Liver fibrosis leading to cirrhosis is one of the major health burdens worldwide with currently limited therapeutic options available. Long noncoding RNAs (lncRNAs) play important roles in various biological and pathological processes in a cell- or tissue-specific manner. However, there is still an important gap in the understanding of the role of hepatocyte-specific lncRNAs in liver fibrosis.Methods: The expressions of lnc-Hser in human and mice fibrotic livers as well as primary hepatocytes (HCs) of mice developing liver fibrosis were determined by real-time RT-PCR. The roles and mechanisms of lnc-Hser in HCs and liver fibrosis were determined in vitro and in vivo.Results: In this study, we have identified a hepatocyte-specifically expressed lnc-Hser, which was reduced in human and mice fibrotic livers as well as primary HCs of mice developing liver fibrosis. We have shown that silencing lnc-Hser aggravated liver fibrosis both in vitro and in vivo through inducing the epithelial-mesenchymal transition (EMT) and the apoptosis of HCs. In addition, knockdown of lnc-Hser promoted hepatic stellate cells (HSCs) activation through the signals derived from injured HCs. Mechanistically, we have revealed that lnc-Hser inhibited HCs apoptosis via the C5AR1-Hippo-YAP pathway and suppressed HCs EMT via the Notch signaling.Conclusions: Our work has identified a hepatocyte-specific lnc-HSER that regulates liver fibrosis, providing a proof that this molecule is a novel biomarker for damaged HCs and a potential target for anti-fibrotic therapy. Liver fibrosis is a common pathological consequence of a sustained wound healing response to chronic liver injury, including viral hepatitis, alcohol abuse and autoimmune disease, characterized by abnormal deposition of extracellular matrix (ECM) and diminished hepatocytes (HCs) regeneration that is responsible for compromised liver function accounting for death Many molecules and signalings have been documented to initiate HCs apoptosis and EMT in liver fibrosis, including TGFβ1, Notch and Hippo pathways. TGFβ1, a known pro-apoptotic cytokine in mature HCs, is capable of mediating phenotypic changes and plasticity in the process of EMT, resulting in ECM deposition Long noncoding RNAs (lncRNAs) represent a group of transcripts that are above 200 nucleotides in length but without protein coding potential  in vitro and in vivo through inducing the EMT and the apoptosis of HCs. In addition, knockdown of lnc-Hser promoted HSCs activation through the signals derived from damaged HCs. we have also revealed that lnc-Hser inhibited HCs apoptosis via the C5AR1-Hippo-YAP pathway and suppressed HCs EMT through the Notch signaling. All these data suggest that lnc-HSER is a novel biomarker for damaged HCs and a potential target for anti-fibrotic therapy.In the present study, we have identified a HC-specifically expressed lnc-Hser, which was reduced in human and mice fibrotic livers as well as primary HCs of mice developing liver fibrosis. We have shown that silencing lnc-Hser aggravated liver fibrosis both2. For co-culture experiment, lnc-Hser-silenced and lnc-Hser-overexpressed AML12 cells and the controls were washed with PBS after 24 - 48h of lentivirus infection. The cells were subsequently incubated in DMEM supplemented with 10% FBS for 48h and the supernatants were centrifuged at 1100 rpm for 5 min and mixed with DMEM containing 10% FBS at 1:1 ratio for preparing conditioned medium (CM). Cells were treated with the C5AR1 inhibitor PMX 205  or γ-secretase inhibitor RO4929097  for 24 hours at concentrations of 5 μM. The antibodies were α-SMA , Collagen1 , TGFβ , MMP2 , TIMP1 , Notch2 , Notch3 , Hes1 , phospho-YAP (Ser-127) , total YAP/TAZ , phospho-MST1/2 , total MST1 , phospho-LATS (Ser-909) , total LATS1 , C5AR1 , Ki67 , Cleaved Caspase3 , Caspase3 , BAX , N-Cadherin , E-cadherin , β-Catenin , Vimentin , Snail , rabbit IgG , goat anti rabbit IgG , goat anti mouse IgG .The non-tumorigenic mouse hepatocyte cell line AML12 was maintained in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum , 1 × insulin-transferrin-sodium selenite media supplement , dexamethasone (40 ng/ml), penicillin (100 U/ml) and streptomycin (100 μg/ml). The human hepatocyte cell line L02 and HEK293T were cultured in DMEM supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml). All cells were cultured at 37°C in an atmosphere containing 5% COhttp://crispr.mit.edu/) or CHOPCHOP (https://chopchop.rc.fas.harvard.edu/) and cloned into lenti-CRISPRv2. Oligos encoding shRNA specific for lnc-Hser and the negative control shRNA were ligated into pSUPER.retro.puro, and the fragment containing the H1 promoter and hairpin sequences was subcloned into the lentiviral shuttle pCCL.PPT.hPGK.GFP.Wpre (lnc-Hser-shRNA and Negative Control (NC)). The full-length lnc-Hser cDNA was sequentially amplified by PCR and ligated into the lentiviral shuttle pCCL.PPT.hPGK.IRES.eGFP/pre to generate the over-expression plasmid (LV-lnc-Hser and the empty plasmid as the LV-Control). These plasmids were used to produce lentivirus in HEK-293T cells with the packaging plasmids pMD2.BSBG, pMDLg/pRRE and pRSV-REV. Infectious lentiviruses were harvested at 36 h and 60 h after transfection and filtered through 0.45 μm PVDF filters. Recombinant lentiviruses used in vivo were concentrated 100-fold by ultracentrifugation . The virus-containing pellet was dissolved in PBS and injected in mice within 48 h. The primer sets used are shown in Table gRNA design was based on CRISPR design (4)-induced mouse liver fibrosis model, forty Balb/c mice were randomly divided into four groups: Mice were treated with olive oil in combination with injection of lenti-NC , CCl4 in combination with injection of lenti-NC , oil in combination with injection of lenti-lnc-Hser-shRNA  and CCl4 in combination with injection of lenti-lnc-Hser-shRNA . The lentivirus was injected via the tail vein 2 weeks after the first injection of CCl4 (1 × 109 pfu/mouse). Mice in NC + CCl4 group and lnc-Hser-shRNA + CCl4 group were administered 5% CCl4 (v/v) dissolved in olive oil (0.2 ml/kg body weight) twice per week for additional 4 weeks via intraperitoneal (ip) injection after the lentivirus was injected. NC and lnc-Hser-shRNA group animals were injected with an equivalent volume of olive oil. After treatment with CCl4 for 6 weeks, all of mice were sacrificed under anesthesia with 3% sodium pentobarbital . Liver specimens and serums were obtained for analyses.Animal protocols were approved by Tianjin Medical University Animal Care and Use Committee. The methods were carried out in accordance with the approved guidelines. All Balb/c mice aged at 8 weeks were obtained from Institute of Laboratory Animal Sciences, CAMS & PUMC . For bile duct ligation (BDL) -induced mouse liver fibrosis model, mice were treated with sham operation or BDL operation for 3 to 21 days and all of mice were sacrificed under anesthesia with 3% sodium pentobarbital . For carbon tetrachloride  for 5 min at 108 °C and pretreated with 3% H2O2 for 15 min at room temperature followed by washing with PBS. Slides were subsequently incubated in normal goat serum for 20 min to block the nonspecific immunoreactivity. Next, the slides were treated with primary antibody α-SMA (1:50), Col1α1 (1:1000) or TGFβ (1:50) overnight at 4°C. In addition, tissue sections were processed omitting the primary antibody as the negative control. The slides were incubated with secondary antibody (1:500) (HRP-conjugated anti-rabbit IgG) and the reaction products were visualized using diaminobenzidine (DAB) and monitored by microscopy.The specimens were sequentially fixed in 10% formalin for two days, transferred to ethanol of different concentration and embedded in paraffin in preparation for histopathological analysis. Thin sections (5 μm) were stained with HThe TGFβ level in cell culture media and the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the serum were assessed using commercial assay kits  according to the manufacturer's protocols.Total collagen content was tested by measuring the amount of hydroxyproline in liver tissue using commercially available hydroxyproline detection kits  according to the manufacturer's instructions.in situ perfusion with 30 ml SC1 solution and 30 ml 0.05% Collagenase IV solution sequentially. HCs were then pelleted by centrifugation 50 g for 4 minutes three times. Cell viability was determined by the trypan blue exclusion method.Primary mouse HCs, HSCs, LSECs and KCs were isolated by pronase/collagenase perfusion digestion followed by subsequent density gradient centrifugation, as previously described 2O2 and subsequent proteinase K permeation. Pretreatment with DNase I served as a positive control and TUNEL reaction mixture lacking terminal transferase (TdT) as a negative control. Samples were analyzed by light microscopy (×400 magnification).For TUNEL staining, we used an in-situ cell detection kit (Roche) according to the manufacturer's protocol. After dewaxing and rehydration, we pretreated tissue sections with 3% HCytoplasmic and nuclear RNA isolation were performed with PARIS™ Kit  following the manufacturer's instruction, as previously described We used the 5'-RACE and 3'-RACE analyses to determine the transcriptional initiation and termination sites of lnc-Hser using a SMARTer™ RACE cDNA Amplification Kit  according to the manufacturer's instructions. In brief, RNA was isolated from primary HCs and 3'- and 5'-RACE-Ready cDNA were synthesized using SMART Scribe Reverse Transcriptase. The obtained band was gel purified and cloned with the lineareized pRACE vector. The obtained band was then sequenced. The gene-specific primers used for the PCR of the RACE analysis are provided in Table The apoptosis of cells with different treatment was analyzed using a FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer's instruction, as previously described Total RNA was extracted from livers or cells with Trizol reagent  and nuclear and cytoplasmic RNA were prepared using PARIS™ Kit . All RNAs were digested with DNase I . Briefly, the first-strand cDNA was synthesized using AMV Reverse