Extensive biochemical and molecular experiments showed that the COPI retrograde complex regulates ligand-mediated AR transcriptional activation, which correlated with the mobilization of the Golgi-localized ARA160 coactivator to the nuclear compartment of prostate tumor cells. Collectively, this study provides a bioanalytical strategy to validate the AR-interactome and define novel AR-interacting proteins involved in ligand-mediated AR activation in prostate tumor cells. Moreover, we describe a cellular system to study how compartment-specific AR-interacting proteins influence AR activation and contribute to aberrant AR-dependent transcription that underlies the majority of human prostate cancers.Aberrant androgen receptor (AR)-dependent transcription is a hallmark of human prostate cancers. At the molecular level, ligand-mediated AR activation is coordinated through spatial and temporal protein-protein interactions involving AR-interacting proteins, which we designate the “AR-interactome.” Despite many years of research, the ligand-sensitive protein complexes involved in ligand-mediated AR activation in prostate tumor cells have not been clearly defined. Here, we describe the development, characterization, and utilization of a novel human LNCaP prostate tumor cell line, N-AR, which stably expresses wild-type AR tagged at its N terminus with the streptavidin-binding peptide epitope . A bioanalytical workflow involving streptavidin chromatography and label-free quantitative mass spectrometry was used to identify SBP-AR and associated ligand-sensitive cytosolic proteins/protein complexes linked to AR activation in prostate tumor cells. Functional studies verified that ligand-sensitive proteins identified in the proteomic screen encoded modulators of AR-mediated transcription, suggesting that these novel proteins were putative SBP-AR-interacting proteins in N-AR cells. This was supported by biochemical associations between recombinant SBP-AR and the ligand-sensitive coatomer protein complex I (COPI) retrograde trafficking complex  TMPRSS2-ERG) that place the ETS family of oncogenic transcription factors (i.e. ERG and ETV1) under the direct control of AR (–i.e. metastatic) castration-resistant prostate cancers. To date, multiple mechanisms are known to elicit aberrant AR activity and thereby facilitate the proliferation and survival of castration-resistant prostate cancers in the context of castration levels of androgens. These include the expression of constitutively active AR splice variants, gain-of-function AR mutations, increased expression of androgen-biosynthesis genes, ligand-independent AR activation, aberrant AR coregulator expression, gain-of-function mutations in steroidogenesis enzymes, and activation of the glucocorticoid receptor bypass pathway , which target the AR signaling axis to disrupt aberrant AR activity in early and late stage human prostate cancers , cellular proteins of diverse function that coactivate or corepress AR-mediated transcription (e.g. PTEN and HIP1), and specific transcription factors (e.g. ERα and FOXA1)  and liganded (i.e. androgen-stimulated) states in LNCaP prostate tumor cells. Label-free directed mass spectrometry (dMS) facilitated the identification and quantitation of ligand-sensitive proteins. The proteomic data set enriched for the AR-interactome and functional studies verified that ligand-sensitive proteins encoded modulators of AR-mediated transcription in LNCaP cells. Further exploration of ligand-sensitive proteins showed that the coatomer protein complex I (COPI) retrograde complex encoded novel SBP-AR-interacting proteins that are functionally linked to AR-mediated transcription in LNCaP cells. Moreover, biochemical studies showed that AR was localized to the Golgi-enriched protein fraction (GEPF) in a ligand-sensitive manner in LNCaP cells. Interestingly, whereas AR-dependent transcription was attenuated by chemical or genetic disruptions of the COPI complex, androgen-mediated nuclear localization of AR was unperturbed in the context of these treatments. In contrast, the nuclear accumulation of the Golgi-localized coactivator ARA160 was disrupted under the same experimental conditions. These results demonstrated that androgen-mediated nuclear mobilization of ARA160 was required for AR-dependent transcription. This study provides a molecular framework for defining compartment-specific, ligand-sensitive AR-interacting proteins involved in androgen-mediated AR activation in prostate tumor cells.To this end, we developed a cellular system to identify ligand-sensitive AR-interacting protein complexes in prostate tumor cells using quantitative mass spectrometry. More specifically, streptavidin chromatography was used to affinity-purify streptavidin-binding peptide-tagged wild-type AR (SBP-AR) from the cytosolic compartment in the unliganded (A) (dK = 2.5 nm) to facilitate the isolation of SBP-AR-interacting proteins from N-AR cells using streptavidin affinity chromatography . The commercial monoclonal AR antibody AR441, which binds to a conserved epitope in AR-T887A and SBP-AR, confirmed that AR immunoreactivity was higher in N-AR cells than in LNCaP and NC cells . Overall, these results validated SBP-AR expression in N-AR cells.The AR-interactome represents a diverse population of proteins that regulates AR function at the molecular level . This prFig. 1A) , 19. Thetography . WesternC). SBP-AR staining was restricted to the cytoplasmic space in vehicle-treated cells, phenocopying the subcellular localization of AR-T877A in LNCaP cells  (m R1881) , demonstrating that the ligand-dependent cytoplasmic-nuclear translocation of SBP-AR was preserved in N-AR cells. Concordant with the IF results, a cytoplasmic decrease and a corresponding nuclear increase in SBP-AR were observed in androgen-treated N-AR cells . These results showed that in N-AR cells, SBP-AR undergoes a ligand-dependent translocation from the cytoplasm to the nucleus that is identical to that of AR-T877A in LNCaP prostate tumor cells  . In contor cells .AR to selectively knock down AR-T877A. SBP-AR-dependent expression of the luciferase reporter could then be measured in N-AR cells in the absence of AR-T877A. As predicted, siRNAs that targeted the AR coding regions greatly attenuated the expression of SBP-AR and AR-T877A in N-AR cells . In contrast, 3′-UTR AR siRNAs exclusively attenuated AR-T877A expression in N-AR cells . These results demonstrated that SBP-AR transcriptional activity could be measured in N-AR cells through selective knockdown of AR-T877A. To measure SBP-AR transcriptional activity in N-AR cells, LNCaP and N-AR cells were co-transfected with the probasin-luciferase reporter and control siRNAs or siRNAs targeting either the coding sequence of AR or its 3′-UTR (F and G). As predicted, AR-dependent luciferase activity was strongly attenuated in LNCaP cells co-transfected with either the coding sequence- or 3′-UTR AR-directed siRNAs . Similarly, and as predicted, coding sequence-directed AR siRNAs strongly attenuated luciferase activity (i.e. ∼5%) in N-AR cells . However, attenuated luciferase activity was less pronounced (i.e. ∼40%) in N-AR cells transfected with the 3′-UTR AR siRNAs . This result suggested that ∼35% of AR transcriptional activity was dependent on SBP-AR after efficient knockdown of AR-T877A in N-AR cells. Overall, these results showed that SBP-AR undergoes ligand-dependent cytoplasmic-nuclear translocation, is transcriptionally activated by androgens in N-AR cells, and phenocopies AR-T877A functions in prostate tumor cells. Therefore, N-AR cells represent a valid cellular system in which to study SBP-AR-dependent functions in prostate tumor cells.We then wanted to determine whether SBP-AR was functionally active and mediates transcription in N-AR cells. Therefore, N-AR cells were transfected with the androgen-responsive rat probasin luciferase reporter and treated with androgens to determine whether SBP-AR could mediate luciferase reporter expression similar to that of AR-T877A in LNCaP prostate tumor cells . To ablai.e. 96 h) N-AR cells that were challenged (i.e. 1 h) with vehicle  or androgen . In essence, SBP-AR would be purified from the AD (i.e. inactive) and AS (i.e. active) cytosolic protein extracts to facilitate the purification of SBP-AR-interacting protein complexes under these treatment conditions. For the SBP-AR purification experiment, equal amounts of the AD and AS cytosolic protein extracts were subjected to streptavidin affinity chromatography involving low-stringency washes (to preserve the association of low-affinity SBP-AR-interacting proteins and protein complexes). The streptavidin affinity-purified samples were eluted, quantified, and subjected to Western blotting analysis for purification of SBP-AR. As shown in Fig. 3A, SBP-AR was efficiently purified from AD and AS cytosolic protein extracts. The remaining purified AD and AS samples were subjected to filter-assisted sample preparation to remove mass spectrometry-incompatible analytes .