PCGEM1, PEG3, EPHA3, and EFNB2) or other types of human cancers , and genes that are diagnostic/prognostic biomarkers of prostate cancer .Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2–8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer ( Androgen signalling through the androgen receptor (AR) is essential for human prostate growth and function; however, excessive AR-signalling activity in the prostate is well-known to be involved in prostate cancer development and progression . TherefoAR gene is located at Xq11-13 and contains eight canonical exons (exons 1–8) and at least seven “cryptic” exons  [AR gene . Isoform 1 (NM_000044.3) consists of canonical exons 1–8 and encodes the full-length wild-type AR . Isoform 2 (NM_0010116445.2) comprises exon 1b spliced to exons 2–8 and encodes AR45 , a variant AR protein that is highly expressed in the heart [AR gene, generating approximately 20 variant AR transcripts  [The exon 1b) ,7. There exons 1– and at lm 2 NM_00116445.2 nd AR23) ,13. Mostnd AR23) ,15,16 orm 2 NM_00116445.2 Breast cancers are highly heterogeneous diseases. Apocrine breast cancers, a subset of ER-negative and AR-positive breast cancers, show androgen-stimulated growth ,18. As sAs mentioned earlier, both AR transcript isoforms 1 and 2 contain exons 2–8 but differ in the use of the canonical exon 1 or exon 1b. Splicing of exon 1 or exon 1b has been considered as mutually exclusive . The preAs mentioned above, AR isoforms 1 and 2 differ in that isoform 1 contains the classical exon 1 and isoform 2 contains the alternative exon 1b  Figure . The splNext, RT-PCR was used to examine the expression of the type 1, 2 and 3 AR transcripts in prostate cancer cell lines  and normal breast and prostate tissues. Primers A were deQuantitative RT-PCR for the type 3 AR transcript (E1E1b) and for the type 1 AR transcript (E7/E8) indicated that type 3 AR was expressed at a level approximately 2%–3% of type 1 AR in breast and prostate tissues and cancer cell lines E. This lSequencing of the RT-PCR products that were amplified using primers that span the exon 1/exon 1b junction (E1E1b) from three cell lines revealed two overlapping sequences following the last nucleotide G of exon 1 A. To defInterrogation of genomic sequence showed that the novel exon 1b (termed exon 1b/TAG) begins three nucleotides (TAG) upstream of exon 1b A. Both eType 1 and types 3a and 3b transcripts have the same translation start site in the canonical exon 1 ; however, the open reading frame of the type 3a (or 3b) transcript reads through exon 1b (1b/TAG) and 33 nucleotides into exon 2 before reaching a premature stop codon B. The diThe full-length coding sequences of the type 3a and type 3b AR transcripts were cloned into the expression vector pcDNA3 generating “pcDNA3/type 3a AR” and “pcDNA3/type 3b AR” as described in Materials and Methods. The expression constructs were transfected into prostate cancer Du145 and breast cancer MDA-MB-231 cells. Expression constructs for wild-type AR (pcDNA3/type 1 AR) or the well-characterized AR-V7 variant (pcDNA3/AR-V7)  and emptTo determine the biological activity of type 3a and type 3b AR variants, co-transfection experiments were performed with luciferase reporters carrying known AR-responsive enhancers (pGL3/PSA enhancer or pGL3/TMPRSS2 enhancer) in prostate  and breast (MDA-MB-231) cancer cell lines. The transactivation function of the type 3a and type 3b variants was compared to that of AR-V7, and transfected cells were treated with either 1 nM dihydrotestosterone (DHT) or vehicle to determine whether activity was ligand dependent or independent.PSA enhancer reporter in LNCaP and VCaP cells in the absence of androgen . Collectively, these data reveal cell context-specific androgen-independent activation of AR-responsive reporters by type 3a and type 3b AR in prostate and breast cancer cells.Although type 3 AR variants lack the DBD (encoded by exons 2–3) and the bipartite nuclear localization signal (NLS) (encoded by exons 3–4), we showed their androgen-independent activation of androgen-responsive reporters. This observation prompted us to define the cellular localization of type 3 AR proteins by expression of GFP/type 3 AR fusion proteins. To avoid potential interference from the endogenously expressed wild-type AR, AR-negative PC3 cells were used for these experiments. As shown in The potential effect of type 3 AR variants on prostate cancer cell growth was assessed using LNCaP cells with stable heterologous expression of type 3a AR or type 3b AR. Heterologous expression of the AR variants in these cell lines was confirmed at both mRNA levels by RT-qPCR A and proPCGEM1, BCHE, EFNB2, PSMD5, MBNL2, GSTA1, TLL1, and ZNF608, has-miR-3689a, and has-miR-3689b) were down-regulated at similar levels by type 3a AR independent of DHT treatment. These results suggest that type 3a AR represses these genes in an androgen-independent manner. PEG3 was the only gene that was upregulated by type 3a AR both with and without DHT treatment. Three genes  were downregulated by type 3a AR only in the absence of DHT, and six genes  were downregulated by type 3a AR only in the presence of DHT. One gene, EPHA3, was upregulated by type 3a AR only in the presence of DHT. Collectively, overexpression of type 3a AR altered the expression of 21 genes including two microRNAs in an androgen-dependent and/or -independent manner in LNCaP cells.Whole-genome gene expression profiling was conducted using Affymetrix Gene Arrays to compare gene expression levels between LNCaP cells stably expressing type 3a AR and control LNCaP cells, with and without DHT treatment. As shown in COL12A1, GRIN3A, and PCGEM1)   in the absence of DHT  following DHT stimulation. These results indicate that type 3a AR represses the expression of these androgen-downregulated genes under androgen-depleted condition.The expression levels of 12 of the genes identified as type 3a AR targets by Gene Array analysis were further measured by quantitative real-time RT-PCR (qRT-PCR) using gene-specific primers . As show PCGEM1) C or down LRRTM3) D by DHT  3/5) [617617RKCYEAGMTLGARKLKK633) that is essential for androgen-induced nuclear localization [BRCA1 has recently been shown to mediate nuclear localization of proteins encoded by BRCA1 splice variants lacking the classical NLS [We showed that GFP-tagged type 3a and 3b AR proteins were present in both cytoplasm and nuclei of PC3 cells under androgen-depleted conditions and that this cytoplasmic/nuclear localization pattern was not significantly affected by DHT. The latter result is consistent with the lack of the LBD in the variants. Nuclear localization signals (NLS) are short basic amino acid-rich  peptide motifs involved in the nuclear import of proteins >45 kDa) via their binding to importins through the nuclear pore complexes ,29. ClasK/R)3/5) . The willization . The fir kDa via lization ,31. The lization . Both tyical NLS . The prePreviously described AR variants that lack the LBD, such as AR-V7, contain the DBD that is involved in binding to target genes and androgen-independent activation ,7,10. Dev567es) has been shown to promote prostate cancer growth both in cell lines and in mouse xenograft models under androgen-depleted conditions [v567es) at low levels stimulated LNCaP cell growth, but at higher levels (higher than endogenous wild-type AR protein) the variants inhibited cell growth [The functional significance of type 3 AR variant proteins may depend on the level of their endogenous expression. Western blotting assays with the AR antibody N20) using long exposure times revealed low levels of endogenous proteins with the same molecular weight as heterologously expressed type 3 AR proteins in LNCaP and MDA-MB-231 cells. However, the endogenous expression levels of most AR variant proteins, including AR45, remain poorly defined due to the lack of such variant-specific antibodies ,20, and  using londitions ,15,16,21nditions ,35. Morenditions ,37. Chanl growth . We showPCGEM1 [BCHE [PEG3 [GSTA1 [GRIN3A [LRRN1 [MBNL2 [EFNB2 [SLITRK3 [EPHA3 [TOX3 [COL12A1 [PCGME1, PEG3, EPHA3, and EFNB2) are of particular interest as they are known to modulate cancer cell proliferation and/or survival.Twelve of the genes regulated by type 3a AR in prostate cancer LNCaP cells  have beePCGEM1 , BCHE [3M1 [BCHE ,40, PEG3HE [PEG3 ,41, GSTA3 [GSTA1 , GRIN3A  [GRIN3A ,44, LRRNA [LRRN1 ,39, MBNL1 [MBNL2 , EFNB2 [2 [EFNB2 , SLITRK3[SLITRK3 , EPHA3 [3 [EPHA3 , TOX3 [4A3 [TOX3 , and COL[COL12A1 . Four ofPCGEM1) encodes a prostate-specific long non-coding RNA (lncRNA) that has oncogenic activity in prostate cancer [PCGEM1 have been linked to prostate cancer risk [PCGEM1 stimulated cell growth while knockdown of PCGEM1 inhibited growth [PCGEM1 suggests the possibility that AR variants can modulate each other’s biogenesis. It is also possible that PCGEM1 influences splicing of type 3 AR, and this remains to be investigated. PEG3  has a critical role in apoptosis, promoting the p53-mediated cell death pathway [Prostate cancer gene expression marker 1  that discriminated clinically relevant high-grade (Gleason score ≥ 4 + 3) tumours from low-grade (Gleason score ≥ 3 + 4) tumours using urine sediments [Three type 3a AR-regulated genes are reported to be useful diagnostic or prognostic biomarkers of prostate cancer, although their molecular mechanisms remain unclear. Bianchi-Frias et al recently reported over-expression of ediments . Advanceediments  and BCHEediments . Multivaediments .S-transferases (GSTs) are involved in detoxification of a variety of carcinogens and thus prevent carcinogenesis [RTN4, SLITRK3, and SPON2) found to define a cell differentiation signature for LNCaP cells [N-Acetyl-Neuraminide α-2,8-Sialyltransferase 4) synthesizes polysialic acid that is present on the embryonic neural cell adhesion molecule (N-CAM). siRNA knockdown of N-CAM and ST8SIA4 abolished pancreatic cancer cell aggregation and migration [TOX3 gene encoding a TOX (thymocyte selection-associated high mobility group box)-family transcription factor [In addition to the seven genes discussed above, other type 3a AR-regulated genes are linked to prostate cancer or other types of cancer. Glutathione ogenesis . Low levogenesis ,65,66,67ogenesis , lymphomogenesis , and gasogenesis , and wasaP cells . ST8SIA4igration . A misseigration . The TOXn factor , residesn factor ,77,78,79n factor . The rolTotal RNA of normal breast tissue was purchased from Clontech . Total RNA of normal prostate tissue was purchased from Ambion™ .2) containing 50 units of superscript® III reverse transcriptase, random hexamer primers, dNTPs, 50 units of RNaseOut recombinant ribonuclease inhibitor. The resultant cDNA was diluted five times with water, and 3 µL of each diluted cDNA sample (~60 ng) was used as template for each standard polymerase chain reaction (PCR).Prostate  and breast  cancer cell lines were purchased from American Type Culture Collection (ATCC). Cell lines were maintained in either Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS) (VCaP) or Roswell Park Memorial Institute (RPMI) 1640 