Transcriptase . For real-time PCR, all reactions were performed in triplicate with SYBR Green master mix  according to the manufacturer's instructions. The expression level of housekeeping gene GAPDH was used to normalize the expression level of the genes-of-interest. The sequences of primers for real-time PCR are listed in Table Immunoblotting analysis was performed as described previously Primary HSCs, HCs and AML12 cells were re-plated on poly-lysine pre-coated glass coverslips, and were sequentially fixed with 4% paraformaldehyde in PBS overnight at 4 °C, permeabilized with 1% Triton X-100 in PBS for 0-60 min and blocked using 5% bovine serum albumin (BSA) in TBST with 0.1% Tween-20 for 30-60 min at room temperature. Next, the cells were incubated with primary antibodies against α-SMA (1:300), Col1α1 (1:500), Cleaved Caspase3 (1:400), N-Cadherin  and Ki67 (1:250), overnight at 4 °C and an irrelevant isotype rabbit IgG was used as a negative control. Cells were then incubated with Alexa Fluor 488 (1:400 for Cleaved Caspase3) or Alexa Fluor 594  in PBS away from light for 1 h at room temperature and the nuclei were stained with DAPI (5 μg/ml). Finally, the slides were washed with PBS and the coverslips were mounted with an anti-fade Mounting Medium . The immunofluorescence was then visualized by a confocal microscope (LSM 700) or a fluorescence microscope.In total, 28 human fibrotic livers and 6 human healthy livers from patients with hepatic haemangioma were obtained from surgical resections without preoperative treatment at Tianjin Third Central Hospital . For histological scoring of liver fibrosis, we stained paraffin embedded 5 μm liver sections with Sirius red, H&E and Masson's trichrome staining. Hepatic fibrosis was scored (stages F0-F4) according to the METAVIR fibrosis staging system by three hepatopathologists blinded to the study protocol and stratified as normal liver (F0), mild fibrosis (F1-F2) or advanced fibrosis (F3-F4). All subjects were of the same ethnicity. Clinical and pathological characteristics including age, gender, ALT, AST and etiologies were recorded and summarized in Table t-test (two-group comparison) or one-way analysis of variance (more than two groups) followed by post hoc comparison, and differences with p<0.05 were considered significantly.Data were expressed as mean ± SD. All the statistical analyses were performed with the SPSS 13.0 . Statistical analyses were performed using either Student's hepatocyte specific expressed lncRNA). We have subsequently performed 5'- and 3'- RACE assay with primary HCs and found lnc-Hser was a 588-nucleotide transcript with poly (A) tail, inconsistent with the ensemble database   Figure C, D. We in vivo function of lnc-Hser in hepatic fibrogenesis, we generated lnc-Hser down-expressed mouse models via tail vein injection of lenti-lnc-Hser-shRNA (lnc-Hser-shRNA) or lenti-negative control virus (NC) 2 weeks after the first injection of CCl4. After 6 weeks of CCl4 treatment, lnc-Hser silencing was confirmed by qRT-PCR in whole liver extracts  developed liver fibrosis. However, administration of lnc-Hser-shRNA in CCl4 group (lnc-Hser-shRNA-CCl4) developed more severe phenotypes of fibrosis as demonstrated by both the staining of H&E, Sirius red, Masson's trichrome and the IHC for α-SMA, Collagen1 and TGFβ  for over-expression in primary HCs and AML12 cells. Recombinant TGFβ was then used to treat the cells after infection with LV-lnc-Hser or LV-Control, and total RNA was extracted for detecting the expression of apoptotic and EMT-related genes. The results showed that the expression of the pro-fibrotic genes α-SMA, Col1α1 and MMP2, the pro-apoptosis genes BAX and BAD, and the mesenchymal marker genes N-Cadherin, Vimentin, Fibronectin, Twist and Snail1 was markedly increased, whereas the expression of the anti-apoptosis gene Bcl2, the pro-proliferation genes Ki67 and Pcna, and the epithelial marker gene E-cadherin was significantly decreased upon TGFβ treatment. However, forced lnc-Hser expression abrogated TGFβ-induced dysregulation of these genes in primary HCs and AML12 cells assessed by qRT-PCR and western blot  from control, lnc-Hser-silenced or lnc-Hser-over-expressed AML12 cells to treat primary HSCs. Compared with the control cells, treatment with the CM from lnc-Hser-silenced AML12 cells significantly promoted the expression of the fibrotic genes including α-SMA and Col1α1, but the administration with CM from lnc-Hser-over-expressed AML12 cells repressed the expression of these genes assessed by western blot, qRT-PCR and confocal microscopy Figure C-E. Morein cis when their effects are restricted to the chromosome from which they are transcribed, and work in trans when they affect genes on other chromosomes in cis. The expression of Cntrl and C5, both of which are lnc-Hser neighboring genes, and C5AR1 which is a member of GPCRs family and plays an essential role in the early events leading to HCs proliferation and liver fibrosis  in vivo models  and 12 advanced fibrotic livers (F3-F4). The results showed that the transcript of ENST00000466280, ENST00000460578 and ENST00000480188 was enriched in healthy livers, but the level of ENST00000460578 and ENST00000480188 was significantly reduced in fibrotic livers Figure A-D. Take in vitro, and aggravated CCl4-induced liver fibrosis in vivo. Although the in vivo data showed that simply knockdown of lnc-Hser did not induce liver fibrosis, the expression of genes related to liver fibrosis including Col4α5, Timp1 and Mmp2, the hepatic inflammation including Tnf-α, Mcp1 and Il-6, the mesenchymal markers N-Cadherin and β-Catenin was increased in livers of lnc-Hser-shRNA mice versus NC mice. The reason for lnc-Hser silencing did not induce significant liver fibrosis may be due to the fact that liver fibrogenesis is a complex process involved in many key factors, and the time period for hepatic fibrogenesis of the mice was only four weeks during which the liver had significant regeneration and self-renewal capacity to counteract the injury caused by silencing lnc-Hser. In addition, knockdown of lnc-Hser promoted HSCs activation through the signals derived from injured HCs. Mechanistically, we demonstrated that lnc-Hser inhibited HCs apoptosis via the C5AR1-Hippo-YAP pathway and suppressed HCs EMT via the Notch pathway. All these data support our conclusion that lnc-HSER is a novel biomarker for damaged HCs and a potential target for anti-fibrotic therapy.Liver fibrosis is a progressive pathologic process that involves deposition of ECM leading to distorted architecture and culminating in cirrhosis, which is one of the major health burdens worldwide 4in vivo4-induced liver fibrosis  in vitro in vitro. On the other hand, the expression of the pro-apoptosis genes and the mesenchymal marker genes in the livers and primary HCs isolated from lnc-Hser-shRNA-CCl4 mice exhibited a profound increase, while the epithelial marker gene E-cadherin was not decreased. The reason for this could be when EMT initiates the epithelial cells to gain some phenotypes of the mesenchymal cells, for instance, up-regulated expression of N-cadherin and ECM, but still keeps some phenotypes of the epithelium. This is the so-called partial EMT. In this study, when EMT initiates during hepatic fibrogenesis, a reversible wound healing process, the HCs have to keep the cellular junctions to maintain the architecture of the hepatic lobule by normal expression of E-cadherin. However, the HCs have gained some mesenchymal phenotypes including over-expressed N-cadherin, β-catenin and Vimentin. Interestingly, our in vivo and in vitro results further revealed that the apoptosis and EMT of HCs were not co-localized, suggesting when lnc-Hser was silenced there are two populations of the cells among which one undergoes apoptosis and the other survived one undergoes EMT simultaneously. Hence, the inhibition of HCs apoptosis and EMT may be a therapeutic approach for the resolution of liver fibrosis.It is well accepted that the apoptosis of HCs is a critical initiator of fibrosis by triggering HSCs activation either directly by the phagocytosis of the apoptotic bodies which promote the secretion of pro-inflammatory and pro-fibrogenic cytokines, or indirectly by the generation of damage-associated molecular patterns in vitro cultured primary HCs could be blocked in the presence of several inhibitors including a Notch inhibitor in vivo and in vitroAccumulating progresses have been made in better understanding the underlying mechanisms of liver fibrosis with recent major investigations focused on the pathways including TGFβ1, Notch and Hippo, involved in initiating HCs apoptosis and EMT in the pathogenesis of liver fibrosis. Notably, it has been reported that Notch signaling contributes to the progression of liver fibrosis via inducing the EMT LncRNAs represent a group of transcripts longer than 200 nucleotides that are 5' capped and polyadenylated like most mRNAs, yet this class of transcripts has limited coding potential. Emerging evidence has demonstrated that lncRNAs were expressed in a cell- or tissue-specific manner and involved in a variety of cellular processes including differentiation, proliferation, and apoptosis in cis when their effects are restricted to the chromosome from which they are transcribed, and work in trans when they affect genes on other chromosomes  in cis. Moreover, lnc-Hser could also inhibit the expression of Notch2, Jagged1, Hes1 and C5AR1 which is a GPCR family member and functions upstream of the Hippo pathway in trans. Since lnc-Hser was mainly located in the nucleus of HCs, we also performed RIP assay in AML12 cells and livers to pull down endogenous RNAs associated with SUZ12, an important subunit of PRC2, which physically associates with twenty percent of all human lncRNAs It is well known that lncRNAs work In conclusion, our data identified lnc-HSER as a novel regulator of C5AR1-Hippo-YAP and Notch pathways in liver fibrosis, suggesting that lnc-HSER might be a novel biomarker and a candidate of anti-fibrotic targets.Supplementary figures and tables.Click here for additional data file.