–25.22–25–analytes . The samB). This included 1,741 proteins in the AD sample and 2,100 proteins in the AS sample (B). A total of 727 proteins overlapped between the AD and AS samples, whereas 1,014 and 1,373 proteins were unique to the AD and AS samples, respectively (B). The results demonstrated a low degree of proteomic overlap between the AD and AS samples. Several factors may have contributed to this proteomic observation. First, cytosolic SBP-AR probably interacts with different types of proteins/protein complexes under the AD versus AS conditions because endogenous AR is localized primarily to the cytosolic compartment under androgen-depleted growth conditions, whereas AR undergoes dynamic cytoplasmic-nuclear recycling under androgen-stimulated growth conditions (i.e. targeted sequencing of preferred list peptide ions) of the dMS approach for comprehensive identification of proteins in complex peptide mixtures (i.e. AS/AD ratio ≤0.5 or AS/AD ratio ≥2) relative to “ligand-insensitive” (i.e. 0.5 < AS/AD ratio < 2) proteins. A total of 493 and 234 proteins were identified as ligand-sensitive and ligand-insensitive, respectively (C). The breakdown of ligand-sensitive proteins included 349 proteins enriched in the AS sample and 144 proteins enriched in the AD sample (C).The dMS analyses resulted in 3,114 non-redundant protein identifications  across the AD and AS samples B. This iS sample B. A totaectively B. The remixtures , 25, 28.ectively C. The brD sample C.D) (D). With the exceptions of the androgen receptor signaling and parkin-ubiquitin proteasomal system protein networks, a greater level of enrichment in the four top-ranked protein networks was observed in the population of ligand-sensitive proteins (D). Overall, these findings suggested that the expression levels of putative SBP-AR-interacting proteins functionally representative of anabolic/catabolic processes were strongly influenced by acute exposure to androgens in N-AR cells.The purpose of our initial bioinformatic analysis was to determine whether there were any differences in functional protein networks represented by the ligand-sensitive and ligand-insensitive population of proteins. Therefore, both populations of proteins were uploaded into the WebGestalt bioinformatic program and subjected to WikiPathway analysis D 29). W. WD) (29D) D. With tproteins D. OveralE). With the exception of the androgen receptor signaling pathway in the six top-ranked pathways, a greater level of enrichment of ligand-sensitive proteins was observed in the AS sample relative to the AD sample. Importantly, enriched networks in the AS sample were concordant with known biochemical processes that modulate AR function(s) at the molecular level in prostate tumor cells. For example, the proteasome system has an active role in androgen-dependent AR transcription, AR trafficking, and AR metabolism in prostate tumor cells (i.e. within hours) stimulate ribosomal RNA synthesis and ribosome biogenesis through an AR-dependent mechanism in prostate tumor cells . Similar to ligand-sensitive overlapped proteins, the top-ranked six pathways included cytoplasmic ribosomal proteins, mRNA processing, translation factors, proteasome degradation, parkin-ubiquitin proteasomal system, and androgen receptor signaling (F). Although the rank order for protein networks encoded by translation factors and mRNA processing were reversed relative to ligand-sensitive overlapping proteins (E), the bioinformatic analysis demonstrated that the composition of enriched protein networks that copurified with SBP-AR was unaffected by the inclusion of unique proteins in either the AD or AS sample. Therefore, ligand-sensitive proteins, both overlapping and unique in the AD and AS samples, were included in subsequent protein network analyses.To expand our understanding of the functional protein networks beyond the existence of overlap in the proteins identified in the proteomic screen, we performed a WikiPathway analysis on unique proteins observed in the AD and AS samples. This bioinformatic analysis tested whether the functional protein networks among the unique proteins were conserved with or distinct from those represented by the ligand-sensitive, overlapped proteins F. Similaignaling F. Althouproteins E, the bii.e. HPRD, BIOGRID, McGill, and STRING) , with 52 enriched in the AS sample, 41 enriched in the AD sample, and 20 categorized as androgen-insensitive  –16. Our B). The six top-ranked pathways identified by the WikiPathway analysis included regulation of androgen receptor activity, androgen-mediated signaling, androgen receptor, proteoglycan syndecan-mediated signaling, IGF1, and plasma membrane estrogen receptor signaling (B). The AR-interactome was enriched at higher levels for all of the top-ranked networks relative to the subpopulation of the AR-interactome identified in the proteomic screen. Thus, the subpopulation of the AR-interactome detected in the proteomic screen was not enriched for protein networks related to AR function relative to the entire AR-interactome.Next, the top-ranked pathways between the AR-interactome and the subpopulation of the AR-interactome identified in the proteomic screen were compared to determine whether specific functional protein networks related to AR function were selectively enriched in the proteomic screen B. The siignaling B. The ARi.e. AD sample) or androgen-stimulated (i.e. AS sample) conditions. WikiPathway analysis of the six top-ranked pathways identified regulation of androgen receptor activity, androgen-mediated signaling, androgen receptor, coregulation of androgen receptor activity, IGF1, and plasma membrane estrogen receptor signaling (C). There was greater enrichment for the top-ranked pathways in the AD sample, which suggests that these protein networks may be active under conditions of androgen depletion to modulate AR function in prostate tumor cells (C). This result was concordant with the enrichment of the androgen signaling pathway among ligand-sensitive proteins in the AD sample (E and F). Overall, the bioinformatic analyses demonstrated that the proteomic screen enriched for the AR-interactome and molecular pathways related to AR function in prostate tumor cells.Last, we examined whether specific protein networks related to AR function were selectively enriched under the androgen-depleted  and androgen-stimulated (i.e. AS sample) conditions. PPI networks of the AR-interactome and proteins in the proteomic screen were constructed using the Protein Interaction Network Analysis (PINA) program and visualized with the Cytoscape software program (–A), whereas the proteomic screen contained 1,455 nodes with 8,358 edges (A and B). NetworkAnalyzer (A), whereas the proteomic screen contained ∼11 neighbors/node (B). This finding showed that there was a higher degree of connected neighbors in the PPI network of the AR-interactome relative to proteins detected in the proteomic screen. We constructed PPI networks of ligand-insensitive and ligand-sensitive interactomes to further characterize the molecular topology of PPIs among this subpopulation of proteins in the proteomic screen (C–E). Furthermore, the ligand-sensitive interactome was separated into the AD and AS interactome to provide greater resolution of PPI networks between the androgen-depleted and androgen-stimulated conditions (D and E). The ligand-insensitive interactome contained 120 nodes with 384 edges with ∼3 neighbors/node (C). Similarly, ∼4 neighbors/node were computed for the AD interactome, which consisted of 364 nodes and 787 edges (D). In contrast, ∼14 neighbors/node were detected in the AS interactome, which displayed 844 nodes and 6,243 edges (E). These results showed that the AS interactome contained a higher degree of connected edges and highlighted the complexity of PPIs between the AD and AS interactomes in the proteomic screen. Overall, the results suggested that androgens facilitated the recovery of more highly connected PPI networks among proteins detected in the proteomic screen.Next, we sought to elucidate the molecular topology of protein-protein interaction (PPI) networks represented by the AR-interactome relative to proteins identified in the proteomic screen. This bioinformatic analysis was expected to achieve three goals. First, it would resolve the molecular composition and highlight the connectivity between the AR-interactome and proteins identified in the proteomic screen. Second, it would facilitate the comparative analysis of PPI networks between the AR-interactome and proteins identified in the proteomic screen. Third, it would enable us to explore differences in the molecular topology of PPI networks related to AR function between the androgen-depleted  (A and B). Interestingly, the AS sample contained a single PIN module , and it was identical to the PIN module detected in the AD sample (B). To elucidate biological pathways associated with each PIN module, the proteins represented by PIN module 160, detected in the AD sample, and PIN module 39, detected in both the AD and AS samples, were subjected to WikiPathway analysis (A and B). The pathways enriched in PIN module 160 of the AD sample included prostate cancer/AR signaling pathways and cell cycle/DNA replication pathways (A). These same pathways were enriched in PIN module 39, but this module was also enriched for the proteasome/ubiquitin and mRNA processing pathways (B). These results were concordant with the finding that under androgen-stimulated conditions (i.e. AS sample), protein modules related to the proteasome/ubiquitin, mRNA processing, and AR signaling pathways were enriched in the proteomic screen (E and F).Next, ligand-sensitive PPIs with SBP-AR were explored to determine whether protein interaction network (PIN) modules functionally linked to androgen-mediated AR activation were enriched in the proteomic screen. Therefore, the WebGestalt Protein Interaction Network Module Analysis program was used to identify statistically significant PIN modules enriched in either the AD or AS samples . PIN mod 0.0257) , A and BD sample B. To eluanalysis , A and Bpathways A. These pathways B. These c screen , E and Fi.e. hsp90 and immunophilins)  (i.e. tubulin and filamin)  and regulatory  complexes of the 26S proteasome were