Androgen Receptor Splice Variants Research Articles

Targeting androgen receptor phase separation to overcome antiandrogen resistance.

Patients with castration-resistant prostate cancer inevitably acquire resistance to antiandrogen therapies in part because of androgen receptor (AR) mutations or splice variants enabling restored AR signaling. Here we show that ligand-activated AR can form transcriptionally active condensates. Both structured and unstructured regions of AR contribute to the effective phase separation of AR and disordered N-terminal domain plays a predominant role. AR liquid-liquid phase separation behaviors faithfully report transcriptional activity and antiandrogen efficacy. Antiandrogens can promote phase separation and transcriptional activity of AR-resistant mutants in a ligand-independent manner. We conducted a phase-separation-based phenotypic screen and identified ET516 that specifically disrupts AR condensates, effectively suppresses AR transcriptional activity and inhibits the proliferation and tumor growth of prostate cancer cells expressing AR-resistant mutants. Our results demonstrate liquid-liquid phase separation as an emerging mechanism underlying drug resistance and show that targeting phase separation may provide a feasible approach for drug discovery.

Targeting L-type amino acid transporter 1 in urological malignancy: Current status and future perspective

Amino acid transporters are responsible for the uptake of amino acids, critical for cell proliferation. L-type amino acid transporters play a major role in the uptake of essential amino acids. L-type amino acid transporter 1 (LAT1) exerts its functional properties by forming a dimer with 4F2hc. Utilizing this cancer-specificity, research on diagnostic imaging and therapeutic agents for malignant tumors targeting LAT1 progresses in various fields. In hormone-sensitive prostate cancer, the up-regulation of L-type amino acid transporter 3 (LAT3) through the androgen receptor (AR) has been identified. On the other hand, in castration-resistant prostate cancer, the negative regulation of LAT1 through AR has been determined. Furthermore, 4F2hc: a binding partner of LAT1, was identified as the specific downstream target of Androgen Receptor Splice Variant 7: AR-V7. LAT1 has been suggested to contribute to acquiring castration resistance in prostate cancer, making LAT1 a completely different therapeutic target from anti-androgens and taxanes. Increased expression of LAT1 has also been found in renal and bladder cancers, suggesting a contribution to acquiring malignancy and progression. In Japan, clinical trials of LAT1 inhibitors for solid tumors are in progress, and clinical applications are now underway. This article will summarize the relationship between LAT1 and urological malignancies.

The Crucial Role of AR-V7 in Enzalutamide-Resistance of Castration-Resistant Prostate Cancer.

Simple SummaryAndrogen receptor splice variant 7 (AR-V7) has always been considered a key driver for triggering enzalutamide resistance of castration-resistant prostate cancer (CRPC). In recent years, both the homeostasis of AR-V7 protein and AR-V7’s relationship with LncRNAs have gained great attention with in-depth studies. Starting from protein stability and LncRNA, the paper discusses and summarizes the mechanisms and drugs that affect the CRPC patients’ sensitivity to enzalutamide by regulating the protein or transcriptional stability of AR-V7, hoping to provide therapeutic ideas for subsequent research to break through the CRPC therapeutic bottleneck.Prostate cancer (PCa) has the second highest incidence of malignancies occurring in men worldwide. The first-line therapy of PCa is androgen deprivation therapy (ADT). Nonetheless, most patients progress to castration-resistant prostate cancer (CRPC) after being treated by ADT. As a second-generation androgen receptor (AR) antagonist, enzalutamide (ENZ) is the current mainstay of new endocrine therapies for CRPC in clinical use. However, almost all patients develop resistance during AR antagonist therapy due to various mechanisms. At present, ENZ resistance (ENZR) has become challenging in the clinical treatment of CRPC. AR splice variant 7 (AR-V7) refers to a ligand-independent and constitutively active variant of the AR and is considered a key driver of ENZR in CRPC. In this review, we summarize the mechanisms and biological behaviors of AR-V7 in ENZR of CRPC to contribute novel insights for CRPC therapy.

