Prostate cancer is the second leading cause of death from cancer among males in Western countries. It is also the most commonly diagnosed male cancer in Japan. The progression of prostate cancer is mainly influenced by androgens and the androgen receptor (AR). Androgen deprivation therapy is an established therapy for advanced prostate cancer; however, prostate cancers frequently develop resistance to low testosterone levels and progress to the fatal stage called castration-resistant prostate cancer (CRPC). Surprisingly, AR and the AR signaling pathway are still activated in most CRPC cases. To overcome this problem, abiraterone acetate and enzalutamide were introduced for the treatment of CRPC. Despite the impact of these drugs on prolonged survival, CRPC acquires further resistance to keep the AR pathway activated. Functional molecular studies have shown that some of the AR collaborative transcription factors (TFs), including octamer transcription factor (OCT1), GATA binding protein 2 (GATA2) and forkhead box A1 (FOXA1), still stimulate AR activity in the castration-resistant state. Therefore, elucidating the crosstalk between the AR and collaborative TFs on the AR pathway is critical for developing new strategies for the treatment of CRPC. Recently, many compounds targeting this pathway have been developed for treating CRPC. In this review, we summarize the AR signaling pathway in terms of AR collaborators and focus on pyrrole-imidazole (PI) polyamide as a candidate compound for the treatment of prostate cancer. Prostate cancer is the major cause of death from cancer among males in Western countries. For example, the American Cancer Society has estimated 180,890 new cases of prostate cancer and 26,120 deaths from the disease in the United States in 2016. The Australian Institute of Health and Welfare estimated 18,138 new diagnoses and 3398 deaths from prostate cancer in 2016. This amounts to 21.4% and 12.8% of all male deaths from cancer in each country in 2016. In Japan, although prostate cancer is the seventh-leading cause of cancer death, recently both the number of cases and the mortality rate due to prostate cancer have increased significantly. The increased population of older males is presumed to be one of the contributors in Japan.The androgen receptor (AR) signaling pathway plays an integral role in the progression of prostate cancer. The AR is a member of the steroid hormone receptor superfamily. The AR is activated by ligands, such as dihydrotestosterone (DHT), and then functions as a transcription factor to modulate the expression of its target genes. Approximately 80%–90% of prostate cancers are androgen-dependent at the time of diagnosis ,2,3,4,5.The extragonadal androgens synthesized in adrenal or CRPC cells are one of the key mechanisms for sustaining AR signaling in CRPC. They activate the cytochrome P450 (CYP) family, which facilitates the unusual conversion of cholesterol to androgen under low testosterone conditions. Thus, the expression of androgen-dependent genes is induced by a very small amount of androgens under castration . AbirateThis article reviews the AR signaling pathway in CRPC as well as the development of novel therapeutic medicines targeting AR collaborators, especially collaborative DNA binding transcription factors (TFs).The AR contains an N-terminal domain , a DNA-binding domain , a hinge region, and a ligand binding domain  . The NTDThe increased frequency of functional AR mutations in CRPC enhances resistance to ADT. In addition, ADT drugs mediate a conformational change in the AR ,28. The Previous reports have shown that constitutively active AR isoforms (splice variants: ARVs) were detected in CRPC cell lines and patient tissues . These APSA, a classical AR-regulated gene. The AR and coactivator complex first occupies the PSA enhancer region and then bridges to the promoter, which allows Rpol II to track to this region [The regulation of AR-targeted gene expression requires the recruitment of coregulators to regulatory regions of the AR protein. Coregulators promote (named coactivators), or inhibit (named corepressors) AR transactivation. Although coregulators do not need to bind DNA, they recruit general TFs associated with RNA polymerase II (Rpol II) to gene promoters . The acts region . Since ts region ,41,42,43s region .In addition to AR coregulators, TFs that collaborate with AR are also important for androgen responsive gene expression. Generally, most genes are packed and condensed into nucleosomes by being wound around the four core histones . Thus, nGATA and FoxA family members are known to play important roles in liver and gut development in mouse embryos . In vivoConsistent with the results of liver developmental studies, one member of the FoxA family, FOXA1, works as a pioneer factor in the AR and estrogen receptor (ER) pathways in prostate cancer and breast cancer cells ,53,54. IGATA family proteins are also recruited to compact chromatin . GATA2 aTGM2 and C20orf77 expression by inhibiting AR activity [PARP1), and metabolic genes [ACSL3) [ACSL3 expression in prostate cancer tissues was associated with poor patient prognosis [OCT1 acts downstream of these pioneer factors. For prostate cancer cells, GATA2 and OCT1 work in a hierarchical network as GATA2 is recruited with AR, followed by OCT1 binding to its motifs . OCT1 isactivity ,65. Thesactivity ,67. Tantactivity  showed tic