Medium containing 5% FBS . For RNA extraction, cells were cultured in 6-well plates to 90% confluence and then harvested for RNA extraction using RNeasy Mini Kits  according to the manufacturer’s instructions. Reverse transcription (RT) was conducted as previously reported using reagents from Invitrogen . BrieflyGGATCCATGGAAGTGCAGTTAGGGCT-3′ and reverse primer: 5′-AAATGCGGCCGCTCACTGGGTGTGGAAATAG-3′) were designed to amplify the full-length coding region of the AR gene from cDNA samples of VCaP, LNCaP, or MDA-MB-453 cells. The initiation (ATG) and stop (TCA) codons of the AR gene in the forward and reverse primers respectively are in italics and BamHI and XhoI sites (underlined) used for cloning in the forward and reverse primers respectively are underlined. PCR was conducted by an iCycler  in 50 µL of 1× Phusion GC buffer containing 1 unit of Phusion® High-Fidelity DNA polymerase  and 100 ng of cDNA from VCaP, LNCaP, or MDA-MB-453 cells. Resultant PCR products were then cloned into the TA vector . Inserts were sequenced bidirectionally using primers T7 and Sp6 by the Flinders Sequencing Facility .Primers (forward primer: 5′-CTTACt−ΔΔ method [Quantitative RT-PCR (qRT-PCR) was performed using a RotorGene 3000 instrument  in a 20-µL reaction of 1xQuantiTect SYBR Green PCR master mix (QIAGEN) containing ~60 ng of cDNA sample and target gene-specific primers . The mRNt method . Quantift method . The prit method . For quaFurther primers as shown in Ct−ΔΔ method [To quantify the mRNA levels of type 3a AR target genes in stably transfected LNCaP cells, gene specific primers were generated and qRT-PCR was performed as described above except that relative quantitation was performed using the 2t method  with 18SGGATCCATGGAAGTGCAGTTAGGGCT-3′ paired with a reverse primer specific for AR-V7 (5′-ATATGCGGCCGCTCAGGGTCTGGTCATTTTGA-3′) or wild-type AR(5′-AAATGCGGCCGCTCACTGGGTGTGGAAATAG-3′), and Phusion® High-Fidelity DNA polymerase (Thermo Scientific). The initiation (ATG) and stop (TCA) codons of the wild-type AR or AR-V7 in the forward and reverse primers respectively are in italics and BamHI and NotI sites (underlined) used for cloning in the forward and reverse primers respectively are underlined. The resultant PCR products were digested with BamHI and NotI and subsequently cloned into BamHI and NotI sites of pcDNA3 vector to generate two expression vectors: pcDNA3/AR-V7 and pcDNA3/type 1 AR. The identities of the constructs were verified by sequencing.The coding region of type 1 AR (wild-type AR) and AR-V7 were respectively amplified from the pCMV/AR3.1 vector containing the full-length wild-type AR and from a lentiviral expression vector containing the full-length AR-V7  using a common forward primer 5′-CTTAGGATCCATGGAAGTGCAGTTAGGGCTGGGAA-3′) and reverse (5′-GTCCGGAGTAGCTATCCATCCAGGGGCCCATTTCGCTTTTGA-3′) primers. The exon 1b/exon 2-encoded sequence of type 3a AR was amplified from VCaP cDNA using the forward (5′-TAGCTACTCCGGACCTTACGGGGACATGCGGTGCTGCGA-3′) and reverse (5′-CATGCTCGAGCGGCCGCTGGGGTGGAAAGTAATAGTCAATGG-3′) primers. PCR was carried using Phusion® High-Fidelity DNA polymerase (Thermo Scientific). In-Fusion HD Cloning Kits (Clontech) used a 10 µL ligation mixture that contained 2 µL of each of the two PCR amplicons, 1 µL of BamHI/NotI-digested pcDNA3 vector, 2 µL of 5× In-fusion HD enzyme premix, 3 µL of water. Ligation was carried out at 50 °C for 15 min. Positive clones were sequenced bidirectionally using primers T7 and Sp6 by the Flinders Sequencing Facility . The pcDNA3/type 3b AR construct was then generated by insertion of TAG between exon 1 and exon 1b via site-directed mutagenesis using a complementary pair of primers  (inserted TAG underlined).The In-Fusion HD Cloning Kit (Clontech) was used to assemble the coding sequence of type 3a AR in the pcDNA3 vector. The assembly approach was used because GC-rich repetitive sequences within exon 1 reduced the efficiency with which the full length AR sequence could be amplified and could lead to PCR-generated deletion artefacts. Furthermore, type 3 AR transcripts use a stop condon at nucleotides 34–36 of exon 2 precludes use of a reverse primer that is specific to type 3 AR. The coding sequence of exon 1 was amplified from pCMV/AR3.1 using the forward (5′-TACCGAGCTCPSA (prostate specific antigen) gene from nucleotides −3822 to −4301 relative to its initiation ATG codon [ACGCGTATCTTGTAGGGTGACCAGAGCA-3′/reverse, 5′-ATACTCGAGAGTATTTAATGGTGGAACGT-3′) and Phusion® High-Fidelity DNA polymerase. Similarly, the androgen-regulated enhancer of the TMPRSS2  gene from nucleotides −13,180 to −14,044 relative to its initiation ATG codon [ACGCGTCACTGATCTTATCTTGTAGGC-3′/reverse, 5′-ATAGCTCGAGCTTCTCCTCCCACCTCACTGCT-3′). The MluI and XhoI sites for cloning in the primers are underlined. The amplified PSA or TMPRSS2 enhancer was subsequently cloned into the MluI and XhoI sites of pGL3-basic vector  generating two androgen-responsive luciferase reporters: pGL3/PSA enhancer and pGL3/TMPRSS2 enhancer.The androgen-regulated enhancer of the TG codon  was amplTG codon  was ampl®2000 (Invitrogen). Twenty-four hours post-transfection, cells were cultured in phenol red-free RPMI 1640 medium with 5% dextran-coated charcoal (DCC)-stripped FBS  for 48 h and then treated with either vehicle (0.1% ethanol) or 10 nM dihydrotestosterone (DHT) for 24 h. After treatment, cells were lysed in passive lysis buffer and assayed using the Dual-Luciferase Reporter Assay system (Promega). Firefly  luciferase activity was first normalized using the Renilla (Renilla reniformis) activity and then expressed as-fold change over that of the cells transfected with the empty pcDNA3 vector and treated with ethanol.Cells  were plated into 96-well plates at 50%–60% confluence and incubated overnight in RPMI medium supplemented with 5% FBS. Cells of each well were then transfected with 100 ng of an androgen-responsive reporter (pGL3/PSA enhancer or pGL3/TMPRSS2 enhancer), 100 ng of a pcDNA3 vector  and 0.8 ng of pRL-null vector (Promega) as an internal reference, using Lipofectamine® imaging system  which scanned the wells every 2 h. Cell confluence as a factor of time was calculated using IncuCyte software (2013B).To make LNCaP cells that stably express type 3a or 3b AR and control LNCaP cells expressing no ectopic AR, pcDNA3/type 3a AR, pcDNA3/type 3b AR, or empty pcDNA3 plasmid was transfected into LNCaP cells and 48 h later, cells were treated with Geneticin . The pools of G418-resistant cells remaining 14 days post-transfection represented stably expressing type 3a AR, type 3b AR, or control cells (pools of cells transfected with empty pcDNA3 plasmid). The stable LNCaP cells were seeded in 96-well plates with 8 replicates for each type of cell at 4000 cells/well in RPMI-medium containing 5% stripped FBS and 10 nM DHT. Cells were cultured for 9 days and monitored using the IncuCytep values <0.05 were considered to be significant. Complete lists of the 4 differentially expressed gene sets from the four different comparisons are given in LNCaP cells stably expressing type 3a AR (LNCaP/pcDNA3/type 3a AR cells) or no ectopic AR (LNCaP/pcDNA3 cells) were cultured in 6-well plates in phenol-red-free RPMI containing 5% stripped FBS for 72 h approaching 90% confluence, and then treated in triplicate with vehicle (0.1% ethanol) and 10 nM DHT for 24 h. After treatment, total RNA of treated cells was extracted using RNeasy Mini Kits (QIAGEN). Microarray experiments and data analysis were conducted by Adelaide University Microarray Facilities, Adelaide, SA, Australia), country. Briefly, 300 ng of total RNA was labelled using the Affymetrix WT Sense Target labeling assay as per the manufacturer’s instructions . Samples were hybridised to Affymetrix Human Gene 2.0 ST Arrays for 17 h at 45 °C prior to washing, staining and scanning as per the manufacturer’s instructions. The array data was analysed using Partek Genomics Suite . Briefly, cel files were imported using RMA back-ground correction, Partek’s own GC content correction, and mean probe summarization. Differential gene expression was assessed by ANOVA with the p value adjusted using step-up multiple test correction to control the false discovery rate (FDR) . AdjusteCells were cultured in 6-well plates at 50%–60% confluence and incubated overnight in RPMI medium supplemented with 5% FBS. Cells were then transfected with 2 µg of relevant expression plasmids as indicated in the figures. After 48 h, whole cell lysates were prepared using RIPA buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS). Thirty micrograms of protein for each sample were resolved on 10% SDS-polyacrylamide gels and subjected to standard immunoblot analysis using a primary antibody recognizing the AR  or β-actin (Sigma-Aldrich), and a secondary donkey anti-rabbit antibody conjugated with peroxidase . SuperSignalWest Pico Chemiluminescent substrate (Thermo Scientific) was used for immunodetection, and immuno-signals were photographed by an ImageQuant LAS 4000 luminescent image analyzer . The anti-AR (N20) antibody raised against the N-terminus of the human AR is able to detect wild-type AR (type 1 AR) and three AR variants, AR-V7, type 3a AR, and type 3b AR.ATGGTGAGCAAGGGCGAGG-3′) and reverse (5′-CGTTACTAGTCCTAGGCTTGTACAGCTCGTCCATGCCGAGA-3′) primers. PCR was carried out using Phusion® High-Fidelity DNA polymerase (Thermo Scientific). pcDNA3/type 3a AR, pcDNA3/type 3b AR, or pcDNA3/type 1 AR (AR WT) was digested by BamHI and the digested products were purified. The amplified GFP PCR products were ligated with BamHI-digested pcDNA3 vector using In-Fusion HD Cloning Kits as described above. Expression consutructs were verified by bidirectional sequencing. Glass coverslips were coated with 50 µg/mL rat-tail collagen for 1 h at 37 °C and seeded with PC3 cells in 24-well plates in RPMI media containing 10% FBS. PC3 cells were transfected in duplicate wells using 1.6 µL Lipofectamine 2000 (Invitrogen) and 400 ng of GFP-tagged vector  alone or in combination . Untagged pcDNA3 empty vector was added where necessary to normalize total DNA amounts between conditions. At 24 h post-transfection media was replaced with phenol red-free RPMI containing 10% stripped FBS, and at 48 h post-transfection cells were treated with 10 nM DHT or vehicle for 4 h. After treatment, cells were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature and nuclei were counterstained with 1 µg/mL DAPI for 5 min before mounting. Cells were imaged with an Olympus BX-50 upright microscope and the mean fluorescence intensity of nuclear and cytoplasmic GFP of each cell was quantified using ImageJ (NIH). Background fluorescence was subtracted and results were reported as means ± SEM of ratios of nuclear/cytoplasmic fluorescence from 20 to 35 cells for each experimental condition.The In-Fusion HD Cloning Kit (Clontech) was used to prepare the pcDNA3 vectors that expressed fusion proteins with GFP fused at the N-termimus of wild-type AR (pcDNA3/GFP/AR WT), type 3a AR (pcDNA3/GFP/type 3a AR), or type 3b AR (pcDNA3/GFP/type 3b AR). The full coding sequence of GFP without the stop codon was amplified from pEGFP/AR-V7 using the forward  variant AR proteins that can act as androgen-independent transcriptional activators, in common with other LBD-lacking AR variants. Type 3 AR variants also lack the DBD, but may interact with wildtype AR or other AR variants in order to interact with DNA and modulate gene expression. The mechanism by which these novel variants inhibit androgen-induced LNCaP cell growth, and in particular the roles of identified type 3 AR target genes, will require further study. Similar to other LDB-lacking AR variants, type 3a AR transcripts are expressed at a level of <5% of the wild-type AR in normal breast and prostate tissues and in the cancer cell lines studied here. However, potential upregulation/downregulation of type 3 AR in clinical breast and prostate cancer specimens remains to be investigated.