ETS family genes with the androgen responsive TMPRSS2 gene (ETS+ tumors) provide a robust biomarker for detecting PCa in approximately 70% of patients. ETS+ PCa express high levels of the androgen receptor (AR), yet PCa tumors lacking ETS fusions (ETS−) also express AR and demonstrate androgen-regulated growth. In this study, we evaluate the differences in the AR-regulated transcriptomes between ETS+ and ETS− PCa tumors.Prostate cancer (PCa) tumors harboring translocations of 10,608 patient tumors from three independent PCa datasets classified as ETS+ (samples overexpressing ERG or other ETS family members) or ETS−  were analyzed for differential gene expression using false-discovery-rate adjusted methods and gene-set enrichment analysis (GSEA).Based on the expression of AR-dependent genes and an unsupervised Principal Component Analysis (PCA) model, AR-regulated gene expression alone was able to separate PCa samples into groups based on ETS status in all PCa databases. ETS status distinguished several differentially expressed genes in both TCGA (6.9%) and GRID (6.6%) databases, with 413 genes overlapping in both databases. Importantly, GSEA showed enrichment of distinct androgen-responsive genes in both ETS− and ETS+ tumors, and AR ChIP-seq data identified 131 direct AR-target genes that are regulated in an ETS-specific fashion. Notably, dysregulation of ETS-dependent AR-target genes within the metabolic and non-canonical WNT pathways was associated with clinical outcomes.ETS status influences the transcriptional repertoire of the AR, and ETS− PCa tumors appear to rely on distinctly different AR-dependent transcriptional programs to drive and sustain tumorigenesis. AR gene can occur during ADT and these are major mechanisms that drive androgen-independent PCa progression [The androgen receptor (AR), a nuclear hormone transcription factor, directs the transcription of several genes implicated in the development and progression of prostate cancer (PCa). Given the critical roles AR plays in PCa progression, advanced PCa is often treated with an androgen deprivation therapy (ADT)-based regimen, which depletes the natural ligands of AR, testosterone and dihydrotestosterone. However, several acquired gain-of-function mutations and modifications of the gression . AccordiERG, ETV1, ETV4, ETV5, and FLI1) are early carcinogenic events found in up to 70% of PCa [ETS gene translocations are AR-regulated events, and most frequently this results in overexpression of ETS family genes by placing them under the control of the androgen-responsive promoter TMPRSS2 [TMPRSS2-ETS fusions [Genomic aberrations in members of the E26 transformation-specific (ETS) family of oncogenic transcription factors  , the rolt-SNE (t-distributed stochastic neighbor embedding)  was determined for all PCa samples within the TCGA, GRID, and GRID-prospective datasets Table . Clinicang) Fig. . Thus, kr > 0.8; Supplementary Fig. r ≥ 0.9; Supplementary Fig. To investigate if discrete biological pathways are upregulated in ETS− prostate tumors compared to ETS+ prostate tumors, we identified the genes that are differentially expressed between the ETS+ and ETS− cohorts in the TCGA and GRID dataset. This analysis found 1423  a PCA model was applied to data from the TCGA cohort. The first principal component (PC1) of the non-canonical PCA model explains 55.9% of the variation, which is better than 99.6% of all random gene PCA models. The ration of PC1/PC2 is 3.3 which is better than 99.99% of the random gene model. The corresponding results for the metabolic pathway is 51.5% explained the variance for PC1 (better than 99.99% of the random models), and with a PC1/PC2 ratio of 5.9 (better than 99.99 % of the random models). These results indicate that the generated gene signatures are robust and better than random gene signatures , 18. NotThe systematic analysis of the transcriptional landscape of PCa tumors based on ETS status reported herein reveals important insights into the biology of ETS+ versus of ETS− tumors, and establishes that the repertoire of AR target genes is dramatically affected by ETS fusion oncoproteins. For example, GSEA analysis demonstrated that metabolic pathway genes, including genes involved in xenobiotic and fatty acid metabolism, were preferentially enriched in ETS− but not in ETS+ PCa tumors. Further, although ETS+ and ETS− PCa are both androgen dependent, different sets of androgen response genes are enriched based on ETS status, and analyses of previously published AR ChIP-seq datasets revealed that AR target genes are dysregulated in uniquely different ways in ETS− versus ETS+ tumors. These findings explain the apparently conflicting reports regarding AR activity in ETS− PCa , where AIn an in-depth analysis of clinically relevant ETS-dependent, AR-regulated biological pathways we have identified 5 distinct pathways including the metabolic, non-canonical WNT, differentiation, chemotaxis, and signal transduction and ion transport. ETS+ tumors showed reduced levels of AR target genes within the metabolic pathway that drive polyamine synthesis –21, fattSMS) and Glycine-N-methyltransferase (GNMT), an enzyme involved in S-adenosyl-methionine (SAM) homeostasis. These findings are in accord with those showing that androgen tightly regulates polyamine biosynthesis via control of ornithine decarboxylase (ODC1) and SAM-decarboxylase (AMD1) [Notably, AR regulates the expression of genes involved in polyamine biosynthesis such as spermine synthase (e (AMD1) , 27, ande (AMD1) .ABDH2, promote prostate tumor growth [ABHD2 levels with high Gleason score, pathological nodal stage, low cancer-specific survival rates, and a resistance to docetaxel-based chemotherapy [ALOX15B (arachidonate 15-lipoxygenase type II) known to metabolize arachidonic acid to fatty acid hydroperoxides, is selectively regulated in PCa in an ETS-dependent fashion [CYP2J2, which promotes tumor cell growth by converting arachidonic acid to epoxyeicosatrienoic acids [The expression analyses reveal that AR coordinately induces targets involved in arachidonic acid metabolism in both ETS− and ETS+ PCa tumors. First, it is interesting that glycerol and arachidonic acid produced via r growth . Indeed,otherapy . Second, fashion . Finallyic acids , is alsoRHOU, INPP4B, EFNB2, and CAMKK2 , as ETS fusion events only occur in a minority of these prostate tumors –36. SimiIn conclusion, this study identified distinct androgen-responsive genes in both ETS− and ETS+ prostate tumors, and validated 131 AR-target genes that are regulated in an ETS-specific fashion. Importantly, AR-target genes in ETS− tumors were involved in metabolic and non-canonical WNT pathways, thus reclassifying these genes for use in targeted therapy discovery.A total of 10,608 radical prostatectomy (RP) tumor expression profiles were used in this analysis. RNA-sequencing (RNAseqV2) gene expression data from the TCGA data portal for PCa and adjacent normal prostate tissue samples for 333 primary PCa tumors and associated clinical information, including molecular subtypes, was retrieved from the TCGA PRAD333 study . RetrospERG and the ETS family members ETV1-5 and FLI1. All other tumors were assigned as ETS−. For TCGA the following molecular subgroups were included in the ETS+ group: 1-ERG, 2-ETV1, 3-ETV2, 4-FLI1, and for the ETS− group: 5-SPOP, 6-FOXA1, 7-IDH1, 8-other, which were retrieved from the TCGA PRAD33 study [ERG or ETV1/4/5) subtyping [ETS+ status was assigned to samples that express high levels of ubtyping . For botubtyping .AR targets genes were defined as those genes that were located within 25 kb downstream of an established AR binding site, based on publicly available AR ChiP-seq data from the studies of Massie et al. , Sharma t-SNE (t-distributed stochastic neighbor embedding) was performed in MATLAB using a perplexity value of 30. All pairwise comparisons were performed using a two-tailed Mann–Whitney U test. Multiple testing was adjusted using FDR (q < 0.05). Fold change was also used to define significantly expressed genes  was performed using 101 androgen response genes as defined in the HALLMARK_ANDROGEN_RESPONSE gene-set . The PCAlization . All stalization . The HALlization  and the lization , 18. MedSupplementary Figure and Table LegendsSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Figure S4Table S1Table S2Table S3Table S4Table S5


