Histamine releasing factor/translationally controlled tumor protein (HRF/TCTP) stimulates cancer progression and allergic responses, but the role of HRF/TCTP in rheumatoid arthritis (RA) remains undefined. In this study, we explored the pathogenic significance of HRF/TCTP and evaluated the therapeutic effects of HRF/TCTP blockade in RA. HRF/TCTP transgenic (TG) and knockdown (KD) mice with collagen-induced arthritis (CIA) were used to determine the experimental phenotypes of RA. HRF/TCTP levels in the sera of RA patients were measured and compared to those from patients with osteoarthritis (OA), ankylosing spondylitis, Behçet’s disease, and healthy controls. HRF/TCTP expression was also assessed in the synovium and fibroblast-like synoviocytes (FLSs) obtained from RA or OA patients. Finally, we assessed the effects of HRF/TCTP and dimerized HRF/TCTP-binding peptide-2 (dTBP2), an HRF/TCTP inhibitor, in RA-FLSs and CIA mice. Our clinical, radiological, histological, and biochemical analyses indicate that inflammatory responses and joint destruction were increased in HRF/TCTP TG mice and decreased in KD mice compared to wild-type littermates. HRF/TCTP levels in the sera, synovial fluid, synovium, and FLSs were higher in patients with RA than in control groups. Serum levels of HRF/TCTP correlated well with RA disease activity. The tumor-like aggressiveness of RA-FLSs was exacerbated by HRF/TCTP stimulation and ameliorated by dTBP2 treatment. dTBP2 exerted protective and therapeutic effects in CIA mice and had no detrimental effects in a murine tuberculosis model. Our results indicate that HRF/TCTP is a novel biomarker and therapeutic target for the diagnosis and treatment of RA. A protein known to stimulate cancer and allergies has been implicated as an exacerbating factor in rheumatoid arthritis (RA); inhibiting its activity or blocking its production could offer options to prevent or treat RA. Previous research had identified high levels of histamine-releasing factor (HRF), also known as translationally controlled tumor protein (TCTP), in patients with RA. Its role in the destruction of joint tissue seen in RA was investigated by researchers in South Korea led by Sang-Il Lee at Gyeongsang National University, Jinju, and Kyunglim Lee at Ewha Womans University, Seoul. They examined HRF/TCTP’s activity and effects in fibroblast-like synoviocytes from RA patients and in a mouse model of RA and confirmed it can cause joint inflammation and damage. A molecule that inhibits HRF/TCTP activity reduced the severity of RA-like effects, suggesting potential new therapies. Our understanding of RA pathogenesis has improved, and therapeutic strategies against RA have been revolutionized by the development of targeted drugs such as tumor necrosis factor inhibitors, interleukin-6 (IL-6) blockers, B-cell depletion agents, and inhibitors of T-cell costimulation and Janus kinases5. Nevertheless, an appreciable percentage of patients experience refractory disease and interrupt treatment due to the risk of infection and malignancy7. Meanwhile, autoantibodies may be present long before the onset of clinical arthritis; thus, recent investigations have focused on RA in preclinical stages9. Although several strategies are currently being tested, there are still no effective methods of RA prevention. Thus, the investigation of new cytokines or cellular mechanisms that could be potential targets for RA treatment or prevention remains of high importance.Rheumatoid arthritis (RA), a chronic inflammatory and systemic autoimmune disease that affects 1% of the general population11. Fibroblast-like synoviocytes (FLSs), which are the major components of hyperplastic pannus, resemble cancer cells in their resistance to apoptosis, increased migratory and invasive properties, and ability to escape contact inhibition. FLSs can destroy bone and cartilage even after their removal from the autoimmune milieu14. The connective-tissue origin of FLSs may render them intrinsically resistant to current immunosuppressive drugs. Thus, recurrent or refractory RA likely depends on the persistent immunopathologic activity of resident RA-FLSs within pannus15. Disrupting FLS-specific immunopathology may prevent joint destruction by mechanisms that do not contribute to systemic immunosuppression and increased infection risk18. Accordingly, interfering with RA-FLS proliferation and invasion may contribute to effective and long-term disease control.Pannus at the interface between the synovium, cartilage, and bone is a characteristic of RA. Therefore, pannus provides a microenvironment that sustains autoimmunity and supports cellular and cytokine activities crucial for joint destruction20. HRF/TCTP triggers histamine release from basophils; however, HRF/TCTP also functions similarly to proinflammatory cytokines in several inflammatory and allergic diseases, including asthma, atopic dermatitis, and eosinophilic pneumonia23. A previous study demonstrated that HRF/TCTP inhibits p53-mediated apoptosis, indicating that HRF/TCTP plays a distinctive role in tumorigenicity24. This notion is supported by our previous study, which shows that HRF/TCTP induces epithelial-mesenchymal transition (EMT) and promotes the pulmonary metastasis of melanoma cells25. Therefore, HRF/TCTP is a promising target for prostate, breast, and lung cancer therapies26. Because RA is a representative inflammatory disease and RA-FLSs exhibit p53 dysfunction and undergo a tumor-like EMT process, HRF/TCTP may potentially serve as a novel therapeutic target in the treatment of RA.Histamine releasing factor (HRF) was initially classified as a tumor protein  in the 1980s, but its function remains unclear27. This finding strongly suggests that HRF/TCTP expression contributes to RA pathogenesis; however, no other studies have examined the functions or relevance of HRF/TCTP in RA. Here, we explored the role of HRF/TCTP in RA using HRF/TCTP transgenic (TG) and knockdown (KD) mice with collagen-induced arthritis (CIA). We also assessed the levels of HRF/TCTP in the sera, joint fluid, synovial tissue, and FLSs of patients with RA and controls and examined how HRF/TCTP expression correlates with RA disease activity. Additionally, we evaluated the inhibitory effects of HRF/TCTP-binding peptide on the RA-FLS phenotype and assessed the preventive and therapeutic effects of this peptide in mice with CIA.An earlier study showed that HRF/TCTP is present and localized in the joints of only patients with RA and not those of healthy controls. In particular, HRF/TCTP expression was more pronounced in the FLSs of severely destroyed regions of the pannus in RA samples28. RA disease activity was determined with the disease activity score (DAS28) using clinical and laboratory data29. Patients with osteoarthritis, ankylosing spondylitis, and Behçet’s disease were classified according to specific disease classification criteria32. All patients provided written informed consent. The biospecimens used in this study were provided by Gyeongsang National University Hospital, a member of the Korea Biobank Network. This study was approved by the Institutional Review Board of Gyeongsang National University Hospital (permit No: GNUH 2014-02-013).Patients with RA were classified according to the ACR/EULAR 2010 Rheumatoid Arthritis Classification CriteriaAll mouse experimental procedures were approved by the Institutional Animal Care and Use Committee of Gyeongsang National University (permit No: GNU-150907-M0049). HRF/TCTP transgenic (TG) mice were generated as described in the “Materials and methods”. HRF/TCTP knockdown (KD) mice, heterozygous for deletion of the HRF/TCTP allele, were kindly provided by Hsin-Fang Yang-Yen . The generation of HRF/TCTP KD mice is described in the “Materials and methods” section.34. Wild-type (WT), TG, or KD mice  were immunized with 150 μg of chicken type II collagen  emulsified in an equal volume of 4 mg/ml complete Freund’s adjuvant . The day of initial immunization was designated day 0. Immunization was boosted by an equal volume of emulsion of CII and incomplete Freund’s adjuvant  on day 21 and the intraperitoneal (i.p.) injection of lipopolysaccharide  on day 28. Clinical arthritis scores, ankle thickness, and micro-computed tomography (micro-CT) imaging were evaluated as described in the “Materials and methods”.A previously established protocol with slight modifications was used to induce CIA using C57BL/6 strain miceTo evaluate the preventive and therapeutic effects of dimerized HRF/TCTP-binding peptide (dTBP2) in the CIA model, dTBP2 was prepared as described in the “Materials and methods”. DBA/1 mice  were immunized with 100 μg of bovine type II collagen  emulsified with an equal volume of 2 mg/mL CFA. Immunization was boosted with an equal amount of BII emulsified in IFA on day 21. For the preventive approach, CIA mice were administered daily i.p. injections of vehicle or dTBP2  from day 21 until the end of the experiment. For the therapeutic approach, following the development of overt arthritis (average score of 5) on day 41, CIA mice were administered daily i.p. injections of vehicle or dTBP2 (5 or 25 mg/kg) from day 41 until the end of the experiment. Measurement of the arthritis score and ankle thickness and histopathological examination were carried out as described in the “Materials and methods”.35, and were then backcrossed with C57BL/6 mice. Mice heterozygous for deletion of the TCTP allele, TCTP+/−, were then backcrossed with C57BL/6 mice. HRF/TCTP KD mice heterozygous for deletion of the HRF/TCTP allele were kindly provided by Hsin-Fang Yang-Yen . After the construction and electroporation of the targeting vector into R1 ES cells, the positive clones were transiently transfected with a cytomegalovirus promoter-driven Cre expression vector to obtain subclones either with the floxed HRF/TCTP allele or in which the HRF/TCTP was absent. The obtained ES cell clones were microinjected into mouse blastocysts, and the mice were backcrossed to generate HRF/TCTP KD mice.HRF/TCTP TG mice were generated in the C57BL/6 and CBA hybrid background using the targeting cDNA construct pCAGGS-HRF/TCTP, which contained the CMV-IE and chicken β-actin promoter, as previously describedClinical arthritis scores on a scale of 0–4 were evaluated for each limb . The maximum possible score for each mouse was 16. Hind paw thickness was measured using an electric caliper placed at the widest point across the ankle joint.A SkyScan 1076 micro-CT apparatus  was used to evaluate structural changes in the ankle joints of CIA mice. Ankle joints were scanned and reconstructed into a 3-dimensional structure with a voxel size of 18 μm. The projections were reconstructed into 3-dimensional images using NRecon software (version 1.6.1.5) and CT Analyzer (version 1.10.0.1) (both from Bruker).36. In brief, Escherichia coli BL21 (DE3) pLysS cells were transformed using pRSET A/mutated d-H/F, and the overexpressed d-H/F was then purified using a Ni2+-charged His-Bind column according to the manufacturer’s protocol . NH2-terminal fusion proteins of d-H/T were separated by fast protein liquid chromatography on a HiTrap Q column  using a NaCl gradient. Synthetic dTBP2 at a purity of over 98% was provided by A & PEP Inc .d-H/T was prepared as described previously2. The dissociated cells were then