The Androgen Receptor (AR) plays a key role in prostate biology and in the progression of prostate cancer (PCa) to castration resistance. The role of microRNAs (miRNAs) in aberrant AR signaling have not been fully characterized. Here we screened a library of 810 miRNA mimics to identify miRNAs that alter AR activity in complementary functional assays including protein lysate microarray (LMA) quantification of AR and PSA protein levels, AR transcriptional reporter activity, and AR-positive PCa cell viability. Candidate AR-regulating miRNAs were verified through AR transcriptional reporter and cell viability assays. MiRNA binding sites were found within the AR 3′-untranslated region (UTR) and within the AR and AR-V7 coding regions. MiRNA activity was characterized by western blotting, 3′-UTR reporter assay, and AR-GFP and AR-V7-GFP reporter assays. Results uncovered miR-30 family members as direct AR inhibitors. Inhibition of endogenous miR-30b-3p and miR-30d-5p enhanced AR expression and androgen-independent cell growth. Droplet digital RT-PCR quantification of miR-30c-5p and miR-30d-5p revealed significantly reduced levels in metastatic castration resistant PCa (CRPC), when compared to healthy prostate tissues. MiR-30d-5p levels were inversely correlated with AR activity, as measured by PSA mRNA, in metastatic CRPC. Collectively, these studies provide a comprehensive evaluation of AR-regulating miRNAs in PCa. The Androgen Receptor (AR) is a ligand-activated nuclear receptor and transcription factor that is required for the natural development of the prostate . The semMiRNAs belong to a class of non-coding RNAs that post-transcriptionally regulate gene expression through complementary seed-pairing interactions within target mRNAs . MiRNA eA library of 810 human miRNA mimics was transfected into three AR-positive PCa cell lines  and miRNA-induced changes in AR protein expression were interrogated by protein LMA. Replicate protein LMAs were also probed for PSA protein expression, to detect potential miRNA-regulation of AR transcriptional activity. The mimic library was further used to screen for miRNA-induced changes in AR transcriptional activity, by using an AR-responsive promoter and reporter assay . FinallyFour complementary screens were performed to identify miRNAs capable of altering AR expression and activity. First, protein lysates were generated from three androgen-responsive PCa cell lines  that had been separately transfected with each of 810 different miRNA mimics Figure . PSA expMetridia luciferase as a bioluminescent reporter that is representative of viable cell density [Two additional functional screens were performed using LNCaP cells and the same library of 810 miRNA mimics Figure . The fou density . An endp density . NotablyThe results from the AR protein LMA, AR transcriptional activity screen, and PSA protein LMA screens were analyzed to identify candidate mimics that inhibited AR expression and activity in multiple assays and cell lines. The top 25% of inhibitory mimics, based on fold signal change, were chosen from each screen and cell line. The resulting data was compared to identify 43 candidates which inhibited AR transcriptional activity, as well as AR and PSA protein level in at least two cell lines. Five additional candidates were considered from the most potent inhibitors of AR transcription activity. A heat map, in Figure MiRNA mimics from 39 candidates were separately obtained for verification analyses. In addition, 5 independent mimics from a previous DNA repair screen  were anaRenilla luciferase vector for normalization. 15 of the 29 mimics significantly suppressed AR 3′UTR reporter activity, when compared to a negative control miRNA  or in androgen deprived media . Inhibition of miR-30b-3p and miR-30d-5p induced LNCaP cell growth under androgen deprived conditions Figure . QuantitTo investigate the four candidate miR-30 family members in human disease, miR-30a-5p, miR-30b-3p, miR-30c-5p and miR-30d-5p levels were measured in 15 primary PCa samples, 15 adjacent normal prostate samples, and 15 metastatic CRPC samples by droplet digital RT-PCR. MiRNA expression levels were normalized to RNU6B for each sample. Notably, miR-30d-5p levels were significantly lower in primary and metastatic CRPC, when compared to normal healthy tissue Figure . MiR-30cLevels of PSA mRNA, an AR-induced gene, were quantified by qRT-PCR as a surrogate marker for AR transcriptional activity in the metastatic CRPC samples. A statistically significant inverse correlation  was found between miR-30d-5p and PSA mRNA levels Figure . SimilarAberrant AR expression and activity are associated with PCa development and its progression through all stages of the disease . AlteredPrevious studies have reported direct miRNA suppression of the AR. One of the first reported was miR-488* (miR-488-5p), which targets the AR through its 3′UTR . Our stuÖstling and colleagues demonstrated the value of protein LMAs for the identification of functional miRNA mimics . Our scrIn addition to these reported miRNAs, our high throughput analyses have uncovered some novel AR-regulating miRNAs. MiR-101 is a tumor suppressive miRNA that is commonly lost during PCa progression and it is associated with over-expression of its target gene, EZH2 . Here weThere have been relatively few studies analyzing miRNA expression in advanced PCa, such as metastatic CRPC, where altered AR expression and activity would be most expected. Analysis of primary tumors isolated by surgery or transurethral resection of the prostate (TURP) have found reduced expression of AR-targeting miRNAs and corresponding elevated AR protein expression or AR transcriptional activity , 36, 45.High throughput gene expression studies have also evaluated miRNA expression in CRPC tissues , 46, 47.In summary, we have applied a series of complementary functional miRNA mimic screens to identify multiple miRNAs capable of inhibiting or activating the AR signaling axis. The results demonstrated a common suppressive nature for miRNAs across many assays. At the same time, potent and specific AR regulating miRNAs were identified and verified in multiple models, assays and clinical samples. These results provide a comprehensive evaluation of AR-regulating miRNAs and AR function in PCa cell models. Importantly, these results implicate miR-30 family members as possible regulators of the AR signaling axis in CRPC. Additional and detailed studies are required to further determine the clinical significance of these miRNAs in PCa development and progression.Materials. Material details and catalog numbers are provided in Metridia Luciferase (MLuc) driven by the β-actin promoter and enhancer [2. Cells verified mycoplasma free annually by MycoAlert Mycoplasma Detection Kit . LNCaP, LAPC4, DU145 and PC3 were authenticated by DDC medical , November of 2014.Human PCa cell lines LNCaP, DU145 and PC3 were purchased from ATCC . LAPC4 and VCaP cells were provided by John Isaacs . Cells were obtained between 2006-2010. LNCaP-MLuc and PC3-MLuc cells stably express enhancer . ConditiThe Dharmacon miRIDIAN® microRNA Mimic Library was purchased from the Johns Hopkins High Throughput Biology - HIT Center . Cells were Lipofectamine  transfected with each mimic (20 nM) in 24 well plates. Protein lysates were harvested 48 hours later and transferred to 384 plates. Dilutions  were generated by Microlab STARlet liquid handling robotics system . Triplicate spots were printed on nitrocellulose-coated Fast slides  by a 4-tip GeSim Nano-Plotter 2 robotics system . Each LMA included control mimic transfection and PC3 and DU145 as AR and PSA negative controls. LMAs were immunoblotted for AR (AR441), PSA (A0562), and GAPDH (G9545) overnight at 4°C and detected by LI-COR Odyssey imaging system . Antibody dilutions were established in pilot studies . For eacRenilla luciferase  in 96 well plate format in phenol-red-free medium supplemented with 10% charcoal stripped serum. Anti-AR and control FlexiTube siRNA  were included (20 nM) in each plate. 48 hours later, cells were treated with 5 nM R1881 and firefly luciferase activity determined 24 hours later, an established endpoint for androgen-induced transcriptional activity [Renilla normalized activity was quantified by a non-commercial dual luciferase assay [LNCaP cells were Lipofectamine reverse-transfected in triplicate with the miRNA library (20 nM), an AR-responsive firefly luciferase reporter (pBK-PSE-PBN-Luc) driven by the probasin (PBN) promoter and the PSA enhancer (PSE), and pRL-CMV se assay , on a WaRenilla buffer by Wallac Microbeta luminometer. Cell viability 11 days post transfection has been previously reported [Cell viability was determined by MLuc Viability Assay as previously described . Brieflyreported . Fold chSDS-PAGE and immunoblotting were performed as previously described . Anti-ARRenilla luciferase [AR 3′UTR regions were PCR amplified  from LNCciferase , 49.Renilla normalized firefly luciferase activity in cells Lipofectamine co-transfected with mimics or siRNAs, pBK-PSE-PBN-Luc, and pRL-CMV in RPMI 1640 plus charcoal stripped FBS. 48 hours later, cells were treated with 5 nM R1881 and AR transcriptional activity determined 24h later by dual luciferase enzyme assay [pEGFP-AR and pEGFP-AR-V7 have been previously described . Plasmidme assay , 49, as Total RNA from normal prostate (n=15), primary PCa (n=15) and metastatic CRPC (n=15) was provided by the Prostate Cancer Biorepository Network . CRPC (n=15) were isolated from adrenal (n=1), lymph node (n=4), diaphragm (n=2), lung (n=1) and liver (n=7) metastases. TaqMan MicroRNA RT  for miRNAs and RNU6B was followed by ddPCR on Bio-Rad's QX200 ddPCR System, according to manufacturer protocol. Expression was RNU6B normalized. For PSA, cDNA was generated using QuantiTect Reverse Transcription Kit  and qPCR was performed using Invitrogen SYBR GreenER qPCR SuperMix  on the ABI 7900 HT Real Time PCR System. β-actin was used for normalization. Primers are included in Differences between groups were evaluated by Student's t-test, relative to negative or untreated control. Spearman correlation calculations were applied to gene expression studies. Statistical significance was assigned with p < 0.05.