AR-Vs and other AR aberrations to better understand the emergence of CRPC.A significant subset of prostate cancer (PC) patients with a castration-resistant form of the disease (CRPC) show primary resistance to androgen receptor (AR)-targeting drugs developed against CRPC. As one explanation could be the expression of constitutively active androgen receptor splice variants (AR-Vs), our current objectives were to study We analysed specimens from different stages of prostate cancer by next-generation sequencing and immunohistochemistry.AR mutations and copy number variations were detected only in CRPC specimens. Genomic structural rearrangements of AR were observed in 5/30 metastatic CRPC patients, but they were not associated with expression of previously known AR-Vs. The predominant AR-Vs detected were AR-V3, AR-V7 and AR-V9, with the expression levels being significantly higher in CRPC cases compared to prostatectomy samples. Out of 25 CRPC metastases that expressed any AR variant, 17 cases harboured expression of all three of these AR-Vs. AR-V7 protein expression was highly heterogeneous and higher in CRPC compared to hormone-naïve tumours.AR-V3, AR-V7 and AR-V9 are co-expressed in CRPC metastases highlighting the fact that inhibiting AR function via regions common to all AR-Vs is likely to provide additional benefit to patients with CRPC. AR gene amplification,2 point mutations in AR gene4 and induction of steroidogenesis in CRPC cells.7AR gene amplification has been demonstrated in approximately 30% of CRPCs.1 Cancers with AR amplification have been shown to respond better to second-line maximal androgen blockade compared to tumours without the amplification, however, the response was short-lived.8 Also AR mutations are rare even at CRPC stage being present in approximately 10–30% of cases.4 These mutations are almost always associated with diverse gains-of-function and about 45% of the mutations occur in the ligand-binding domain.9AR mutations can broaden ligand specificity to alternative steroid hormones, hypersensitise the receptor to castrate levels of androgens or lead to resistance to current forms of treatment making AR active even in the presence of anti-androgens.10Prostate cancer (PC) is the most common malignancy and third most common cause of cancer-related death among men in Europe. Androgens are required for the normal development of prostate tissue and exert their effects through androgen receptor (AR)-mediated signalling, but also have important role during PC emergence and progression. Most PCs grow slowly and are curable by surgery and radiation when confined to the prostate. In contrast, treatment of PCs that have spread outside the prostate usually includes manipulation of the AR signalling axis; androgen-deprivation therapy (ADT) either by surgical or chemical castration. However, during currently available ADT, lethal castration-resistant form of PC (CRPC) will eventually emerge after a variable period of time. Even though the exact mechanism by which CRPC develops remains to be fully understood, several mechanisms of castration resistance have been identified such as 12 The established concept of sustained AR signalling during CRPC has led to the clinical development of second-generation AR-targeting drugs enzalutamide and abiraterone that target the ligand-binding domain of AR directly and indirectly, respectively. Enzalutamide is an AR antagonist, whereas abiraterone is a CYP17 inhibitor approved by the US Food and Drug Administration (FDA) for the treatment of metastatic CRPC. Several studies have shown that presence of AR amplification or AR mutations in plasma samples is associated with worse outcome with enzalutamide and abiraterone.17 Furthermore, a significant subset of patients show primary resistance to these agents with respect to PSA (prostate-specific antigen) level,18 and among patients who initially respond, nearly all eventually develop acquired resistance.Importantly, androgen signalling remains active even in the CRPC stage.AR mRNA usually resulting in truncated AR protein product. The key domains shared among wild-type full-length AR (AR-FL) and all AR-Vs are the NH2-terminal transactivating domain (NTD) and DNA-binding domain (DBD). However, AR-Vs lack variable portions of the COOH-terminal domain including the ligand-binding domain (LBD).21 In spite of the fact that AR-Vs are unable to bind a ligand, they are constitutively active as transcription factors and capable of activating target genes.22One potential explanation for the resistance to first-generation and second-generation AR-targeted therapies is the presence of AR splice variants (AR-Vs). AR-Vs are alternatively spliced isoforms of the 23 AR-V7 is the most clinically relevant variant as it is most frequently observed and the most abundant AR-V in clinical specimens. In addition, AR-V7 is the only variant that can be detected reproducibly at both the mRNA and protein levels. Moreover, detection of AR-V7 mRNA in circulating tumour cells (CTCs) and peripheral whole blood from CRPC patients treated with enzalutamide or abiraterone has been implicated in primary resistance and shorter progression-free and overall survival.27 Interestingly, the prevalence of AR-V7 was shown to be higher in enzalutamide-treated men who had previously received abiraterone and in abiraterone-treated men who had previously received enzalutamide.25 These findings were supported by an independent study that also utilised CTC-based RT-PCR assay.28 In this prospective study, it was shown that PSA response rate to abiraterone or enzalutamide was 7% among AR-V7-positive patients and 63% among AR-V7-negative patients. Another recent study demonstrated that AR-V7 detection in plasma-derived exosomal RNA strongly predicts resistance to enzalutamide or abiraterone in CRPC patients.29 Although these studies implicate that AR-V7 could be used as a treatment-specific biomarker, it is likely that other AR-Vs also play a role in the development of CRPC. For example, it was recently reported that AR-V9 is often co-expressed with AR-V7 in CRPC metastases, and predicts primary resistance to abiraterone.30To date, at least 22 AR-Vs have been discovered in CRPC specimens.AR (AR-GSRs) were established as a new class of AR gene alteration occurring in one third of CRPC-stage specimens.31 This work showed that the presence of AR-GSRs at high variant allele frequency was associated with outlier, tumour-specific expression of rearrangement-dependent AR-V species that displayed androgen-independent and enzalutamide-resistant transcriptional activity. However, contrary to the prior studies in cell lines,33AR-GSRs were not associated with the AR-V7 expression levels in metastatic CRPC tissue.31 Another recent study utilising peripheral blood collected from patients with CRPC detected intra-AR structural variation in 15/30 patients of whom 14 expressed AR-Vs.34 Of note, most of the AR-V -positive patients expressed multiple AR-Vs, with AR-V7 being the most frequently occurring splice variant. However, AR-V3 was the most abundantly expressed AR splice variant. According to this study the presence of any AR-V was associated with shorter progression-free survival after second-line endocrine treatment compared to patients that did not harbour AR-Vs.34 Furthermore, in another recent investigation AR-GSRs in circulating tumour DNA were shown to associate with primary resistance to enzalutamide or abiraterone also in treatment-naïve CRPC patients with metastatic disease.13Recently, genomic structural rearrangements of AR splice variants, rearrangements, mutations and copy-number variations (CNVs) in different stages of PC to better understand the emergence of CRPC. We used multiple sample cohorts representing hormone-naïve PCs and lymph node metastases as well as locally recurrent and metastatic CRPCs. We first employed whole-genome and whole-transcriptome sequencing followed by targeted AR sequencing panels allowing deeper sequence coverage. In particular, our aim was to confirm whether AR-Vs are expressed in higher levels in CRPC samples compared to earlier stage cancers. In addition, we wished to elucidate to what extent AR-V expression is due to the aberrant splicing and, on the other hand, AR gene rearrangements. We also wanted to study the association between AR-V and AR-FL expression; and to find out whether AR-V expression affects the expression of AR-regulated genes.Our aim was to study n = 12), hormone-naïve PC (n = 30) and locally recurrent CRPC (n = 13) with clinicopathological characteristics of PC cases and prior treatments of CRPC cases being shown in Supplementary Table Two different sample sets utilised in the study are shown in Table The sample set 2 consisted of 24 additional hormone-naïve PCs removed by prostatectomy, of which six specimens were also included in sample set 1, eight lymph node metastases obtained at lymphadenectomy and 30 metastatic CRPC specimens obtained at autopsy  according to manufacturer’s instructions. The integrity of isolated RNA was confirmed using Fragment Analyzer .Portions of the metastatic cancer tissue from pelvic lymphatic metastasis obtained at lymphadenectomy were used for this study. None of the eight patients had undergone ADT, chemotherapy or radiation therapy prior to this surgery. Precise histological control was achieved for all tissues studied in this group using the following protocol. Serial cryostat sectioning was used to identify portions of the sample containing a lower fraction of tumour cells. These areas were manually microdissected from the tissue block every 300 µm based on H&E stained slide visual analysis. The tumour cell fraction was 70% or greater by histologic visual estimation. DNA purification was performed as described previously.Metastatic CRPC specimens were obtained from 30 men who participated in the PELICAN  integrated clinical-molecular autopsy study of metastatic PC (a detailed sample list is shown in Supplementary Table 36Low-coverage (4–6×) whole-genome DNA sequencing and whole-transcriptome sequencing (applied to sample set 1) have been described before.AR exons and introns. In addition, FOXA1  exons  and SPOP exons 6-7 were included in the panel. Targeted sequence enrichment was performed using the SureSelectXT Target Enrichment System  according to manufacturer’s instructions. Briefly, 200 ng of genomic DNA was fragmented using Covaris®  to yield a fragment size of 150–200 bp. End repair, addition of the 3’-dA overhang, ligation of indexing-specific adaptors, hybridisation to custom RNA baits, hybrid capture selection and index tagging were performed according to the Illumina paired-end sequencing library protocol. All recommended quality controls were performed between steps. The multiplexed samples were sequenced on the Illumina Miseq platform using 150 bp paired-end reads.A custom DNA sequencing panel was designed to cover all AR and five androgen-responsive genes  were targeted for capture and sequencing. In addition, three house-keeping genes TBP, STARD7 and DDX1 were included for normalisation purposes. This custom RNA sequencing panel was designed to cover all AR exons and nonrepetitive intronic regions to enable investigation of most common AR splicing variants ; other genes were covered less intensively (one or five amplicons per gene). Targeted sequence enrichment was performed using the SureSelectXT RNA Target Enrichment System  according to manufacturer’s instructions. Briefly, poly(A) RNA was purified from 1 µg of total RNA and fragmented chemically. In the following steps, samples were prepared using SureSelect Strand-Specific RNA Library Prep Kit to obtain adaptor-ligated cDNA library amplicons. Finally, hybridisation to custom RNA baits, hybrid capture selection and index tagging were performed. All the AMPure XP bead purification steps were conducted as instructed. The multiplexed samples were sequenced on the Illumina Miseq platform using 150 bp paired-end reads. The following modifications were made to the protocol if RNA was highly degraded  as recommended by Agilent Technologies: (1) Instead of poly(A) RNA purification from 1 µg total RNA, Ribo-Zero Gold Magnetic Kit  was used to remove rRNA from 2 µg of total RNA. (2) Instead of fragmenting the purified RNA at 94 °C for 8 min, RNA was denatured at 65 °C for 5 min. (3) All AMPure XP bead purification steps were performed using 1.8:1 bead volume to sample volume ratio. (4) Instead of 13 cycles in the pre-capture PCR, the number of cycles was increased to 14.AR aberrations in comparison to our previously published whole-genome DNA-seq data38 and whole-transcriptome RNA-seq data from this study. There was a good concordance in mutation detection between SureSelect DNA panel and previously analysed data from 22Rv1 cell line sample and metastatic CRPC samples from patients A21, A22 and A24. SureSelect DNA-seq detected the previously found H875Y mutation from 22Rv1 cell line, L702H mutation from liver metastasis from patient A21 as well as T878A mutation from a pelvic lymph node metastasis from patient A22, and from a right rib metastasis from patient A24.38 The data from AR splicing variant analysis also showed good accordance between SureSelect RNA panel and whole-transcriptome RNA-seq in three PC cell lines and two patient samples  genome using Bowtie2.AR copy numbers were analysed by calculating aligned read counts within overlapping 400 bp windows along the targeted regions using bedtools.42 The median of all AR bait coverage ratios that were obtained by dividing each normalised bait coverage value by the median of all values was used as the estimate of AR copy number. Chromosomal rearrangements were called using the in-house Breakfast algorithm that looks for paired-end reads and individual mates overlapping a chromosomal breakpoint.AR splice variant analysis using targeted or WTS RNA-seq data, Illumina MiSeq reads or HiSeq reads were aligned to an indexed reference fasta file containing unique signature sequences for various AR-Vs and AR-FL. The signatures consisted of 130 bp of the 3’ end of upstream exon and 130 bp of the 5’ end of downstream exon of a given unique splice junction  at +98 °C for 15 min. The staining was performed by Lab Vision Autostainer . The primary antibody Anti-Androgen Receptor (AR-V7 specific) Rabbit Monoclonal Antibody [RM7]  and secondary antibody  were used. ImmPACT DAB  was used as a chromogen. The sections were counterstained with haematoxylin and mounted with DPX mounting medium (Sigma-Aldrich). The percentage of AR-V7-positive cells between PC and CRPC groups was statistically assessed with Mann-Whitney test.Formalin-fixed, paraffin-embedded tumour microarrays of hormone-naïve PC, locally recurrent CRPC and metastatic CRPC  were used. Immunohistochemistry for AR  has been previously described.AR splicing variants and other AR aberrations in tandem during different stages of PC. For this purpose, we performed low-coverage whole-genome DNA sequencing and whole-transcriptome sequencing in sample set 1 that included BPH specimens, hormone-naïve PC from prostatectomies and locally recurrent CRPCs  of locally recurrent CRPC specimens and in 2/23 (9%) of metastatic CRPC specimens. L702H mutation that converts glucocorticoids to AR agonists49 was found in 3/23 (13%) of metastatic CRPC specimens. Indeed, all three patients harbouring L702H mutation had been treated with glucocorticoids (Supplementary Table AR mutations). Copy-number gains (>1 copy of AR) or amplifications (>2 AR copies) were observed in 4/9 (44%) of locally recurrent CRPC specimens and in 19/23 (83%) of metastatic CRPC specimens, respectively .First, we wanted to analyse the status of ely Fig. . It shouAR-Vs that were detected from the RNA-seq data by aligning the reads against indexed AR-V signature sequence file containing exon-exon junction sequences unique to every AR-V under investigation  in terms of reliable detection of AR variants, we also performed targeted RNA sequencing of the AR, which provided an average coverage range from 95× to 2247× utilising sample set 2  in sample set 2. There was a modest correlation when CNV status was compared to the combined expression levels of AR-V3, AR-V7 and AR-V9 .Next we studied the presence of known omy Fig.  or when AR-FL expression was threefold higher in metastatic CRPC compared to hormone-naïve PC from prostatectomy specimens, whereas in sample set 1, AR-FL was expressed fivefold higher in CRPC lesions than in prostatectomy samples. Notably, the expression of AR-V3, AR-V7 and AR-V9 was strongly associated with the levels of full-length AR in sample set 2   lymph node metastases and in 1/23 (4%) CRPC metastases, whereas SPOP mutations were detected in 1/8 (13%) lymph node metastases and in 2/23 (9%) CRPC metastases  were recently identified as a novel class of AR alteration using both autopsy CRPC specimens and peripheral blood collected from CRPC patients.34 More importantly, the presence of AR-GSRs was associated with expression of AR-Vs in both studies. To this end, we analysed AR DNA-seq data with our structural variant detection pipeline to identify AR-GSRs, defined as events having at least one breakpoint detected within the AR gene region. Average per-base sequence coverage of the AR gene region ranged from 114×–3358× and on average 78% of AR was covered by at least 10 reads (range 76–82%). We detected putative AR-GSRs in 5/30 metastatic CRPC patients who all harboured a highly amplified AR .n = 146), locally recurrent CRPCs (n = 97) and metastatic CRPC samples . We also studied immunohistochemistry of AR . As a positive control we used a sample of 22Rv1 cell line known to contain high AR-V7 expression  and detected in 23/29 and 22/29 CRPC metastases, respectively. Our finding that AR-V3, AR-V7 and AR-V9 are present at varying levels also in benign prostate tissue and hormone-naïve primary PCs is in line with previous reports.50 Furthermore, our whole-genome and targeted DNA sequencing results were in accordance with previous reports demonstrating that AR mutations and amplifications are rare in early stages of untreated PC, but occur much more frequently in patients affected by metastatic CRPC.50 In our study, no AR mutations or copy number changes were detected in untreated cases; they were observed only in locally recurrent and metastatic CRPC specimens. In CRPC metastases, 5/23 cases harboured an AR mutation and 19/23 cases had a copy number gain or amplification. Out of 22 cases of metastatic CRPC of which both DNA- and RNA-seq data were available, all but one patient (patient A5) had at least one AR aberration underlining the crucial role of AR in the disease progression. Figure AR detected in this study during different stages of PC.In CRPC metastases, the expression of AR-Vs. It has been demonstrated earlier using several PC cell lines that inhibition of the full-length AR protein via castration, antiandrogen treatment or siRNA induced the expression of AR-V7, although concomitant, yet lesser increases in full-length AR were also observed.52 It has also been shown that ADT does not directly regulate levels of AR-V7, but rather enhances AR gene transcription rate and splicing factor recruitment to AR pre-mRNA, thus elevating AR-V7 levels.53 Accordingly, we also showed that the expression levels of AR-V7 as well as levels of AR-V3 and AR-V9 were strongly associated with the levels of full-length AR, indicating that the AR-V expression is dependent on transcription rate of AR locus.It is noteworthy that lymph node metastasis specimens from patients who had not undergone any ADT did not show elevated levels of AR-V7 was strongly co-expressed with AR-V9 in sample set 2, which is in line with a recent report also demonstrating simultaneous expression of AR-V7 and AR-V9 in CRPC metastases.30 Moreover, our data showed that AR-V7 was co-expressed with AR-V3; and there was also a clear positive correlation between expression of AR-V9 and AR-V3. It should be noted that out of 25 CRPC metastases that expressed any AR variant, as many as 17 cases harboured expression of all three of these AR-Vs. Since AR-V3, AR-V7 and AR-V9 are constitutively active, it is reasonable to expect that their combined contribution to PC progression might be greater than what could be expected when their effects are studied separately. In our data, AR-V expression levels were above 5% when compared to overall AR transcript expression levels in 11 metastatic CRPC specimens (sample set 2). Since metastatic CRPC specimens harboured three times higher expression of AR-FL in comparison to hormone-naïve prostatectomy samples it would mean that 5% AR-V fraction does not yet bring the AR-V levels to corresponding levels of AR-FL in hormone-naïve PC. However, the levels of AR-V required to drive an androgen-independent transcriptome are unknown.Interestingly, we observed that KLK3, TMPRSS2 and FKBP5 in the absence of androgens or AR-FL.55 Therefore, we interrogated the levels of classical AR-regulated genes in our sample sets, and calculated the summed z-score of