genes . PARP1 pic genes . These dic genes . These d [ACSL3)  as the m [ACSL3) . In addirognosis .In addition to these primary factors, several groups have subsequently identified ETS proto-oncogene 1, transcription factor (ETS1), ERG, ETS transcription factor (ERG), CCAAT/enhancer binding proteins (C/EBPs), nuclear factor I (NFI), NK3 homeobox 1 (NKX3-1), runt related transcription factor 1 (RUNX1), and forkhead box P1 (FOXP1) as other AR collaborative TFs ,76,77,78ETS1 expression is related to high-grade prostate cancer and the resistance to flutamide in prostate cancer cell lines. In addition, ETS1 directly interacts with AR and stimulates NKX3-1 expression [ETS1 is a member of the ETS (v-ets erythroblastosis virus E26 oncogene) family. Massie et al.  reportedpression ,84.NKX3-1 gene region is frequently lost in prostate cancer and this leads to increase vascular endothelial growth factor-C (VEGF-C) expression, NKX3-1 is known as a tumor suppressor gene [NKX3-1 is an AR response gene as well as an AR collaborating TF [RAB3B, a member of the RAS oncogene family [The NKX family belongs to the homeodomain class of TFs, which are critical regulators of whole organ development . The rolsor gene ,87. On tating TF . This ste family .Similar to ETS1, ERG belongs to the class I ETS family  and possesses oncogenic properties, which activate the phosphoinositide 3-kinase (PI3K) pathway to promote prostate cancer progression ,89. On tPSA, solute carrier family 45 member 3: SLC45A3, microseminoprotein beta: MSMB, and secretoglobin family 1D member 2: SCGB1D2). In other words, these genes are prostate epithelial differentiation markers [EZH2). TMPRSS2:ERG expression decreases during ADT, but is reactivated in the castration resistant state [DAB2IP) was inhibited by EZH2/PRC2 [EZH2 is also overexpressed in hormone-refractory metastatic prostate cancer, suggesting EZH2 promotes AR independent growth [RUNX1 functions as an AR collaborative factor to maintain AR activity. In addition, EZH2 is recruited to the RUNX1 promoter to repress its expression [RUNX1 expression level in clinical prostate cancer tissues is negatively associated with EZH2 expression, and decreased RUNX1 expression is correlated with poor prognosis [EZH2 expression in androgen-independent prostate cancer inhibits RUNX1 and the negative effect of RUNX1 on prostate cancer progression. In addition to EZH2, Ma et al. [Unlike ETS1, ERG has a unique role in the AR signaling pathway. Yu et al.  showed t markers . Yu et a markers  suggest nt state . EZH2, wnt state . This meZH2/PRC2 . EZH2 ist growth . Furthert growth  has showpression . The RUNrognosis . These da et al.  showed tInterestingly, previous reports have shown that high dose testosterone supplementation of castrate-resistant cells inhibits their proliferation ,102. ThiMYC), and signal transducer and activator of transcription (STAT) motifs compared to those in ADT naïve and prostate cancer cell lines [AR binding regions might keep changing with prostate cancer progression under a low testosterone environment. Recently, Sharma et al.  elucidatll lines .1–S transition and DNA synthesis and induces cell cycle progression [E2F-1 is regulated by the tumor suppressor gene RB transcriptional corepressor 1 (RB1). RB1 inhibits G1–S transition related gene expression by directly obstructing the transactivation domain of E2F and the promoter activity of these genes [RB1 loss is frequently observed in CRPC, the RB1/E2F-1 complex could play a significant role in tumor progression. A previous report suggested that loss of RB1 enhances AR activity via E2F-1 activation to induce resistance to ADT [E2F-1 activates genes related to Ggression . The expse genes . Since Re to ADT .c-MYC gene in 72% of CRPC [Bin1 and MXI1, were inactivated in advanced prostate cancer [c-MYC and represses prostate epithelial differentiation genes.c-MYC is known as an oncogenic transcription factor that regulates ribosomal RNA expression, glutamine metabolism, and energy and reactive oxygen species ,108,109. of CRPC ,112. Some cancer ,114. Cone cancer  about the cancer  also repSTAT3 is regulated by the Janus kinase (Jak) family/interleukin 6 (IL-6) and is also oncogenic, promoting cytosolic dimerization, nuclear translocation and DNA binding ,117,118.Collectively, these studies suggest that the role of the AR signaling pathway in prostate cancer progression is more complicated than expected, because AR collaborating TFs are entangled with each other and have differing effects on AR activity depending on testosterone levels and the duration of anti-androgen drug treatment.N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids, the side by side pairings of which recognize and attach to the minor groove of DNA with high affinity and sequence specificity [Different classes of drugs are under investigation to inhibit AR collaborative TFs. In this section, we review the development of one new class of compounds, pyrrole-imidazole (PI) polyamides, before discussing specific examples of compounds that target AR collaborative TFs in the following section. PI polyamides are small synthetic molecules made up of cificity ,124,125.cificity ,127.In addition, the C-terminal β-alanine residue next to dimethylpropylamine (Dp) and the γ-aminobutyric acid turns a unit, which enforces an antiparallel hairpin configuration and enhances both DNA binding affinity and specificity ,128,129.The pharmacokinetics of PI polyamides provide promise for future clinical applications. Previous reports have shown that PI polyamides are not absorbed from the intestine . After tFOXP1 acts as a negative AR collaborative transcriptional factor, and represses tumor activity by binding to adjacent regions to AREs [FOXA1 expression and inhibits breast cancer growth via cooperation with BRCA1 [Targeting the pioneer factor FOXA1 showed contradictory results for AR activity and prostate cancer prognosis . Increas to AREs ,140. Intth BRCA1 . Recentlth BRCA1  elucidatth BRCA1 . Whilst th BRCA1 . Previouth BRCA1  suppressth BRCA1 . Althougth BRCA1 ,146,147.Whilst many studies have focused on FOXA1 and GATA2, OCT1 is often overlooked, so we have developed a novel drug targeting Oct1/AR using PI polyamides. A previous report showed that a PI polyamide targeting AREs suppressed androgen-responsive gene expression in LNCaP cells . This soThere is one report of an ETS-1 inhibitor using double-strand oligodeoxynucleotides (ODNs) that represses gastric cancer cell proliferation . ODNs miTMPRSS2 and ERG gene loci to repress TMPRSS2:ERG expression and prostate cancer cell growth [Since ERG was shown to be an oncogenic protein, ERG target drugs became attractive agents for prostate cancer. PARP inhibitors, a direct ERG binding small molecule (YK-4-279), a DNA-binding inhibitor targeting ETS consensus sites (DB1255), and a drug that enhances ERG ubiquitination (WP1130) are all promising compounds for prostate cancer ,153,154.l growth . Furtherl growth .NKX3-1 expression by RNAa formulated in lipid nanoparticles significantly inhibited prostate tumor growth both in vitro and in vivo [Ren et al.  develope in vivo .C/EBPα repressed the proliferation of pancreatic ductal adenocarcinoma cells [C/EBPα is underway for severe liver cancer (NCT02716012).Although the role of the C/EBP family in prostate cancer is still unknown, a recent report showed that RNAa targeting ma cells . In addiNigella sativa seeds for prostate cancer. Interestingly, thymoquinone inhibited the tumor growth of CRPC xenografts and repressed E2F-1 and AR expression [E2F-1 consensus sequence. Treatment of mice with this peptide encapsulated in PEGylated liposomes inhibited the growth of an AR negative prostate cancer cell line without toxicity [Several studies of E2F-1 inhibitors have been reported. Kaseb et al.  studied pression . Xie et pression  also devtoxicity .Like ERG, there are several agents targeting c-MYC ,165,166.STAT3 using ODNs repressed head and neck cancer cell growth. In addition, Hedvat et al. showed favorable results in prostate cancer for a STAT3 inhibitor, AZD1480, which is a potent ATP competitive inhibitor of Jak2 kinase [STAT3 expression, but did not find a survival improvement in patients with advanced prostate cancer.Leong et al.  showed t2 kinase . However2 kinase  reportedThe information about AR collaborative TFs and related drugs discussed in this section is summarized in AR collaborators, such as collaborative TFs, are important in the extraordinary hypersensitivity of the AR in CRPC. In addition, activation of AR-regulated genes promotes prostate cancer progression. Over the last decade, sophisticated technologies for investigating transcriptional networks have broadened our understanding of AR signaling in prostate cancer. Various functional studies, including our own work, have elucidated the complicated influence that AR collaborators have on prostate cancer progression. These reports provide fundamental evidence to support the premise that developing novel drugs against AR collaborators could provide promising strategies to treat CRPC. Thus, further studies of these novel candidate compounds with pre-clinical drug screening models will be crucial for developing new strategies to treat CRPC ,173,174.


Ovarian cancer is the major cause of death in women with gynecologic malignancies. There is emerging evidence that Androgen/androgen receptor (AR) signaling plays a critical role in the etiology and progression of this disease. Androgen receptor is frequently expressed in various subtypes of ovarian cancers and androgen/AR signaling has been shown to promote proliferation, migration, and invasion of ovarian cancer cells. Furthermore, shorter AR CAG repeats length and increased AR activity are associated with increased ovarian cancer risk and may be a useful prognosticator under certain circumstances. Here, we summarize current findings regarding the role of the AR in ovarian cancer and discuss agents that target this pathway as potential therapeutics for ovarian cancer. Ovarian cancer is the most lethal gynecologic malignancy with an estimated 22,280 new cases and 14,240 deaths in 2016 in the United States . For theSince HA Risch 1998) first put forward a hypothesis for the pathogenesis of ovarian cancer relating to the role of androgens in stimulating epithelial cell proliferation , a large first puThe effects of androgens are mediated through the androgen receptor (AR), a steroid hormone receptor that belongs to the nuclear receptor superfamily . In its In this article, we provide a comprehensive overview on the role of AR in ovarian cancer carcinogenesis and progression. We also summarize and discuss key data describing clinical implications in ovarian cancer. A better understanding on the activity and clinical relevance of the steroid hormone receptor