z15 comparable AR inhibition (A) Dual-luciferase reporter assay to measure PSA-luc reporter luciferase activities in LNCaP cells stimulated by 5 nM DHT, and treated with different concentrations of indicated compounds for 24 hr. (B) AR transcription inhibition (C) Western blot analysis of PSA and AR protein levels of LNCaP cells treated with indicated concentrations of Z15 and its in the presence of 5 nM DHT for 24 hr. Results are shown as mean ± sd. Experiments were performed in triplicate. Discussion SGAs are more in the clinical treatment of patients with CRPC. However, drug resistance by AR mutation, AR amplification, and has been reported to restrict the clinical of these therapies (Buttigliero et al., 2015; Robinson et al., 2015). AR a crucial target for CRPC therapeutic development of its key function in the progress of CRPC. In this study, we identified a compound Z15 that selectively inhibited AR transcriptional activity and significantly downregulated AR target genes at the mRNA and protein levels. studies proved that Z15 could bind directly to both AR LBD and AR AF1, as to androgen-induced AR nuclear which Z15 as an AR Moreover, Z15 could also degrade AR and ARV7 through the proteasome pathway (Figure several Z15 exhibited AR inhibition and downregulation potency than Z15, that Z15 is a promising lead compound for further chemical structure Figure Download asset Open asset The of Z15 inhibits the AR pathway and antiandrogen resistance. Z15 binds to both AR LBD and AR AF1, AR nuclear AR promotes AR and ARVs degradation through the proteasome as to overcome AR mutation, AR overexpression, and ARVs-induced antiandrogen resistance. AR amplification is a common in CRPC patients antiandrogens treatment. data showed that ENZa could hardly DHT-induced PSA levels in VCaP cells, which that AR is to overcome the drug resistance by AR rational to this

The rational drug design combining the bioassay identified a novel selective androgen receptor (AR) degrader for both AR and AR-VRs and illustrated the synergistic importance of AR antagonism and degradation in advanced prostate cancer treatment.

The rational drug design combining the bioassay identified a novel selective androgen receptor (AR) degrader for both AR and AR-VRs and illustrated the synergistic importance of AR antagonism and degradation in advanced prostate cancer treatment.

In advanced prostate cancer, cells become androgen independent and therefore resistant to androgen ablation therapy. One mechanism by which prostate cancer bypasses androgen ablation therapy is acquisition of androgen receptor (AR) mutations some of which render it promiscuous enabling activation by a broad group of steroids such as estrogen, progesterone, and even anti-androgens. Genistein, an exogenous steroid (phytoestrogen) and isoflavone found in soy, has estrogenic activity. Most studies have shown that genistein has anti-proliferative effects on prostate cancer cells without regard for the status of the AR. It has been suggested that activation of the mutant AR (MT-AR) by genistein could lead to increased cellular proliferation. Previous studies examining genistein's effect on AR expression used prostate cell lines (i.e. LNCaP) with ARs carrying the promiscuous T877A mutation, without comparison to cells with wild type (WT) AR. Genistein does not bind WT-AR, and thus, may not have the same effect on AR expression as it does in the presence of MT-AR. We set out to compare the effects of genistein in the presence of WT-AR versus MT-AR, using human prostate cancer cell lines with WT-AR (LAPC-4) and MT-AR (LNCaP). Cells were treated with increasing concentrations (0, 0.5, 1, 10, 25, and 50 μM) of genistein. Real time PCR, Western blot analysis, PSA luciferase assay, cell counting by hemocytometer, and MTS proliferation assays were used to determine levels of AR mRNA, protein, transcriptional activity, and cell proliferation, respectively. Genistein caused an androgen-independent biphasic changes in AR mRNA, protein levels, and transcriptional activity, and in cell proliferation in LNCaP cells, which reached a maximum at 1μM of genistein (40-45% increase in AR protein and mRNA expression, 42% increase in PSA luciferase activity, and 2-fold increase in cell proliferation compared to controls). These effects reversed at a concentration of 10 μM. In contrast, in LAPC-4 cells AR mRNA and protein levels and transcriptional activity as well as cell proliferation decreased linearly with increasing dose of genistein, without showing the stimulatory bi-phasic trend observed in LNCaP cells. These decreases were significant, beginning at 1 μM genistein, at which concentration AR mRNA was reduced by 30%, protein expression by 22%, PSA luciferase activity by 40%, and cell proliferation by 56%. These results demonstrate that at physiological concentrations, genistein can exert a mitogenic effect in the presence of MT-AR. Our findings highlight the significance of promiscuous mutations such as T877A for the AR response to genistein treatment and indicate that men with advanced prostatic cancers which carry such AR mutations could be adversely affected by genistein. (Supported in part by Grant No. CA116195) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1864. doi:10.1158/1538-7445.AM2011-1864

Autoregulation of androgen receptor (AR) mRNA was investigated using Northern blot analysis with AR cDNA fragments as probes. The amount of AR mRNA increased 2- to 10-fold with androgen withdrawal and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney, and brain, and in a human prostate cancer cell line, LNCaP. In rat ventral prostate, AR mRNA increased 2- to 3-fold within 24 h after castration and remained elevated for 4 days. Treatment with testosterone propionate beginning 24 h after castration reduced ventral prostate AR mRNA 4-fold within 8 h of androgen replacement. Administration of estradiol 24 h after castration had no significant effect on prostatic AR mRNA. Androgens, including testosterone and the synthetic androgen methyltrienolone (R1881), or the antiandrogen cyproterone acetate down-regulated AR mRNA in vitro in LNCaP cells, whereas estradiol was without effect. Administration of testosterone propionate to rats with androgen insensitivity did not decrease AR mRNA. Down-regulation of AR mRNA by androgen is therefore a receptor-mediated process which occurs in vivo in rat tissues that differ in androgen responsiveness and in cultured human prostate cells.