five androgen responsive genes . There was no association between the expression levels of AR-V3, AR-V7 or AR-V9 and z-score in sample set 2. Likewise, no correlation was detected when KLK3 expression was compared to the expression levels of these AR-Vs in CRPC metastases. One explanation for this discrepancy could be the fact that metastatic CRPC samples expressing the highest levels of AR-Vs were taken at autopsy making it highly unlikely that AR-regulation was anymore classical at the late stage of the disease. In addition, it has previously been demonstrated that bone metastases with high AR-V levels did not show high levels of KLK3, KLK2, FKBP5, TMPRSS2 and NKX3-1, whereas the levels of other transcripts known to be positively regulated by AR were elevated .56 Therefore, it seems that the expression profile induced by AR-Vs can be context-dependent and might not correspond to the effects observed in cell lines.It has been demonstrated in several cell line studies that AR-Vs are able to induce the expression of AR-controlled genes such as As protein expression does not always fully correlate with mRNA expression, and as the AR-Vs may also be regulated post-transcriptionally, it is of importance to study their expression also at the protein level. We assessed the expression of AR-V7 by immunohistochemistry in hormone-naïve PC, locally recurrent CRPC and metastatic CRPC samples. Although a third of CRPCs in this cohort were found negative for AR-V7 protein, the results support the view that AR-V7 expression increases during castration resistance, and that the protein is present in most PC metastases. It is noteworthy that expression of AR-V7 is highly more heterogeneous than that of AR overall. This indicates either differences in transcriptional expression of AR-V7 between tumour cells, or heterogeneous post-transcriptional regulation of it within tumour cell populations.AR aberrations occurring at a considerable frequency in CRPC material.34 Henzler et al. studied AR-GSRs in 30 rapid autopsy CRPC soft tissue metastases obtained from 15 patients and found that 10/30 metastases (6/15 patients) displayed at least one AR-GSR event. Instead, De Laere et al. utilised liquid biopsies from 30 chemotherapy pretreated or chemo-naïve CRPC patients and detected at least one AR-GSR in 15/30 patients. We observed AR-GSRs in 5/30 patients with metastatic CRPC, which is considerably less when compared to these prior findings. The reason for this discrepancy is unclear, but it can be at least partly due to the fact that there were more uncovered regions in our assay (76–82% of AR covered) than in Henzler et al. assay (83–89% of AR covered). Interestingly, AR-GSRs were not detected in the context of AR missense mutations in both our and Henzler et al. sample cohorts.31 Furthermore, in our material, the break fusion junctions of AR-GSRs were variable, demonstrating several types of rearrangements, but none of the AR-GSRs were associated with the expression of previously known AR-Vs. Patient A27 was the only one whose AR-GSR was also detected by RNA-seq and his variant allele fraction was also the highest being 10.9%. For other patients with AR-GSRs the variant allele fractions ranged from 2.6 to 8.6%. In CRPC metastases, half of the AR-GSR-positive patients expressed AR-Vs,31 whereas all but one of AR-GSR-positive patients who were liquid-biopsied harboured AR-V expression.34 Together, these results demonstrate that the connection of AR-GSRs and the expression of AR-Vs is highly variable in different sample cohorts. It is also noteworthy that in the study of Henzler et al., the only previously reported variant that was associated with the presence of AR-GSRs were AR-V7 and AR-V12 (ARv567es), but the data from De Laere et al. showed that the majority of AR-GSR-positive patients expressed multiple previously reported AR-Vs. AR-GSRs were restricted to CRPC specimens in both our and Henzler et al. data, suggesting that they are yet another means of CRPC to retain AR signalling.Genomic structural rearrangements (GSRs) have recently been shown to define a class of AR-V expression levels increase in patients treated with androgen-deprivation therapy might indicate that there is a clonal selection pressure on the different tumour clones in order to maintain functional AR signalling independent of the androgen levels. We provide evidence that AR-V3, AR-V7 and AR-V9 are co-expressed in metastatic CRPC highlighting the fact that targeting of the AR ligand-binding domain might not be sufficient to achieve clinically relevant treatment responses. Consequently, inhibiting AR function via regions common to all AR-Vs is likely to provide additional benefit to patients with CRPC.In conclusion, the finding that Supplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Figure S4Supplementary Figure S5Supplementary Figure S6Supplementary Figure S7Supplementary Figure S8Supplementary Figure S9Supplementary methodsSupplementary files


Breast cancer (BC) is a hormone-related tumor. Despite the progress in BC therapy, this disease still remains the most common cancer amongst women around the world. This is likely due to the amazing BC heterogeneity. Accumulating evidence suggests a role for androgen signaling in BC. Nevertheless, a precise understanding of the mechanism of androgen action in this disease remains a challenging puzzle. Androgen receptor (AR) is often expressed in BC and several studies suggest that its role depends on the tumor microenvironment as well as the relative levels of circulating estrogens and androgens. However, the AR function in BC is still conflicting. Although AR expression is often associated with a favorable prognosis in EREstradiol Receptorα-positive (ERα +) BC, many findings suggest that, in some instances, high levels of AR can contribute to the therapy-resistance. Again, in ERα negative BC (ERα –), AR is mainly expressed in tumors with apocrine differentiation and a lower Nottingham grade. Moreover, AR stimulates cellular proliferation in triple negative breast cancer . This finding is substantiated by the observation that high levels of circulating androgens are associated with an increased risk of developing BC in post-menopausal woman. Treatment of ERα- BC with AR antagonists, such as bicalutamide or enzalutamide, reduces, indeed, the tumor growth. In this review, we will analyze the putative role of AR in BC. Emerging therapies based on the use of new agonists or antagonists or inhibitors will be here discussed. In this scenario, the androgen receptor (AR) is emerging as a new marker and a potential new therapeutic target in the treatment of BC .Circulating androgens are detected at physiological conditions in women, and their levels change during life. Many studies have tried to understand the relationship between levels of androgens and breast cancer risk in women but the results are conflicting.This paper is aimed to provide an overview of the role of androgen/AR axis in various BC subtypes, with particular focus on TNBC.AR belongs to the steroid receptor superfamily and is classically considered a hormone-regulated transcription factor made up of 919-aminoacids encoded from a 180 kb gene located at the chromosome Xq11-12 . The recThe AR-DNA binding domain is responsible for the binding to DNA that take place through the binding with a palindromic consensus sequence 5′-GGTACAnnnTGTTCT-3′ called androgen response element (ARE) and is only recognized by AR. Between the DBD and the hinge region, is localized the nuclear localization signal (NLS), responsible for AR nuclear import . The carDepending on the subtype, the wild type AR (AR wt) is expressed in 50–70% of BC. In MCF-7 cells and in T47D cells has been reported the expression of a membrane androgen receptor (mAR) and in estrogen receptor negative breast cancer MDA-MB 453 cells, showing a molecular apocrine differentiation, is expressed a mutated form of AR with a glutamine to histidine substitution, called Q865H . This muIn sum, the presence of AR and/or its variants makes more complex the molecular scenario of BC.To date, there is not a clear relationship between levels of circulating androgens and BC risk. In women, circulating androgens are dehydroepiandrosterone-sulfate (DHEAS), dehydro-androstenedione (DHEA), androstenedione (A4), testosterone, and DHT. DHEA, DHEAS, A4 are secreted by adrenal glands, while testosterone, DHEA and A4 are produced by ovaries . This oHowever, the findings reported above cannot allow concluding that AR exerts univocally a protective, anti-proliferative effect in ER+ BC. In fact, AR in concert with the lysine-specific demethylase 1 (LSD1) also induces the epithelial-to-mesenchymal transition (EMT) in BC cells in an ER alpha independent way. Upon DHT stimulation, AR and LSD1 interact to AREs localized at the promoters of E-cadherin and vimentin to regulate their expression, thus promoting metastasis formation .In addition to classic AR, mediating transcriptional and non-transcriptional actions in BC, expression of mAR has been also reported in both MCF-7 and T47D , 40. ThiFurthermore, some studies indicate that AR is responsible for BC chemoresistence , 42. In In conclusion, whatever the mechanism, AR stimulates or inhibits cellular proliferation, promotes metastatization or resistence to therapies in ER-positive BC cells. These opposing actions could depend on the multitude of proteins interacting with AR.ER-negative BCs might express or not HER2 . RecentTreatment of HER2+/ER–/AR+ BC cells (LAR-BC cells), MDA-MB453, with DHT stimulates cell growth by activating the oncogenic Wnt and the HER3 signaling pathway. In these cells, AR transcriptionally regulates the levels of Wnt7b, which in turn activates the Wnt pathway and stimulates the nuclear translocation of β-catenin that interacts with