Here, we investigated the role of ar in zebrafish using CRISPR/Cas9 gene targeting technology. Targeted disruption of ar in zebrafish increases the number of female offspring and increases offspring weight. In addition, ar-null male zebrafish have female secondary sex characteristics. More importantly, targeted disruption of ar in zebrafish causes male infertility via defective spermatogenesis and female premature ovarian failure during growth. Mechanistic assays suggest that these effects are caused by fewer proliferated cells and more apoptotic cells in ar-null testes. Moreover, genes involved in reproductive development, estradiol induction and hormone synthesis were dys-regulated in testes and ovaries and the reproductive-endocrine axis was disordered. Our data thus suggest that the zebrafish ar is required for spermatogenesis and maintenance of ovarian function, which confirms evolutionarily conserved functions of ar in vertebrates, as well as indicates that ar-null zebrafish are a suitable model for studying pathologic mechanisms related to androgen disorders.The androgen receptor (AR) is a nuclear receptor protein family member and inducible transcription factor that modulates androgen target gene expression. Studies using a mouse model confirmed the need for  Upon androgen binding, the ar forms a dimer, translocates to nucleus and recruits a wide variety of co-regulators to modulate androgen target genes , 69ar ged affair .ar is evolutionarily conserved because targeted disruption of ar in zebrafish caused defects in spermatogenesis and maintenance of ovarian function, which were similar to ar-null mice. In addition, late onset of obesity phenotypes as indicated by fatty abdominal cavities were also evident in ar-null zebrafish, similar to that of ar-null mice [ar-null zebrafish, it might also cause zebrafish to gain weight.It appears that the function of ull mice . Thus, iar caused defects in the formation of breeding tubercles (BTs), which confirmed the role of androgen action on development of zebrafish secondary sex characteristics. However, we observed no defects of pectoral fin regeneration in ar-null male zebrafish, which is reported to be controlled by androgen/GSK3 signaling [Androgen action on zebrafish organogenesis has been described –73 and aignaling . Such inignaling .ar disruption on reproductive development have been described in a mouse model, the underlying mechanisms are unknown. Once androgens bind to their ar, a signaling cascade is initiated that directly or indirectly stimulates reproductive development, but how these genes work and how deregulation of the genes in ar-null mice (zebrafish) cause a series of defects is unclear. We noted expression of meiosis-related genes (ccnd2a and gsdf) were dysregulated in ar-null testes and fewer proliferated cells and more apoptotic cells were present. This may partially explain the effect of ar on zebrafish spermatogenesis. Also, we noticed that expression of genes involved in reproductive development (sox9a and amh) [cyp19a1a and hsd17b1) and hormone synthesis (star and cyp17a1a) were deregulated in testes and ovaries after ar disruption. In addition, in ar-null male zebrafish, the level of testosterone was decreased, the level of 11-ketotestosterone was increased. By contrast, in ar-null female zebrafish, the level of androgenic sex hormone was increased dramatically, but the level of estrogenic sex hormone was decreased significantly. These phenomena not only suggested that the feminization of ar-null zebrafish was mediated by estrogen signaling, but also indicated that the importance of androgen signaling in maintenance of ovary function. The disorder of sexual hormone in ar-null zebrafish also confirmed that in zebrafish the synthesis of sexual hormone was inter-conversed during they were synthesized from cholesterol. In ar-null zebrafish, when testosterone was reduced, estradiol was increased or vice versa [ar disruption could explain phenotypes seen in ar-null zebrafish.Even though the effects of and amh) , 60, 61,ce versa . Therefoar-null male zebrafish and zebrafish with testis androgen deprivation using trilostane. In zebrafish with testis androgen deprevtion, Leydig cell marked gene (star) was increased, but the Sertoli cell gene marked genes  were not changed significantly. It seemed that androgen deprivationmainly affected androgen synthesis in Leydig cells [ar-null zebrafish, 11-KT was increased and the expression of genes related to steroidogenesis was increased in Leydig cells. In addition, the expressions of Sertoli cell marked gene (amh), and the spermatogonia-stimulating gene (gsdf) were also increased. Given that 11-ketotestosterone (11-KT) instead of testosterone serves as the prominent circulating androgen in zebrafish, the germ cell activities modulated by androgen action might mediate by paracrine factors.Intriguingly, we noticed some differences between ig cells . Howeverar-null zebrafish may be a potential model for revealing pathological mechanisms of diseases related to ar defects. Using genetic approaches to understand mechanisms involved in ar-modulated reproductive development in a zebrafish model may help to elucidate the role of ar in hormone-dependent physiological development and pathogenesis, such as testicular feminization mutation syndrome (Tfm), oligozoospermia and stein-leventhal symdrome.Taken together, Danio rerio; AB strain) were used for generating ar mutant lines. The guidelines for zebrafish nomenclature  were followed for naming the two mutants (inb1225/ihb1225ar  and inb1226/ihb1226ar ) (https://zfin.org/action/feature/view/ZDB-ALT-161228-1 and https://zfin.org/actin/feature/view/ZDB-ALT-170119-1).The wild type zebrafish , heterozygous (ar+/−) and homozygous (ar−/−).The zebrafish-Codon-Optimized Cas9 plasmid  was digetemplate . The gentemplate . If the ar−/− and wildtype (ar+/+) siblings, we set 5 groups for ar−/− or ar+/+ female respectively. One ar−/− or ar+/+sibling female was mated with two wildtype males (AB line) every day for 2 weeks. The egg numbers were counted and the survival rate was calculated.Egg production assessment was conducted as described previously . To eval2, 1.0 mM MgSO4, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 4.2 mM NaHCO3, and 5.55 mM glucose, pH 7.2) and then were crushed using a forceps. Simultaneously, the eggs were obtained from wildtype female zebrafish (AB line). Artificial insemination was carried out according to the method reported previously [For artificial insemination, at 3 mpf, the sperm was obtained by crushing the dissected testes. Briefly, the testes were dissected from mutant male zebrafish under a stereo microscope and transferred to a culture dish. The testes were cut into small pieces in 100 μl of Hanks’ balanced salt solution  at 40×magnification. The motility value was presented as the percentage of sperms with active movement, the sperms only with vibration were not counted as motile sperms. For each sample, the sperm motility was measured in three different fields at least 2 times.ar+/+) and homozygous (ar−/−) zebrafish, we firstly distinguished them by morphology. Female zebrafish had a big abdomen and prominent genital pore. To ensure the accuracy, we eventually verified their sex by dissection.For sex determination of the wildtype (Zebrafish (4.5 mpf) were weighted, photographed and dissected after anesthetization. Intact testes and ovaries from zebrafish (1.5 to 5 mpf) were dissected and fixed in 4 % PFA  over night at 4°C. Samples were dehydrated and embedded in paraffin, and cut into 4 μm. Hematoxylin-Eosin (H.E) staining was performed as described previously , 80. TolBlood samples were collected from 5 month-old zebrafish as described . For eacImmunofluorescent staining for germ cells was performed using anti-Vasa antibody (DEAD-box helicase 4 (Cag_Vasa) antibody; Cat# Cag_Vasa, RRID:AB_2631966) as described previously , 84. ImmApoptotic cells were detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay .ar+/+ and ar−/− zebrafish (5 months) with similar weight were injected with 10 μl each (100nm/L). After 12 hours of intraperitoneal injection, intact ovaries from adult zebrafish were dissected after anesthetization, and total RNA was extracted from the ovaries for semi-quantitative RT-PCR assays.Dihydrotestosterone (DHT) (Sigma-Aldrich) were prepared in the vehicle dimethoxyethane (DME)  and diluted in Sodium Chloride solution at 100 nM concentration. Three fActb1 (β-actin) was used as an internal control [Total RNA was extracted using RNAiso Plus  following the protocol provided by the manufacturer. cDNAs were synthesized using the Revert Aid First Strand cDNA Synthesis Kit (Fermentas). SYBR Green mix (Roche) was used for quantitative RT-PCR assays. The gene names and the primers are listed in  control , 86.t-test) (GraphPad Software Inc). Data are reported as means ± S.E.M. of three independent experiments performed in triplicate. The difference was considered to be significant if the p values were less than 0.05. The p values are summarized with the following symbols: * p<0.05; ** p<0.01; ***p<0.001.Statistical analysis for sex ratio was performed using Microsoft Excel 2007, other statistical analysis was performed using GraphPad Prism, v5 (unpaired