in ovarian carcinogenesis is essential for the development of potential prognostic marker and therapeutic target in this disease.A large body of evidence has consistently vindicated over-expression of AR in ovarian cancers. In an early report, Hamilton et al.  demonstrAbout 90% of primary malignant ovarian tumors are epithelial ovarian cancer and can be further classified into five histopathological types: low-grade serous carcinomas, high-grade serous carcinoma, clear cell carcinoma, endometrioid carcinoma, and mucinous carcinoma , 5. It iThe association between AR expression and other clinical pathological characteristics such as tumor stages and grades has also been evaluated , 24, 26.in vivo studies about androgen promoting tumor growth. Using guinea pigs, Silva et al. . Th. Thvia apromoter  showed tThe epidermal growth factor receptor (EGFR) is over-expressed in 30-98% of epithelial ovarian carcinomas, and the activation of signaling cascades is linked to cell proliferation, migration and invasion, and angiogenesis, as well as resistance to cell apoptosis . AR was Using a cDNA microarray, Sheach et al.  identifiThe AR also works in concert with AR coactivators to promote tumorgenesis. For instance, AR-associated protein 70 (ARA70) is a reported AR coactivator that enhances the transactivation potential of the AR up to 10-fold. ARA70 transcripts were negative in the normal ovarian surface epithelium, whereas it was highly expressed in 17 out of 20 ovarian carcinomas of various histological types . P44/Mepvia interacting with a number of key components including the TGF-beta pathway, IL-6/IL-8, epidermal growth factor receptor, cell cycle regulators, and AR coactivators. . Nevertheless, Lee et al. and Toledo et al. pointed out that AR expression was not associated with survival [Given the tumor-promoting role of AR, several other studies evaluated the potential prognostic value of AR in ovarian cancer patients Table . In a prsurvival , 25. Consurvival  reportedsurvival  in high-survival . CollectIn addition to AR expression, the effects of AR alleles on prognosis have also been evaluated. Li et al.  reportedAR is expressed in a majority of human ovarian cancers and androgen/AR signaling stimulates proliferation, cell migration and invasion of ovarian cancer cells. Shorter AR CAG repeats length and increased AR activity predict a high risk of ovarian cancer. Clinical data suggests that anti-androgen treatment in ovarian cancer may be effective for some patients. Since agents used for AR blockade in ovarian cancers are known to be weak AR antagonists, novel promising treatments that are more potent suppressors of the AR axis should be evaluated in clinical trials for ovarian cancers. Further, the use of tumor genomic expression profiling and the detection of patient AR response biomarkers may be very important in selecting appropriate patients for anti-androgen therapeutics, monitoring the bioactivity of new drug candidates, and predicting therapy outcomes and adverse events. In addition, AR expression is involved in response to chemotherapy and could be a potential biomarker to predict patient outcome to treatment. Increased AR expression likely predicts a favorable prognosis in epithelial ovarian cancer.


Prostate cancer (PCa) is one of the most common cancers and among the leading causes of cancer deaths for men in industrialized countries. It has long been recognized that the prostate is an androgen-dependent organ and PCa is an androgen-dependent disease. Androgen action is mediated by the androgen receptor (AR). Androgen deprivation therapy (ADT) is the standard treatment for metastatic PCa. However, almost all advanced PCa cases progress to castration-resistant prostate cancer (CRPC) after a period of ADT. A variety of mechanisms of progression from androgen-dependent PCa to CRPC under ADT have been postulated, but it remains largely unclear as to when and how castration resistance arises within prostate tumors. In addition, AR signaling may be modulated by extracellular factors among which are the cysteine-rich glycoproteins WNTs. The WNTs are capable of signaling through several pathways, the best-characterized being the canonical WNT/β-catenin/TCF-mediated canonical pathway. Recent studies from sequencing PCa genomes revealed that CRPC cells frequently harbor mutations in major components of the WNT/β-catenin pathway. Moreover, the finding of an interaction between β-catenin and AR suggests a possible mechanism of cross talk between WNT and androgen/AR signaling pathways. In this review, we discuss the current knowledge of both AR and WNT pathways in prostate development and tumorigenesis, and their interaction during development of CRPC. We also review the possible therapeutic application of drugs that target both AR and WNT/β-catenin pathways. Finally, we extend our review of AR and WNT signaling to the mammary gland system and breast cancer. We highlight that the role of AR signaling and its interaction with WNT signaling in these two hormone-related cancer types are highly context-dependent. For the men in the United States, prostate cancer (PCa) is not only one of the most commonly diagnosed cancers, but also one of the most predominant causes of death from cancer ,2. The AWNT signaling is an evolutionary highly conserved signaling system throughout the eukaryotic kingdom. During embryonic and postnatal development, WNT