203 (PB083) - Molecular characterization of next generation AR-NTD inhibitors

Background: Inhibition of the androgen receptor (AR) is the mainstay treatment for advanced prostate cancer. While initially effective, the disease ultimately progresses to metastatic castration-resistant prostate cancer (mCRPC), which is lethal. All current AR-targeted therapies directly or indirectly target the ligand binding domain (LBD). We have developed the EPI and sintokamide (SINT) compounds which target the AR N-terminal domain (NTD), allowing for sustained inhibition in the context of constitutively active AR-splice variants and mutations which often drive resistance to current AR-targeted therapies.

Choice of antibody is critical for specific and sensitive detection of androgen receptor splice variant-7 in circulating tumor cells

Androgen receptor variant 7 (AR-V7) is an important biomarker to guide treatment options for castration-resistant prostate cancer (CRPC) patients. Its detectability in circulating tumour cells (CTCs) opens non-invasive diagnostic avenues. While detectable at the transcript level, AR-V7 protein detection in CTCs may add additional information and clinical relevance. The aim of this study was to compare commercially available anti-AR-V7 antibodies and establish reliable AR-V7 immunocytostaining applicable to CTCs from prostate cancer (PCa) patients. We compared seven AR-V7 antibodies by western blotting and immmunocytostaining using a set of PCa cell lines with known AR/AR-V7 status. The emerging best antibody was validated for detection of CRPC patient CTCs enriched by negative depletion of leucocytes. The anti-AR-V7 antibody, clone E308L emerged as the best antibody in regard to signal to noise ratio with a specific nuclear signal. Moreover, this antibody detects CRPC CTCs more efficiently compared to an antibody previously shown to detect AR-V7 CTCs. We have determined the best antibody for AR-V7 detection of CTCs, which will open future studies to correlate AR-V7 subcellular localization and potential co-localization with other proteins and cellular structures to patient outcomes.

Androgen Receptor Splice Variants Contribute to the Upregulation of DNA Repair in Prostate Cancer.

Simple SummaryLigand-independent androgen receptor splice variants emerge during androgen deprivation therapy and are suspected to render prostate carcinomas castration-resistant. In a retrospective analysis of a large cohort of primary and advanced prostate tumors, we observed increased expression of androgen receptor splice variants in therapy refractory tumors. Our hypothesis was that AR splice variants exert their tumor-promoting activity by modulating the intrinsic DNA repair machinery. In the sequence from primary over advanced tumors under androgen-deprivation therapy to castration resistance, AR splice variant expression increases and is linked to increased expression of DNA repair genes. This effect of AR splice variants appeared independent of their known impact on tumor cell proliferation. These clinical findings were validated in an androgen-sensitive prostate cancer cell line that mimics a castration-resistant phenotype by overexpression of AR-V7. Modulated DNA repair gene expression in the presence of AR splice variants is linked to increased DNA repair activity, pointing at a novel therapeutic approach for castration-resistant prostate cancer.Background: Canonical androgen receptor (AR) signaling regulates a network of DNA repair genes in prostate cancer (PCA). Experimental and clinical evidence indicates that androgen deprivation not only suppresses DNA repair activity but is often synthetically lethal in combination with PARP inhibition. The present study aimed to elucidate the impact of AR splice variants (AR-Vs), occurring in advanced or late-stage PCA, on DNA repair machinery. Methods: Two hundred and seventy-three tissue samples were analyzed, including primary hormone-naïve PCA, primary metastases, hormone-sensitive PCA on androgen deprivation therapy (ADT) and castration refractory PCA (CRPC group). The transcript levels of the target genes were profiled using the nCounter platform. Experimental support for the findings was gained in AR/AR-V7-expressing LNCaP cells subjected to ionizing radiation. Results: AR-Vs were present in half of hormone-sensitive PCAs on androgen deprivation therapy (ADT) and two-thirds of CRPC samples. The presence of AR-Vs is highly correlated with increased activity in the AR pathway and DNA repair gene expression. In AR-V-expressing CRPC, the DNA repair score increased by 2.5-fold as compared to AR-V-negative samples. Enhanced DNA repair and the deregulation of DNA repair genes by AR-V7 supported the clinical data in a cell line model. Conclusions: The expression of AR splice variants such as AR-V7 in PCA patients following ADT might be a reason for reduced or absent therapy effects in patients on additional PARP inhibition due to the modulation of DNA repair gene expression. Consequently, AR-Vs should be further studied as predictive biomarkers for therapy response in this setting.