An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor.The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å in vivo.The androgen receptor is crucial for the development and physiology of reproductive organs. Here the authors present the structure of the androgen receptor ligand-binding domain bound to dihydrotestosterone, identifying a homodimerization interface that is crucial for receptor activity  AR gene have been reportedAR mutations have been associated with castration-resistant prostate cancer (PCa), one of the leading causes of cancer death in men worldwide6The androgen receptor AR/NR3C4) belongs to the steroid receptor subfamily of nuclear receptors (NRs), which also includes the glucocorticoid receptor GR/NR3C1), mineralocorticoid receptor MR/NR3C2), progesterone receptor (PR/NR3C3) and oestrogen receptors α and β . Steroid receptors are major therapeutic targets, due to their pivotal role in a number of endocrine-related diseases, mineral, progestC4 belongStructurally, the AR is similar to other NRs consisting of an N-terminal domain, followed by an almost strictly conserved DNA-binding domain (DBD), an interdomain linker or hinge, and a C-terminal ligand-binding domain (LBD). The LBD contains the internal ligand-binding pocket (LBP) and two major solvent-exposed surfaces responsible for interaction with coregulators, activation function 2 (AF-2) and binding function 3 (BF-3)8910121314151720212225AR mutations linked to AIS or PCa have been found to cluster at this interface providing significant in vivo support for the current homodimeric AR-LBD structure. Disease-associated mutations were found to affect the dimer interface and lead to functional dysregulation of key AR actions, corroborating the physiological significance of this protein–protein interaction site.Here we present the crystal structure of the human AR-LBD homodimer bound to its natural agonist, dihydrotestosterone (DHT) and provide in addition strong evidence for its crucial role in receptor functioning. Most importantly, over forty published 59TESLQFLLDTCKV72 (S59-V72), was found by surface plasmon resonance (SPR) to bind with high affinity to liganded AR-LBD  was identified in yeast two-hybrid screens to bind to DHT-bound AR-LBD, used as bait against human adult brain and prostate cDNA libraries. UBA3 has previously been shown to interact directly with ERα ref. . The ande AR-LBD , similar12121), and feature a monomer in the asymmetric unit (ASU)2021222 of solvent-exposed surface from each molecule (All AR-LBD structures deposited in the Protein Data Bank (PDB) to date belong to the same crystal form . Significant structural differences were limited to the more N-terminal residues (E669-F674) and to some loops that were mobile or even partially disordered in most monomeric AR-LBD structures. (Numbering refers to the recently revised sequence of full-length human AR). This is the case of L1–3 (E682-S697), but in particular of the basic L9–10 (C845-N849), which is clearly defined by electron density in the current structure . The staCore dimer: the two monomers in the AR-LBD core dimer are arranged ‘head-to-head' around a local pseudo twofold axis with both AF-2 pockets facing opposite directions and separated by over 60 Å . In esseπ-stacking interactions of residues W752 and F755 rigidify the H5 helices, which appears to be essential for this H5-H5′ interface. Particularly strong van der Waals (vdW) interactions are formed between the F755-P802 pairs  × 103 M−1 s−1) and dissociation phases (kd=0.072±0.002 s−1), from which an affinity constant (KD)=8.8±0.08 μM could be calculated , and thus capable of reacting with sulfhydryl-reactive small molecules. Further, inspection of the homodimer structure immediately reveals that only residues C687 from the two core monomers are located close enough to be simultaneously engaged by the short-arm crosslinker, bis-maleimidoethane BMOE; . As expeIn addition, we assessed the possible relevance of the H6-BF-3 interaction in solution by taking advantage of the presence of a salt bridge between residues K778* and E830 see . We concThe experiments described above were conducted using highly purified, isolated AR-LBD proteins. To study AR-LBD dimerization in living cells, we performed acceptor-bleaching fluorescence resonance energy transfer microscopy (FRET) experimentsin vivo, we introduced either a mutation predicted to favour homodimer formation  or the CAIS-associated mutation, P767A, in both EYFP- and ECFP-tagged AR-LBD fusions  or both AIS and PCa (Y764C), or that represent more drastic replacements of naturally occurring variants . With exception of the Y764C exchange, all mutations were anticipated to have a negative effect on homodimer stability.50, while for R761E the response was reduced two-fold. Interestingly, the maximal response of the Y764C variant previously shown to retain the homodimerization ability almost doubled that of wild-type AR. These dramatic effects on transactivation activity caused by mutations that affect the dimer interface were not due to large differences in ligand affinity and binding capacity, as evaluated in whole-cell binding assays for the full-length AR variants W752R, Y764C and P767A  compares well with other solved structures of NR homo- and heterodimers, and is significantly larger than the previously reported GR-LBD and PR-LBD homodimers323334in vivo, the isolated AR-LBD was shown by FRET to dimerize also in a more complex, cellular environment. Importantly, the control of dimerization by AR agonists and antagonists correlates with the well-known effect of these compounds on the activity of the full-length receptor. Finally, the CAIS-associated mutation, P767A, which is predicted to impair dimerization because of its negative impact on vdW interactions, disrupts DHT-induced homodimer formation.The contribution of LBD dimerization to the physiological activity of the AR has remained controversial for a long time. Here we present the crystal structure of an AR-LBD homodimer, along with biochemical and functional evidence of its relevance The role of the AR-LBD interface in the functioning of the full-size receptor was further verified through functional analysis of carefully selected mutations of residues exposed on the contact surface. Replacements were predicted to either disrupt  or enhance dimerization (Y764C) without major impact on the 3D structure of the AR-LBD and without altering other important functions . In factInterestingly, the Y764C variant showed a higher transactivation potential than the wild-type receptor. A similar higher activity has been reported for another mutation, T756A, which would also stabilize the dimer according to our analysesAltogether, the current findings demonstrate that the LBD dimerization surface is critical for the transcriptional activity of the AR and for androgen physiology. There is now evidence for inter- and intramolecular interactions at the levels of the N/C interactionsAR mutations that affect residues buried in the AR-LBD dimer interface  map to the LBP. Mutations in residues that line AF-2 and BF-3 grooves explain further 28 (22%) and 21 (16%) variants, respectively202122ace 33%; . This isWe used various bioinformatics tools for a systematic in-depth rationalization of the impact of disease-linked point mutations on AR protein folding as well as on homodimer formation and/or stability . InteresWhile the physiological relevance of the core LBD dimer for AR functioning seems to be solidly demonstrated by a wealth of structural and functional data, including a large number of naturally occurring point mutations, there are certain more speculative issues raised by the current structure that we would like to address below.Signal transduction in NRs at the molecular level is mediated by long-range communication between topographically distinct (non-overlapping) binding sites . These allosteric transitions may involve subtle, reversible conformational changes that are still under intense investigation. How allosteric effects are propagated across the different intra- or inter-functional surfaces  are still not fully elucidated8424344202122At the same time, LBP occupancy in one LBD may influence the ligand-binding capacity of the second monomer through the H5-H5′ interface. Whether this allosteric effect fully explains the reduction in ligand affinity of the P767A variant remains to be investigated. Along these lines, it is noteworthy that binding of antagonist R-bicalutamide destabilizes helix H5 in monomeric LBD, as indicated by significantly higher B factorsThe LBP-to-LBP′ allosteric connection could be expected to synchronize the AF-2 and BF-3 grooves from the two interacting partners , in line84243454647The LBDs of the subfamily of oxosteroid NRs  differ in several key structural features from the ER subclass and the RARα-RXRα heterodimers322 buried surface area versus 1,000 Å2 in the case of the AR homodimer  are highly conserved in all oxosteroid receptors. Indeed, the current AR-LBD homodimer shows some resemblance to a previously reported crystal structure of homodimeric GR-LBDomodimer . A hypotomodimer . Notably2 of solvent accessible surface3351Similarly, our modelling experiments also indicate that head-to-head dimers are perfectly compatible for the PR and MR LBDs. We notice that a topologically unrelated configuration has been reported for the PR-LBD homodimer that includes H12 and is formed upon exclusion of 700 Åin vivo and allows for a deeper understanding of the mechanisms of action of this important transcription factor. We provide structure-function insights into how pathogenic mutations that cluster at the dimer interface alter key functions of the AR. Furthermore, the current structure suggests an elegant map of allosteric connections between major AR functional sites with important implications for signal transmission across the LBD. How this dimeric structure fits in the context of the AR full-length dimer and its exact roles in spatial-temporal control of gene transcription remain major intellectual challenges with important clinical implications. Even though caution must be exercised when extrapolating the current results to other NRs, the head-to-head structure of the AR-LBD homodimer might represent a common conformation for both homo- and heterodimers of other members of the subfamily of oxosteroid receptors and perhaps for other NRs as well. Moreover, despite the inherent challenges to develop protein–protein disruptors, our structural investigation opens up avenues for therapeutic intervention, as small molecules that may interfere with AR dimerization could be potentially useful for PCa treatment.In summary, the crystal structure of the AR-LBD homodimer presented here appears to represent a physiologically relevant conformation of the dimeric receptor 59-V72 of human UBA3 was custom-synthesized at Pepmic. Recombinant human AR-LBD (residues 662–919) was expressed as a fusion protein with thioredoxin and purified to homogeneity using standard purification methodsA peptide corresponding to residues S59S–72V peptide and incubated overnight at 4 °C. Drops of the AR-LBD-UBA3 mixture were equilibrated against 0.1 M HEPES, pH 7.5, 1.32 M ammonium sulfate using the sitting drop vapor-diffusion method. Diffraction data were collected at 100 K at ALBA CELLS synchrotron and processed using MOSFLM (http://www.mrc-lmb.cam.ac.uk/harry/mosflm/) and CCP4 (http://www.ccp4.ac.uk/). The crystal structure was solved and refined using MOLREP, REFMAC5 and COOT from the CCP4 package. Crystal packing analysis was performed using PISA (http://www.ebi.ac.uk/), model quality was assessed with MolProbity (http://molprobity.biochem.duke.edu/) and figures were prepared with PyMOL (http://www.pymol.org).Purified, concentrated DHT-bound human AR-LBD (residues 662–919) was combined with a threefold molar excess of synthetic UBA3 2SO4, 5% glycerol, 1 mM DTT, 5 mM DHT. Sensorgrams were analysed with the BIAcore T200 Evaluation