Androgen and estrogen receptor mRNA status in apocrine carcinomas.

Sex differences in androgen receptor mRNA levels and regulation in hamster facial motoneurons.

The effects of androgen withdrawal and replacement on the concentrations of androgen receptor (AR) protein and AR mRNA were investigated in rat ventral prostate and seminal vesicles and in cultured human hepatoma (HepG2) cells. AR mRNA concentrations were determined by Northern blotting with single stranded AR cRNA as the hybridization probe, whereas antibodies raised against two synthetic 17-amino acid long peptides corresponding to the N-terminal and steroid-binding regions of the AR were employed in immunological receptor assays. AR mRNA levels in both prostate and seminal vesicles increased about 2-fold within 24 h after castration and continued to rise within the next 48 h to values that were 9- to 11-fold higher than those in intact controls. Administration of pharmacological doses of testosterone (400 micrograms steroid/day) to 1-day castrated animals for 24-48 h brought about a decrease in AR mRNA levels in accessory sex organs to levels in intact controls. Similar results were obtained in cultured HepG2 cells where a switch to serum- and steroid-free medium elicited a rapid increase (approximately 4-fold in 10 h) in the AR mRNA level, which was prevented by inclusion of 10(-7) M testosterone in culture medium. Similar, but quantitatively less marked, changes occurred in the AR protein concentration in prostate, seminal vesicles, and HepG2 cells, as determined by immunoblotting using antibodies against AR peptides. In addition, immunohistochemical studies showed that AR is a nuclear protein of the prostatic epithelial cells in both intact and castrated rats, and suggested that short term castration increases the concentration of nuclear AR in the prostate. Taken together, these data indicate that androgens down-regulate the concentration of AR protein and AR mRNA in a variety of target tissues.

Background: Docetaxel (doc) is currently used as first-line therapy in castration-resistant prostate cancer (CRPC). However, a significant number of patients are irresponsive to the drug. To better understand this inter-individual variability, we studied the impact of c-jun phosphorylation, a major pathway of doc-induced apoptosis, in prostate cancer (PCa) cells. Furthermore, we validated the effect of doc treatment on the androgen receptor (AR) and c-jun gene alteration. Methods: All in vitro experiments were performed on human PCa cell lines- PC-3, LNCaP and LNCaP cells cultured in bicalutamide for 4 weeks (LNb4). To examine the role of c-jun in doc therapy, AR-negative PC-3 cells were transfected with c-jun or c-jun, mutated at serine 63/73 (c-junA) or co-transfected with AR and c-jun or c-junA. Quantitative real time PCR (qPCR) of c-jun, AR, PSA, KLK2, NKX3.1 and c-Myc mRNA was used to determine the impact of doc treatment on gene expression. Proliferation and Western blotting assays were performed to analyze the treatment efficacy and protein expression, respectively. Results: A pronounced increase in cell proliferation, in c-junA-transfected cells, in presence or absence of doc was observed, suggesting that phosphorylation of c-jun at Ser63/73 might be necessary to respond to doc treatment. Moreover, cells that were co-transfected with AR seemed to be more responsive to doc treatment compared to cells transfected with c-junA or c-jun alone. Western blot analysis revealed that cells which were co-transfected with AR and c-jun exhibit a strong band for AR compared to other transfection groups. Time-dependent gene expression alterations after exposure to doc were observed in LNCaP and LNb4 cells. In LNCaP cells, doc treatment lead to a decreased in AR, but an increased in PSA mRNA. In contrast, a significant increase in AR mRNA was observed in LNb4 cells, whereas PSA mRNA levels gradually decreased after an initial flare. Expression pattern of KLK2 mRNA was similar to that of PSA in both cell types. NKX3.1 mRNA expression in LNCaP cells showed a significant increase after 48h, whereas in LNb4 cells, NKX3.1 expression significantly increased already after 6h but then gradually decreased throughout 48h of exposure to doc. No significant changes in the mRNA expression of c-jun or c-Myc were observed. Conclusions: Our results indicate functional significance of phosphorylated c-jun and AR in doc treated PCa cells. Moreover, we also provide further evidence of AR role in treatment resistance mechanisms. Conclusively, our study shows promising results that merits further in-depth investigation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C226. Citation Format: Nishtman Dizeyi, Martina Viktoria Tinzl, Shao-yong Chen, Julius Semenas, Per-Anders Abrahamsson. Phosphorylation of ser63/73 in c-jun is required to respond to docetaxel treatment in prostate cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C226.

Lipid nanoparticles to silence androgen receptor variants for prostate cancer therapy.

Heat shock proteins (HSP) are involved in processes of folding, activation, trafficking and transcriptional activity of most steroid receptors including the androgen receptor (AR). Accumulating evidence links rising heat shock protein 27 (HSP27) levels with the development of castration-resistant prostate cancer. In order to study the functional relationship between HSP27 and the AR, we modulated the expression of the small heat shock protein HSP27 in human prostate cancer (PC) cell lines. HSP27 protein concentrations in LNCaP and PC-3 cells were modulated by over-expression or silencing of HSP27. The effects of HSP27 on AR protein and mRNA levels were monitored by Western blotting and quantitative RT-PCR. Treatment for the AR-positive LNCaP with HSP27-specific siRNA resulted in a down-regulation of AR levels. This down-regulation of protein was paralleled by a decrease in AR mRNA. Most interestingly, over-expression of HSP27 in PC-3 cells led to a significant increase in AR mRNA although the cells were unable to produce functional AR protein. The observation that HSP27 is involved in the regulation of AR mRNA by a yet unknown mechanism highlights the complexity of HSP27-AR signaling network.