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

Advanced-stage prostate cancer remains an incurable disease with poor patient prognosis. There is an unmet clinical need to target androgen receptor (AR) splice variants, which are key drivers of the disease. Some AR splice variants are insensitive to conventional hormonal or androgen deprivation therapy due to loss of the androgen ligand binding domain at the C-terminus and are constitutively active. Here we explore the use of RNA interference (RNAi) to target a universally conserved region of all AR splice variants for cleavage and degradation, thereby eliminating protein level resistance mechanisms. To this end, we tested five siRNA sequences designed against exon 1 of the AR mRNA and identified several that induced potent knockdown of full-length and truncated variant ARs in the 22Rv1 human prostate cancer cell line. We then demonstrated that 2'O methyl modification of the top candidate siRNA (siARvm) enhanced AR and AR-V7 mRNA silencing potency in both 22Rv1 and LNCaP cells, which represent two different prostate cancer models. For downstream in vivo delivery, we formulated siARvm-LNPs and functionally validated these in vitro by demonstrating knockdown of AR and AR-V7 mRNA in prostate cancer cells and loss of AR-mediated transcriptional activation of the PSA gene in both cell lines following treatment. We also observed that siARvm-LNP induced cell viability inhibition was more potent compared to LNP containing siRNA targeting full-length AR mRNA (siARfl-LNP) in 22Rv1 cells as their proliferation is more dependent on AR splice variants than LNCaP and PC3 cells. The in vivo biodistribution of siARvm-LNPs was determined in 22Rv1 tumor-bearing mice by incorporating 14C-radiolabelled DSPC in LNP formulation, and we observed a 4.4% ID/g tumor accumulation following intravenous administration. Finally, treatment of 22Rv1 tumor bearing mice with siARvm-LNP resulted in significant tumor growth inhibition and survival benefit compared to siARfl-LNP or the siLUC-LNP control. To best of our knowledge, this is the first report demonstrating therapeutic effects of LNP-siRNA targeting AR splice variants in prostate cancer.

Antagonizing CD105 and androgen receptor to target stromal-epithelial interactions for clinical benefit

Androgen receptor signaling inhibitors (ARSIs) are standard of care for advanced prostate cancer (PCa) patients. Eventual resistance to ARSIs can include the expression of androgen receptor (AR) splice variant, AR-V7, expression as a recognized means of ligand-independent androgen signaling. We demonstrated that interleukin (IL)-6-mediated AR-V7 expression requires bone morphogenic protein (BMP) and CD105 receptor activity in both PCa and associated fibroblasts. Chromatin immunoprecipitation supported CD105-dependent ID1- and E2F-mediated expression of RBM38. Further, RNA immune precipitation demonstrated RBM38 binds the AR-cryptic exon 3 to enable AR-V7 generation. The forced expression of AR-V7 by primary prostatic fibroblasts diminished PCa sensitivity to ARSI. Conversely, downregulation of AR-V7 expression in cancer epithelia and associated fibroblasts was achieved by a CD105-neutralizing antibody, carotuximab. These compelling pre-clinical findings initiated an interventional study in PCa patients developing ARSI resistance. The combination of carotuximab and ARSI (i.e., enzalutamide or abiraterone) provided disease stabilization in four of nine assessable ARSI-refractory patients. Circulating tumor cell evaluation showed AR-V7 downregulation in the responsive subjects on combination treatment and revealed a three-gene panel that was predictive of response. The systemic antagonism of BMP/CD105 signaling can support ARSI re-sensitization in pre-clinical models and subjects that have otherwise developed resistance due to AR-V7 expression.