centrifuged at 1000 × g, resuspended in DMEM supplemented with 10% fetal bovine serum (FBS) and 2.5 µg/ml amphotericin B, and seeded in 75 cm2 flasks . After overnight culture, nonadherent cells were removed, and adherent cells were cultured in DMEM supplemented with 10% FBS. The cultures were maintained at 37 °C in 5% CO2, and the medium was replaced every 3 d. The purity of the cells was tested by flow cytometric analysis with fluorescein isothiocyanate phycoerythrin-conjugated anti-CD3, anti-CD14, anti-CD19 , or anti-CD90  monoclonal antibodies. A FACSCalibur flow cytometer (488Ex/620Em) was used for the analysis. Among the RA-FLSs from ten different donors, we selected those from 3, and the cells from passages 3–8 were used for each experiment.Fibroblast-like synoviocytes (FLSs) were isolated from the synovial tissues of ten RA or three OA patients during total knee replacement surgery. The FLSs of OA patients were used as a control for the detection of monomeric and dimeric HRF/TCTP. The synovial tissues were minced and treated for 4 h with 2.5 mg/mL type I collagenase  in Dulbecco’s modified Eagle’s medium (DMEM) at 37 °C in 5% CO3 cells/well in 96-well plates. After incubation for 72 h, the media were replaced with DMEM containing 0.1% FBS supplemented with 75 nM d-H/T or 75 nM d-H/T plus 75 nM dTBP2. The rate of cell proliferation was measured using the Cell Counting Kit-8  assay according to the manufacturer’s instructions. Wound closure and cell invasion assays were performed as described previously . In brief, RA-FLS monolayers grown to confluence on 60-mm culture dishes were scratched with a sterile 200-μl pipette tip and then treated with 75 nM d-H/T or 75 nM d-H/T plus 75 nM dTBP2. The width of the wounded area was imaged and measured under an inverted phase-contrast microscope (Nikon) at ×50 magnification to assess cell migration at 0 and 48 h after scratching. To examine cell invasiveness, Transwell migration assay chambers  with an 8-μm pore size were used for a two-chamber migration assay. The upper surfaces of the Transwell inserts were coated with Matrigel , and RA-FLSs were seeded using serum-free medium. Complete growth medium supplemented with 75 nM d-H/T or 75 nM d-H/T plus 75 nM dTBP2 was added to the lower chamber as a chemoattractant. After 24 h, cells that had invaded and passed through the bottom surface of the insert were fixed with a 10% formalin solution, stained with propidium iodide , and quantified by counting the cells in 10 randomly selected viewing fields.FLSs were obtained from the synovial tissues of patients with RA or OA as described in the “Materials and methods”. Dimerized HRF/TCTP (d-H/T) and dimerized HRF/TCTP-binding peptide (dTBP2) were prepared as described in the “Materials and methods“. RA-FLSs were seeded at 5 × 10GAPDH , and relative expression was calculated using the 2−ΔΔCt method.Total RNA extracted from frozen joint tissues using TRIzol  was subjected to cDNA synthesis using the iScript cDNA synthesis kit  according to the manufacturer’s instructions. qRT-PCR was performed on a ViiA 7 real-time PCR system  using TaqMan Universal PCR Master Mix (Applied Biosystems) and TaqMan primer/probe sets  for IL-1β, IL-6, MMP-1, MMP-3, MMP13, and CCR2 . Data were normalized to the expression of 37.Ankle joint tissues were harvested, fixed in 10% formalin, decalcified for 4 weeks in 10% EDTA, embedded in paraffin, and sectioned into 5 μm sections. The sections were then stained with hematoxylin and eosin (H&E), safranin O, or tartrate-resistant acid phosphatase (TRAP). Synovial inflammation, cartilage damage, and bone erosion were evaluated, and TRAP-positive multinucleated osteoclasts (with ≥3 nuclei) were counted as described previouslyFor immunohistochemical staining, human synovial tissue sections were incubated with an anti-HRF/TCTP antibody  overnight at 4 °C. Sections were then incubated with biotinylated anti-rabbit IgG  for 1 h at room temperature. HRF/TCTP-positive cells were detected using a Peroxidase-Conjugated Avidin-Biotin Complex kit (Vector Laboratories), followed by staining with 3′3-diaminobenzidine tetrahydrochloride . For immunofluorescent staining, tissue sections were incubated with monoclonal anti-CD55 antibody  plus anti-HRF/TCTP antibody  overnight at 4 °C. Each slide was then incubated with Alexa Fluor 594-conjugated anti-rabbit IgG or Alexa 488-conjugated anti-mouse IgG . Fluorescence images were obtained using a fluorescence microscope  at excitation/emission wavelengths of 488/520 nm (for Alexa Fluor 488) or 594/617 nm (for Alexa Fluor 594).The levels of HRF/TCTP , anti-collagen IgG, IgG1, and IgG2a  in the sera, synovial fluid, and FLS culture supernatants were measured using ELISA kits according to the instructions of the corresponding manufacturer.Total proteins were extracted from FLSs with RIPA lysis buffer supplemented with protease inhibitor cocktail . Nuclear fractionation was performed using NE-PER Nuclear and Cytoplasmic Extraction Reagents . Total protein lysates or nuclear extracts were separated by SDS-PAGE and transferred onto a membrane. For dimerized HRF/TCTP detection, proteins were fractionated by nonreducing SDS-PAGE. The membranes were blocked with 5% skimmed milk and incubated with primary antibodies against the following: HRF/TCTP , β-actin , ERK, phospho-ERK, phospho-IκBα, Lamin A/C , GAPDH , and p65 . Signals were visualized using the corresponding HRP-conjugated secondary antibodies and substrates. Bands were normalized using a loading control and quantified by densitometry using ImageJ 1.48 software.Mtb H37Rv (ATCC 27294) was purchased from American Type Culture Collection . The original stock was streaked on a plate containing 7H11 medium to which OADC had been added and incubated at 37 °C. After 3–4 weeks, a single colony was picked and incubated for 2 weeks in a culture flask containing 7H9 broth supplemented with OADC. To this culture flask, 7H9 broth containing OADC was added, after which the flask was incubated at 37 °C for 7‒10 days. The flask was incubated for another 7‒10 days, after which single cells were prepared. The seed lots for each strain were stored in small aliquots at −80 °C until use.Specific pathogen-free 6-week-old C57BL/6J female mice were purchased from Japan SLC, Inc.  and strictly maintained under barrier conditions in a Biosafety Level 3 (BSL-3) facility at the Avison Biomedical Research Center at the Yonsei University College of Medicine . All animal studies were performed in accordance with Korean Food and Drug Administration (KFDA) guidelines. The experimental protocols used in this study were approved by the Ethics Committee and Institutional Animal Care and Use Committee of the Laboratory Animal Research Center at Yonsei University College of Medicine (permit number: 2017-0342).38. The mice were exposed to the Mtb strain H37Rv for 60 min in the calibrated inhalation chamber of an airborne infection apparatus that delivers a predetermined Mtb dose , and ~150 viable bacteria were delivered into the lungs. To confirm the initial bacterial burden, the mice were euthanized one day later, and ~150 viable bacteria were delivered to the lungs of each mouse. Mouse anti-TNF-α monoclonal antibody  in PBS was administered intraperitoneally  for neutralization once before infection and once every week for 4 weeks after Mtb infection. dTBP2 (50 mg/kg) in PBS was administered intraperitoneally (200 µl/mouse) twice per week for 4 weeks after infection. All mice were euthanized 30 d after Mtb infection.Mice were infected with Mtb strain H37Rv via aerosol10 colony-forming units (CFU) per organ.Disease phenotype and severity were evaluated through histopathology and by measuring bacterial growth in the lung and spleen. To determine how well they had been protected, the organs were removed 30 d after infection. For lung histopathology, the right superior lobes were fixed overnight in 10% formalin and embedded in paraffin. Lungs were sectioned at a thickness of 4–5 μm and stained with H&E. For bacterial growth analysis, the lung and spleen were homogenized, and serially diluted samples were plated onto Middlebrook 7H10 agar plates  supplemented with 10% OADC , 2 μg/ml 2-thiophenecarboxylic acid hydrazide  and amphotericin B (Sigma-Aldrich). After 4 weeks of incubation at 37 °C, bacterial colonies were counted. Data are presented as logt-test or one-way ANOVA with Tukey’s post hoc test. Data are expressed as the means ± SEMs, and a p-value of less than 0.05 was used to indicate statistical significance.Data were analyzed using two-way analysis of variance (ANOVA) followed by Fisher’s post hoc analysis. The significance of differences between groups was determined using Student’s unpaired We established a CIA model in transgenic (TG) mice to examine the role of HRF/TCTP overexpression in RA. CIA was significantly more severe in TG mice than in WT littermates Fig. . AssessmTo further determine the role of HRF/TCTP in human RA, we next assessed HRF/TCTP levels in human blood and joint fluid. While HRF/TCTP levels in other inflammatory diseases were similar to those observed in healthy individuals and patients with osteoarthritis (OA), only patients with advanced RA exhibited high serum levels of HRF/TCTP in the present study Fig. . AdditioNext, we examined the effects of HRF/TCTP on the cancer-like phenotype of RA-FLSs. Our results show that d-H/T, an active dimerized form of HRF/TCTP, stimulated the migration, invasion, and proliferation of RA-FLSs. The blockade of d-H/T via dTBP2, an HRF/TCTP-binding peptide, decreased the aggressiveness of d-H/T-stimulated RA-FLSs Fig. . TreatmeBased on our in vitro data showing that dTBP2 dramatically inhibited the inflammatory and cancer-like characteristics of RA-FLSs, we next evaluated whether dTBP2 could prevent the development of autoimmune arthritis in a CIA mouse model. Mice with CIA treated with 12.5 or 25 mg/kg dTBP2 showed a significantly decreased clinical score and ankle thickness compared to those of CIA mice injected with the vehicle Fig. . Histolo). Consistent with these results, dTBP2 treatment led to a decrease in the serum levels of anti-CII IgG, IgG1, and IgG2a  infections is an important factor in the safety of RA therapeutics. In Mtb infection and treatment experiments aimed at ruling out these clinical concerns, dTBP2 decreased the severity of TB infection more than anti-TNF–α monoclonal antibody, which is used in RA treatment, and showed a phenotype similar to that of the vehicle-treated groups  were injected with the vehicle or 5 or 25 mg/kg dTBP2. Mice treated with 5 mg/kg dTBP2 showed partially improved clinical scores until 54 weeks. In particular, CIA mice treated with 25 mg/kg dTBP2 showed a greater reduction in arthritic phenotypes than mice treated with the vehicle or 5 mg/kg dTBP2 Fig. . Mice tr2fG2a Fig. . Furtherups Fig. . Collect39, in the present study, we have shown the novel role and clinical implication of HRF/TCTP expression in the pathogenesis of RA. Of multiple inflammatory diseases, HRF/TCTP is highly expressed in only RA, and its serum levels are correlated with RA disease activity. This result suggests that HRF/TCTP levels can be used as a biomarker to diagnose RA and predict its severity. In RA, HRF/TCTP is mainly produced by RA-FLSs and plays a pivotal role in conferring RA-FLSs with