enriched in the AD and AS samples (A). The molecular chaperones interact with unliganded SHRs in the cytosolic compartment, as inactive protein complexes, to modulate SHR function at the molecular level (B). The next class of ligand-sensitive proteins included the karyopherins, which mediate the transport of molecules between the cytoplasm and the nucleus (C) and are known to mediate cytoplasmic nuclear AR trafficking (39–D and E). The last group of proteins examined included the motor proteins, which mediate vesicle trafficking and organelle transport but have also been linked to ligand-mediated SHR activation (F). Notably, the dynein isoforms, which are minus-end-directed motor proteins, were enriched in the AD and AS samples. The discordant enrichment of dynein protein isoforms was also observed for the plus-end-directed motor proteins kinesin and myosin (F). Of note, myosin VI (MYO6) is a minus-end-directed motor protein that binds AR, regulates AR stability, and modulates AR-dependent transcription in prostate tumor cells  , cytoske myosin) , 39, cytfilamin) –42, and ilamin) –, 43–46. min) –43–. Previoumin) –43–, 47, 48. samples . Notably samples , was enr samples A. The moar level . Many ofar level B. The ne nucleus . Both ka nucleus C and arefficking , 52. Thecking 39–. Interescking 39–, D and Etivation , 39, 53.tivation F. Notabld myosin F. Of notor cells . Overalli.e. attenuation or potentiation) on AR-dependent transcription (i.e. ethanol) or androgen (i.e. 1 nm R1881) for 18 h, and measured luciferase activity was compared between control and target siRNA knockdown cells . As expected, luciferase activity was strongly attenuated in cells transfected with AR siRNAs, which demonstrated that probasin-luciferase expression/activity was AR-dependent in LNCaP cells . Similar to AR siRNA-transfected cells, the majority of experimental siRNAs tested . However, a number . For example, siRNAs directed against COPI, which controls retrograde protein trafficking . In contrast, siRNAs targeting the deubiquitinases had the opposite effect and potentiated luciferase activity. Additionally, siRNAs that targeted enzymes involved in the maturation and post-translational processing of both plasma membrane and membrane-associated receptors strongly modulated luciferase activity . For example, cells transfected with siRNAs against mannosyltransferase POMT1, which mediates serine and threonine protein mannosylation . As expected, more in depth molecular studies will be required to establish a direct functional link between protein trafficking and AR-mediated transcription in prostate tumor cells. Nonetheless, the siRNA-based transcriptional screen revealed that the expression of ligand-sensitive proteins was required for optimal AR transcriptional activity in LNCaP prostate tumor cells.Next we wanted to test whether ligand-sensitive proteins could modulate AR-dependent transcription because this group of proteins is likely enriched for SBP-AR-interacting proteins. Thus, these molecules are predicted to modulate AR transcriptional activity in prostate tumor cells. Therefore, an siRNA-based transcriptional screen in LNCaP cells was utilized to test whether ligand-sensitive proteins had any effect . Moreover, COPI siRNAs strongly attenuated AR-dependent transcription in LNCaP cells (B). Interactions between SBP-AR and the COPD and COPE subunits were selected for further study due to the availability of antibody reagents to detect their expression in LNCaP cells (B). An in vitro binding assay was developed to validate ligand-sensitive interactions between SBP-AR and the components of the COPI complex. For the assay, recombinant SBP-AR (rSBP-AR) was incubated with cytosolic extracts derived from AD and AS cells and subjected to streptavidin affinity chromatography, as performed in the original proteomic experiment . Western blotting analysis showed that COPD and COPE levels were equivalent between the AD and AS samples before the addition of rSBP-AR . However, after the samples were subjected to streptavidin affinity chromatography, both COPD and COPE subunits were enriched in the AS sample relative to the AD sample (D and E). These results showed that rSBP-AR interactions with the COPE and COPD subunits are enhanced in the presence of androgens (D and E), and thus a ligand-sensitive interaction exists between rSBP-AR and components of the COPI complex. They further suggest that the proteomic screen identified ligand-sensitive protein interactions with SBP-AR.Next, experiments were undertaken to validate interactions between SBP-AR and ligand-sensitive proteins observed in the proteomic screen. Components of the COPI complex were selected for further study because each subunit was enriched in the AS sample A. MoreovaP cells B. InteraaP cells B. An in periment . As expeA). Western blotting analysis showed the COPE subunit associated with the Golgi-enriched protein fraction , which validated the biochemical integrity of the Golgi-enriched protein in this sample. Notably, COPE levels were unchanged between the AD and AS sample in both LNCaP and N-AR cells , demonstrating that acute androgen exposure had no measurable effect on COPE levels in the GEPF. Surprisingly, AR was also detected in the GEPFs of LNCaP and N-AR cells , suggesting that it may be associated with the Golgi compartment in prostate tumor cells. Moreover, AR levels in the AS sample were noticeably reduced relative to those in the AD sample , which also showed that AR levels at the GEPF were sensitive to androgens.A biochemical association between SBP-AR and the COPI subunits might suggest that the AR cytoplasmic-nuclear translocation process was physically coupled to the COPI retrograde trafficking complex. Although AR co-localization to the Golgi apparatus has yet to be established, the protein ARA160 has been shown to colocalize to the Golgi and the nucleus , and it Fig. 10m sucrose protein fraction , the 1.2 m sucrose protein fraction (i.e. soluble cytosolic and associated with the heavier membranes of the ER and nucleus), and pellet protein fraction (i.e. nucleoplasmic) were subjected to Western blotting analysis to verify subcellular marker protein expression in LNCaP and N-AR cells (C and D). As expected, the Golgi marker protein GM130 was detected in the GEPF , the molecular chaperone Hsp90 was detected in the 0.8 and 1.2 m sucrose protein fractions , and the nuclear marker histone H3 was restricted to the pellet sucrose protein fraction . These results verified the subcellular composition of the discontinuous sucrose gradient fractions. Interestingly, robust COPE expression was detected in the 0.8 m sucrose protein fraction, with expression nearly undetectable in the GEPF . This comparative Western blotting analysis demonstrated that only a small fraction (i.e. ∼8–13%) of COPE is present in the GEPF compared with the 0.8 m sucrose protein fraction (B to those in C and D). Western blotting analysis of ARA160 showed that it was distributed across the 0.8 m, GEPF, and pellet sucrose protein fractions (C and D). Interestingly, higher levels of ARA160 were detected in the AS sample, which showed that ARA160 levels and/or subcellular compartmentalization were androgen-sensitive in LNCaP and N-AR cells (i.e. 1 h) . Similar to COPE, AR was nearly undetectable in the GEPF  but was robustly detected in the 1.2 m and pellet sucrose protein fractions . These findings showed that AR was present in the GEPF, albeit in a smaller quantity  than the AR present in the 1.2 m and pellet sucrose protein fractions. Moreover, as was the case for ARA160, levels of AR were higher in the nuclear pellet of the AS sample. This finding was in agreement with androgen's role in increasing AR levels in the nuclear compartment through AR cytoplasmic-nuclear translocation. Overall, our biochemical findings are suggestive of a ligand-dependent association between AR and the Golgi compartment in prostate tumor cells.Next, AR levels in the GEPF and other sucrose gradient-derived protein fractions were compared to determine whether the subpopulation associated with the Golgi in LNCaP and N-AR cells is relatively large or small. In addition to the GEPF, the 0.8 AR cells , C and Dractions , C and Di.e. 50 μm) for 30 min, washed to remove BFA, and then challenged with vehicle (i.e. ethanol) or androgen (i.e. 1 nm R1881) for 4, 8, or 12 h. As predicted, androgens increased luciferase activity in a time-dependent manner in vehicle-pretreated cells (A). However, in BFA-pretreated cells, luciferase activity was completely abolished in androgen-treated cells (A). These results showed that the time-dependent increase in androgen-mediated AR transcriptional activity was disrupted by BFA in LNCaP cells. These findings support a functional role of the COPI complex in the process of androgen-mediated AR-dependent transcription in LNCaP prostate tumor cells.Next, we wanted to test whether disruption of the Golgi apparatus had any effect on AR-mediated transcription in prostate tumor cells. First, pharmacological disruption of protein trafficking at the Golgi apparatus would be tested to determine whether it had any impact on AR transcriptional activity in LNCaP cells. The drug brefeldin A (BFA) was selected for this experiment because it binds to and inhibits the activation of ARF1-guanine exchange factors to promote the disassembly of the COPI coat and the subsequent disruption of COPI retrograde trafficking to the Golgi . The exped cells A. Howeveed cells A. These B and C). For the IF experiments, 72-h androgen-depleted LNCaP cells were incubated with BFA for 30 min, washed, and challenged with vehicle or androgen (i.e. 1 nm R1881) for 1 h. The cells were processed for IF analyses and stained for the Golgi marker protein GM130 and AR. As expected, GM130 staining was predominantly perinuclear in vehicle-treated cells, which suggested that it was localized to the Golgi in LNCaP cells . Similar to vehicle-treated cells, the GM130 perinuclear staining pattern was preserved in androgen-treated cells . ARF1 inactivation by BFA promotes dissolution of the Golgi apparatus at the cellular level . Instead, the staining pattern for GM130 was predominantly cytosolic . This staining pattern was preserved in androgen-treated cells . These results suggested that COPI retrograde protein trafficking was compromised in BFA-treated cells. In contrast to the Golgi localization of GM130 in vehicle-treated cells, AR localization was predominantly cytosolic and nuclear under the same conditions . These IF results validate previous studies showing that unliganded AR is primarily cytosolic in prostate tumor cells , which showed that the cytosolic-nuclear AR translocation process was unperturbed in LNCaP cells. Surprisingly, in cells pretreated with BFA, cytosolic and nuclear AR staining was increased in vehicle or androgen-treated cells . These results showed that the cytoplasmic-nuclear AR translocation process was unaffected in BFA-treated cells and that BFA influenced AR expression in LNCaP cells.Next, we explored whether cytoplasmic-nuclear AR translocation was disrupted in BFA-treated cells because this molecular process could be functionally coupled to COPI-mediated retrograde protein trafficking. If these molecular pathways were coupled directly or indirectly, it could explain why BFA strongly attenuated AR transcriptional activity by disrupting AR nuclear localization in LNCaP cells. IF microscopy experiments were performed to determine whether androgen-mediated cytoplasmic-nuclear AR trafficking was disrupted in BFA-treated cells , B and CaP cells B, I-I. Sed cells B, II-I. ar level , and, asor cells . As anti+/K+ ATPase and the nuclear protein marker histone H2A authenticated the purity of subcellular protein fractions because both markers were restricted to the membrane and nuclear protein fractions, respectively (D). As predicted, in vehicle-pretreated cells, unliganded AR was predominantly localized to the cytoplasmic space encompassing the cytosolic and membranous protein fractions . In contrast, liganded AR was primarily localized to the nuclear protein fraction in vehicle-pretreated cells . Similar to vehicle-pretreated cells, unliganded AR was restricted to the cytosolic and membranous protein fractions in BFA-pretreated cells . Importantly, AR levels were reduced in BFA-pretreated cells relative to vehicle-pretreated cells treated with vehicle or androgen . However, similar to the IF results, AR levels in the nuclear compartment increased in BFA-pretreated cells treated with androgens . Again, this result demonstrated that the cytoplasmic-nuclear AR translocation process was unaffected by BFA. Furthermore, because AR levels were noticeably reduced across all protein fractions in BFA-pretreated cells, ARA160 expression was probed across these same protein fractions. In vehicle-pretreated cells, ARA160 was detected only in the nuclear protein fraction of cells challenged with vehicle or androgens . However, in BFA-pretreated cells, a noticeable reduction in ARA160 levels was detected in the nuclear protein fraction . Moreover, detectable levels of ARA160 were observed in the cytosolic and membrane protein fractions of BFA-treated cells . These results demonstrated that BFA changed ARA160 intracellular localization in LNCaP cells.To determine whether BFA had any effects on AR levels in LNCaP cells, crude subcellular fractionated protein extracts were subjected to Western blotting analyses to determine AR levels across the cytosolic, membrane, and nuclear protein fractions of BFA-treated cells. Western blotting analysis of the membrane protein marker Naectively D. As preA). Briefly, 24-h androgen-depleted LNCaP cells were cotransfected with the probasin-luciferase reporter and siRNAs targeted against AR, COPA, COPE, COPG, COPD, and ARF1. The cells were challenged with vehicle (i.e. ethanol) or androgen (i.e. 1 nm R1881) for 18 h, and luciferase activity was determined between control and target siRNA knockdown cells . Quantitative RT-PCR confirmed greater than ∼70% knockdown of COPI mRNAs  in siRNA-transfected cells . Notably, luciferase activity was strongly attenuated in COPA and COPD knockdown cells . Furthermore, siRNAs directed against ARF1, which coordinates the assembly of the COPI complex, strongly attenuated luciferase activity in LNCaP cells . Overall, these results showed that optimal AR transcriptional activity required COPI coatomer expression.The redistribution of nuclear ARA160 to the cytosolic and membrane protein fractions in BFA-treated cells suggested that the nuclear localization of ARA160 was regulated by the COPI complex. ARA160 is a critical coregulator of AR-mediated transcription , and, asFig. 12C–G). For this experiment, 24-h androgen-depleted LNCaP cells were transfected with siRNAs directed against AR, COPA, COPD, COPE, and COPG for 72 h. The cells were challenged for 1 h (i.e. acute) with vehicle (ethanol) or androgen (100 nm R1881), and the cells were subjected to subcellular fractionation to produce crude cytosolic, membrane, and nuclear protein fractions. Western blotting analyses demonstrated the effectiveness of the subcellular fractionation because the membrane marker Na+/K+ ATPase and the nuclear marker histone H2A were primarily restricted to the membrane and nuclear protein fractions in COPI coatomer knockdown cells (C–G). As expected, the cytoplasmic-nuclear AR translocation process remained intact in control knockdown cells because androgens decreased cytosolic levels and increased the nuclear levels of AR . Furthermore, AR levels were undetectable in the cytosolic, membrane, and nuclear protein fractions of AR knockdown cells (C). Notably, AR levels were relatively unchanged in the nuclear protein fraction of coatomer knockdown cells, demonstrating that cytoplasmic-nuclear translocation of AR was unperturbed in these cells (C–G). Next, Western blotting analysis revealed that ARA160 levels were uniformly decreased in the nuclear protein fraction of androgen-treated coatomer knockdown cells . These results suggested that COPI expression was required for optimal nuclear levels of ARA160 in androgen-treated LNCaP cells. Overall, these findings demonstrated that the process of translocating AR from the cytoplasm to the nucleus was unaffected by chemical or genetic disruptions in COPI-mediated retrograde protein trafficking and that the COPI complex regulated nuclear levels of ARA160 in LNCaP prostate tumor cells.Next, we wanted to determine whether disrupted AR transcriptional activity in COPI knockdown cells was correlated with a defect in androgen-mediated ARA160 nuclear localization. Thus, ARA160 levels and subcellular localization were examined in COPI coatomer knockdown cells , C–G. Fown cells , C–G. Aswn cells C. Notablse cells , C–G. NeA), we wanted to determine COPI coatomer expression across normal prostate tissue, localized prostate cancers, and metastatic prostate cancers. Gene expression changes in COPI coatomers  were queried with the Oncomine database (A) –75A), weAR, prostate-specific antigen (PSA), fatty acid synthase (FASN), NK3 homeobox 1 (NKX3.1), and transmembrane protease serine 2 (TMPRSS2) (B). Interestingly, COPI coatomer knockdown had a discordant effect on ARG expression. For example, whereas AR and TMPRSS2 expression was potentiated in COPI coatomer knockdown cells, the opposite effect was observed for PSA and FASN (B). Surprisingly, COPI coatomer knockdown had minimal effect on NKX3.1 expression (B). Interestingly, Western blotting analyses showed a reduction in AR protein in COPD, COPE, and COPG knockdown cells (C). This was an unanticipated finding because it represented a discordant relationship between expression of the AR mRNA and protein in COPI coatomer knockdown cells. As expected, the decrease in levels of the PSA protein was similar to that in of the PSA mRNA in COPI coatomer knockdown cells (C). This result demonstrated a congruent relationship between expression of the PSA mRNA and PSA protein in COPI coatomer knockdown cells. Collectively, these results show that COPI coatomers modulate the expression of AR and PSA in prostate tumor cells.These results prompted us to establish a functional link between COPI coatomer expression and androgen-regulated gene (ARG) expression in prostate tumor cells. Therefore, we wanted to extend these findings by exploring whether COPI coatomer knockdown had any effect on the expression of a subset of ARGs. The ARGs measured included TMPRSS2) , 77. As TMPRSS2) B. Intereand FASN B. Surpripression B. Interewn cells C. This wwn cells C. This re.g. EGFR and glutamate receptor) (D). Densitometry analysis revealed minimal increases in calreticulin and ERGIC-53 levels in AR and COPI coatomer knockdown cells (D). However, measurable decreases in calreticulin and ERGIC-53 were observed in COPE < COPD < COPA < COPG knockdown cells (D). The functional significance of decreased calreticulin and ERGIC-53 in COPI coatomer knockdown cells has yet to be determined. Overall, these results show that the effects of COPI coatomer knockdown on AR-dependent transcription are independent of the UPR pathway in LNCaP prostate tumor cells.Many signaling pathways are connected through COPI retrograde protein trafficking (eceptor) , 79, andeceptor) , are