software 3.0 and Scrubber2, and fitted according to the 1:1 Langmuir model.SPR analyses were performed at 25 °C in a BIAcore T200 instrument . Highly purified, DHT-bound recombinant AR-LBD was diluted in 10 mM sodium acetate, pH 4.7 and directly immobilized on a CM5 chip  by amine coupling. Two different ligand densities were used: ∼300 resonance units (RU) for AR-LBD self-association experiments, and ≈6,800 RU for studying the binding of the UBA3-derived peptide to AR-LBD. As a reference, one of the channels was also amine-activated and blocked in the absence of protein. Alternatively, in some experiments the unrelated NR, TLX/NR2E1, was coupled in the reference channel. The running buffer was 50 mM HEPES pH 7.2, 50 mM LiPurified recombinant human AR-LBD (33 μM) was incubated either with glutaraldehyde  for 10 min, or with 200 μM bis(maileimido)ethane  or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride  for 1 h at 37 °C following the manufacturer's instructions. Samples of the reaction mixtures were boiled in the presence of reducing Laemmli sample buffer, and separated by SDS-PAGE.Proteins were resolved on 10% SDS-polyacrylamide gels, which were then either stained with Coomassie Brilliant Blue or electroblotted onto PVDF membranes. Western blot analysis of recombinant AR-LBD was performed using a commercially available anti-AR-LBD antibody . For verifying expression levels of full-length AR we used an in-house antibody targeting the N-terminal domain of the protein−1 using a nanoAcquity Ultra Performance LCTM chromatographic system (Waters). Peptides were separated using a C18 analytical column  with a 120-min run, comprising three consecutive linear gradients: from 1 to 35% B in 100 min, from 35 to 50% B in 10 min and from 50 to 85% B in 10 min . The column outlet was directly connected to an Advion TriVersa NanoMate (Advion) fitted on an LTQ-FT Ultra mass spectrometer (Thermo), which was operated in positive mode using the data-dependent acquisition mode. Survey MS scans were acquired in the FT with the resolution (defined at 400 m/z) set to 100,000. Up to six of the most intense ions per scan were fragmented and detected in the linear ion trap. The ion count target value was 1,000,000 for the survey scan and 50,000 for the MS/MS scan. Target ions already selected for MS/MS were dynamically excluded for 30 s. Spray voltage in the NanoMate source was set to 1.70 kV. Capillary voltage and tube lens on the LTQ FT were tuned to 40 and 120 V, respectively. The minimum signal required to trigger MS to MS/MS switch was set to 1,000 and activation Q was 0.250. Singly charged precursors were rejected for fragmentation.Coomassie Brilliant Blue-stained bands corresponding to monomeric and dimeric AR-LBD were excised from the gels and subjected to in-gel digestion following standard protocolsversus dimeric AR-LBD conformations: CUPSAT  and were authenticated by short-tandem repeat DNA profiling by Genetica. PC-3 and COS-7 cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% foetal calf serum, while Hep3B cells were cultured in minimum essential medium (MEM)-α medium with GlutaMAX supplement and without nucleosides (Gibco).AR gene was genetically labelled with EYFP  or ECFP . In addition, P767A and Y764C mutations were introduced in these constructs. Acceptor photobleaching (abFRET) was performed following standard protocols2 25 times at 514 nm at high laser power. After photobleaching, a second EYFP and ECFP image pair was collected. Apparent FRET efficiency was calculated using the equation:To enable detection of AR-LBD dimerization in living cells, a fragment coding for residues 612–919 of the human before and EYFPbefore are the mean prebleach fluorescence intensities of ECFP and EYFP, respectively, in the area to be bleached (after subtraction of background), and ECFPafter and EYFPafter are their mean postbleach fluorescence intensities, in the bleached area. The apparent FRET efficiency was finally expressed relative to control measurements in cells expressing either free ECFP and EYFP (abFRET0) or the ECFP-EYFP fusion protein (abFRETECFP-EYFP fusion):where ECFPPC-3 cells were seeded in 96-well plates  and transfected with 100 ng of reporter construct, 10 ng of AR expression vector and 5 ng of pCMV-β-gal expression vector. Hormone treatments were done in triplicate; transfections were done in biological quadruplicates. After transfection, the cells were stimulated with increasing concentrations of DHT. The following day, cells were harvested in Passive Lysis buffer and luciferase as well as β-galactosidase activity were measured with a Luminoskan luminometer3H]-labelled mibolerone and increasing concentrations of unlabelled DHT (0.1 nM–10 μM). After incubation at 37 °C for 90 min, cells were washed three times with ice cold PBS and lysed in Passive Lysis buffer. Radioactivity present in these extracts was determined by liquid scintillation counting.COS-7 cells were seeded in 48-well plates at a density of 30,000 cells per well and transfected with 375 ng of AR and 75 ng of pCMV-β-gal expression vectors, respectively. On the next day, cells were incubated with 1 nM [3H]-labelled mibolerone or with the same concentrations of the unlabelled ligand. After incubation at 37 °C for 90 min, cells were washed three times with ice cold PBS and lysed in Passive Lysis buffer. Radioactivity present in these extracts was determined by liquid scintillation counting. Maximal binding was determined by incubating the cells with 300 nM labelled mibolerone.COS-7 cells were seeded in 48-well plates at a density of 30,000 cells per well and transfected with 375 ng of AR and 75 ng of pCMV-β-gal expression vectors, respectively. On the next day, cells were incubated with a range of concentrations of [www.rcsb.org) and the accession code assigned is 5JJM. The PDB accessibility has been designed ‘for immediate release on publication'. The following PDB accession codes were used in this work: 1I38, 1T76, 1T63, 1XQ3, 1T5Z, 1Z95, 1XNN, 2IHQ, 2HVC, 2PIX, 2PIU, 2PIR, 2PIO, 3L3Z, 4OH5, 1M2Z, 1A28, 3D57, 4IQR, 1DKF and 3E00. The proteomic data sets have been deposited in the PRIDE repository and the data set is available via ProteomeXchange with the data set identifier PXD005575. The authors declare that all the data supporting the findings of this study are available within the article and its The atomic coordinates and structure factors have been deposited in the PDB .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Notes, Supplementary References.


Within 2 to 3 years of ADT, disease recurs—at which time, patients are considered to have castration-recurrent PCa (CR-PCa). A universal mechanism by which PCa becomes resistant to ADT has yet to be discovered. In this review article, we discuss underlying molecular mechanisms by which PCa evades ADT. Several major resistance pathways center on androgen signaling, including intratumoral and adrenal androgen production, AR-overexpression and amplification, expression of AR mutants, and constitutively-active AR splice variants. Other ADT resistance mechanisms, including activation of glucocorticoid receptor and impairment of DNA repair pathways are also discussed. New therapies have been approved for treatment of CR-PCa, but increase median survival by only 2-8 months. We discuss possible mechanisms of resistance to these new ADT agents. Finally, the practicality of the application of “precision oncology” to this continuing challenge of therapy resistance in metastatic or CR-PCa is examined. Empirical validation and clinical-based evidence are definitely needed to prove the superiority of “precision” treatment in providing a more targeted approach and curative therapies over the existing practices that are based on biological “cause-and-effect” relationship.Prostate cancer (PCa) is the most widely diagnosed male cancer in the Western World and while low- and intermediate-risk PCa patients have a variety of treatment options, metastatic patients are limited to androgen deprivation therapy (ADT). This treatment paradigm has been in place for 75 years due to the unique role of androgens in promoting growth of prostatic epithelial cells  Prostate cancer (PCa) is the most common male cancer in the Americas, Caribbean, Western and Northern Europe, Australia, New Zealand, and several countries in sub-Saharan Africa . The Amevia the transrectal ultrasound-guided (TRUS) method [Serum PSA and DRE are used in combination to determine if a biopsy should be performed, usually ) method . Other i) method . Further) method , 14. Des) method . Importa) method . Along w) method , 17. The) method  and are ) method , 20. Thi) method , 20. SinFollowing diagnosis, PSA, Gleason score, and general tumor staging are used to assess overall prognosis , 22. WhiMen who are diagnosed with intermediate-risk PCa are a more heterogeneous population than those in the low-risk group . TypicalIn the most simplistic of paradigms, PCa cases are considered high-risk with a Gleason score 8-10, PSA ≥ 20 ng/mL, and indications of high tumor volume with or without regional metastases  treatment of the castrate-recurrent CWR-R1 cell line cultured in low-androgen conditions promotes phosphorylation of AR at serine 515 (Ser515) and serine 578 (Ser578), mitogen activated protein kinase (MAPK) and protein kinase C (PKC) consensus sites, respectively [Post-translational modification of AR by phosphorylation plays a role in its protein stability, transcriptional activity, and nuclear localization , 117. Phectively . AR Ser5ectively . Phosphoectively . Overexpectively . These dectively , 120. Inectively . Since Iectively . More reectively . Howeverectively . For insectively . Since 2ectively . When phectively . In a laectively . No correctively . These dectively , 123. Ovectively , 117, a As discussed in the previous section, AR mutations clinically relevant to therapy resistance are located in the LBD, demonstrating that alternations in this domain can drive CR-PCa . Furthervia an unbiased method with a protocol using in vitro transcription and not primer-directed PCR [There is also evidence of AR variant transcripts derived from exon skipping. With primers against AR exon 2 and 8, RT-PCR using cDNA generated from LuCaP xenografts produced a short amplicon lacking exons 5, 6, and 7 . ARv567ected PCR . This mected PCR . Three ncted PCR . Of thescted PCR . Furthercted PCR . Based oLuciferase assays were utilized to characterize the activity of AR variants in many studies, but these data represent AR transcriptional activity at only exogenously-introduced promoters. When compared to the LNCaP cells treated