tumor-like capabilities, including increased migratory capacity, invasiveness, and proliferation. Experimental arthritis was promoted in HRF/TCTP TG mice and suppressed in KD mice. Inhibition of HRF/TCTP activity via dTBP2 shows preventive and therapeutic potential because it downregulates the synthesis of numerous proinflammatory cytokines and antibodies in CIA.In agreement with previous findings indicating that HRF/TCTP contributes to allergic inflammation and tumorigenesis23. Interestingly, histamine is present in both the synovial tissue and joint fluid of patients with RA and recognized as a mediator of joint inflammation42. Other studies, however, indicated that histamine levels were lower in the blood and joint fluid of patients with RA than in those of controls. Additionally, prolonged treatment with anti-TNF-α significantly increased the levels of circulating histamine, suggesting that histamine is not useful as a biomarker to assess RA43. Conversely, a study using preclinical models of arthritis reported that both histamine H4 receptor (H4R)-deficient mice and mice treated with an H4R antagonist showed reduced arthritic severity44. However, a clinical trial examining toreforant, a selective H4R antagonist, was terminated prematurely due to patient fatality and the lack of significant RA alleviation46. In contrast, HRF/TCTP exhibits cytokine-like upstream functions; while it can thus potentiate histamine release, it also induces IL-8 and GM-CSF secretion, augments B-cell proliferation, and increases immunoglobulin22. This upstream stimulatory activity supports the correlation of HRF/TCTP expression with the clinical and inflammatory RA activities observed in our study. The targeting of HRF/TCTP may also aid in guiding future successful therapeutic approaches for RA that are similar to the use of Syk and JAK inhibitors, which target upstream signaling cascade kinase inhibitors47.HRF/TCTP, as its name suggests, participates in histamine release and is mainly responsible for allergic diseases48. Elevated levels of HRF/TCTP are found in the sera, bronchoalveolar lavage (BAL), and skin blister fluids of patients with asthma and various allergies22. In our study, increased levels of HRF/TCTP were found in the sera and joint fluid of patients with RA. HRF/TCTP levels were approximately 10-fold higher in the joint fluid than in the sera, which was unexpected. This finding suggests that joint stromal cells are the main source of HRF/TCTP in RA. Once activated, RA-FLSs acquire robust proliferative capacity, demonstrate tumor-like behavior, and become the most abundant stromal cell type in the inflamed joint synovium. Consistent with a previous report showing the presence of HRF/TCTP in the synovial pannus and FLSs of patients with RA27, our study also showed that HRF/TCTP expression was higher in the synovium and FLSs of patients with RA than in those of patients with OA. Furthermore, our results showed that IL-1β stimulation increased the production of HRF/TCTP by RA-FLSs. In agreement with previous reports, our results demonstrate that HRF/TCTP is generated mainly by RA-FLSs.In humans, HRF/TCTP is generated by peripheral blood mononuclear cells, in addition to other cell types, such as T and B lymphocytes, platelets, vascular endothelial cells, and alveolar macrophages residing in tissue compartments36. The results of our present study showed that after stimulation with IL-1β, the levels of dimerized HRF/TCTP were increased, and this active form drove the transformation of RA-FLSs into an aggressive inflammatory phenotype. MAPK and nuclear factor κB (NFκB) are involved in regulating the aggressiveness of RA-FLSs12. Our previous study demonstrated that dimerized HRF/TCTP induced the increased expression of IL-8, an inflammatory cytokine, via MAPK and NF-κB signaling in human bronchial epithelial cells36. In our present study, dimerized HRF/TCTP increased the phosphorylation of ERK and IκBα and nuclear translocation of p65 in the NFκB pathway. Thus, consistent with previous studies highlighting the importance of HRF/TCTP activity in cancer cells26, our results indicate a link between HRF/TCTP and the cancer-like behavior of RA-FLSs, which crucially contributes to the development and progression of RA.Our previous studies, as well as those of others, have recently demonstrated the presence of a dimerized form of HRF/TCTP in the sera and BAL fluids of patients and mice with atopy and asthma; additionally, dimerization appears to be essential for the proinflammatory activity of HRF/TCTP at sites of inflammation39. In our present study, dTBP2 markedly inhibited the aggressiveness of RA-FLSs exacerbated by dimerized HRF/TCTP. Via both preventive and therapeutic approaches, dTBP2 robustly blocked the development and progression of arthritis in mice with CIA. The CIA model offers an opportunity to evaluate preventive treatments in RA at preclinical stages, since the prevention of RA is rather difficult to study in humans49. In assessing the effectiveness of new drug candidates in animal RA models, a therapeutic, rather than preventive, approach is more predictive of a drug’s clinical efficacy in human patients with RA51. In our present study, dTBP2 exerted protective and therapeutic effects in CIA mice. Thus, our results strongly suggest that the use of dTBP2 is an attractive new strategy for the prevention and treatment of RA.Because dimerized HRF/TCTP plays such a critical role in inflammatory diseases such as RA, blocking dimerized HRF/TCTP may offer a rational strategy for the treatment of RA. For this reason, we previously searched for dimerized HRF/TCTP-binding peptides (dTBPs) by screening a phage-displayed 7-mer peptide library. One peptide, designated dTBP2, showed a stronger affinity for dimerized HRF/TCTP than for the inactive monomeric form, and dTBP2 alleviated several allergy-related symptoms in animal models of rhinitisIn summary, our study shows that HRF/TCTP levels were increased in samples obtained from patients with RA and correlated well with RA disease activity. HRF/TCTP was found to be generated mainly by RA-FLSs, and the active dimerize form of HRF/TCTP drives RA-FLSs toward a cancer-like phenotype. Consistent with these in vitro findings, HRF/TCTP overexpression led to the exacerbation of arthritis, while HRF/TCTP knockdown attenuated arthritic manifestations in mice with CIA. dTBP2, a peptide that blocks HRF/TCTP, abolished the pathologic effects of RA-FLSs and dramatically inhibited the development and progression of arthritis in mice with CIA. Additionally, dTBP2 did not aggravate TB, while treatment with anti-TNF-α mAb aggravated TB by causing a significant increase in bacterial burden along with excessive lung inflammation in a murine TB model. Together, our findings suggest that HRF/TCTP is an attractive target and that dTBP2 has preventive and therapeutic applications in patients with RA.Supplemental information


Brain metastases remain a major problem in patients with advanced non-small cell lung cancer (NSCLC). The permeability of the blood–brain barrier (BBB) is highly increased during lung cancer brain metastasis; however, the underlying mechanism remains largely unknown. We previously found that lnc-MMP2-2 is highly enriched in tumor growth factor (TGF)-β1-mediated exosomes and regulates the migration of lung cancer cells. This study aimed to explore the role of exosomal lnc-MMP2-2 in the regulation of BBB and NSCLC brain metastasis. Here, using endothelial monolayers and mouse models, we found that TGF-β1-mediated NSCLC-derived exosomes efficiently destroyed tight junctions and the integrity of these natural barriers. Overexpression of lnc-MMP2-2 in human brain microvascular endothelial cells increased vascular permeability in endothelial monolayers, whereas inhibition of lnc-MMP2-2 alleviated these effects. Furthermore, lnc-MMP2-2 knockdown markedly reduced NSCLC brain metastasis in vivo. Mechanistically, through luciferase reporter assays, RNA pull-down assay, and Ago2 RNA immunoprecipitation assay, we showed that lnc-MMP2-2 served as a microRNA sponge or a competing endogenous RNA for miR-1207-5p and consequently modulated the derepression of EPB41L5. In conclusion, TGF-β1-mediated exosomal lnc-MMP2-2 increases BBB permeability to promote NSCLC brain metastasis. Thus, exosomal lnc-MMP2-2 may be a potential biomarker and therapeutic target against lung cancer brain metastasis. Lung cancer is the most common cancer in men and is associated with a high mortality rate worldwide . DistantExosomes have been recently a topic of interest owing to their role in multiple diseases –8. A prePrevious studies have demonstrated that lncRNAs exhibit highly cell type-specific expression and function and have roles in carcinogenesis, metastasis, and drug resistance –15. HoweAntibodies against N-cadherin (22018-1-AP), ZO-1 (21773-1-AP), occludin (13409-1-AP), CD63 (25682-1-AP), CD81 (27855-1-AP), Alix (12422-1-AP), GAPDH (60004-1-Ig), and anti-Ago2 (10686-1-AP) were purchased from Proteintech . Antibodies to claudin-5 (343214) and EPB41L5 (614203) were purchased from Zenbio . Anti-VE-cadherin antibodies (2500) were obtained from CST . Rhodamine B isothiocyanate-dextran was purchased from Sigma-Aldrich . Exo-Fect Exosome Transfection Kit was purchased from System Biosciences . Recombinant human transforming growth factor β1 (TGF-β1) was purchased from PeproTech .2. The lnc-MMP2-2 overexpression and silencing lentivirus and their control lentivirus were packaged by Genomeditech . EPB41L5-targeting siRNA and scramble control siRNA were obtained from Ribobio . The EPB41L5-targeting sequences were as follows: siRNA#1, 5′-GGATCACGATTTA GATATA-3′; siRNA#2, 5′-GTCCTGAACTTGTCTCAGA-3′; siRNA#3: 5′-CGACTATTTTGGTCTGAG A-3′. The overexpression plasmid (pcDNA3.1-EPB41L5) and the empty vector (pcDNA3.1) were obtained from Genomeditech . miR-1207-5p antagomir, antagomir control, and miR-1207-5p agomir and agomir control were purchased from Ribobio . Cell transfection was performed using Lipofectamine 3000  following the manufacturer’s instructions. For exosome transfection, lnc-MMP2-2 Smart Silencer  were loaded in exosomes using the Exo-Fect Exosome Transfection Kit.The NSCLC cell line A549 was obtained from Cell Bank of the Chinese Academy of Sciences . Non-fetal-derived human brain microvascular endothelial cells (HBMECs) were purchased from Bioleaf Biotechnology . A549 and HBMECs were grown in Roswell Park Memorial Institute-1640 medium  containing 10% fetal bovine serum at 37 °C with 5% COExosomes were isolated according to our previous method . BrieflyqRT-PCR assay was performed as described previously . β-actinIF and western blotting assays were performed to measure the expressions of VE-cadherin, N-cadherin, occludin, claudin-5, ZO-1, CD63, CD81, Alix, and EPB41L5 according to our previous method .A total of 2000 HBMECs in full medium were seeded on Costar Transwell inserts  for 24 h. Rhodamine B isothiocyanate-Dextran (400 µg/ml) was then added to the upper wells. After 2 h of additional incubation at 37 °C, the media in the lower wells were collected, and the fluorescence intensity was measured at excitation and emission wavelengths of 485 and 535 nm, respectively, using a FlexStation 3 microplate reader (USA).The BBB integrity was evaluated by using Evans blue (EB) and rhodamine B isothiocyanate-Dextran injection. EB  or rhodamine B (100 mg/kg) was injected into the mouse tail vein pretreated with exo or Texo for 24 h. After 3 h of circulation, the mice were anesthetized, and the heart was perfused. The mouse brains injected with rhodamine B were embedded into frozen blocks and sectioned into 15 μm by cryostat sectioning. The distribution of rhodamine B in the cerebra was observed through fluorescence microscopy. The EB fluorescence detection assay was performed according to a previous method .The lnc-MMP2-2 overexpression, silencing lentivirus, and the empty vector were packaged by Genomeditech . The lnc-MMP2-2-silencing targeting sequences were as follows: shControl (NC), 5′-TTCTCCGAACGTGTCACGT-3′; shRNA#1, 5′-GCTGCAAGAAACATCTCTTGC-3′; shRNA#2, 5′-GGATGATCT AGTTCTCCATCC-3′; shRNA#3: 5′-GCCTCCTGC AAATCTCCCAAT-3′. Cell transduction was performed following the manufacturer’s instructions. Stable cells were selected using medium containing 0.5 µg/ml puromycin.Fluorescence in situ hybridization (FISH) assay was performed according to a previous method . The proFor the luciferase reporter assay, we used the pGL3 luciferase reporter plasmids for lnc-MMP2-2 and EPB41L5 designed by Genomeditech . Two reporter plasmids or vector control and control agomir or miR-1207-5p agomir were co-transfected into HBMECs. The luciferase activity was measured as per our method previously described [RNA immunoprecipitation (RIP) was performed using the EZMagna RIP kit  following the manufacturer’s protocol. Briefly, HBMECs were lysed in complete RIP lysis buffer, after which 100 μl of whole cell extract was incubated with RIP buffer containing magnetic beads conjugated with human anti-Ago2 antibody or control IgG. Samples were incubated with Proteinase K (1X), and then immunoprecipitated RNA was isolated. The RNA concentration was measured using a NanoDrop (Thermo Scientific). Moreover, purified RNA was subjected to qRT-PCR analysis to demonstrate the presence of the binding targets using respective primers.HBMECs were transfected with biotinylated wild-type (Wt) miR-1207-5p, mutant miR-1207-5p . First, cell lysates were harvested 48 h after transfection and incubated with Dynabeads M-280 Streptavidin  for 3 h at 4 °C according to the manufacturer’s protocol. Then, the beads were washed three times with ice-cold lysis buffer and once with high-salt buffer according to a previous method . The bou6 cells in 100 μl PBS) and were monitored for brain metastasis using an IVIS Imaging System. Approximately 3 weeks post-injection, all the mice were euthanized, and whole brains were collected. The presence of brain metastases was confirmed by fluorescence photography and hematoxylin-eosin staining.All experimental protocols were approved by the Laboratory Animal Ethical Committee at Chengdu Medical College. Female nude mice  were injected intracardially with control A549, lnc-MMP2-2-knocked down A549, TGF-β1-pretreated (10 ng/ml for 24 h) control A549, or TGF-β1-pretreated lnc-MMP2-2-knocked down A549 cells . Statistical significance was assigned at p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***).Each in vitro experiment was performed independently at least three times. The results are presented as means ± SD. Student’s Our previous studies have shown that TGF-β1-mediated A549 cell exosomes may increase lung vascular EC permeability and downregulate its tight junctions . Figure + ECs  were employed to analyze the potential targets of lnc-MMP2-2. The results revealed that miR-1207-5p had putative lnc-MMP2-2 binding sites Fig. . The qPCIn addition, the anti-Ago2 RIP assay further confirmed that miR-1207-5p was a target of lnc-MMP2-2 in HBMECs Fig. . SubsequNext, we confirmed the roles of miR-1207-5p in regulating HBMECs monolayer permeability. miR-1207-5p silencing and knockdown following transfection was confirmed via qRT-PCR Fig. S. IF staiTo elucidate the underlying molecular mechanism by which miR-1207-5p exerts its effects on HBMECs, bioinformatic tools (microRNA.org and miRBase) were used for searching the candidate targets of miR-1207-5p. As shown in Fig. EPB41L5 has been confirmed to play an oncogenic role in glioblastoma , gastricTo further elucidate the effect of lnc-MMP2-2 in NSCLC brain metastasis in vivo, a brain metastasis mouse model was established by implanting stably transfected A549 cells with control or shlnc-MMP2-2 lentivirus treated with or without TGF-β1. lnc-MMP2-2 knockdown significantly inhibited the occurrence of brain metastasis Fig. .Fig. 7lnBBB permeability is highly increased during lung cancer brain metastasis, but the mechanism still remains largely unknown. The current study found that TGF-β1-mediated NSCLC-derived exosomes promote EndoMT, downregulate the expression of tight junction proteins, increase HBMECs monolayer permeability, and alter the integrity of BBB in vivo. Inhibition of exosomal lnc-MMP2-2 reversed this effect. lnc-MMP2-2 overexpression and knockdown assays showed that lnc-MMP2-2 remarkably promotes EndoMT, destroys tight junctions, and induces HBMECs monolayer permeability.Despite recent advances in treatment modalities, lung cancer remains the leading cause of cancer-related deaths worldwide, and metastasis remains the major determinant of poor outcomes . A previHuman brain microvascular ECs is the main component of BBB. Thus, the cell morphology and tight junctions between HBMECs is essential for BBB permeability. EndoMT is a specific form of epithelial-to-mesenchymal transition characterized by a loss of endothelial features and the acquisition of mesenchymal features. Studies have shown that TGF-β1 is a potent inducer of EndoMT , 26. TGFOne of the most well-known function of lncRNAs is that they function as competing endogenous RNAs (ceRNAs) to sponge microRNAs , 31. TheEPB41L5 belongs to the NBL4 subgroup of the band 4.1 superfamily, which has a conserved 4.1-ezrin-radixin-moesin (FERM) domain at the N-terminus and a nonhomologous sequence at the C-terminus . Recent Furthermore, this study has some limitations. Endothelial glycocalyx, a carbohydrate-rich layer that lines the luminal side of the vascular endothelium, is essential in vascular permeability and is regarded as the first BBB barrier. . NumerouIn conclusion, we identified a novel ceRNA regulatory pathway in which lnc-MMP2-2 upregulates EPB41L5 expression by sponging miR-1207-5p. EPB41L5 directly promotes EndoMT, destroys tight junctions, increases BBB permeability, ultimately promoting brain metastasis in NSCLC Fig. . These fSupplemental Figure legendsSupplemental Figure 1Supplemental Figure 2


The androgen receptor (AR) is a central driver of aggressive prostate cancer. After initial treatment with androgen receptor signaling inhibitors (ARSi), reactivation of AR signaling leads to resistance. Alternative splicing of AR mRNA yields the AR-V7 splice variant, which is currently an undruggable mechanism of ARSi resistance: AR-V7 lacks a ligand binding domain, where hormones and anti-androgen antagonists act, but still activates AR signaling. We reveal PKCβ as a druggable regulator of transcription and splicing at the AR genomic locus. We identify a clinical PKCβ inhibitor in combination with an FDA-approved anti-androgen as an approach for repressing AR genomic locus expression, including expression of AR-V7, while antagonizing full-length AR. PKCβ inhibition reduces total AR gene expression, thus reducing AR-V7 protein levels and sensitizing prostate cancer cells to current anti-androgen therapies. We demonstrate that this combination may be a viable therapeutic strategy for AR-V7-positive prostate cancer. Androgen receptor signaling inhibitors (ARSi) are currently the primary treatment regimen for advanced prostate cancer. These therapies work either by directly antagonizing the AR at its ligand-binding domain (LBD) or by inhibiting androgen synthesis. Such treatments are generally initially successful, but many patients eventually relapse and develop lethal, metastatic castration-resistant prostate cancer (CRPC), which thrives even in a reduced-hormone environment . CRPC prThe presence of the AR-V7 splice variant is associated with resistance to ARSi therapies and clinically poor outcomes . AR-V7 iTargeting AR-V7 is currently an active and dynamic area of drug discovery. Drugs with several conceptually distinct approaches have been identified and pursued: (1) small molecules that bind to the AR-V7 protein despite its lack of known small-molecule-targetable features –13, (2) The AR has a well-established role as an androgen-dependent transcriptional activator, and the mechanistic details for the activation of its targets are well studied –29. But On the basis of this hypothesis, we evaluated potential kinase targets and selected the protein kinase PKCβ1 for investigation. PKCβ1 is reported to be associated with active transcription in prostate cancer and phosphorylates histone H3T6 . H3T6 phThe VCaP prostate cancer cell line is a metastatic CRPC model that expresses full-length wild-type (WT) AR and the alternatively spliced AR-V7 isoform , 7–9. Th(top), suppressing AR gene expression . Our. Our6]. . Further, when cells are treated with enzastaurin alone, a decrease in AR-V7 protein levels relative to MDV alone is observed, demonstrating that this effect is fully dependent on the PKCβ inhibitor  in combination with MDV, full-length AR mRNA transcript levels were reduced 1.7-fold and AR-V7 mRNA transcript levels were reduced 3.6-fold relative to vehicle when the low-androgen stress response is established Fig. , and fulNext, we evaluated our combination in another AR-V7 positive cell line that is resistant to anti-androgen treatment. The 22RV1 prostate cancer cell line expresses full-length AR and AR-V7 splice variant and includes a drug-binding-resistant somatic mutation in the full-length AR LBD (H875Y). The cell line also contains a 35 kb intragenic tandem duplication of the AR gene that results in deregulation of AR splicing and contributes to AR alternative splicing and AR-V7 mRNA transcripts. This duplication event encompasses exon 3 and the neighboring sequences, including cryptic exon 3, which is found in AR-V7 , 42. In ). Interestingly, targets suppressed by our combination include proteins involved in genome replication (MCM2 and MCM7), in DNA damage response, and the repair of double-stranded breaks (FANCI and RAD51AP1), and metabolic enzymes involved in androgen synthesis (AKR1C3). The data indicates that combining enzastaurin with MDV can also mitigate the increases in transcription observed in response to MDV at other AR-repressed genomic loci. Finally, we evaluated expression of the AR target genes KLK2, KLK3, TMPRSS2, and ZBTB16, which demonstrate androgen-dependent activation . Importantly, our data indicate that repression of AR-V7 by enzastaurin does allow for MDV-dependent growth inhibition. Overall, we show that our combination improves the response of AR-V7 positive prostate cancer cells to MDV.Next, we assessed our combination by Gaddum’s non-interaction in 22RV1 prostate cancer cells. The 22RV1 cells display uninhibited growth in the presence of MDV despite the antagonism of full-length AR androgen-dependent activation, however, knockdown of AR-V7 has been shown to sensitize this cell line to MDV . In our Fig. . Importa50 value. We suspect this to be due to the resistant nature of the cell line.Finally, we assessed our combination by Gaddum’s non-interaction in the PC-3 and LNCaP prostate cancer cell lines. The PC-3 cell line is AR-null, and as expected, neither MDV nor our combination of MDV with enzastaurin showed an effect on cell viability Fig. . The LNC). We, therefore, wished to determine if our combination strategy with enzastaurin could effectively reduce AR-V7 splice variant mRNA transcripts in the presence of an AR degrader. First, we synthesized a biologically evaluated AR degrader (AR PROTAC 2b) reported previously   supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Axenia BioLogix). 