Identification of the gossypol derivatives as androgen receptor inhibitor

Prostate cancer (PCa) is the most frequently diagnosed male malignant tumor and remains the second leading cause of male cancer mortality in western countries. The development of novel antiandrogens to circumvent the drug resistance in anti-PCa treatment is highly demanded. Herein, we identified that gossypol (GOS) specificly inhibited the AR signaling. Further optimization of GOS derivatives led to the discovery of compound XY-32. XY-32 efficiently inhibits AR signaling with the IC50 of 1.23 μM. XY-32 downregulates both the full-length AR and the AR variable splice AR-V7 via suppressing the mRNA expression. It inhibits the proliferation of CRPC cells such as the LNCaP cells, the PC-3 cells, and enzalutamide resistance 22Rv1 cells. The work demonstrates the GOS derivatives represent a novel series of anti-androgen to conquer the acquired AR mutations or AR splice variants induced drug resistance of mCRPC.

Radium-223 for patients with metastatic castration-resistant prostate cancer with asymptomatic bone metastases progressing on first-line abiraterone acetate or enzalutamide: A single-arm phase II trial

PurposeThe paper aims to evaluate the efficacy and safety of 223Ra in patients who progressed after first-line androgen deprivation therapy. Patients and methodsEXCAAPE (NCT03002220) was a multicentre, single-arm, open-label, non-controlled phase IIa trial in 52 patients with metastatic castration-resistant prostate cancer and asymptomatic bone metastases who have progressed on abiraterone acetate or enzalutamide, up to six doses of 223Ra (55 kBq/kg of body weight per month). The primary end-point was radiographic progression-free survival (rPFS). Secondary end-points included rPFS based on androgen receptor splice variant 7 (AR-V7) expression in circulating tumour cells (CTCs), overall survival, and safety. ResultsMedian rPFS was 5.5 months (95% CI 5.3–5.5). Median rPFS of patients with AR-V7(−) CTCs was longer than that of patients with AR-V7(+) CTCs (5.5 versus 2.2 months, respectively; P = 0.056). Median overall survival was 14.8 months (95% CI 11.2–not reached) and was significantly greater for AR-V7(−) patients than for AR-V7(+) patients (14.8 months versus 3.5 months, respectively; P < 0.01). 223Ra was well tolerated; anaemia and thrombocytopenia were the most common grade 3/4 adverse events (5.8% and 11.5%, respectively). Conclusions223Ra seems to be a reasonable treatment for patients with metastatic castration-resistant prostate cancer and asymptomatic bone metastases progressing on novel hormonal therapy and had an acceptable safety profile.

De Novo Design of an Androgen Receptor DNA Binding Domain-Targeted peptide PROTAC for Prostate Cancer Therapy.

Androgen receptor splice variant‐7 (AR‐V7), one of the major driving factors, is the most attractive drug target in castration‐resistant prostate cancer (CRPC). Currently, no available drugs efficiently target AR‐V7 in clinical practice. The DNA binding domain (DBD) is indispensable for the transcriptional activity of AR full length and AR splice variants, including AR‐V7. Based on the homodimerization structure of the AR DBD, a novel peptide‐based proteolysis‐targeting chimera (PROTAC) drug is designed to induce AR and AR‐V7 degradation in a DBD and MDM2‐dependent manner, without showing any activity on other hormone receptors. To overcome the short half‐life and poor cell penetrability of peptide PROTAC drugs, an ultrasmall gold (Au)‐peptide complex platform to deliver the AR DBD PROTAC in vivo is developed. The obtained Au‐AR pep‐PROTAC effectively degrades AR and AR‐V7 in prostate cancer cell lines, particularly in CWR22Rv1 cells with DC50 values 48.8 and 79.2 nM, respectively. Au‐AR pep‐PROTAC results in suppression of AR levels and induces tumor regression in both enzalutamide sensitive and resistant prostate cancer animal models. Further optimization of the Au‐AR pep‐PROTAC can ultimately lead to a new therapy for AR‐V7‐positive CRPC.

Androgen receptor splicing variant 7 (ARv7) promotes DNA damage response in prostate cancer cells.