signaling controls many cellular processes, including proliferation, survival and differentiation ,24,25,26Wingless (Wg) and integration 1 (Int1) are homologues in Drosophila and mouse, respectively [Wg was genetically characterized as a segment polarity gene in Drosophila in 1980 by Nüsslein-Volhard and Wieschaus [Int1 was first identified in 1982 by Nusse and Varmus as a preferential site for proviral integration of the mouse mammary tumor virus (MMTV) in a mouse mammary cancer model [Wnt1, genome sequencing has revealed the existence of 19 Wnt genes in mammals. All WNT proteins share common features that are essential for their function, including a signal peptide for secretion, many potential glycosylation sites, and WNT ligands interact with seven-pass transmembrane receptors of the Frizzled (FZD) family and/or single-pass transmembrane co-receptors, such as lipoprotein receptor-related protein 5/6 (LRP5/6), ROR2, and RYK [+  and WNT/JNK , WNT/Rho pathways, are β-catenin-independent and activate a variety of downstream intracellular signaling cascades [The term Wnt is an amalgam of Wg and Int , as the ectively ,46. Wg wieschaus . The proer model . Since t and RYK ,52,53,54 and RYK ,56,57. Rcascades ,58,59,60cascades ,24,25,26In this review, we will discuss the multifaceted manner with which both the canonical and non-canonical WNT pathways influence and modulate AR signaling in CRPC development. We will consider the possible therapeutic application of drugs that target both pathways. We will also discuss these under the context of recurrent mutations in both pathways identified from PCa genomes. Finally, we will extend our review of these two pathways to the mammary gland system and breast cancer.CTNNB1 gene [AR gene [http://www.stanford.edu/group/nusselab/cgi-bin/wnt/.The known molecular components and the cascade of the canonical WNT signaling pathway are summarized in NB1 gene , is a meNB1 gene . The N-tNB1 gene . β-CatenNB1 gene ,65,66,67NB1 gene ,70,71,72NB1 gene . CanonicNB1 gene ,75,76,77NB1 gene ,79. In cNB1 gene ,81. The NB1 gene ,65,66 anNB1 gene  and by rNB1 gene ,84, PygoNB1 gene ,86, CREBNB1 gene ,88 or HyNB1 gene , ensurinNB1 gene , Cyclin NB1 gene ,92, urokNB1 gene , CD44 [9NB1 gene , Cox-2 aNB1 gene , and the[AR gene ,97, as wLGR5 is a WNT target gene and although originally discovered as an intestinal stem cell marker [In addition to promoting the WNT activity, a series of biochemical experiments indicated that R-spondins (RSPOs) are able to synergize with the WNT pathway in the presence of canonical WNT ligands . Similarl marker , it has l marker ,100,101.l marker . Close rl marker ,102,103.The activation of canonical WNT signaling can also be blocked by extracellular proteins. These include the sFRP family  , WIF  and G protein, which activates phospholipase C (PLC), leading to production of 1,2-diacylglycerol (DAG); 1,2-DAG then activates protein kinase C (PKC), and inositol 1,4,5-triphosphate (IP3), thereby triggering intracellular calcium release from the endoplasmic reticulum [2+/calmodulin-dependent protein kinase II (CAMKII), which increase expression of nuclear factor of activated T cells (NFAT)-dependent genes and inhibit canonical WNT signaling through nemo-like kinase (NLK), respectively [In the WNT/Ca2eticulum ,113. Calectively ,115. Actectively ,117,118.ectively . In contectively ,60. In aectively ,121. Thiectively .In both human and rodents, the prostate gland surrounds the urethra at the base of the bladder and functions by contributing secretory proteins to the seminal fluid. In men, the prostate gland is a walnut-sized tissue with a zonal architecture, corresponding to central, periurethral transition, and peripheral zones, together with an anterior fibromuscular stroma . ImportaIn all species, formation of the prostate gland initiates during embryogenesis. During mid-gestation, the primitive urogenital sinus (UGS) is separated from the terminal region of the hindgut through division of the cloaca by the urorectal septum. The most rostral region  of the primitive UGS forms the urinary bladder, whereas the most caudal region  forms the penile urethra. The prostate gland originates from a sub-compartment of the lower urogenital tract (LUT), known as the definitive UGS ,128. TheSfrp2, Wnt4, Wnt5a, Wnt11, Fzd1, Fzd7, Fzd10, Lrp5, Axin1, Lef1, Nkd1, and RhoA [Sfrp1-overexpressing transgenic mice and Sfrp1-null mice confirmed that this WNT modulator stimulates prostate branching morphogenesis, epithelial cell proliferation and secretory gene expression [Wnt5a was found to be indispensable during the UGS development. High levels of Wnt5a expression has been observed at the distal tips and along the centro-distal periductal mesenchyme during the period of postnatal branching morphogenesis, with a rapid decline thereafter in the VP but not the DP and LP [Wnt5a impeded buds branching during morphogenesis [The use of transgenic mice combined with molecular analyses have demonstrated the importance of several developmental signaling pathways during prostate organogenesis, including bone morphogenetic protein (BMP), transforming growth factor beta (TGFβ), Notch, sonic hedgehog (SHH), and WNT pathways . Evidencand RhoA ,149. Accpression . Additiopression . This wapression . In addiP and LP . Anotherogenesis .Ctnnb1) in the mouse prostate during embryonic stages results in significantly decreased prostatic budding and abrogates prostatic development [R-spondin1-4 (Rspo1-4) in developing and neonatal mouse LUT. They found that Rspo3, together with Wnt4, Wnt10b, Wnt11 and Wnt16, appear to be more abundant in male versus female UGS and they stimulate prostatic development [β-Catenin has been identified in both epithelial and mesenchymal structures that undergo a budding program; its activation is necessary and/or sufficient for specification of hair follicle, mammary gland and tooth buds ,155,156.elopment . Furtherelopment . By in selopment .