22RV1 cells were cultured in 5% CO2 at 37 °C with Roswell Park Memorial Institute medium (RPMI)  supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Axenia BioLogix). Cellular assays were performed in RPMI medium supplemented with 5% (v/v) charcoal stripped fetal bovine serum (CSS) . Cells were periodically tested for contamination using the MycoAlert Plus Mycoplasma Detection Kit (Lonza). Dihydrotestosterone was purchased from Sigma Aldrich. Enzalutamide (MDV) was purchased from MedChem Express. Enzastaurin was purchased from both Selleck Chemicals and MedChem Express. All reagents used for synthesis of the AR degrader were obtained from Sigma Aldrich, Acros Organics, Cayman Chemicals or AstaTech. The AR degrader was synthesized as described [All cell lines in this study  were obtained from the American Type Culture Collection (ATCC), with the exception of the 22RV1 cell line, which was provided to us by Felix Feng. VCaP, PC-3 and LNCaP cells were cultured in 5% COescribed .Treated cells  were lysed with RIPA buffer or with 20 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 10% Glycerol and 0.5% IGEPAL CA-630 supplemented with phosphate inhibitors  and protease inhibitors , and protein concentration was determined by either a Bradford Assay  or a bicinchoninic acid assay . Protein lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad) and blocked using either 5% milk or 5% BSA in TBST buffer , 0.1% Tween-20). Nitrocellulose membranes were immunoblotted with antibodies against AR , AR-V7 ; PKCβ1 , H3T6ph antibody , Histone H3 , α-tubulin  and GAPDH . Following the primary antibodies, nitrocellulose membranes were incubated with IRDye secondary antibodies (LI-COR Biosciences) and analyzed on an Odyssey Imaging System (LI-COR Biosciences) according to manufacturer instructions.N = 3) were isolated using the RNeasy Plus Mini Kit (QIAGEN) according to manufacturer instructions. Reverse transcription was performed using the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Life Technologies) according to manufacturer instructions. The reverse transcription products were evaluated by qRT-PCR using the Maxima SYBR Green qPCR Master Mix (Life Technologies) on a Bio-Rad CFX Touch Real-Time PCR system according to manufacturer instructions. GAPDH served as a reference gene. All samples were evaluated using the ΔΔCq method under the gene expression tab in the Bio-Rad CFX Maestro for Mac 1.1 software. Primer sequences are as follows: AR forward: 5’-TCT TGT CGT CTT CGG AAA TGT-3’, AR reverse: 5’-AAG CCT CTC CTT CCT CCT GTA-3’; AR-V7 forward: 5’-CAG GGA TGA CTC TGG GAG AA-3’, AR-V7 reverse: 5’-GCC CTC TAG AGC CCT CAT TT-3’; GAPDH forward: 5’-GGA CCT GAC CTG CCG TCT AG AA-3’, GAPDH reverse: 5’-GGT GTC GCT GTT GAA GTC AGA G-3’; MCM2 forward: 5’-ATT TCG TCC TGG GTC CTT TC-3’, MCM2 reverse: 5’-GCT GGT AGT TCT GAT AGA TGG T-3’; MCM7 forward: 5’-GGA TGC CAC CTA TAC TTC TGC-3’, MCM7 reverse: 5’-CCT TTG ACA TCT CCA TTA GCC T-3’; FANCI forward: 5’-CAA TGA GGA ACA GAG TGG TGA-3’, FANCI reverse: 5’-GCC TAG TTC ATA GTC CAA TTT GAT G-3’; LMNB1 forward: 5’-GGA AAT CAG TGC TTA CAG GAA AC-3’, LMNB1 reverse: 5’-CTT GAG GAT GCT CGG GAT AC-3’; RAD51AP1 forward: 5’-GTC TTC AGA TAC CAC TAG GAA ACC-3’; RAD51AP1 reverse: 5’-CTG CTG CTA CTT CTG CTA CC-3’; OPRK1 forward: 5’-TCA TCA ATA TCT GCA TCT GGC T-3’; OPRK1 reverse: 5’-AAG GAG CAC TCA ATG ACA TCG-3’; AKR1C3 forward: 5’-GGC CAC TTC ATG CCT GTA-3’, AKR1C3 reverse: 5’-GAA CCC AGC TTC TAT TGC TAA-3’.RNA from cells  treated in biological replicates  supplemented with 5% (v/v) charcoal stripped fetal bovine serum (CSS)  for 48 h. Plates were then treated in biological replicates with vehicle, 5 μM MDV or 5 μM MDV + 10 μM Enzastaurin for 24 h. Samples were subsequently processed using the Zymo-Spin ChIP Kit (D5209) and either a H3T6ph antibody , H3K4Me2 , H3K4Me1 , LSD1  or a rabbit IgG antibody . The precipitated DNA was evaluated by qRT-PCR using the Maxima SYBR Green qPCR Master Mix (Life Technologies) on a Bio-Rad CFX Touch Real-Time PCR system according to manufacturer instructions. Data is reported as percent of input. Primer sequences are as follows: ARBS2d’ forward: GCT CAG AGA GGT TTT AGT TGT G, ARBS2d’ reverse: CAA AAT GTC TAA GCT GGA AGC AC; ARBS2b’ forward: GTC TTG CTT TCC TAG AAG GTG AC; ARBS2b’ reverse: CAA GGA GAA AAT CTG AGT CCT GAG; ARBS2b forward: CAC ATG GAG TGC TGT TTG GT, ARBS2b reverse: GTA AAC ATC AGT GAG GAT GGT G; ARBS2e forward: GCA GAG AGT TTT TGG TGC ATA TC, ARBS2e reverse: CAA AGA TAC CTG ATG AAG GCT CTG; ARBS2g forward: CAG ACT TTA GAT TTA GGG GTT GG, ARBS2g reverse: GTC TAT GGC TGC TTT CAT CCT AC.N = 3) accordingly: Gaddum’s non-interaction: In the presence of 0.1 nM DHT, MDV in a nine-point threefold dilution series at Enzastaurin concentrations of 5 μM, 3 μM, 1 μM or 0 μM; Checkerboard assay: In the presence of 0.1 nM DHT, MDV and Enzastaurin checkerboarded in a threefold dilution series; Chou-Talalay combination indices: In the presence of 0.1 nM DHT, MDV and Enzastaurin as monotherapies or as a 1:1 concentration ratio combination in a nine-point threefold dilution series. Cell viability was assessed after three or five days using a CellTiter-Glo luminescence-based assay (Promega). The CellTiter-Glo reagent was diluted fivefold in PBS and added to cells in a 1:1 ratio with the cellular growth medium. Plates were incubated with shaking at room temperature for 20 min and then the luminescence signal was recorded on a Tecan Spark plate reader. Bliss synergy scores were calculated using https://synergyfinder.fimm.fi/. Chou-Talalay combination indices were determined using CompuSyn 1.0 .Cells were seeded into white 96-well clear flat bottom plates  in RPMI 1640  supplemented with 5% (v/v) charcoal stripped fetal bovine serum (CSS)  for 48 h. Cells were then treated in biological replicates , MDV – 10 mg/kg , Enzastaurin – 50 mg/kg BID  and MDV + Enzastaurin. Tumor volumes and body weights were collected biweekly over the course of the six week study. Tumor volumes were assessed by 2D caliper measurements and volume was calculated according to the volume of an ellipsoid (V = 0.52 x (width)2 x length). The xenografts were collected and flash frozen upon termination of the time course study. Pieces of the xenografts were crushed under liquid nitrogen, and the tissue was lysed with RIPA buffer containing 1X PhosSTOP, 1X PIC and 1 mM PMSF on ice with occasional vortexing. Samples were analyzed according the protocol outlined in the SDS Page and Immunoblotting section.All mouse manipulations were performed in accordance with the University of California, San Francisco’s Institutional Animal Care and Use Committee. All animals were housed in specific pathogen-free conditions and cared for according to the International Association for Assessment and Accreditation of Laboratory Animal Care policies and certification (IACUC protocol ANI179937). All surgeries were performed under isoflurane anesthesia. Six- to eight-week-old male NSG mice  were bred in house and housed with ad libitum food and water on a 12 h light cycle at the UCSF Preclinical Therapeutics Core vivarium. VCaP xenografts were introduced to the right flanks of mice by subcutaneous injection . Mice were castrated on a rolling basis as the VCaP xenografts reached sizes of ~100 mm50 values were determined in Graphpad Prism 8 using a log(inhibitor) vs response – variable slope (four parameter) model. The Bliss synergy grid was modeled using https://synergyfinder.fimm.fi/. Chou-Talalay combination indices were calculated using CompuSyn 1.0. P-values for ChIP-qPCR assay calculated in Microsoft Excel using the two-tail Student’s T-test function assuming equal variance. P-values for the xenograft studies were calculated by two-way ANOVA in Graphpad Prism 8.qRT-PCR ΔΔCt values were calculated using the Bio-Rad CFX Maestro software and then plotted using Graphpad Prism 8 as the mean ± SD with individual data points shown, and the Tukey P-values are reported from the ANOVA tab. Immunoblots were processed with Image Studio Lite 5.2.5 (LI-COR). Gaddum’s non-interaction data is represented as mean ± SD, and ICSupplementary TablesSupplementary Figures


Canthon balteatus, Dichotomius problematicus, and Onthophagus confusus are abundant in disturbed sites, where soils are generally more compact and less fertile but contain a greater food supply. These findings can help decision makers to identify disturbed areas and to implement adequate policies for sustainable environmental management.This study analyzed the abundance and diversity of dung beetle communities at several disturbed sites in a tropical dry forest ecosystem in southern Ecuador. Dung beetle community diversity indices with different land uses were related to environmental variables , soil physicochemical properties, and food supply (manure). The results indicated that the species p value < 0.001), besides the food availability (dung). The highest abundance and diversity  was found in highly disturbed sites, where soils were generally more compacted, but with a greater food supply due to the introduced farm animals. At highly disturbed sites, the species Canthon balteatus, Dichotomius problematicus, and Onthphagus confuses were found specifically, which makes them useful as bio-indicators for disturbed dry forest ecosystems in southern Ecuador.This study evaluated the effects of land-use change (L-UCH) on dung beetle community structure (Scarabaeinae) in a disturbed dry ecosystem in southern Ecuador. Five different L-UCH classes were analyzed by capturing the dung beetle species at each site using 120 pitfall traps in total. To determine dung beetle abundance and diversity at each L-UCH, a general linear model (GLM) and a redundancy analysis (RDA) were applied, which correlated environmental and edaphic conditions to the community structure. Furthermore, changes in dung-producing vertebrate fauna were examined, which varied significantly between the different L-UCH classes due to the specific anthropogenic use or level of ecosystem disturbance. The results indicated that soil organic matter, pH, potassium, and phosphorus (RDA: component 1), as well as temperature and altitude (RDA: component 2) significantly affect the abundance of beetles (GLM:  Loss of tropical forests due to land-use changes (L-UCH) is a major environmental concern at local, regional, and national scales, especially in developing countries . ApproxiApis mellifera), ants (Formicidae), and beetles (Scarabaeus sp.) are especially affected [The estimated environmental and socio-economic trends for tropical forests indicate that large areas of the remaining natural