In the treatment of patients with locally advanced prostate cancer (PCa), androgen deprivation therapy (ADT) significantly enhances the efficacy of radiotherapy by weakening the DNA damage response (DDR) pathway. Recently, several studies have suggested that androgen receptor splicing variants (ARvs) may mediate a compensatory DDR pathway when canonical androgen receptor (AR) signaling is inhibited, thus contributing to the resistance of some patients to this combinational treatment. However, the specific roles of certain ARvs as well as the detailed mechanism of how ARvs regulate the DDR are not well understood. Here, we demonstrated that AR splicing variant 7 (ARv7), which is the most abundant form of ARvs, significantly promotes the DDR of PCa cells under severe DNA damage independent of its parental AR by using the ionizing radiation (IR) and doxorubicin (Dox)-treated cell models. Mechanistically, ARv7 is sufficient to upregulate both the homologous recombination (HR) and the nonhomologous end joining (NHEJ) pathways by forming a positive regulatory loop with poly ADP-ribose polymerase 1 (PARP1). Moreover, the presence of ARv7 impairs the synergistic effect between AR antagonists and poly ADP-ribose polymerase (PARP) inhibitor, which has been recently shown to be a promising future treatment strategy for metastatic castration resistant prostate cancer (mCRPC). Combined, our data indicate that constitutively active ARv7 not only contributes to radioresistance after ADT, but may also serve as a potential predictive biomarker for assessing the efficacy of novel PARP inhibitor-based therapy in PCa.

New diterpenes from the marine sponge Spongionella sp. overcome drug resistance in prostate cancer by inhibition of P-glycoprotein

Spongian diterpenes are a group of marine natural compounds possessing various biological activities. However, their anticancer activity is still poorly studied and understood. We isolated six spongian diterpenes from the marine sponge Spongionella sp., including one new spongionellol A and five previously known molecules. The structures were elucidated using a detailed analysis MS and NMR spectra as well as by comparison with previously reported data. Two of them, namely, spongionellol A and 15,16-dideoxy-15α,17β-dihydroxy-15,17-oxidospongian-16-carboxylate-15,17-diacetate exhibited high activity and selectivity in human prostate cancer cells, including cells resistant to hormonal therapy and docetaxel. The mechanism of action has been identified as caspase-dependent apoptosis. Remarkably, both compounds were able to suppress expression of androgen receptor (AR) and AR-splice variant 7, as well as AR-dependent signaling. The isolated diterpenes effectively inhibited drug efflux mediated by multidrug-resistance protein 1 (MDR1; p-glycoprotein). Of note, a synergistic effect of the compounds with docetaxel, a substrate of p-glycoprotein, suggests resensitization of p-glycoprotein overexpressing cells to standard chemotherapy. In conclusion, the isolated spongian diterpenes possess high activity and selectivity towards prostate cancer cells combined with the ability to inhibit one of the main drug-resistance mechanism. This makes them promising candidates for combinational anticancer therapy.

Targeting the radiation-induced ARv7-mediated circNHS/miR-512-5p/XRCC5 signaling with Quercetin increases prostate cancer radiosensitivity

BackgroundRadiation therapy (RT) with androgen deprivation therapy (ADT) is an effective therapy to suppress the locally advanced prostate cancer (PCa). However, we unexpectedly found that RT could also induce the androgen receptor splice variant 7 (ARv7) expression to decrease the radiosensitivity.MethodsThe study was designed to target ARv7 expression with Quercetin or ARv7-shRNA that leads to enhancing and increasing the radiation sensitivity to better suppress the PCa that involved the modulation of the circNHS/miR-512-5p/XRCC5 signaling.ResultsMechanism studies revealed that RT-induced ARv7 may function via altering the circNHS/miR-512-5p/XRCC5 signaling to decrease the radiosensitivity. Results from preclinical studies using multiple in vitro cell lines and in vivo mouse models concluded that combining RT with the small molecule of Quercetin to target full-length AR and ARv7 could lead to better efficacy to suppress PCa progression.ConclusionTogether, these results suggest that ARv7 may play key roles to alter the PCa radiosensitivity, and targeting this newly identified ARv7 mediated circNHS/miR-512-5p/XRCC5 signaling with Quercetin may help physicians to develop a novel RT to better suppress the progression of PCa.