acteceptor) , was proeceptor) D. Densitwn cells D. Howevewn cells D. The fui.e. androgen-depleted) and liganded (i.e. androgen-stimulated) states in prostate tumor cells. Despite years of experimental research, the biochemical composition of AR-interacting protein complexes in the unliganded and liganded states remains incomplete (––i.e. >350 proteins) that comprise the AR-interactome has made it difficult to understand how AR interacts with so many different types of proteins at the molecular level to mediate AR-dependent processes at the cellular level. AR has a finite number of protein interaction interfaces through which to mediate direct interactions between AR and the AR-interactome. Therefore, AR lacks the capacity to simultaneously bind to every member of the AR-interactome as a single supramolecular protein complex. Instead, it is reasonable to expect that both spatial and temporal constraints will determine what members of the AR-interactome bind to AR at the molecular level. This concept is underscored by the important experimental reality that the majority of the AR-interactome was discovered through the application of binary protein interaction assays   is nearly equivalent to those of mouse monoclonal antibodies for their target antigens (i.e. AR-interactome). Last, the dMS approach utilized in this study facilitated an in depth proteomic analysis of the complex peptide samples representative of “streptavidin-copurified” proteins. The dMS approach is based upon principles of a targeted MS/MS acquisition scheme that selectively targets lower abundance ions for MS/MS. Previous studies have shown that targeted LC-MS/MS experiments are more sensitive and robust for the identification of proteins than traditional data-dependent LC-MS/MS experiments  –16. Althisplay) –. Moreoveisplay) –. First, antigens , 88. Thiantigens . Also, santigens . Additioeriments , 25, 90.eriments . Future D and E). These findings validate previous reports of a biochemical interaction between AR and specific subunits of the COPI complex in prostate tumor cells (i.e. NCOR and SMRT) in response to androgens. Additional experiments are warranted to test this hypothesis further. The COPI complex has been indirectly linked to the pathobiology of prostate tumor cells . Interestingly, some of the results of the siRNA luciferase screen were discordant with the purported function of the E3 ligases and deubiquitinases on AR-mediated transcription . For example, MDM2 was shown to promote AR protein degradation . Instead, AR transcriptional activity was attenuated in MDM2 siRNA-transfected cells . Assuming that MDM2 promotes AR protein degradation/turnover, our results suggest that this process is required for normal ligand-dependent AR transcriptional activity in prostate tumor cells. This outcome is supported by a previous study showing that AR turnover and transcriptional activity were blocked in LNCaP cells pretreated with the proteasomal inhibitor MG132 . This protein had initially been identified as an AR coactivator of the PSA gene in prostate tumor cells . Interestingly, RNF6 is also an AR corepressor . USP10 was shown to act as an AR coactivator in WT AR-expressing PC3 cells when AR transcriptional activity was measured using the mouse mammary tumor virus luciferase reporter  that are commonly mutated in human prostate cancers . Amplified DNA was cloned in-frame into the 3′-end of the SBP and FLAG pcDNA3 plasmid, thus generating a pcDNA3-SBP-FLAG-AR plasmid. The DNA was cloned into the 5′ EcoRI and 3′ XhoI restriction sites of the pcDNA3 SBP vector. The SBP sequence used was 5′-ATGGACTACAAGGACGACGAC-3′. The oligonucleotide primers (Invitrogen) used for cloning pcDNA3-SBP-FLAG-AR were as follows: 5′ primer, 5′-GATCGATATCATATGGAAGTGCAGTTAGGGCTGGGAAGGGTCTAC-3′; 3′ primer, 5′-GATCCTCGAGTCACTGGGTGTGGAAATAGATGGGCTTGACTTTCCCA-3′. All constructs were confirmed by sequencing the coding region using both gene-specific and vector-specific primers.LNCaP prostate cancer cells (American Type Culture Collection) were cultured in phenol red-deficient RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum , 1× Glutamax, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). For the generation of cell lines, individual pcDNA3-SBP-FLAG  and pcDNA3-SBP-FLAG-AR cDNAs (N-AR cell line) were transfected into LNCaP cells using the Lipofectamine LTX reagent (Invitrogen) following the manufacturer's instructions. Two days after transfection, cells were selected in Geneticin . STR analysis was used to authenticate the genotype of all human prostate cancer cell lines  .m Tris, pH 7.5, 150 mm NaCl) + 0.1% (v/v) Tween 20)) for 1 h at room temperature and then incubated in TBST containing 5% bovine serum with one of the following antibodies: rabbit polyclonal AR (N-20) antibody ; mouse monoclonal AR  antibody ; mouse monoclonal Na+/K+ ATPase antibody ; rabbit polyclonal PSA antibody ; H2A antibody ; or a mouse monoclonal ARA160 antibody . After three 5-min washes with TBST, the membranes were incubated in TBST containing 5% BSA with goat anti-mouse or goat anti-rabbit HRP-conjugated secondary antibodies  for 1 h at room temperature. After three 5-min washes with TBST, immunoreactive bands were developed and visualized using the ECL reagent kit (Thermo Fisher Scientific), and the membranes were exposed to Hyperfilm ECL film  for <5 min.Total protein extracts were quantified using the Pierce BCA protein assay kit (Thermo Fisher Scientific). 4 μg of total protein lysates were subjected to 4–12% SDS-PAGE (Invitrogen) and transferred to a PVDF membrane (Bio-Rad). Membranes were incubated with Blotto (4% (w/v) nonfat milk in TBST  for 1 h. The medium was removed, and the cells were fixed in DPBS containing 4% formaldehyde for 20 min at room temperature. After three washes with DPBS, nonspecific protein binding sites were blocked with Blotto (4% (w/v) nonfat milk in TBS plus 0.1% (v/v) Triton X-100) for 1 h at room temperature and then incubated with mouse anti-SBP monoclonal antibody (1:50 dilution) for 1 h at room temperature. After washing three times with Blotto + Triton X-100, cells were incubated with Alexa 488 goat anti-mouse antibody, phalloidin, and DAPI nuclear dye (Invitrogen) for 1 h at room temperature, washed three times with DPBS, and mounted in ProLong Gold (Invitrogen). All cells in three randomly chosen fields in three independent samples were imaged using a digital camera at ×10 magnification on an Olympus IX70 inverted microscope. Post-imaging processing was performed using Adobe Photoshop software, taking care to maintain any linear differences in signal intensities present in the original samples.N-AR cells were cultured in androgen-depleted medium (phenol red-deficient RPMI 1640 medium (Invitrogen) supplemented with 10% charcoal-stripped fetal bovine serum , 1× Glutamax, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen)) for 96 h and then treated with vehicle (ethanol) or androgen (100 nm) for 30 min. After one wash with DPBS, cells were treated with vehicle (ethanol) or androgen (100 nm R1881) for 1 h and subjected to fixation and IF labeling with rabbit anti-AR polyclonal antibody  and Alexa 488 goat anti-rabbit antibody as described above.LNCaP cells were cultured in androgen-depleted medium for 96 h and subsequently treated with vehicle (ethanol) or BFA (50 μm R1881) for 1 h. Cytoplasmic and nuclear proteins were isolated from the cells using the Subcellular Protein Fractionation Kit for Cultured Cells according to the manufacturer's guidelines (Thermo Fisher Scientific).NC and N-AR cells were cultured in androgen-depleted medium for 96 h and subsequently treated with vehicle (ethanol) or androgen  or androgen (100 nm R1881) for 1 h. Cells were then harvested and incubated in hypotonic solution  for 10 min and passed through an 18-gauge syringe 15 times. Nuclei were pelleted by centrifugation at 600 × g for 20 min at 4 °C and resuspended in nuclear extraction buffer . The supernatant was then subjected to ultracentrifugation at 100,000 × g for 3 h at 4 °C to separate the membranes (crude microsomes) from the cytosol.LNCaP cells were cultured in androgen-depleted medium for 96 h and subsequently treated with vehicle (ethanol) or 50 μm) was performed using Oligofectamine (Invitrogen), and cells were harvested 72 h post-transfection with lysis buffer . The isolated protein extracts were subjected to Western blotting analysis with antibodies to SBP and AR.Validated siRNAs targeting the coding region or 3′-UTR of AR  were transfected into LNCaP and N-AR cells. Scrambled siRNA was used as a control. Transfection of siRNAs (100 nm) was performed using Oligofectamine  for 72 h, cells were treated with vehicle (ethanol) or androgen (100 nm R1881) for 1 h and subjected to subcellular fractionation and Western blotting analysis as described above.LNCaP cells cultured in androgen-depleted medium for 24 h were transfected with validated siRNAs targeting AR, COPA, COPD, COPE, and COPG (Qiagen). Scrambled siRNA was used as a control. After transfection of siRNAs  with Oligofectamine for 72 h. The isolated total protein extracts were subjected to Western blotting analysis with antibodies to AR, calreticulin, ERGIC-53, and PSA.Ct method . Strand cDNA synthesis was performed using reverse transcription protocols detailed in the SuperScript® III first-strand synthesis kit (Invitrogen). Real-time quantitative PCR was carried out in a reaction containing cDNA, respective primer pairs, and SYBR Green PCR Master Mix (Applied Biosystems). GAPDH was used as an internal control for normalization. Relative expression values were calculated using the comparative t