AR. In nuclei, the AR/β-catenin complex in concert with the transcription factor FOXA1 recognizes the regulatory regions of HER3 and increases its transcription thereby promoting cell growth . It has The receptor, however, also influences the growth of HER2+ ER– BC cells in non-genomic way, since in these cells, inhibition of AR by Enzalutamide decreases the HER2 phosphorylation without affecting the total level of HER2 or HER3, and treatment of cells with a combination of enzalutamide and trastuzumab, an approved HER2 target drug, potentiates the inhibitory effect on cell growth due to a single inhibiting drug . GiovannIn conclusion, it appears that in ER-negative BC cells, AR acts in a more homogeneous way as compared to ER-positive BC cells. In these tumors the receptor clearly promotes cell proliferation and spreading by acting at different levels. This evidence depicts AR as a therapeutic target, potentially very exploitable, for TNBC and provides new opportunities for the treatment of this deadly kind of breast cancer.Although is not completely clear the influence of circulating androgens in breast cancer development, the role of AR in BC progression seems to be undeniable. In this review we have presented and discussed many findings showing that ligand activation of AR inhibits the cellular proliferation in most of ER-positive BC and induce the cancer growth in most of ER-negative BC. This scenario makes plausible the use of AR modulators or blockers in BCs as shown in Figure In ER-positive BCs and in a subset of ER-negative BCs in which AR activation inhibits tumor growth, natural and synthetic steroidal androgens –53 have The most used therapy for advanced BC (Tamoxifen-resistent-BCs and TNBCs) is based on the use of AR antagonists, such as bicalutamide and enzalutamide, a first and second generation AR antagonist, respectively [, 42 and Preclinical and clinical findings, however, have indicated that AR stimulates the growth of TNBCs or HER2 + BC in combination with other effectors. Some of them are directly involved in cell cycle progression CDK4/6), some others  command the most important intracellular circuits leading to survival, proliferation, invasiveness and drug-escape. Therefore, optimal results might be obtained by approaches in which AR antagonists are used in combination with inhibitors of these patways /6, some –64.BC is a heterogeneous disease and, in addition, became frequently resistant to the therapies so far used. Abeit the circulating levels of androgens do not allow a precise understanding of the influence of these hormones in BC development and progression, preclinical and clinical findings have highlighted the role of AR in BC pathogenesis. The concept that AR modulates the growth and progression of BC is currently undeniable. The findings so far reported in literature have also enabled the discovery of new strategies to control this pathology. AR acts in a genomic and in non-genomic way, both in ER-positive and negative BC. It appears, however, that AR can either inhibit or promote the ER+ BC cell growth, while it predominantly stimulates the cellular proliferation and the spreading of ER-BC. AR might act alone or in combination with other effectors participating in intracellular signaling pathways. High-throughput approaches have identified several druggable targets in BC, including the effectors of Src, PI3-K- or Ras-dependent pathways. Currently, targeted agents under investigation include Src, or PI3-K or MEK inhibitors or their combination with hormone therapies or chemotherapeutic agents. Preclinical evidence supports their use in various clinical trials and offers excellent opportunities for the development of new strategies, especially in combination with standard-of-care treatments.PG and AM: conceptualization and supervision. PG and MD: writing—original draft. AM, PG, MD, AB, ED, and GG: review and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite allergic rhinitis (AR) being a highly prevalent disease, according to literature, it is often underdiagnosed or undertreated.This study explored the disease characteristics of AR in the Saudi community and the non-conventional coping measures used to alleviate symptoms.The study was a nationwide cross-sectional study, using a self-report electronic survey distributed via social media networks in 2018. The survey included an explanatory letter and consent. The sample size comprised 3,458 participants and 2,849 adults had at least one of the four signs of AR, i.e. watery-nose, sneezing, nasal obstruction, itchy nose, watery eyes, in the past year, not related to cold/flu. The outcomes of the study were the triggers, pattern, severity and the classification of AR (2016 ARIA guidelines) and coping measures. Descriptive statistics, univariate analytic statistics and binary logistic regression analyses were conducted. The P-value was considered statistically significant at <0.05.The leading reported trigger of AR was dust , followed by pollen , mold  and fur . The prevalence of intermittent AR was , while that of persistent AR was . Almost one-third  complained of mild forms of AR, while others complained of moderate to severe forms . The coping measures were mainly shower/humidification 368(12.9%) and herbal hot drinks 266(9.3%). Older participants (adj.OR = 1.3[1.1–1.5]) and overweight participants, (adj.OR = 1.2[1.1–1.4]) reported more persistent forms of rhinitis compared to their counter groups, (adj.P<0.001 and adj.P = 0.032) respectively. Female participants (adj.OR = 0.8[0.7–0.9]) had significantly milder forms of AR, in comparison to males, adj.P = 0.006.This study presented the disease characteristics of self-reported AR and its associated factors in Saudi Arabia. Special attention should be paid to older age groups and overweight patients who reported persistent forms of AR. Males reported more severe and persistent forms of AR. Allergic rhinitis (AR) is an inflammatory disease of the nasal mucosa caused by indoor or outdoor allergens . In termIn persons with AR, there is an abundance of dendritic cells (DCs) that form a network, localized within epithelium and submucosal nasal/upper respiratory tract. In addition, there is an increased number of CD1a1 and CD11c1 DCs in the epithelium and lamina propria of the nasal mucosa clustered with CD41 T lymphocytes and eosinophils. With the presence of an antigen, DCs can polarize naive T cells into either Th1 or Th2 cells according to their own phenotype and with signals received from processed antigens and from the tissue microenvironment during antigen presentation. Within minutes of contact with allergens, the IgE–allergen interaction takes place, leading to mast cell and basophil degranulation and the release of preformed mediators such as histamine and tryptase .The targets of these mediators vary. For example, histamine activates H1 receptors on sensory nerve endings and causes sneezing, pruritus, and reflex secretory responses, but it interacts with H1 and H2 receptors on mucosal blood vessels, leading to vascular engorgement  and plasma leakage. The symptoms produced immediately after exposure to allergen reach their peak within a few minutes and tend to dissipate within 1 hour. Some individuals continue experiencing symptoms for several hours; others enter a quiescent phase and their symptoms recrudesce after several hours. The nature of the late symptoms is somewhat different from that of the acute symptoms in that sneezing and pruritus are not prominent, whereas nasal congestion is. Overall, these late symptoms occur in approximately 50% of people and, because their relative indolence resembles the clinical presentation of chronic rhinitis, the late phase is of particular scientific interest as a model of chronic allergic disease .Despite the fact that AR is a very prevalent disease, according to literature, it is often underdiagnosed or undertreated by physicians . The preThe burden of AR on patients is significant; it negatively affects their social life, school performance, work productivity, and the quality of sleep . TreatmeDespite the spread of AR worldwide and in particular within high risk countries such as Saudi Arabia, there is a dearth of studies investigating disease characteristics and local coping measures . Saudi AThe study had a cross-sectional design.Between July 15 and August 7, 2018, an anonymous, Arabic language electronic survey was distributed nationwide through social media networks. These networks were mainly those attracting general public interest, for example community health, sports, political shows, entertainment to a smaller degree social groups or gatherings. A rapid growth in the usage of internet has been witnessed in Saudi Arabia between 2000 and 2012, where more than 84% are internet users , 15. In This convenient sampling method maximized the exposure of the survey to participants from the various regions in Saudi Arabia and boosted the representativeness of the sample. The distribution of the survey was done in three rounds. Incomplete questionnaires were dropped out. It included an explanatory letter that explained the purpose of the study and clarified some of the clinical terms such as the AR sign and symptoms. Participants were requested to report their nationality and region upon submission.Eligibility criteria included adults who had at least one of the four signs of AR, i.e. a watery runny nose, sneezing (violent/in bouts), nasal obstruction (inability to breath), itchy nose; watery/red itchy eyes, during the past year and not related to an episode of cold/flu . ParticiThe data collection tool comprised of two main domains. It was developed based on the commonly reported exposures in literature (participants’ characteristics) , 19, 20 The English and Arabic versions of the survey are presented