Interferon signalling contributes to therapy‐resistant prostate cancer progression

Interferon signalling contributes to therapy‐resistant prostate cancer progression

AR-V7 exhibits non-canonical mechanisms of nuclear import and chromatin engagement in castrate-resistant prostate cancer.

Expression of the AR splice variant, androgen receptor variant 7 (AR-V7), in prostate cancer is correlated with poor patient survival and resistance to AR targeted therapies and taxanes. Currently, there is no specific inhibitor of AR-V7, while the molecular mechanisms regulating its biological function are not well elucidated. Here, we report that AR-V7 has unique biological features that functionally differentiate it from canonical AR-fl or from the second most prevalent variant, AR-v567. First, AR-V7 exhibits fast nuclear import kinetics via a pathway distinct from the nuclear localization signal dependent importin-α/β pathway used by AR-fl and AR-v567. We also show that the dimerization box domain, known to mediate AR dimerization and transactivation, is required for AR-V7 nuclear import but not for AR-fl. Once in the nucleus, AR-V7 is transcriptionally active, yet exhibits unusually high intranuclear mobility and transient chromatin interactions, unlike the stable chromatin association of liganded AR-fl. The high intranuclear mobility of AR-V7 together with its high transcriptional output, suggest a Hit-and-Run mode of transcription. Our findings reveal unique mechanisms regulating AR-V7 activity, offering the opportunity to develop selective therapeutic interventions.

Comparison of circulating tumor cells and AR-V7 as clinical biomarker in metastatic castration-resistant prostate cancer patients

Biomarker in metastatic castration resistant prostate cancer (mCRPC) treatment are rare. We aimed to compare the clinical value of circulating tumor cells (CTCs) and androgen receptor splice variant 7 (AR-V7) as biomarker in mCRPC patients undergoing androgen receptor-targeted agent (ARTA) treatment. Overall cohort (65 patients) was stratified regarding either CTC or AR-V7 status followed by further sub-stratification of the respective other marker. Subsequently, prostate specific antigen (PSA) response, progression free survival (PFS) and overall survival (OS)) of subgroups was compared. CTCs and AR-V7 were detected in 54 (83%) and 33 (61%) patients, respectively. All AR-V7 + were CTC +. We detected PSA response in all subgroups. For PFS and OS, biomarker stratification revealed differences between all subgroups. Interestingly, no significant differences of AR-V7 transcript copy numbers were detected between responding and non-responding patients. Additionally, multivariable analysis revealed no independent prognostic value of AR-V7 positivity. Both biomarkers show clinical value in prognosticating clinical outcome. Nonetheless, AR-V7 stratification underestimates the heterogenous subgroup of CTC − and CTC + patient, the latter requiring more intense clinical surveillance. Additionally, AR-V7 level does not correlate with clinical response. Thus, the value of AR-V7 as a clinical biomarker must be considered skeptically.

MO20-3 Caffeic acid phenethyl ester suppresses drug resistance of enzalutamide or abiraterone in prostate cancer cells

Enzalutamide (ENZ) and Abiraterone acetate (AA) are drugs targeting androgen receptor axis for treatment of metastatic castration-resistant prostate cancer (mCRPC). However, a majority of patients receiving these treatments will eventually acquire drug resistance, which is caused by multiple mechanisms including the activation of AR splice variant 7 (AR-V7). We determined if caffeic acid phenethyl ester (CAPE) can suppress the signaling of AR-V7 and tumor growth of ENZ-resistant or AA-resistant mCRPC cells.