+ cells [+CD133+ basal cells also correspond to an enriched stem cell fraction in the human prostate epithelium [−Sca-1+CD133+CD44+CD117+ cells, which are predominantly basal in mice and are exclusively basal in humans [Nkx3-1 expressing cells (CARNs)) are bipotential and can self-renew in vivo [High cells, enriched in adult PSCs; (iii) Sca-1Low cells, that represent transit-amplifying cells; and (iv) Sca-1Negative cells representing terminally differentiated population with no regenerative potential [+ human cord blood-derived cells [−Sca−CD49fhigh mouse prostate stem and progenitor cells has been provided in the study by Lukacs et al. [Although development of the adult prostate is largely completed at puberty, it must possess a mechanism to assure the homeostasis of its epithelium. To achieve this, prostate, similar to other epithelial organs, sets aside a life-long reservoir of somatic stem cells that retain self-renewal. The regenerative capacity of prostate epithelial stem cells (PSCs) has been shown in the experiment with repeated rounds of androgen ablation and restoration; thus PSCs are androgen-sensitive but not dependent, are capable of self-regeneration, and give rise to transit-amplifying cells that differentiate into various specialized epithelial cells of the prostate . To date+ cells ,166,167.ithelium . Finallyn humans . In addi in vivo . Neverth in vivo ,171. To  in vivo . By usin in vivo . Of noteotential . Upregulotential . In anototential . Similarotential . Interesotential . This ised cells  and it sed cells . Furthers et al. . This grs et al. .The most critical molecular component of the androgen signaling pathway is the AR protein. Upon activation by androgens, AR mediates transcription of target genes that modulate growth and differentiation of prostate epithelial cells. AR plays a vital role in the development of male reproductive organs. Of note, its dysregulation contributes to the male pattern of baldness, development of prostatic hyperplasia, and later in life to PCa.AR gene is located on chromosome Xq11-12. It consists eight exons that encode an 110 kDa nuclear receptor that is a unique member of the nuclear steroid receptor gene family , Hsp40 (Ydj1), Hop (p60), Hsp90 and p23. The main role of this complex is to maintain AR in a conformation capable of ligand binding and to protect it from proteolysis ,187,188.Regulation of the AR activity occurs, in part, by posttranslational modifications, such as phosphorylation at several serine residues with or without a bound ligand . PreciseAR found in PCa, important recent studies have shown that AR can drive expression of oncogenes such as those encoding the ETS transcription factors  as a consequence of gene rearrangements [TMPRSS2-ERG gene fusion, resulting in expression of an N-terminally truncated ERG protein under the control of the androgen-responsive promoter of TMPRSS2  that miss the C-terminal LBD were detected in CRPCs [Prostate gland development and PCa are critically dependent on AR signaling. The ADT remains the most widely used treatment for patients with advanced PCa. In fact, androgen deprivation causes reduced AR expression, apoptosis and decreased tumor cell volume; however most PCas eventually develop the capacity for recurrent growth in the absence of testicular androgen  ,209,210.isoforms B. Anothe Xq11-12 ,220,221. tissues . This amy number . Therefoin CRPCs ,223,224.in CRPCs . Interesin CRPCs . Furtherin CRPCs . Of notein CRPCs .loss-of-function mutations in the tumor suppressor APC gene [APC or CTNNB1 (β-catenin) genes, which lead to constitutive activation of WNT signaling, similar to those found in colon cancer, have also been identified [The paradigm that PCa development and emergence of therapy resistance are a consequence of the restoration of embryonic developmental programs  has shed a new light on understanding the molecular mechanisms underlying epithelial invasion in prostate development and development of CRPC. While the (aberrant) AR signaling pathway is considered as the most critical player in CRPCs, as intracellular signaling pathways are often interconnected, other pathways, in particular, the WNT pathway, can also play key roles. As noted in the previous section, considerable evidence indicates that the WNT pathway plays a central role in the development of prostate tissues, by providing developmental growth inductive signals during embryonic/neonatal organogenesis. In PCa, studies by Schaeffer et al. have reported that androgen exposure regulates genes previously implicated in prostate carcinogenesis; these genes included those related to developmental pathways, such as WNT signaling, along with cellular programs regulating such “hallmarks” of cancer as angiogenesis, apoptosis, migration and proliferation . This obAPC gene ,231,232.entified ,235,236.