method .2. The cells were cultured in androgen-depleted medium for 24 h and co-transfected with siRNAs targeting the coding region or 3′-UTR of AR  along with pGL4.10-Luc2-probasin (10 ng) and pRLSV40-Renilla (25 ng). After transfection was performed using Lipofectamine 2000 (Invitrogen) for 48 h, the cells were treated with vehicle (ethanol) or androgen (1 nm R1881) for 18 h and harvested for luciferase activity using the Dual-Luciferase reporter assay system  according to the manufacturer's protocol. Analysis of variance was used to determine significant differences between experimental and control siRNA-transfected cells .LNCaP and N-AR cells were seeded into Falcon (BD Biosciences) 48-well tissue culture dishes at a density of 30,000 cells/cmRenilla (25 ng) for 48 h and then treated with vehicle (ethanol) or BFA (50 μm) for 30 min. The cells were then washed with DPBS once and treated with vehicle (ethanol) or androgen (1 nm R1881) for 4, 8, or 12 h. Luciferase activities were then quantified as described above.LNCaP cells were seeded as described above. Transfections were carried out in triplicate with pGL4.10-Luc2-probasin (10 ng) and pRLSV40-m) along with pGL4.10-Luc2-probasin (10 ng) and pRLSV40-Renilla (25 ng).LNCaP cells were seeded as described above, with the exception that the cells were co-transfected with validated siRNAs targeting COPG, COPE, COPA, ARF1, COPD, and AR  was used to transfect the cells.LNCaP cells were seeded as described above, with the exception that the cells were co-transfected with validated siRNAs targeting proteins identified from the proteomic analysis. 62 total siRNAs were purchased as a Flexiplate from Qiagen , and a mix of four siRNAs targeting a single gene  for 1 h. The cells were then harvested and incubated in hypotonic solution  with 5 mm DTT and 1× protease inhibitor mixture (Thermo Fisher Scientific) for 10 min and subjected to nitrogen cavitation at 100 psi for 5 min. Lysed samples were centrifuged at 600 × g for 20 min at 4 °C to pellet the nuclei, and the resulting supernatant was centrifuged at 100,000 × g for 3 h at 4 °C to remove the microsomes (pellet) from the cytosolic proteins. 10 mg of cytosolic proteins were used for the affinity purification of AR protein complexes by incubating overnight with a 250-μl bed volume of UltraLink Plus streptavidin beads (Thermo Fisher Scientific) in AR purification buffer  with 5 mm DTT (Sigma), 1× protease inhibitor mixture (Thermo Fisher Scientific), 5 mm ATP (Sigma), and 0.025% Nonidet P-40 (Sigma) with the addition of vehicle (ethanol) or androgen (100 nm R1881) for AD and AS samples, respectively, at 4 °C. The next day, samples were centrifuged at 500 × g for 1 min to cluster the beads, which were then washed three times with 1 ml of wash buffer . Proteins were eluted with 8 m urea, 50 mm Tris, 1% SDS, 10 mm DTT, and 5 mm Biotin, pH 8.5, at room temperature for 1 h. The samples were dialyzed against 8 m urea, 50 mm Tris, and 100 mm β-mercaptoethanol, pH 8.5, with 10 kDa cut-off dialysis cassettes (Thermo Fisher Scientific) to remove detergent before mass spectrometry analysis. To measure purification efficiency, 1% of the input, void (unbound proteins), wash, and eluate were analyzed by Western blotting with antibodies to SBP and AR.N-AR cells were grown in androgen-depleted medium for 96 h and treated with vehicle  or androgen (100 nm DTT (Thermo Fisher Scientific) for 1 h at 37 °C, alkylated in 55 mm iodoacetamide (Thermo Fisher Scientific) for 1 h at room temperature in the dark, and then digested with trypsin  in 0.5 m urea. Next, each sample was added to a tryptic digest of BSA containing iodoacetic acid alkylated cysteine residues  at a 1:75 BSA/protein molar ratio. Samples were acidified before being desalted on Vydac C18 spin columns  and then subjected to strong cation exchange fractionation on polysulfoethyl A packed spin columns (Nest Group). Briefly, desalted samples were dissolved into strong cation exchange buffer A  and loaded onto strong cation exchange spin columns. Peptides were eluted from the strong cation exchange spin columns using a six-step  KCl elution gradient developed from a mixture of buffer A and buffer B . Salt-bumped, eluted fractions were desalted, dried, and redissolved in mass spectrometry loading buffer .Samples were first reduced in 10 mm/z and acquisition rate of 1 spectrum/s. From these data, an ion preferred list was generated with Agilent MassHunter qualitative software with settings of 400–1,250 m/z, 2+ and 3+ charge states, and spectra with 2 or more ions. The dMS was performed with the following settings: a maximum of 10 ions/cycle, a narrow isolation width (∼1.3 atomic mass units), precursor masses dynamically excluded for 30 s after 8 MS/MS in a 30-s time window, and use of the preferred ion list. Mass spectrometry capillary voltage and capillary temperature settings were set to 1,800 V and 330 °C, respectively. The infused reference mass of 1,221.9906 was used to correct precursor m/z masses for each LC-MS/MS experiment.The samples were analyzed by nano-liquid chromatography-tandem mass spectrometry using an Agilent 6520 Accurate-Mass quadrupole time-of-flight mass spectrometer interfaced with an HPLC Chip Cube. The samples were loaded onto an Ultra High Capacity Chip . LC-MS/MS analysis was performed using a 180-min gradient ranging from 8 to 35% buffer B . Full MS (MS1) data were acquired with a mass range of 400–1250 supplemental Table 1 and Table 4.The raw data files were searched against the UniProt human database using SpectrumMill software version B.04.00.127 and the following settings: precursor mass tolerance of 25 ppm, product mass tolerance of 200 ppm, and a maximum of two trypsin miscleavages. Search modifications included a static carbamidomethylation on cysteine residues (C = 57.02146 atomic mass units), and differential modifications for oxidized methionine (M = 15.9949 atomic mass units), phosphorylated serine, threonine, and tyrosine (STY = 79.9663 atomic mass units), and ubiquitinated lysine (K = 114.0429 atomic mass units) were used for post-translational modifications. A false discovery rate of <1% was accepted for this analysis. The identified proteins are presented in m R1881) for AD and AS samples, respectively. The samples were subjected to streptavidin affinity chromatography as performed in the original proteomic experiment and described under “Streptavidin Affinity Chromatography.”rSBP-AR was generated from the TNT® Quick Coupled Transcription/Translation Reticulocyte Lysate System (Promega). Equal amounts of SBP-AR, as determined by Western blotting, were incubated with 200 μg of cytosolic protein extracts isolated from AD and AS LNCaP cells and 25 μl of UltraLink Plus streptavidin beads with the addition of vehicle (ethanol) or androgen   .Protein interaction networks were visualized with Cytoscape version 3.1.0 . Known im R1881; AS) for 1 h. The cells were then harvested and incubated in hypotonic solution  with 5 mm DTT and 1× protease inhibitor mixture for 10 min, followed by nitrogen cavitation at 100 p.s.i. for 5 min. Ice-cold 2.3 m sucrose containing 10 mm Tris-HCl, pH 7.4, was added to 6 ml of crude homogenate to a final 1.4 m sucrose concentration. The mixture was loaded into a clear plastic SW27 centrifuge tube , and overlaid with 14 ml of 1.2 m sucrose  and then 9 ml of 0.8 m sucrose  for a final volume of 35 ml. The gradients were centrifuged at 90,000 × g for 2.5 h with a SW27 rotor. A turbid band containing the Golgi membrane formed at the 0.8 m/1.2 m sucrose interface and was collected for downstream analysis.LNCaP and N-AR cells were grown in androgen-depleted medium for 96 h and treated with vehicle  or androgen (100 nJ. J. H. conducted most of the experiments, analyzed results, and co-wrote the paper. M. M. S., B. H. N., and J. L. performed experiments and bioinformatic analyses. M. E. W. conducted experiments, developed reagents, analyzed results, and co-wrote the paper.


Decoys did not reduce levels of AR protein or prevent nuclear localization of the AR. Nor did decoys interact directly with the AR. Thus decoys did not inhibit AR transactivation by a dominant negative mechanism. This work provides evidence that the AR NTD plays an important role in the hormonal progression of prostate cancer and supports the development of AR antagonists that target the AR NTD.Androgen receptor (AR) is a member of the steroid receptor family and a therapeutic target for all stages of prostate cancer. AR is activated by ligand binding within its C-terminus ligand-binding domain (LBD). Here we show that overexpression of the AR NTD to generate decoy molecules inhibited both the growth and progression of prostate cancer in castrated hosts. Specifically, it was shown that lentivirus delivery of decoys delayed hormonal progression in castrated hosts as indicated by increased doubling time of tumor volume, prolonged time to achieve pre-castrate levels of serum prostate-specific antigen (PSA) and PSA nadir. These clinical parameters are indicative of delayed hormonal progression and improved therapeutic response and prognosis. Decoys reduced the expression of androgen-regulated genes that correlated with reduced  The prostate is an androgen-dependent tissue that requires androgen for the growth and survival of epithelial cells. Androgen receptor (AR) is a transcription factor that mediates the effects of androgen. It is composed of functional domains that include a C-terminal ligand-binding domain (LBD) that contains transactivation function-2 (AF-2), a DNA-binding domain (DBD), and an N-terminal domain (NTD) that harbors AF-1 with two transcriptional