with R1881, LNCaP cells expressing ARV7 had equivalent induction of 20 different androgen-regulated genes (ARGs); providing evidence of ARV7's constitutive activity in endogenous gene transcription . Similarhttps://clinicaltrials.gov/). In addition, several clinical trials are already using ARV7 expression analysis in CTCs to stratify their CR-PCa patient populations and assess effectiveness of treatments, including cabazitaxel  and galaterone  based on ARV7 expression status. The results of the clinical trials aimed to assess ARV7 as a biomarker and determine the effectiveness of specific therapies in ARV7-expressing CR-PCa patients are awaited in anticipation to determine the true clinical utility of AR splice variants in governing patient decision-making.Several studies have shown that AR splice variants are expressed in PCa tumor samples Table . Semi-quWhile understanding the role of androgen signaling and AR activity has been at the forefront of research identifying molecular drivers of CR-PCa, there are also mechanisms that possibly govern resistance to ADT that do not directly involve androgen signaling. For the purpose of this review article, we will limit our discussion of those pathways considered clinically relevant, including activation of glucocorticoid receptor (GR), impairment of DNA repair pathways, and expression of microRNAs.Multiple treatment regimens for PCa include glucocorticoid administration . As discIt has been known since the early 2000s that glucocorticoids promote PCa cell proliferation. Treatment with either cortisol, the primary glucocorticoid in circulation, or cortisone, a metabolite of cortisol, increased proliferation of the recurrent MDA-PCa 2b cell line . In conthttps://clinicaltrials.gov/). It is the hope that a different glucocorticoid regimen will be able to suppress the side effects of abiraterone, but also limit a patient's exposure to these steroids that enable survival of PCa cells in an androgen-deprived environment.The amino acid sequences of the DBDs of AR and GR are 80% homologous ; therefoin vitro assay showed that microsatellite instability was increased in DU145 and PC3 cells compared to HeLa cells [It has been known since the mid-1990s that genomic abnormalities are associated with PCa –146. SpeLa cells . TogetheLa cells . SpecifiLa cells , loss orLa cells . These dvia ADT may impair the activity of Ku70. Al-Ubaidi et al. performed immunohistofluorescence for Ku70 before and after castration from the same tumor, which showed that Ku70 nuclear staining was decreased in castrated tissue [In addition to playing a role in initial development of PCa, DNA repair pathways in PCa tissues are inhibited by ADT. Al-Ubaidi et al. performed prostate biopsies on PCa patients following initial diagnosis and after short-term ADT  to assess whether castration affects the activity of non-homologous end-joining (NHEJ), one pathway responsible for repairing double stand breaks (DSBs) . Their hd tissue . When thd tissue . To deted tissue , a histod tissue . Quantifd tissue . TogetheThe intent of the research described above was to explain why outcomes for PCa patients receiving ADT and radiotherapy were better than those receiving radiotherapy alone . In addiWhile deficiency in the DNA repair response is advantageous during PCa radiotherapy, data suggests that long-term impairment of DNA repair pathways by ADT may contribute to resistance in advanced/metastatic PCa. Genomic alterations in genes associated with the DNA damage response have been found in 22.7% of metastatic CR-PCa samples . These gMicroRNAs (miRs) are endogenously expressed short non-coding RNA molecules that negatively regulate gene expression by binding to mRNAs and inhibiting translation . A comprThe study of miRs in PCa pathogenesis and development of CR-PCa has shown that specific miRs can act either as tumor suppressors or oncogenes, where tumor suppressor-miRs are decreased and oncogenic-miRs are increased in expression , 163. ThAbiraterone and enzalutamide are recently approved therapies for CR-PCa that target androgen signaling Table , the mecvia the enzymes CYP11A1 and HSD3B1 or 2, not CYP17A1, it was postulated that survival of C4-2 cells with abiraterone is dependent on expression of AR T878A [Abiraterone targets androgen biosynthesis by inhibiting CYP17A1, the role of which is to convert pregnenolone to 17α-OH-pregnenolone and 17α-OH-pregnenolone to DHEA during steroidogenesis . Cai et AR T878A . A speciAR T878A . Nested AR T878A . The numAR T878A . These dAR T878A . InteresAR T878A . The secAR T878A . FinallyAR T878A . Altogetin vitro and preclinical data suggest that AR F877L contributes to enzalutamide resistance via its ability to be activated, rather than inhibited, by this second generation ADT drug [As discussed in detail above, several lines of evidence suggest that resistance to enzalutamide may be governed by increased GR expression and GR-dependent activation of ARGs . EnzalutADT drug . In a reADT drug ; howevervia personalized oncology rests in the identification of biomarkers or gene panels that are biological relevant and development of clinical tests that are technically feasible in a routine setting. Obtaining sufficient material to perform genomic analysis and standardization of procedures both remain dilemmas in the field of personalized oncology [In this review article, we describe the mechanisms by which advanced/metastatic PCa becomes resistant to approved therapies, giving special attention to the molecular biology methods used to study this continuing challenge in urologic oncology. In the era of high-thoroughput DNA sequencing, many researchers and clinicians in the field have postulated that “personalized medicine” may achieve curative therapy for metastatic PCa. Personalized medicine is a hypothesis derived from the idea that molecularly-targeted therapies based on a patient's specific genetic or molecular profile will achieve more affective clinical outcomes , 180. Inoncology . The useoncology . Even asoncology  and AR Loncology , verificoncology . In addioncology . Altogetoncology . We beli


Molecular classifications of breast cancer have resulted in improved treatments. However, treatments for triple negative breast cancer (TNBC) are lacking. Analysis of molecular targets for TNBC is a priority. One potential candidate is androgen receptor (AR) phosphorylation. This study assessed the role of AR phosphorylation at ser81/ser515 and their two upstream effectors, cyclin-dependent kinase 1 (pCDK1) and extracellular-regulated kinase 1/2 (pERK1/2) in 332 ductal breast cancer patients by immunohistochemistry.p = 0.038), decreased size (p = 0.001), invasive grade (p < 0.001), necrosis (p = 0.003), b-lymphocytes (p = 0.020), molecular subtype (p < 0.001) and estrogen receptor (ER)/progesterone receptor (PR)-status (p < 0.001). The cohort was therefore stratified into ER+ve and ER-ve patients. In ER+ve tumours, pERK1/2 combined with AR-515 associated with improved CSS (p = 0.038), smaller size (p = 0.004), invasive grade (p = 0.001), decreased b-lymphocytes (p = 0.013) and increased plasma cells (p = 0.048). In contrast, in TNBC patients, phosphorylation of AR-515 associated with poorer CSS (p = 0.007). pERK1/2 combined with AR-515 associated with decreased inflammation (p = 0.003), increased tumour stroma (p = 0.003) and tumour budding (p = 0.011), with trends towards decrease CSS (p = 0.065) and macrophage levels (p = 0.093).pERK1/2 combined with AR-515 associated with improved cancer-specific survival (CSS, In Conclusions, AR-515 may be an important regulator of inflammation in breast cancer potential via ERK1/2 phosphorylation. AR-515 is a potential prognostic marker and therapeutic target for TNBC. Approximately 1.7 million cases of breast cancer are diagnosed every year. About 522,000 die of breast cancer yearly making it the 5th most common cause of cancer death globally . In the The majority of breast cancers comprise of luminal A and luminal B subtypes. These subtypes display oestrogen (ER) and/or progesterone receptors (PR) rendering these cancer cells susceptible to endocrine therapies e.g. anti-oestrogen therapies (Tamoxifen) , 8. In HIt has been shown that TNBC patients displaying pathological complete response (pCR) had better outcomes in terms of progression-free survival than patients who had not achieved pCR . pCR is Although numerous studies on AR have been published, the exact role of AR as tumour-promoting or anti-tumorigenic remains undecided. It is known that AR signalling plays a role in regulating cancer cell proliferation and apoptosis –19. AR aIt was also reported that when Tamoxifen-resistant breast cancer cells that expressed high levels of AR were exposed to bicalutamide (anti-androgen), cell growth was shown to decrease as well as reversing Tamoxifen resistance , indicatThe MAPK/ERK signalling cascade is heavily involved in the regulation of cancer cell proliferation, growth and differentiation. The stimulation of this pathway leads to the activation of a cascade of kinases , which ultimately leads to the activation of ERK1/2, which is associated with poor prognosis in breast cancer . One funin vitro kinase assays demonstrated an increase in ser-308 phosphorylation by CDK1 is responsible for Serine–81 phosphorylation [In contrast, AR phosphorylation at ser-81 is the prototypical site for dihydrotestosterone (DHT) activation, which is thought to be facilitated by activated Cdk1, as observed by Chen et al where Cdk1 inhibitors decreased ser-81 phosphorylation of AR in LNCaP cells and similarly decreased AR protein expression and transcriptional activity. Whereas transfected Cdk1 stimulated AR phosphorylation at ser-81 and increased AR protein expression . This surylation . Cdk1 phrylation .This study aims to further understand the role of androgens and AR activation in breast cancer by determining its effect on patient survival. This is followed by the determination of the role of upstream pathways (i.e. Cdk1 and ERK1/2 pathways) on AR activation and their effects on cancer specific survival (CSS). We aim to confirm that the phosphorylation of AR at either ser-515 or ser-81 is a surrogate for AR activation and assess if the pathways responsible for this phosphorylation are potential targets for therapeutic intervention.n = 332, Figure Only patients with staining for AR, AR-81 and AR-515 were included in the analysis (p = 0.842) nor AR-515 (p = 0.563) were associated with CSS alone. Therefore, upstream effectors of AR phosphorylation were investigated. AR-81 significantly correlated with nuclear CDK1 (p < 0.001) and AR-515 correlated