in English version  and ArabData were entered and analyzed using SPSS statistical software . Descriptive statistics such as the mean, standard deviation, frequency, and percentages were used to present the participants’ and disease characteristics. Disease characteristics were analyzed as categorical variables using Pearson’s Chi-square. Two binary logistic regression analyses were conducted to determine factors associated with study outcomes and control for any potential confounding effect. The adjusted Odds Ratio and 95% confidence interval (adj.OR[95%CI)] were presented and tabulated, with a P-value statistically significant at <0.05. The sample was later stratified by the smoking status, to determine if smoking had a confounding effect on the pattern and severity of self-reported AR .A self-explanatory letter of invitation to participate was presented electronically to each of the participants. All participants had given informed consents for their participation. Participants consented by ticking “agree”, indicating their agreement to provide their feedback for this research study. The Institutional Review Board of the Saudi Ministry of National Guard Health Affairs, Riyadh, Saudi Arabia (Protocol # RSS18-019R), approved this study. Studies conducted followed the recommendations of the International Conference on Harmonization for Good Clinical Practice and the Uniform Requirements for manuscripts submitted to biomedical journals.Participants with self-reported symptomology of AR were . The majority of participants 2,786(97.8%) had at least two signs of AR (including ocular symptoms), while 63(2.2%) reported only watery/red itchy eyes which might indicate a co-exiting allergic conjunctivitis. The highest prevalence was in Central Saudi Arabia (53.4%), followed by the Western region (22.1%), the Eastern region (12.4%) and both the Northern/Southern regions (12.1%). Of the 2,849 cases, 59.4% were females with 40.6% males, with a mean±standard deviation of age 29±10 years. Almost 6% were underweight (BMI<18.5), 38% had a normal weight (BMI = 18.5–24.9) and 58% pre-obese/obese (BMI≥25). Smokers comprised 13.2% of the sample, and almost 37% had at least one chronic medical condition. Almost 55% admitted to complain of snoring, followed by allergy/asthma (37.2%) and irritable bowel syndrome (23.7%). The age of onset of symptoms was dropped out due to extensive recall bias and lack of accuracy . Two thirds of participants (64.1%) had a positive family history of allergic rhinitis .Triggers of AR, pattern, and levels of severity are presented in In terms of conventional therapies, more than half of the participants took antihistamine pills (55.1%), followed by a visit to health care clinics (1.9%) and usage of inhalers (1.1%). The most frequently reported unconventional coping measures were shower/humidification (12.9%) and herbal hot drinks (9.3%). Other types of measures are enlisted in a descending order .Males were more likely (20.6%) to have pollen as a trigger for AR, in comparison to females . On the contrary, females were more likely (77.8%) to complain of AR triggered by dust, compared to males . Older age group were also more likely to have AR triggered by pollen (19.8%), compared to younger participants . Younger AR participants were more likely to be affected by dust (77.7%), compared to their counter group . BMI and a family history of AR showed no statistically significant differences across the type of triggers. However, being a smoker was associated with AR triggers such as pollen  and furred animals , while non-smokers were more affected  by dust. Persistent AR was associated with mold . Moderate to severe AR was associated with pollen triggers , with milder forms of AR being associated with dust as a trigger  .Persistent forms of AR were more prevalent in the older age group (49.8%) and pre-obese/obese participants (48.6%), (P<0.001 and P = 0.003 respectively). Moderate-severe forms of rhinitis was more prevalent among male participants (70.7%), older age group (68.6%), and smokers (74.0%),  .Binary logistic regression analyses showed that older AR participants (≥25) and overweight AR participants were significantly 1.3[1.1–1.6] and 1.2[1.1–1.4] times more likely to complain of persistent rhinitis compared to their counter age group, (adj.P<0.001 and adj.P = 0.032) respectively. Female AR participants 0.8[0.7–0.9] were significantly less likely to be associated with moderate-to-severe forms of AR, adj.P = 0.006 .Furthermore, the sample was stratified based on being a smoker or not, to determine if smoking had a certain confounding role on the pattern or severity of AR. Among the non-smoker group, older participants 1.4[1.2–1.6] and heavier participants 1.2[1.1–1.4] reported more persistent forms of AR, adj.P<0.001 and adj.P = 0.04 respectively. Female participants who were non-smokers 0.8[0.7–0.9] reported milder forms of AR, adj.P = 0.009. However, among the smoker participants who reported AR, no statistically significant variables were observed.This epidemiological study presents to the public and healthcare community the the patient and disease characteristics of 2,849 participants complaining of AR in a Middle Eastern country with a relatively harsh climate. While the Prick test remains the most accurate diagnostic test to confirm an allergic trigger, there was a need to launch a less costly preliminary community surveillance to evaluate the general status of AR. Prick tests range in cost between $60-$300 and for research purposes; participants need to be followed-up for further observation. However, at this stage results can be generalized to settings with similar environmental conditions. In addition, it provides insight regarding AR for expatriates who are seeking job opportunities and willing to relocate to Saudi Arabia, as well as those visiting this country on a temporary basis. A multisite study conducted in five Middle Eastern countries revealed that only 10% of the 7,411 self-reported AR cases were medically diagnosed with AR . Based oThe leading self-reported triggers of AR in this setting was dust, followed by pollen, which were different from triggers reported in other Asian countries  . In SoutThe whole region is a common route for sand storms, a natural hallmark of the country . During Authors believe that some participants who complained of AR spend more time outdoors making them at higher risk of exposure to pollen released from certain types of dessert plants. AR triggered by pollen was reported by participants to be moderate to severe, in comparison to that triggered by dust which was reported to be mild. The botanical fauna in Saudi Arabia is pretty distinctive compared to other habitats, being able to tolerate the harsh dessert conditions. The severity of AR associated with pollen triggers was comparable to a study stating that 18.9% of patients exposed to pollen grains developed severe AR . In otheAR triggered by mold tends to be persistent, as mold exists mainly indoors and in places unnoticed by residents. Occupants of damp and/or moldy buildings most frequently and persistently complain of cough, rhinosinusitis, worsening of asthma, and increased susceptibility to respiratory infections . The chaThe least reported AR trigger was furred animals (4%). The popularity of cats and dogs as pets affects the rates of AR among the general population. For instance, in Europe the frequency of cat ownership ranges from 7.2 to 35% and dog ownership from 5.4 to 35%, while in the United States, nearly 40% and 33% of households own dogs and cats, respectively. The overall European cat sensitization rate was 26.3% and for dog allergens, it was 27.2%, ranging from 16.1 to 56% . AlthougA study reported that the prevalence of intermittent AR in Saudi Arabia was 65%, in comparison to the persistent form of AR (35%) . In thisOne of the main outcomes of the study was to determine the coping measures participants adapted to relief AR symptoms. Responses were diverse, yet upon categorizing, the most frequently reported measure related to humidification such as taking a shower. A review paper of the so called “neglected non-pharmacological treatments” of AR recommended by two Persian pioneers Rhazes and Avicenna showed that avoiding overeating and polydipsia, massage of the lower extremities, adjusting the duration and time of sleep, sleeping in the supine position, avoiding exposure of the head to cold air and taking a shower early in the morning all aided in managing AR exacerbations . Nasal sAR is a manifestation of an immunological reaction, and its etiology is probably linked to both genetic predispositions and environmental influences. The scope of this study was beyond a genetic investigation. However, many of the gene-based AR studies revealed no significant association with AR . AllergiThis study presented the disease characteristics and associated factors of AR as reported by the public community in Saudi Arabia. AR was mainly triggered by dust, followed by pollen, mold and fur. More than half of the AR cases in Saudi Arabia suffered from moderate to severe symptoms and intermittent pattern of the disease. Factors associated with triggering AR by pollen were male gender, older age group, smoking and persistent AR. AR triggered by mold was associated with persistent forms of the disease, while AR triggered by fur was associated with smoking. AR triggered by dust was associated with the female gender, younger age, smoking, as well as, mild forms of the disease. Patients with AR are advised to adopt weight control measures as they were more likely to endure persistent forms of the disease if overweight.S1 Survey(DOCX)Click here for additional data file.S2 Survey(DOCX)Click here for additional data file.S1 Dataset(SAV)Click here for additional data file.