ecosystems are endangered by L-UCH . Howeveraffected ,22.Scarabaeinae) [A group of insects which is widely used to analyze the effects of habitat modifications upon biodiversity are dung beetles (baeinae) , becausebaeinae) . In Soutbaeinae) ,25,26, bbaeinae) ,28. Morebaeinae) ,30.Sarabaeinae) are classified into three functional groups: (i) Telecoprids or Rollers , Paracoprids or Tunnellers (tunnels are excavated for food source), and (ii) Endocoprids or Inhabitants (feed and reproduce within the food source). Additionally, dung beetles can be classified into nocturnal or diurnal species .However, the effects of L-UCH upon dung beetle communities, as important soil organisms and in semi-arid regions, have rarely been studied , especiaIn the semi-arid regions of Ecuador, the effect of land-use change (L-UCH) on dung beetle communities has so far not been studied. Only studies with respect to dung beetle abundance and diversity in natural humid and Andean forests have been executed ,47, besiTherefore, the objective of the present research was to analyze the disruptive effects on the abundance and diversity of dung beetle communities in areas with different levels of disturbance (L-UCH). Therefore, edaphological (soil) and environmental factors  were analyzed, applying a general linear model (GLM) and a redundancy analysis (RDA). In addition, the relative manure supply of native and introduced vertebrates was examined to contrast the food availability for dung beetle communities among the different L-UCH classes. The results might help decision makers to identify highly degraded ecosystems, where adequate policies and sustainable environmental management should be implemented .The investigation was carried out in the parish of Malacatos , located between 4°12′24″ and 4° 13′39″ S (south), and between 79°19′12″ and 79°19′21″ W (west) . The altThe natural vegetation in the study area is tropical dry forest (TDF) . However(a)Coffea arabica) is associated with banana (Musa paradisiaca), papaya (Carica papaya), and guavas (Inga feuilleei), but also with native trees species, especially faique (Acacia macracantha), wilco (Anadenanthera colubrina), and fig trees (Ficus sp.). In this L-UCH class, native vertebrates exist, such as deer , squirrels , and anteaters , but also domestic animals, particularly goats .Coffee in an old agroforestry system (CoAS), where cultivation of coffee ((b)Anadenanthera colubrina, with an average DBH of 0.26 m and an average height of 18 m), ceibo , and faique . In this L-UCH class native vertebrates dominate, especially anteaters, deer, skunks , and opossums , while domestic animals are also frequent, such as goats, cows , and horses .Seasonally disturbed dry forest (SDF), in which the dominant tree species are wilco ((c)Beta vulgaris), turmeric (Curcuma longa), cabbage (Brassica oleracea), and carrot (Daucus carota). In this L-UCH class domestic animals are more frequent, such as donkeys  and horses, but some native species, particularly deer and skunk, can also be found.Organic crop area (OCA), which is a polyculture system, including chard ((d)Hyparrhenia rufa) and Cybitax antisyphilitica seedlings. In this L-UCH class generally only domesticated animals exist, such as cows, horses, and donkeys.Grasslands (GLd), mainly composed of yaragua grass ((e)Coffea arabica), apple , and guava (Inga feuilleei), but also young trees, especially arabisco (Jacaranda copaia), faique (Acacia macracantha), and wilco (Anadenanthera colubrina). In this L-UCH class native vertebrates, such as anteaters, skunk, and deer can be found, but the presence of domestic animals is higher, in particular cows and horses.Coffee in a young agroforestry system (CyAS), which were recently planted (max. 3 years ago), including coffee Coffe®, Bourne, MA, USA).Sampling was performed between May 2019 and February 2020. Mean altitude for each L-UCH class was determined using a GPS (Garmin eTrex Legend). Additionally, air temperature (°C) at the individual L-UCH classes was measured during the whole study period by means of a HOBO sensor  was estimated based on the Tyurin method [In the laboratory, first, Bd was determined by means of the cylinder method , for whin method ,62, and n method . Finallyn method .All individual results were averaged to obtain mean values for each L-UCH class , and the standard deviation for each L-UCH was also calculated.To analyze the composition of the dung beetle communities for the different L-UCH classes , two transects were established within each plot, separated by 7.5 m. Then, 4 pitfall traps were installed along each transect, separated evenly by 4 m, which resulted in 8 pitfall traps per plot and a total of 120 pitfall traps for all L-UCH classes . The pitThe sampling was carried out in December 2019 during the rainy season, in which the pitfall traps remained active for 48 h, as recommended by da Silva and Hernández . The traTo obtain information with respect to the type of manure and its amount within the different L-UCH classes, questioning of the land owners (farmers) was executed , besidesp < 0.05). If the statistical analysis was significant, the means were compared to the Tukey’s post hoc test and accepted with a p-value of <0.05 in all cases. The dung beetles captured in the pitfall traps were similarly statistically analyzed (ANOVA and Tukey’s post hoc test), considering the total number of each species and the L-UCH class. All analyses were carried out using SPSS statistical software .The data from physical–chemical soil analysis of the different L-UCH classes were subjected to a one-way analysis of variance  upon the dung beetle communities, a generalized linear model  was appThe average altitude of each L-UCH class, as well as the measured mean air temperature during the study period (May 2019–February 2020), are summarized in –1 to 1050 mm year–1; [With respect to annual precipitation, the differences between the L-UCH classes were negligible , due to year–1; ,79, prec year–1; , which w year–1; .During the study period (May 2019 to February 2020) a total precipitation amount of 550 mm was measured at the Malacatos station, whereas during the beetle collection period  monthly precipitation amounted to approximately 80 mm. This indicated that water stress was unlikely to affect dung beetle abundance nor diversity in this study, as reported by other investigations during dry seasons ,81. Ther−3 and 1.12 g cm−3, with significant differences between the L-UCH classes (−3) and CyAS (1.05 g cm−3), whereas for SDF, the lowest values were measured. This indicates that highly disturbed sites have more compact soils, which is likely due to intensive anthropogenic activities, such as the overgrazing of cattle  or the use of agricultural machinery (CyAS). Soil compaction is especially detectable within the first few centimeters of the soil  at a soil depth of 10 cm varied between 0.71 g cm classes . Bd was b et al. ), which b et al. ,84.−3; CoAS: 0.80 g cm−3, and SDF: 0.71 g cm−3, respectively), which is due to the lower anthropogenic pressure upon these soils [3) can be higher than those obtained in the study area , while the SOM amount was high. This was confirmed by Dam et al. [−3 in sandy-loam soils at depths between 0–10 cm. However, Bd is also influenced by tillage practices. As Kushwaha et al. [−3) compared to conventional tillage (1.40 g cm−3) at sandy-loam sites. This is consistent with the present study, where differences in Bd were detected where tillage was practiced. Furthermore, a recent study in Ecuador showed, if vermicompost was used as an organic amendment within sandy soils, after 45 days of decomposition not only was Bd improved, but also less CO2 was emitted when compared to loamy soils [The dominant soil texture of the L-UCH classes was loam-sandy , for whim et al. , who foua et al.  indicatemy soils . Accordimy soils , these dThe chemical soil properties of the different L-UCH classes are presented in Taking into consideration optimal soil pH ranges , the soiThe SOM (6.7%), TN (0.34%), and SOC (3.9%) were significantly higher in CoAS than in the SDF , OCA , and GLd , whereas the CyAS soil contained the lowest amounts , Dichotomius problematicus (9.9 ± 4.4), and Onthophagus confusus (5.2 ± 2.3). The second L-UCH class with a large number of dung beetles was OCA, where the species C. balteatus (5.5 ± 2.0), D. problematicus (5.7 ± 1.9), and O. confusus (2.3 ± 1.3) were the most abundant. The other L-UCH classes showed a lower quantity of these beetles, with the exception of the species Onthophagus curvicornis (6.1 ± 2.0), which was also found in great numbers at CoAS. This indicated that the dung beetle species C. balteatus, D. problematicus, and O. confusus are more abundant at highly disturbed sites.C. balteatus varied significantly between the L-UCH classes , where the highest values were found in CyAS (24 ± 7.9), whereas in OCA (5.5 ± 2.0) and GLd (5.5 ± 1.9) the number of individuals notably deceased. The lowest numbers of this species were found in CoAS (1.0 ± 0.4) and SDF (0.3 ± 0.6). The same was valid for the species D. problematicus, which showed significant statistical differences between the L-UCH classes . The most individuals of this species were found in CyAS (9.9 ± 4.4) compared to GLD (6.4 ± 1.8), OCA (5.7 ± 1.9), and SDF (4.5 ± 1.7). The third species with a high numbers of individuals at all L-UCH classes was O. confuses, which was more abundant at CyAs (5.2 ± 2.3) and CoAS (4.7 ± 2.8), compared to OCA (2.3 ± 1.3) and GLD (1.1 ± 0.4).Analyzing the species distribution of the individual L-UCH classes , consideOnthophagus curvicornis, Phanaeus achilles, Onoreidium ohausi, and Aphodius sp1, but with mostly insignificant numbers for all the L-UCH classes. These results are consistent with Domínguez et al. [C. balteatus, D. problematicus, and Onthophagus sp. are the species with greatest abundance in neotropical dry forest, whereas the species Ph. achilles and O. ohausi are represented in lower numbers.The other species present in the study area were z et al. , who foup-value < 0.001). Especially at CyAS, species diversity was very low (H ‘= 0.4 ± 0.4), whereas the highest values were calculated for CoAS and SDF (H ‘= 1.1). In contrast, the Simpson index, which indicates habitat diversity, showed the lowest diversity for SDF (0.8 ± 0.3) and highest diversity for OCA (0.3 ± 0.0). This indicated that dung beetle individuals are rare in the SDF, but more numerous in highly disturbed L-UCH classes. This might be due to the continual extraction of timber trees, converting the natural forest into a secondary forest, where faique (Acacia macracantha), in particular, is harvested for charcoal, and wilco (Anadenanthera colubrina) for logs used in house construction [C. balteatus, were found. Furthermore, at disturbed sites, generally greater amounts of food are available due to more intensive livestock grazing [Regarding the Shannon–Wiener index H ‘; , speciestruction . These atruction ). These truction  and Noritruction , who fou grazing . This waIn general, as confirmed by the land owners, the dominant native animals in SDF are anteaters, opossums, skunks, and squirrels. At the same time, domestic animals, particularly goats, graze in large numbers, while fewer numbers of cows, horses, and donkeys are present in SDF. In more