with nuclear pERK1/2 (p < 0.001) as expected. However, AR-81 still showed no associations even when combined with CDK1 in the nucleus (p = 0.344). In contrast, AR-515 was significantly associated with improved CSS when combined with ERK1/2 phosphorylation in the nucleus . When nuclear pERK1/2 was assessed alone it was also significantly associated with improved CSS (p = 0.004). Therefore, further analysis was confined to nuclear pERK1/2 and AR-515.The relationship between nuclear AR activation and CSS is shown in Table p = 0.001), lower grade (p < 0.001), ER/PR+ve tumours (p < 0.001), molecular subtype (p < 0.001), lower necrosis (p = 0.003), weak Klintrup-Makinen (KM) grade (p = 0.004) and lower b-lymphocyte levels (p = 0.020). There was also a trend towards decreased HER2 (p = 0.096) and lower lymphovascular invasion (p = 0.099). When pERK1/2 was combined with AR-515 significant associations were still seen with smaller size (p = 0.017), lower grade (p < 0.001), ER+ve tumours (p = 0.013), PR+ve tumours (p = 0.008), decreased HER2 (p = 0.041), molecular subtype (p < 0.001), lower necrosis (p = 0.006), weak KM grade (p = 0.005) and lower b-lymphocytes (p = 0.023). Significant associations were now also seen with decreased proliferation (p = 0.009), lymphovascular invasion (p = 0.017) and increased tumour budding (p = 0.047).The relationship between pERK1/2, AR-515, clinicopathological characteristics, and local inflammatory response is presented in Table p = 0.008). As pERK1/2 and AR-515 strongly correlated in ER+ve patients (p < 0.001) they were combined. However, when combined the survival effect was weakened (p = 0.038), suggesting that pERK can also act independently of AR-515 in ER+ve breast cancers. When ER+ve patients where stratified further, there was no difference in CSS between luminal A and luminal B subtypes for pERK1/2 alone or combined with AR-515 therefore further analysis was carried out in all ER+ve tumours (n = 228).As pERK1/2 plus AR-515 associated with molecular subtype, we firstly stratified patients into ER+ve and ER-ve tumours . pERK1/2p = 0.004), lower grade (p = 0.001), molecular subtype (p = 0.024), decreased proliferation (p = 0.006), lower b-lymphocytes (p = 0.013) and increased plasma cells (p = 0.048). There was also a trend towards decreased necrosis (p = 0.096). When pERK1/2 was combined with AR-515 significant associations where still seen with lower grade (p = 0.002), molecular subtype (p = 0.001), decreased proliferation (p = 0.001), lower b-lymphocytes (p = 0.006) and increased plasma cells (p = 0.023). Significant associations were now also seen with decreased necrosis (p = 0.009) and lymphovascular invasion (p = 0.029).The relationship between pERK1/2, AR-515, clinicopathological characteristics, and local inflammatory response in ER+ve tumours is presented in Table p = 0.023) may be due to differences between the two ER-ve subtypes. Therefore, ER-ve patients were stratified into HER2-type and TNBC to further investigate these effects (p = 0.010), however no significant effect on CSS was observed for pERK1/2 alone (p = 0.534) and only a trend towards poorer outcome was seen when combined with AR-515 (p = 0.065). However, high levels of AR-515 combined with total AR, representing full AR activation, associated with a significantly poorer outcome  and no significant associations were seen in these patients. However, there was a trend towards improved patient survival with AR-515 phosphorylation (p = 0.056) alone and when combined with pERK1/2 (p = 0.076), confirming our hypothesis that these two ER-ve subtypes respond differently to AR-515 phosphorylation.It was observed in patients with ER-ve breast cancer that high pERK1/2 plus AR-515 had a trend towards an effect on survival rate. It was hypothesised that the loss of significance and weaker correlation between pERK1/2 and AR-515 in ER-ve patients ( effects . In patip = 0.033), lower KM grade (p = 0.004), increased tumour-stroma percentage (TSP) (p = 0.033) and increased tumour budding (p = 0.007). When combined with pERK1/2 associations were still seen with lower KM grade (p = 0.003), increased TSP (p = 0.003) and increased tumour budding (p = 0.011). Trends towards association were also seen for increased age (p = 0.075) and decreased macrophages (p = 0.093).Due to the detrimental effect of AR-515 phosphorylation in TNBC, associations with clinicopathological characteristics were assessed Table . Full acp = 0.002), invasive grade (p = 0.013), lymph node involvement (p = 0.009), necrosis (p < 0.001), proliferation index (p < 0.001), lymphatic invasion (p < 0.001), cytotoxic t-lymphocytes (p = 0.005), TSP (p = 0.009), tumour budding (p = 0.001) and angiogenesis (p = 0.043) were significantly associated with poorer CSS. Lymphovascular invasion (p = 0.054) and b-lymphocytes (p = 0.064) trended towards significance.For ER+ve tumours, since pERK1/2 plus AR-515 had significant associations with improved patient survival, these factors were taken into univariate and multivariate analysis (Table p = 0.036), proliferation index (p = 0.014), cytotoxic t-lymphocytes (p = 0.001), and TSP (p = 0.033) were independent prognostic factors, with lymphatic invasion (p = 0.060), tumour budding (p = 0.084), angiogenesis (p = 0.051) and pERK1/2 (p = 0.073) showing trends towards independence. However, pERK1/2 plus AR-515 does not appear to be independently prognostic in these patients (p = 0.682).On multivariate survival analysis including pERK1/2 plus AR-515; necrosis (p = 0.022), lymph node involvement (p < 0.001), necrosis (p = 0.030), lymphatic invasion (p = 0.001), lymphvascular invasion (p < 0.001), macrophages (p = 0.011) and cytotoxic t-lymphocytes (p = 0.047) were significantly associated with poorer CSS. Age (p = 0.055) and plasma cells (p = 0.055) trended towards significance.For TNBC, since AR-51 plus total AR had significant associations with poorer patient survival, these factors were taken into univariate and multivariate analysis (Table 7). On univariate analysis, size (p = 0.006), lymphovascular invasion (p = 0.002), and macrophages (p = 0.002) were independent prognostic factors. However, AR-515 plus total AR (p = 0.957) nor AR alone (p = 0.801) appear to be independently prognostic in these patients.On multivariate survival analysis including AR-515 plus total AR; lymph node involvement . The study was approved by the Research Ethics Committee of the West Glasgow University Hospitals NHS Trust.Patients presenting with invasive ductal breast cancer at Glasgow Royal Infirmary, Western Infirmary and Stobhill Hospital between 1995 and 1998 with formalin-fixed paraffin embedded blocks of the primary tumour available for evaluation were studied  were retrieved from the routine reports. Tumour grade was assigned according to the Nottingham Grading System. ER and PR status were assessed on tissue microarrays (TMAs) using immunohistochemistry (IHC) with Dako  ER antibody and Leica  PR antibody and scored according to the American Society of Clinical Oncology and College of American Pathologists guidelines with a cut-off value of 1% positive tumour nuclei . Her2 stFull-section haematoxylin and eosin (H&E) slides for the 474 patients were used to score local inflammatory infiltrate according to Klintrup-Makinen (KM) criteria. KM scoring of slides was carried out as previously described. Briefly, tumours were scored on four-point scores based on appearances at the tumour invasive margin. A score of 0 signified that there were no inflammatory cells at tumour's invasive margin; score 1 indicated a mild and patchy inflammatory cells; score 2 denoted a prominent band-like inflammatory reaction at the invasive margin; and score 3 revealed a florid cup-like inflammatory infiltrate at the invasive edge , 44. IndFull-section H&E slides were also used to score the tumour stroma percentage (TSP) as previously reported . BrieflyLymphatic and blood vessel (lymphovascular) invasion were assessed, on 2.5-μm thick sections, using IHC staining with the lymphatic endothelial marker D2-40  diluted 1:100 and vascular endothelial marker Factor VIII  diluted 1:100 as previously described . Ki67 prThe molecular subtypes were defined as follows: Luminal A: oestrogen (ER) and/or progesterone receptor (PR) positive, Her-2 negative or low proliferative index (< 15%); Luminal B: hormone receptor positive, Her-2 positive or high proliferative index (> 15%); Her-2 subtype: Her-2 positive and hormone receptor negative, any proliferative index; and triple negative: Her-2 negative, hormone receptor negative, any proliferative index.2O2. Sections were further blocked using 5% horse serum (10% casein for pERK1/2) in Tris-buffered saline (TBS). Antibodies for pCdk1 (Abcam), pERK1/2 , AR-81 , AR-515  were incubated overnight at 4°C diluted in Dako antibody diluent at 1:150 (Dako UK Ltd.) Total AR antibody (Dako UK Ltd.) was incubated overnight at 4C at 1:4000. Bound antibody complex was visualised using EnVision plus kit (Dako UK Ltd.) followed by 3,3-diaminobenzidine tetrahydrochloride . Nuclei were counterstained with haematoxylin and blued with Scots Tap Water Substitute, dehydrated through graded alcohol and xylene and mounted with Di-N-Butyl Phthalate in xylene (DPX).Antibody validation for AR-81, AR-515, pERK1/2 and pCDK1 was carried out as previously described , 26. ImmTMAs were scanned using the Hamamatsu NanoZoomer  at × 20 magnification, and visualization was carried out using Slidepath Digital Image Hub, version 4.0.1 .Only nuclear expression was scored as this represents the active form of each protein. Tissue staining intensity was scored by two blinded independent observers using a weighted histo-score (H-score) method , 49. Then = 332). Cut-off values for high or low protein expression were determined by the median of the average values of the intensity of staining. The relationships between variables were assessed using contingency table analysis with the X2 test for linear trend. Correlations coefficients were analysed using a Spearman's rho. Kaplan– Meier curves with log rank analysis was used to examine the effect of protein expression on CSS. Univariate survival analysis was performed using Cox proportional hazards regression. Variables with P-value of < 0.05 were entered into a multivariable model using a backwards conditional method for all patients and triple negative patients. All statistical analyses were two-sided and significance defined as P-value < 0.05. All statistical analysis was performed using the SPSS software version 22 .Only patients with a score for AR, AR-81 and AR-515 were included in the analysis (