activation regions [in vivo proof-of-concept for AR NTD as a novel therapeutic target was provided using copies (decoys) of the AR NTD residues 1–558 (AR1-558) [In 2007, the first 1–558 have been described [Male NOD-SCID mice were obtained from the Animal Research Center of the British Columbia Cancer Agency. All animal studies conformed to the relevant regulatory and ethical standards. Analgesic (Metacam) and anaesthesia (isoflurane) were used and all efforts were made to minimize suffering. The University of British Columbia Animal Care Committee approved all experiments involving animals (Permit Number A03-0260). LNCaP cells  were routinely maintained in RPMI 1640 supplemented with 5% (v/v) FBS . LNCaP cells that stably express decoy AR1-558, AR1-233, and AR392-558 plasmids were made by polymerase chain reaction (PCR) amplification of the nucleotides of the cDNA corresponding to the amino acids 1–558, 1–233, and 392–558 of the human AR and cloning the products into the BamHI site of pcDNA3.1/His©A plasmid . The human AR1-558 decoys and lentivirus plasmids have been described [His-tag expression plasmids for ARescribed .3 in size, the animals were randomly divided into four groups . Treatment consisted of injections every 5 days with 1–2 x 107 particles for GFP-AR1–558 and AR1–558 and 1x108 particles for GFP for the duration of the experiment. Tumors were measured weekly. Castration was performed under anesthesia by making a small incision in the scrotum to remove each testicle after ligation of the cord. At 5 days after the last inoculation, mice were sacrificed, and the tumors and major organs were excised and prepared for immunohistochemistry and Western blot analyses.LNCaP xenografts were established subcutaneously (s.c.) in the flanks of 6-week-old male NOD-SCID mice . The lenBlood samples were obtained from mice weekly before and after castration. Serum PSA levels were determined by an enzymatic immunoassay kit with a lower limit of sensitivity of 0.2 μg/liter .® ABC Kit  was used for detection. Peroxidase activity was localized with 3,3-diaminobenzidine, and the sections were counterstained with hematoxyline before dehydration and mounting. The slides were visualized using a Zeiss Axioplan 2 Microscope .Tissue sections (5 μm) were blocked in immunohistochemistry solution  and immunostained with anti-AR LBD , or anti-AR NTD . The Vectastain2 (liquid) then homogenized using a polytron homogenizer in ice-cold RIPA buffer . The protein lysates were treated with Albumin Depletion kit (Millipore). Protein concentrations were determined by RC DC assay (BioRad). Protein samples (40 ug) were loaded on a 10% polyacrylamide gel, transferred on Immobilon™ PVDF membranes , and probed with anti-GFP (Santa Cruz), or anti-AR  antibodies. Whole cell lystes from LNCaP cells transfected with the His-Tag plasmids were loaded on a 12.5% SDS PAGE and analyzed by Western blot analysis using anti-His-probe .Whole cell protein lysates were obtained from frozen xenografts that were ground up in N4) that stably express vector or AR1-558 [LNCaP cells 2.5 x 10.5 x 104 1–558, GFP, and GFP-AR1–558 were flash frozen with liquid nitrogen prior to isolation of total RNA also with Trizol®. Oligo-d(T)-primed total RNAs (5 μg per sample) were reverse-transcribed with SuperScript III (Invitrogen). An appropriate dilution of cDNA and gene-specific primers were combined with iQ SYBR Green Supermix (Bio-Rad) and amplified in an iCycler iQ real-time PCR machine . All qPCR reactions were performed in triplicate. Ct (threshold cycle number) and expression values with standard deviations were calculated using the Gene Expression Macro for Excel (Bio-Rad). Real-time amplification was performed with initial denaturation at 95°C for 2 min, followed by 40 cycles of two-step amplification . The results were normalized for GAPDH as housekeeping gene. Primer sequences for real-time PCRs were as listed in Levels of expression of androgen-regulated genes were analyzed in LNCaP cells that stably express vector or decoy as well as in xenografts derived from mice. LNCaP cells were treated with R1881 (10 nM) for 24 hours and total RNA isolated in Trizol® Reagent (Life Technologies). For androgen-repressed genes, cDNA was generated using the high Capacity RNA-cDNA kit (Applied Biosystems). The appropriate cDNA dilution was mixed with gene-specific primers and Platinum SYBR Green qPCR Supermix–UDG with ROX. Transcript levels were measured using ABI 7900HT qPCR machine. Xenografts from mice with injected empty lentivirus vector, lentivirus vectors for AR6 cells) stably transfected with vector or AR1-558 were plated in 10cm plates . The next day the media was replaced with 9 ml of serum-free RPMI. The cells were treated for 30 mins with R1881 (10nM), cross-linked with 1% formaldehyde and harvested. The cells were lysed in SDS lysis buffer , 10mM EDTA, Complete™ protease inhibitors), and sonicated. The extracts were used for immunoprecipitation with anti-AR antibody AR . PSA primers used for real time PCR with Sybr green qPCR kit from (Invitrogen) were: 5’-GCCTGGATCTGAGAGATATCATC-3’ (forward) and 5’-ACACCTTTTTTTTCTGGA TTGTTG-3’ (reverse) [LNCaP , 1% NonidetP-40, 150 mM NaCl, 0.5% sodium deoxycholate, 1 mM EDTA, 10ug/ml leupeptin, 10ug/ml aprotinin, 1mM AEBSF, 2mM Na3VO4, 10mM beta-glycerophosphate, and Complete™ protease inhibitor cocktail (Roche). Whole cell lysates were centrifuged at 14,000rpm for 20min at 4°C and supernatant was used for immunoprecipitation. Pre-clearance was done by incubation with rabbit IgG (Santa Cruz Biotechnology) and Protein A/G-agarose beads  for 1hour at 4°C with rotation. Protein complexes were incubated with AR antibody(C-19)  for 90 minutes at 4°C and pulled down with Protein A/G agarose beads. After overnight incubation, protein complexes were washed with lysis buffer three times, eluted with SDS-PAGE sample loading buffer and separated by SDS-PAGE. Western blotting was performed using anti-AR(441)  and the protein bands were visualized by chemiluminescence (Amersham Biosciences).LNCaP cells that stably express vector or AR1-558  were cul5 cells/well) were plated on 6-well plates  containing RPMI with 5% FBS. After 24 hours, the medium was removed and transfection was performed by using Lipofectin® Reagent (Invitrogen). The total amount of plasmid DNA was normalized to 3 μg/ well by the addition of pRc/CMV as the control plasmid with no promoter insert. The cells were cotransfected with PSA promoter-luciferase reporter gene and various portions of AR NTD or His-tag. Cells were treated with or without forskolin (50 μM) for 48 h. Cells were harvested in 1×Passive Lysis Buffer  and luciferase activity in the cell lysates was measured using the Luciferase Assay Reagent (Promega). The luciferase activity was normalized to the protein concentration by Bradford assay. The inhibitory effect of each ARN decoy molecule was calculated from the normalized luciferase expression to that of the His-tag control.LNCaP cells (3x101-558 decoys by lentivirus to established LNCaP xenografts in combination with androgen ablation therapy (castration). Once the tumors were 50–100 mm3 in volume, the mice were randomly assigned into 4 groups that were: mock injection (vehicle only); decoy AR1-558; GFP; and GFP-AR1-558. Animals were castrated 5 days after the first injection. Upon castration, there were significant differences in the percent decrease in serum PSA levels (nadir) between animals treated with decoys as compared to the controls. In mock-injected animals, serum levels of PSA dropped by 87% in comparison to precastrate levels, while in decoy AR1-558 injected animals the serum levels of PSA dropped by 99% (p = 0.0063). A similar result was obtained when GFP-AR1-558 chimera proteins were used. GFP injected animals showed a 94% drop in serum levels of PSA compared to GFP-AR1-558 that dropped 100% (p = 0.0271)  . For pat p = 0.003. A simi1-558 injected mice that took 10.3 weeks (p = 0.0296) to progress to castration-recurrence. Similarly, GFP injected animals took 9.1 weeks compared to decoy GFP-AR1-558 injected mice that took 15.5 weeks (p = 0.0245) to progress to castration-recurrence (1-558 in comparison to control tumors (mock and GFP).The time to castration-recurrence is defined here as the time for serum PSA levels to return to pre-castration levels. The longer the time for serum PSA to reach the precastration level suggests a better prognosis. Mock-injected animals took 7.7 weeks compared to decoy ARcurrence . The tim1-558 injected mice that took 8.4 weeks (p = 0.0095). GFP injected animals took 7.1 weeks compared to decoy GFP-AR1-558 injected mice that took 11.5 weeks (p = 0.021) (1-558 decreased staining of the proliferation marker Ki67 (data not shown) in xenografts as previously reported [1-558 can significantly delay the time for prostate cancer to become castration recurrent.The time for the tumor to double in volume also has prognostic value. The longer it takes for the tumor to double in size, the more favorable the prognosis. In examining this parameter for prognosis, mock-injected animals required 4.1 weeks to double in tumor volume compared to decoy AR= 0.021) . Thus, i1-558 decoy does not have effect on tissues that are not dependent upon functional AR. Alternatively the lentivirus may not have been delivered to these other organs. To test this, we employed an antibody to GFP and analysed whole cell lysates from these organs from hosts treated with lentivirus. Western blot analysis showed detection of GFP in xenografts harvested from the host  confirmed that decoy AR1-558 was delivered to the xenograft . One interpretation could be