disturbed areas , domestic animals such as cows, horses, donkeys, and, to a lesser extent, goats predominate, while the presence of native animals is generally rare. In succession areas (CoAS and OCA) less domestic animals are present, and goats predominate in CoAS, with donkeys as well as horses in OCA. Cows are also numerous in CyAS, possibly because this area is linked to pastures (GLd).According to the farmers, the use of fresh dung from domestic animals, as well as human feces, has been implemented in the study area over the last 3 years as an ecological alternative for habitat restoration . Most coC. balteatus, D. problematicus, and O. confusus  and temperature  were positively related to total dung beetle abundance (p < 0.001), CyAS , and OCA , whereas SDF showed a negative relationship , and GLd did not show any relationship . Chemical properties such as pH , SOM , TN , TC , K , P , and C/N ratio  had a negative relationship with dung beetle abundance, while Bd  showed a positive relationship. The three abiotic factors  determined the specific environmental conditions at each site . In these areas the eurytopic species C. balteatus, D. problematicus and O. confusus had the greatest abundance, which is consistent with the work of Brussaard and Slager [C. balteatus, D. problematicus, and O. confusus are found in particularly large numbers at highly disturbed sites  have adapted to dung from domestic animals, and thus are more abundant at highly disturbed sites [Sulcophanaeus imperator and Eucranium arachnoides) to a eurytopic dung beetle species (Digitonthophagus gazella) in a disturbed arid forest in Argentina, and found that the generalist species incorporated a greater amount of organic matter, total nitrogen, and phosphorus into the soil. Furthermore, as Menéndez et al. [C. balteatus, D. problematicus, and O. confusus in the dry forest area of southern Ecuador at highly disturbed sites, because they are tunnellers.Although there is no direct relationship between the total abundance of dung beetles and the chemical properties of the soil , the gened sites , even ined sites  comparedz et al.  demonstrC. balteatus, D. problematicus, and O. confusus  , but neg and SDF b. As expd Slager , who obsOnthophagus curvicornis). These results suggest that anthropogenic land-use change affects the community structure of dung beetles in TDF ecosystems, favoring eurytopic or generalist dung beetle species. The most abundant dung beetle species in the study area were C. balteatus, D. problematicus, and O. confuses, which were captured particularly in highly disturbed sites . Therefore, the abundance and diversity of dung beetles depend on the level of disturbance, which includes food supply, because manure from introduced domestic animals favors the distribution of eurytopic species. These findings can help decision makers to design policies and technical proposals for environmental restoration in the TDFs of southern Ecuador.In this study, three factors were correlated to abundance and diversity of dung beetles in a disturbed TDF region in southern Ecuador: (i) the level of disturbance, represented here by different L-UCH classes ; (ii) abiotic factors , and iii) soil fertility . Due to the altered environmental conditions in all L-UCH classes and the availability of food (dung), most of the species caught were eurytopic. These species inhabit more compact soils (high Bd), which generally occur in highly disturbed anthropogenic sites, due to the use of agricultural machinery (CyAS), as well as high stock density , which leads to overgrazing, especially at pasture sites (GLd). Furthermore, the application of dung and organic fertilizers at these sites improves their food availability. On the other hand, in less disturbed areas (SDF and CoAS) fewer generalist species were found, and native dung beetle species were more numerous (e.g.,


The constitutively active androgen receptor (AR) splice variant, AR-V7, plays an important role in resistance to androgen deprivation therapy in castration resistant prostate cancer (CRPC). Studies seeking to determine whether AR-V7 is a partial mimic of the AR, or also has unique activities, and whether the AR-V7 cistrome contains unique binding sites have yielded conflicting results. One limitation in many studies has been the low level of AR variant compared to AR. Here, LNCaP and VCaP cell lines in which AR-V7 expression can be induced to match the level of AR, were used to compare the activities of AR and AR-V7. The two AR isoforms shared many targets, but overall had distinct transcriptomes. Optimal induction of novel targets sometimes required more receptor isoform than classical targets such as PSA. The isoforms displayed remarkably different cistromes with numerous differential binding sites. Some of the unique AR-V7 sites were located proximal to the transcription start sites (TSS). A de novo binding motif similar to a half ARE was identified in many AR-V7 preferential sites and, in contrast to conventional half ARE sites that bind AR-V7, FOXA1 was not enriched at these sites. This supports the concept that the AR isoforms have unique actions with the potential to serve as biomarkers or novel therapeutic targets. Most CRPC remain AR-dependent. Alterations contributing to AR reactivation include elevated AR expression3, local steroid synthesis4, mutation of the ligand binding domain (LBD)5, altered cell signalling and altered AR splicing. Alternative splicing causes expression of constitutively active AR splice variants that retain the amino-terminal transactivation domain and the DNA binding domain (DBD) but lack the LBD9. The variants are particularly challenging to target therapeutically, since they lack the LBD, which is the primary target for most therapeutic efforts. The best characterized and most widely expressed variant is AR-V7. It contains exons 1, 2, 3 of AR and 16 unique amino acids7 , and whether it only regulates a sub-set of AR targets or also has unique targets have yielded conflicting results16. A major limitation in evaluating the role of AR-V7 is the lack of appropriate cell models. Many studies have relied on two cell lines, LNCaP95 (LN95) and 22RV119, which endogenously express AR-V7, but are both derivatives of tumors that did not originally express AR-V7. LN95 is a derivative of LNCaP cells20 and 22RV1 cells were derived from an androgen dependent patient derived xenograft (PDX) that developed resistance subsequent to castration of the mouse21. LN95 cells express quite low levels of AR-V722 and 22RV1 cells express multiple variants6. The AR-V7 expression levels in cell lines do not necessarily reflect AR-V7 levels in tumors. In the initial identification of AR-V7, at least some tumors were shown to express AR-V7 protein at levels comparable to that of AR in hormone dependent tumors7. Consistent with this, a subsequent study of AR isoform mRNA levels in circulating tumor cells (CTCs) showed that AR-V7 mRNA levels were equivalent to or higher than the AR mRNA levels in many AR-V7 negative CTCs10. Lines engineered to express variants have been generated by others. For example, Roggero et al. used engineered LNCaP cells expressing AR-V7 upon treatment with cumate, but the levels of AR-V7 were much lower than the levels of AR23. Here, to address the capacity of AR-V7 to regulate transcription and chromatin binding relative to AR, LNCaP and VCaP cell derivatives, in which the levels of AR-V7 are controlled by treatment with doxycycline (Dox)24, were used to equalize expression with levels equivalent to LNCaP AR.Primary prostate cancer is androgen receptor (AR) signalling dependent; metastatic disease is treated with some form of androgen deprivation therapy (ADT). Initially responsive, some tumors develop resistance and are termed castration resistant prostate cancer (CRPC)pHAGE AR-V7 (LNCaP AR-V7) and VCaP pHAGE AR-V7 (VCaP AR-V7) cells treated with Dox at levels used in subsequent studies. In the absence of hormone, the VCaP cells express some AR-V7 25 and VCaP AR-V7 (VCaP pHAGE AR-V7) cells in charcoal stripped serum (CSS) treated with vehicle, R1881 or Dox for 24 h prior to harvesting were used for transcriptomic analyses. The reproducibility of the biological replicates prepared in separate experiments is shown in the principal component analysis (PCA) SFig. RNA-seq data from LNCaP AR-V7 cells . There was minimal overlap of any of the signatures  revealed a remarkable preferential enrichment of AR-V7 near the TSS  in LNCaP cells treated with R1881 and AR-V7 binding sites (V7Bs) in LNCaP AR-V7 cells treated with Dox were identified by ChIP-exo. The analyses yielded 87,095 AR binding peaks and 59,852 AR-V7 binding sites with 32,082 common sites detected in both analyses. , and immunoprecipitations performed as described in methods. Complexes were sequentially extracted with RIPA (does not release IgG) to facilitate detection of FOXA1, followed by treatment with SDS sample buffer to release IgG and remaining proteins. Immunoprecipitation with AR441 showed that both AR isoforms can co-IP FOXA1  was detected as enriched in both the AR-V7 specific binding sites and in the sites common to AR and AR-V7, but not in the AR unique peaks or in the overall pool of AR binding sites Fig. a. When A38. Similarly, some investigators have identified unique AR-V7 chromatin binding sites, while others have described these as artifacts/unreliable39. Some investigators have concluded that AR-V7 acts with and/or requires AR for activity whereas others describe AR-V7 actions as independent of AR28. Many of the studies have been done in LN95 and 22RV1 cells14, which endogenously express low level of variants, and/or also express other active variants40, but neither cell line was derived from a tumor expressing variants21. These lines also have undergone changes during their derivation41, so there is no AR only line suitable for a direct comparison. To more precisely compare the capacity of AR and AR-V7 to regulate gene expression and bind to chromatin, LNCaP and VCaP lines that express AR-V7 and an LNCaP line that expresses additional AR in response to Dox treatment were used. Isoform dependent gene expression was examined in these lines as well as in LN95 cells.There is much interest in the role of splice variants, especially AR-V7, in CRPC; AR-V7-expressing tumors typically are refractory to second line ADT and there is a great need to develop therapies that target variants lacking the LBD and to identify variant actions that might be targeted therapeutically. The studies seeking to elucidate variant action have yielded conflicting results. There is good agreement that AR-V7 and other variants can regulate many of the same genes, but there is a substantial debate about whether AR-V7 and other variants also regulate a substantial set of unique targets or have minimal or no unique activities42. Similar results were obtained in the VCaP model  in the LNCaP model exhibit AR-V7 binding at the TSS. Moreover, an analysis of the consensus binding motifs surrounding these AR-V7 binding sites revealed that whereas upregulated genes contained AREs, binding regions in repressed genes were enriched for FOXA1 motifs suggesting additional specificity  have been suggested to play a role in preferential actions of the AR isoformsted Fig. . Interes25 and