P-606 Androgen receptor splice variant lead to PCOS with insulin resistance via IRS1/AKT signaling pathway in mice

Abstract Study question How does the androgen receptor splice variant (ARSV) contribute to insulin resistance in polycystic ovary syndrome (PCOS) patients? Summary answer ARSV inhibited the intracellular signaling pathway of insulin, resulting in decreased glucose transporter type 4 (GLUT4) translocation, impaired cellular glucose uptake, and insulin resistance. What is known already PCOS is the most common endocrine disorder in women of reproductive age and characterized by hyperandrogenism, ovulation dysfunction, and ovarian polycystic changes. In addition, a significant proportion of PCOS patients will experience insulin resistance. Our previous study identified an ARSV in granulosa cells from PCOS patients, which may be associated with higher insulin levels in follicular fluid. Study design, size, duration / Participants/materials, setting, methods Glucose metabolism of a mouse model presenting ARSV by CRISPR-Cas9 was measured by glucose tolerance test and insulin tolerance test. Body composition was measured by a nuclear magnetic resonance system. KGN cell lines stably overexpressing wild type androgen receptor (AR group) or ARSV (ARSV group) were successfully constructed by lentivirus infection. The process of glucose uptake by GLUT4, and the expression of target genes in the glucose uptake pathway were detected in cells. Main results and the role of chance The mice expressing ARSV had higher body weight and body fat content than the wild type mice, while the muscle content was equivalent.ARSV mice displayed diabetes with impaired glucose tolerance and insulin tolerance at 48 week-age. The levels of fasting glucose and insulin levels were significantly higher in ARSV mice. The basal and insulin-stimulated glucose uptake capacity were also found impaired in ARSV group. Immunofluorescence demonstrated that under insulin stimulation, GLUT4 protein was less expressed in the membrane and more expressed in the cytoplasm in ARSV group. Similar results were obtained by detecting the expression of GLUT4 protein after separating the cell membrane and cytoplasmic proteins. Further detection of the upstream signaling molecules of GLUT4 revealed that the phosphorylation levels of insulin receptor substrate 1 (IRS1) at Ser1101 were slightly increased in ARSV group, after stimulation with insulin and DHT. Besides, the phosphorylation levels of protein kinase B (AKT) were also found significantly decreased at Thr308 and slightly decreased at Ser473 in ARSV group. ARSV inhibited the intracellular signaling pathway of insulin by altering the phosphorylation level of IRS1/AKT signaling, resulting in decreased GLUT4 translocation, impaired cellular glucose uptake, and insulin resistance. Limitations, reasons for caution In this study, we failed to obtain clinical samples of PCOS to prospectively verify the association between ARSV and glucose uptake, insulin resistance, and subsequent onset diabetes. Wider implications of the findings This transgenic animal model can simulate the reproductive and metabolic phenotypes of PCOS patients, providing a model for future PCOS research. PCOS patients with ARSV should delay the onset of insulin resistance through lifestyle intervention, exercise, and drug therapy as early as possible. Trial registration number NA

Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer.

Aberrant transcriptional activity of androgen receptor (AR) is one of the dominant mechanisms for developing of castration-resistant prostate cancer (CRPC). Analyzing AR-transcriptional complex related to CRPC is therefore important towards understanding the mechanism of therapy resistance. While studying its mechanism, we observed that a transmembrane protein called neuropilin-2 (NRP2) plays a contributory role in forming a novel AR-transcriptional complex containing nuclear pore proteins. Using immunogold electron microscopy, high-resolution confocal microscopy, chromatin immunoprecipitation, proteomics, and other biochemical techniques, we delineated the molecular mechanism of how a specific splice variant of NRP2 becomes sumoylated upon ligand stimulation and translocates to the inner nuclear membrane. This splice variant of NRP2 then stabilizes the complex between AR and nuclear pore proteins to promote CRPC specific gene expression. Both full-length and splice variants of AR have been identified in this specific transcriptional complex. In vitro cell line-based assays indicated that depletion of NRP2 not only destabilizes the AR-nuclear pore protein interaction but also inhibits the transcriptional activities of AR. Using an in vivo bone metastasis model, we showed that the inhibition of NRP2 led to the sensitization of CRPC cells toward established anti-AR therapies such as enzalutamide. Overall, our finding emphasize the importance of combinatorial inhibition of NRP2 and AR as an effective therapeutic strategy against treatment refractory prostate cancer.