+ PCa cell lines CWR22-Rv1 and LAPC-4 enhanced AR-mediated transcription of its target genes, in an androgen-dependent manner [AR gene expression via the TCF/LEF-1 binding sites in the AR promoter [Accumulating evidence has supported that the WNT/β-catenin pathway plays an important role in CRPC, by interacting with AR signaling ,238,239.t manner . Hence, t manner . Furtherpromoter . Thus, iIn the other hand, the effect of AR signaling on WNT/β-catenin signaling is more complicated. Early studies in gonadotropin-releasing hormone neuronal cells showed that in the presence of DHT, liganded AR repressed β-catenin/TCF-responsive reporter gene activity . In andrAR gene, as well as in genes encoding the main components of the WNT/β-catenin pathway, such as APC, β-catenin and R-spondins, leading to overactivation of WNT/β-catenin signaling [To add another layer of the complexity of interaction between AR and WNT/β-catenin signaling, it was shown that when PCa cells had been adapted to the low androgen environment , β-catenin could act as a co-activator of AR as well to enhance AR transcriptional activity in the presence of androstenedione, a weaker adrenal androgen remaining present in CRPC patients ,242,243.ignaling .WNT5A and WNT7B. Similarly, WNT5A or WNT7B mRNA was detected by RNA-ISH in a subset of CTCs from patients with CRPC [As described in the previous section, WNT ligands are highly conserved secreted molecules that play critical but pleiotropic roles in cell-cell signaling during embryogenesis. Interestingly, expression levels of several WNT ligands were found to be up- or down-regulated in advanced PCa. For instance, Chen et al. demonstrated that high levels of WNT1 and β-catenin expression were associated with advanced, metastatic, hormone-refractory prostate carcinoma, in which they could serve as markers for disease progression . In two ith CRPC . This deith CRPC . Interesith CRPC .WIF1 mRNA appears to be downregulated in a considerable percentage of PCa samples [Secreted WNT antagonists, including the sFRP family, DKK family, and Wnt inhibitory factor-1 (WIF1), are negative modulators of WNT signaling ,254,255. samples . Interes samples ,259. The samples ,255. Alt samples ,261. Alt+/CD133+ CSC-like subpopulation in these PCa cell lines. Previous studies have shown that PCa cells with these markers exhibited tumor-initiating and metastasis-initiation cell properties, although it was not absolutely clear whether the ALDH+/CD133+ subpopulation represented CSCs definitively [+/CD24− populations. Interestingly, they reported that it was the WNT/β-catenin signaling pathway that mediated upregulation of CD44 by DAB2IP knockdown. In this setting, CD44 not only served as a marker for CSCs, but also played a key role in facilitating the onset of prostate CSCs and increasing their chemoresistance [Cancer stem cells (CSCs) have been proposed to contribute to therapy resistance and cancer recurrence . In addinitively ,264,265.nitively , it was sistance . Importasistance . In anotsistance . By usinsistance . Similarsistance . Overallsistance .As to the non-canonical WNT pathway, the most recent clinical studies by Miyamoto et al. have shown that activation of this pathway in CTCs from patients with metastatic CRPC correlates with reduced effectiveness of antiandrogen treatment . In partTaken together, WNT signaling interacts with AR signaling using distinct mechanisms at different stages of PCa progression. In hormone-naïve PCa cells, WNT/β-catenin signaling promotes transcription of AR target genes, whereas AR signaling inhibits the transcription of WNT/β-catenin target genes A. HoweveWNT signaling plays key roles in both mammary gland development and breast cancer (BCa), largely through regulating mammary stem cell maintenance and basal mammary epithelial cell fate determination. An excellent review for this topic was published in this journal recently . As to tUnlike PCa, our understanding of AR signaling in BCa is still at its infancy. Some studies report that overexpression of AR is associated with better outcomes in BCa, while others illustrate a positive correlation of circulating androgens with high risk, recurrence and metastasis of BCa ,278,279.In breast tissues, androgen can be converted to DHT, which subsequently activates AR. The liganded AR direct or indirectly (possibly together with distinct co-regulators under different ERα settings) interacts with either ARE or ERE in its target genes . In the +/PR+/HER2−, ER+/PR+/HER2+, ER−/PR−/HER2+, and ER−/PR−/HER2− . Relating to the ER status, AR likely plays distinct roles in BCa in a subtype-specific manner.BCa is often classified clinically into four subtypes based on expression of ER, progesterone receptor (PR), and human epidermal growth factor receptor 2 : ER+, ER−/HER2+, and TNBC subtypes, respectively [Positive expression of AR was clinically defined as immunohistochemical (IHC) nuclear staining ≥1% or ≥10% according to various studies ,291,292.ectively ,297,298.n = 53) with testosterone therapy in patients with metastatic ER+ BCa [+ BCa, but this study is still at the beginning stage [Modulation of AR signaling, either inhibitory or stimulatory, exhibits somewhat contradictory observations in different subtypes of BCa, particularly when interacting with ER signaling ,299. Whe ER+ BCa . Androgeng stage ,305. Recng stage ,306,307.