INPP4B32), whereas a gene (EDN2)25 that is only induced by AR when FOXA1 is depleted is induced by AR-V7 regardless of FOXA1 status25. One possible explanation for these differences was differential binding of FOXA1 to AR and AR-V7. Our studies show that AR-V7 can interact with FOXA1 as measured by a co-ip assay  and batches with no background activity were used and/or serum was stripped in the lab and lack of activity confirmed in the same manner. The LNCaP (and LN95) AR have a mutant hormone binding domain and respond to a broader range of steroids. Thus, they may be more sensitive to residual steroids than lines with wild type receptor.Our studies in LN95 cells supported the finding of unique isoform dependent activities, but there are differences in the results among investigators who have studied either LN95 or 22RV1 cells. Different approaches have been taken and this may contribute to the different conclusions. Generally, investigators who have used stable expression or depletion approaches to produce separate cell lines that have elevated or reduced levels of AR isoforms and use these for comparisons detect fewer isoform specific differences in gene expression/binding than those who use transient methods (siRNA or PROTAC), so that all comparisons are within the same passages of a single cell line. Another potential confounding factor is the level of steroid depletion in charcoal stripped serum as has been suggested by Liang et al.Our studies utilizing the inducible models are unique in that the levels of AR-V7 were comparable to LNCaP AR levels to determine the capacity of AR-V7 to bind to chromatin and regulate gene transcription. At this level of receptor, AR-V7 shows several unique activities, a capacity to bind to TSS, enrichment of binding to a novel ARE related sequence and differential functional interactions with FOXA1. Although coregulators presumably also play an important role, the vast majority of differentially regulated genes showed differential binding to chromatin implicating interactions with other DNA binding proteins and potentially different DNA sequences in the unique receptor activities. These studies were limited to defined cell lines, but the results suggest that the activities of AR-V7 in CRPC will be heavily dependent on the level of AR-V7 expressed in the tumor and serve as the preclinical foundation for discerning the isoform specific function in disease progression.24 were grown under the same conditions except that G418 was included to prevent loss of lentivirus. LN95 cells were kindly provided by Dr. Jun Luo, Johns Hopkins University, and are grown in phenol red free RPMI 1640 with 10% charcoal stripped serum (CSS). All cell lines have been authenticated by STR analysis and are maintained in culture for up to three months before a new batch is thawed. Mycoplasma testing is done routinely every few months.LNCaP, VCaP, 22RV1, and HEK 293 T/17 cells were obtained from the American Type Culture Collection (ATCC) Manassas, VA and grown as recommended by the provider. The engineered derivatives of LNCaP and VCaPCell culture media were purchased from Gibco, a division of ThermoFisher Scientifiic . Fetal bovine serum (FBS) and CSS were purchased from Sigma Aldrich, St. Louis, MO. In some cases, FBS was stripped with charcoal dextran in the cell culture core to produce CSS. R1881 and doxycycline (Dox) were purchased from Sigma Aldrich and Enzalutamide from Selleck chemical LLC, Houston, TX. If not otherwise indicated, chemicals were reagent grade.An AR antibody (RRID: AB_2793341) that recognises the N-terminus of AR was used by Active Motif for ChIP-exo analysis; this antibody also was used for ChIP-qPCR. Other antibodies used in the ChIP-qPCR assays include, AR-N20 (RRID:AB_1563391) Santa Cruz Biotechnology, Santa Cruz, CA, AR C-19 (RRID:AB_630864), rabbit anti mouse IgG (RRID:AB_228419). These antibodies and FOXA1 (RRID:AB_2104842), HOXB13 , Tubulin (RRID:AB_309885), Actin (RRID:AB_11004139), and AR-V7 (RRID:AB_2631057) antibodies were used for western blotting also. AR441 was used for western blotting and immunoprecipitation. It was grown in the BCM Protein and Monoclonal Antibody Production core, but also is sold as RRID AB_11000751 by ThermoFisher Scientific. A FOXA1 ChIP validated antibody (RRID:AB_2104842) was used for CoIP experiments.SDS sample loading buffer was added to 20 µg of extracted protein, samples were boiled at 100ºC for 5 min and run on a 7.5% SDS-PAGE gel with a set of standards. Proteins were detected by western blotting, using Protran nitrocellulose membrane  and ECL2 western blot substrate  to visualise signals. Each nitrocellulose membrane was split prior to hybridization to process the upper portion with the specified antibody and the lower portion of each membrane, except for the CoIP experiments described below, was processed for Tubulin/Actin. In the supplementary materials, the top, left and right edges of the membranes are shown for every figure except for Fig. 50 by fusing with 3X FLAG sequences at the beginning and an HA-Tag at the end. This was cloned into the pHAGE lentiviral expression vector as previously described25. pCR3.1 AR-V7 has been described previously51. The pLX302_FOXA1-V5 plasmid was purchased from Addgene, Watertown, MA.DH5a and TOP10 competent cells were purchased from Thermo Fisher Scientific, Waltham, MD and used for plasmid transformation and propagation as per manufacturer’s instructions. PCR amplified cDNA of AR (full length) was cloned from an available pCR3.1 expression vector25. Synthesised oligo duplexes were purchased from Sigma-Genosys, The Woodlands, TX (sequences and source listed in STable 2). Cells were transferred to medium containing CSS and transfected with 35–100 nM siRNA using the Lipofectamine RNAiMAX reagent  according to the manufacturer’s directions for a duration of 48–72 h.Knock-downs of FOXA1, HOXB13, AR, and AR-V7 expression were performed using transient transfection of siRNA against the gene in comparison to non-targeted negative control siRNAFor experiments to test AR variant binding and activity, cells were ligand-starved for 24 h using RPMI-1640 base medium supplemented with 10% CSS, before treating with vehicle (0.1% ethanol), 20 ng/mL Dox (LNCaP lines) or 100 ng/mL Dox (VCaP) or 10 nM R1881. All cells were used within 25 passages of thawing and inducible cells were maintained under G418 selection and transferred to medium lacking G418 for individual experiments.42. The LNCaP cell line with inducible expression of flag tagged AR was generated by infection of lentivirus encoding Flag tagged AR, followed by G418 selection in RPMI-1640 medium for two weeks and the cells termed LNCaPpHAGE flAR (LNCaP AR).Lentivirus was prepared using HEK293T/17 cells and a packaging system as reported earlierCells were harvested in PureXtract RNAzol solution , RNA isolated, and cDNA synthesised from 500 µg total RNA using an AmphiRevert cDNA synthesis kit . Primers (STable 2) and SYBR Green PCR Master Mix  were used for q-PCR analysis using the StepOnePlus Real-Time PCR System . All qPCR plots are expression values normalized to 18S on the y-axis and the corresponding treatment shown on the x-axis. Data are plotted as the mean of three biological replicates from the same experiment; error bars are SEM., pCR3.1 AR transfected cells were treated with 10 nM R1881 for 4 h. Biological replicates of cell pellets were pooled together and lysed by incubating in 1X TESH lysis buffer 52 with 0.15 M NaCl on ice for 2 h. Lysates were split into equal fractions of approximately 250 ug protein, and 2 μg of antibody or control IgG were added. Tubes were incubated for 1 h at 4 °C and then protein A/G agarose magnetic beads (Thermo Scientific) were added, and samples incubated for 10 min at room temperature. Beads were washed three times in 1X TESH buffer with 0.15 M NaCl and resuspended in 40 µl of RIPA buffer  on ice for 1 h. The RIPA fraction was transferred to a new tube and a second fraction was extracted from the same beads using 40 µl 2X SDS sample loading buffer and transferred to a separate tube. SDS sample loading buffer was added to the RIPA fraction, samples were boiled at 100 °C for 5 min. RIPA samples were run on a 7.5% SDS-PAGE gel with a set of controls and SDS samples on another gel with a second set of controls and proteins detected by western blotting, using Protran nitrocellulose membrane  and ECL2 western blot substrate  to visualise signals. Each nitrocellulose membrane was split to process the upper portion with AR441 mAb and the lower portions with the FOXA1 antibody.CoIP was performed from biological replicates of HEK293T/17 cells maintained in DMEM with 10% CSS. Briefly, HEK293T/17 cells were transfected with pLX302_FOXA1-V5, pLX302_FOXA1-V5 + pCR3.1 AR or pLX302_FOXA1-V5 + pCR3.1 AR-V7 expression vectors using 15 µl Lipofectamine 3000 Reagent and 10 µl P3000 reagent  for 28 h. After 24 h of treatment25 with the modifications using AR antibody  or AR C-19  as described in the Supplementary methods (SMethods). Data generated by Active Motif using the ChIP-exo method and their AR antibody 53 were used to map AR and AR-V7 binding ChIP-seq and ChIP-exo (STable 1) were analysed as described in SMethods, using the bowtie2  alignment software54 and peaks were called using macs2 55.ChIP assays were performed as previously described56 and STAR57 were used to align raw reads to human genome build hg19 (V24), gene expression was measured using featureCounts58, and DEGs calculated using edgeR59. Potential direct target DEGs were identified by integrating ARBs, V7Bs and DEGs as described in SMethods.LN95 cells were treated with 10 nM R1881 or an equivalent volume of the vehicle control for 24 h prior to harvesting for preparation of RNA. For knock-down experiments cells were treated with control siRNA, siRNA targeting exon7 or siRNA targeting cryptic exon of AR-V7 for 48 h before harvesting and processed as described in SMethods. Total RNA was extracted, and column purified using an RNAeasy mini kit. (Qiagen). The clean RNA samples having RIN more than 8.0 were used for library preparation in the Genomic and RNA Profiling Core, Baylor College of Medicine. Libraries were constructed using a Truseq library preparation kit  following the manufacturer’s instructions. cDNA libraries were sequenced on the Illumina NextSeq-500 or NovaSeq-6000 platform in the Genomic and RNA Profiling Core facility, Baylor College of Medicine. These RNA-seq datasets and the LNCaP AR-V7 and VCaP AR-V7 datasets listed in STable 1 were used to identify the AR and AR-V7 transcriptome. In short HISAT2We utilised public gene expression datasets and ChIP datasets listed in STable 1 for comparisons in this study. Raw sequence data were processed as above and as described in SMethods.t-test is used to calculate difference in mean gene expression between the treatment and control group.Three independent experiments were performed using separate batches of cells with biological triplicates within each experiment for qPCR assays. For western blot, qPCR and ChIP-qPCR assays, representative examples are presented although the experiments were performed a minimum of three times. Representative genes from subset of genes induced or repressed by FOXA1 or HOXB13 were tested using qPCR assays. Student’s Supplementary Information.