that ARthe host , yet levenograft . Levels 1-558. These LNCaP cells do not express detectable levels of AR splice variant protein [1-558, or vector, and treated with androgen for 30 minutes induced the nuclear localization of the GFP-AR regardless of the presence of decoy AR1-558  in comparison to control cells expressing the vector  cells in response to androgen. These studies revealed that the decoys significantly (p<0.05) reduced AR-ARE interaction by 50% in presence of R1881 in cells stably expressing the decoys in comparison to cells untransfected or solely expressing the vector . These data were consistent with reduced expression of PSA mRNA in cells expressing the decoy AR1-558 . This suggests that decoy AR1-558 does not interact with the AR to inhibit the receptor through a dominant negative mechanism.To initiate transcription in response to androgen, dimerization of AR is needed and requires interactions between DNA-binding domains  (for a review see ). Heteroandrogen , we test23FQNLF27 or 429WHTLF433 could repress expression of PSA, we co-transfected LNCaP cells with expression vectors encoding regions with amino acids 1–233 (contains 23FQNLF27) or 392–558 (contains 429WHTLF433) of the AR NTD and PSA promoter-luciferase reporter gene construct prior to treating the cells with forskolin. Consistent with previous reports, forskolin increased PSA-luciferase activity in the absence of serum and androgen [1-558 significantly reduced the induction of PSA-luciferase activity compared to control (positive control labeled “His-tag”) as shown previously [1-558 and AR by co-immunoprecipitation, these data do not support a dominant negative mechanism of decoy AR1-558 to inhibit transcriptional activity of AR.To determine if decoys that contain androgen . Decoy A1-558 did not de-repress the expression of the known androgen-repressed genes that were tested  and Tau-5 (435WHTLF439). The decoys also did not prevent nuclear localization of AR in response to ligand. Thus, overexpression of decoy AR1-558 allows the AR to be in the nucleus yet still prevents the interaction of endogenous AR with AREs to reduce transcription of genes regulated by AR.AR NTD has a high degree of intrinsic disorder and acts as a hub for protein-protein interactions. Not surprising is also the fact that the NTD is highly posttranslationally modified which can alter protein interactions and localization. Within the NTD is AF-1 which is essential for transcriptional activity. Thus targeting AR NTD for therapeutic intervention would be effective in preventing its transcriptional activity regardless of the presence or absence of ligand and LBD. Here we show that in combination with castration, lentivirus delivery of decoys to the AR NTD to established tumors delayed hormonal progression to castration-recurrence. The mechanism of action of the decoys involved decreased expression of genes normally regulated by androgen, and reduced physical interaction of the AR with AREs. The decoys did not reduce levels of endogenous AR protein, nor did decoys inhibit the activity of the AR through a dominant negative mechanism or involve amino acids 1–233 or 392–558 which contain the (F/W)XXLF motifs as well the core sequences for Tau-1 Click here for additional data file.


It has been identified that the synthesis reaction of the block copolymer was living polymerization. The thermo-sensitivity study suggested that N-vinylpyrrolidone (NVP), played a key role on the lower critical solution temperature (LCST) performance.The reversible addition fragmentation chain transfer (RAFT) polymerization method was adopted here to prepare a series of thermo-sensitive copolymers, poly . Their structures, molecular weight distribution and temperature sensitivity performances were characterized by the nuclear magnetic resonance ( BesidesCompared with PNIPAM, a later found out thermo-sensitive polymer, poly  (PDEA), has shown better biocompatible  propertyGenerally, a block copolymer with a well-designed structure is usually prepared by the living radical polymerization method because the living radical polymerization always exists a reactive end groups and can be used to polymerize different monomers. For example, common monomers have styrene, methacrylates, acrylonitrile, vinyl acetate, etc., functional monomers have acrylic acid, sodium styrene sulfonate, dimethylaminoethyl methacrylate, etc. . What’s PDI) were characterized by 1HNMR and GPC, respectively. All results documented that the polymerization was living polymerization. Then, the thermo-sensitive copolymer of DV was characterized, and the result of thermo-sensitivity about NVP showed that increasing the molar ratio of NVP, LCST increased, while the thermo-sensitivity was weakened, and when the ratio of nDEA:nNVP was in the range of , LCST approximated to 36 °C, which was very close to body temperature.Based on the above findings, the preparation of DV using RAFT method was carried out in this work, the process was completed as follows: DV was prepared by polymerized PDEA, DV, in turn, using RAFT polymerization technology, and the structure contained the content of each block, the molecule weight and polydispersity index(2.2.1.Dodecylmercaptan (AR), Sodium hydride(AR), Carbon disulfide (AR), Iodine (AR), Sodium thiosulfate (AR),and Sodium sulfate (AR) were provided by the YantaiShuangshuang Chemical Co. Ltd. Ethyl acetate (AR), Petroleum ether (AR), Ether (AR),n-hexane (AR),and n-decane(AR) were purchased by the Rionlon Bo hua pharmaceutical chemical company. Acenaphthylene(AR) was provided by the Shanghai SA Ren Limited Liability Company. Acetone (AR) was produced by Beijing Chemical Works. All above reagents were used directly. Azobisisobutyronitrile(AR) was provided by YantaiShuangshuang Chemical Co. Ltd and was recrystallizedby95% ethanol before using.2.2.The synthesis theory of trithiocododecanoicacid-2-cyanoisopropyl (CPDTC), a RAFT agent, was shown in Scheme 2.3.1HNMR.N, N- diethylacrylamide and its precursor, acryloyl chloride, were prepared according to literature , and the2.4.2.4.1.Synthesis of PDEA was shown in Scheme 2.4.2.Synthesis of DV was shown in Scheme 2.5.3) solution (10 mg/mL) of CPDTC, PDEA and DV were prepared, respectively. And then1HNMR for each compound was determined by nuclear magnetic resonance spectroscopy , and the molecular structure was characterized according to the chemical shift of hydrogen in each compound.Deuterated chloroform  solution of PDEA and DV were prepared, respectively, and the molecular weight distribution for each compound was detected by a gel permeation chromatograph  which was calibrated with narrowly distributed polymethyl methacrylate. During this process, DMF in which 0.05 mmol/L lithium bromide was contained was adopted as the mobile phase, and the injection volume was 10 μL at 1 mL/min flow rate and 25 °C.2.7.The thermo-sensitive performance of DV was determined by static fluorescence spectroscopy technology using a fluorescence spectrometer . During this process, 0.01 mg/mL of DV aqueous solution was prepared, and operation conditions were set as follows: the excitation wavelength was 290 nm, the scanning speed was 240 nm/min, the excitation and emission slit were10 nm, the scanning emission wavelength was 330–430 nm, and the fluorescent intensity determination wavelength was 380 nm.3.3.1.3.1.1.1HNMR  spectra of CPDTC: 2.84 , 1.88 , 1.72–1.65 , 1.26  and 0.87 .Figure 3.1.2.1HNMR  spectra of DEA:1.16 , 3.40 , 5.65 , 6.32 , 6.53 .Figure 1HNMR  spectra of NVP:2.10 , 2.48 , 3.50 , 4.40 , 7.08 Figure 3.1.3.1HNMR spectra of PDEA. It could be found in a polymer, except for peak at δ = 2.96 in CPDTC and δ = 3.34 in DEA, almost all peaks for both CPDTC and DEA were submersed and could not be identified. The reason could be ascribed to large molecule of PDEA, because of the polymerization, large amounts of DEA were bonded together, and the quantities of hydrogen for a given group would increase hundreds of times. Due to the superposition of each group, and the influence of other groups adjacent to them both in link and in space, peaks related to a given group could not be distinguished clearly, naturally. However, due to the far δ distance of q and x from the others, the influence they suffered was relatively small, and could be conscripted as reference to calculate molecular weight of PDEA as Equation (1):Figure δ(2.96) in 1 mol CPDTC, but 4 mol H at δ(3.34) in DEA, so as 1 mol CPDTC was combined with 1 mol DEA, the integral of H at δ(3.34)would be 2 times that at δ(2.96), 127 was the molecular weight of DEA.where, For example, from Figure PDI) of PDEA as 1.05, which was far smaller than the general free radical polymerization, and it indicated that the process of preparing PDEA was living polymerization.Besides, GPC characterization showed a polydispersity index :Figures δ(3.68) in 1 mol NVP, but 4 mol H at δ(3.31) in DEA, so as 1 mol NVP combined with 1 mol DEA, the integral of H at δ(3.31) would be 2 times that at δ(3.68), 127 was the molecular weight of DEA.where, As the molar ratio of DEA and NVP was obtained, the molecular weight of PVP could also be calculated using Equation (3):M(DEA), M(NVP) and where, After where 1HNMR spectra of the DV2, DV3, DV4 and DV5 were shown as Figure The 3.2.PDI) of DV at different reaction times. It was shown that the molecular weight was linearly increased with the polymerization time, and PDI was . The drift off of experimental results to their related theoretical could be ascribed to the variation of viscosity. With prolonged reaction time, the molecular weight of the polymer increased, and the viscosity augmented correspondently .