+ BCa with a frequency of ~70%–95% [+ BCa in a treatment-dependent manner: some studies indicated that higher AR expression is associated with better therapy outcomes, whereas others have reported that AR plays an oncogenic role in tamoxifen-resistant subjects [+ BCa initially, due to its ability to antagonize the growth-promoting role of ER signaling [+/AR+ BCas [AR is highly expressed in ER~70%–95% ,298,308.~70%–95% . AR and ~70%–95% ,310. AR subjects ,313,314.ignaling . AccordiAR+ BCas . But comAR+ BCas .− BCa and the functional crosstalk between AR and HER2 is critical for the tumor cell survival and expansion [AR transcription in a positive feedback loop [WNT7B, which activates β-catenin, resulting in HER3 transcriptional activation [AR is highly expressed in ERxpansion ,297,315.xpansion ,280. Mecack loop ,317. AR tivation , clinicativation .− breast tumors that were also AR+, was defined based on microarray expression profiling [+ TNBC was reported recently, in which a patient with this BCa subtype had progressive disease following six cycles of cytotoxic chemotherapy, but attained a 100% response to bicalutamide (an antiandrogen) [+ TNBC are being developed [The frequency of AR expression in TNBC is around 10% to 53% ,296,298.rofiling . Later orofiling . AR signrofiling ,321. Therofiling . An encondrogen) . With theveloped ,321.WNT1 is oncogenic for BCa [−/HER2+ BCa subtype. Using gene set enrichment analysis (GSEA), Ni et al. observed that androgen (DHT)-stimulated genes in ER−/HER2+ BCa cells were mainly those involved in WNT signaling [WNT7B transcription in a ligand-dependent manner. WNT7B is a canonical WNT ligand and may play roles in the normal mammary gland development during the stages of ductal formation and involution [WNT7B has been found in ~10% of BCa cases [−/HER2+ BCa cells by upregulating HER3, which encodes a key co-receptor of HER2 in HER2+ BCa [−/HER2+ breast tumor cells in vivo was inhibited [Overexpression of WNT induces aberrant activities of the WNT signaling pathway, which is a main driving force in BCa progression ,324. WNT for BCa . The intignaling . Furthervolution ,327. EleCa cases . In addiER2+ BCa . Importanhibited .+ BCa) and hormone-refractory cancers . Accordingly, both AR agonists and AR (and/or WNT) antagonists may be beneficial for BCa therapy, but in a BCa subtype and therapy stage-dependent manner. In particular, as both the AR and WNT signaling pathways drive progression and maintenance of AR+ TNBCs, inhibitors for these two pathways may prove to be useful for targeting this TNBC subtype. In addition, AR antagonists and anti-HER2 agents may also be used in combination to treat ER−/HER2+ BCa with AR expression, and inhibitors for WNT signaling may offer another therapeutic opportunity, particularly when ER−/HER2+ BCa cells develop resistance to the anti-HER2/AR agents.Thus, in both PCa and BCa, AR signaling appears to regulate distinct sets of target genes in hormone-dependent cancers  are refractory or unresponsive to hormone therapy, a better understanding of roles of AR and WNT pathways and their interactions in these hormone-refractory diseases should open a new avenue for improving their treatment and for combating the inevitable challenge of therapy resistance.As two key pathways regulating both normal development and tumorigenesis in hormone-responsive prostate and mammary glands, the context-dependent interplay of AR and WNT signaling pathways provides a unique opportunity to explore therapeutic options for treating prostate and breast cancers, particularly when under the setting of therapeutic resistance. As both CRPCs and ER


Whether α1-ARs also regulate chemokine receptor function within such heteromeric receptor complexes is unknown. We observed that activation of α1b-AR within the α1b-AR:CXCR4 heteromeric complex leads to cross-recruitment of β-arrestin2 to CXCR4, which could not be inhibited with AMD3100. Activation of CXCR4 did not cross-recruit β-arrestin2 to α1b-AR. A peptide analogue of transmembrane domain 2 of CXCR4 interfered with α1b-AR:CXCR4 heteromerization and inhibited α1b-AR-mediated β-arrestin2 cross-recruitment. Phenylephrine (PE) induced internalization of CXCR4 in HEK293 cells co-expressing CXCR4 and α1b-AR and of endogenous CXCR4 in human vascular smooth muscle cells (hVSMC). The latter was detectable despite blockade of CXCR4 with the neutralizing antibody 12G5. hVSMC migrated towards CXCL12 and PE, but not towards a combination of CXCL12 and PE. PE inhibited CXCL12-induced chemotaxis of hVSMC (IC50: 77 ± 30 nM). Phentolamine cross-inhibited CXCL12-induced chemotaxis of hVSMC, whereas AMD3100 did not cross-inhibit PE-induced chemotaxis. These data provide evidence for asymmetrical cross-regulation of CXCR4 by α1-adrenergic receptors within the heteromeric receptor complex. Our findings provide mechanistic insights into the function of α1-AR:CXCR4 heteromers and suggest alternative approaches to modulate CXCR4 in disease conditions.Recently, we reported that chemokine (C-X-C motif) receptor (CXCR)4 and atypical chemokine receptor 3 regulate α Based on the involvement of CXCR4 in various disease processes, such as cancer metastases, HIV infection or inflammatory diseases, multiple drugs targeting CXCR4 are currently under development and the CXCR4 antagonist AMD3100 is already approved by the Federal Drug Administration to mobilize hematopoietic stem cells in cancer patients5. CXCR4 is a typical G protein-coupled receptor (GPCR)1. Upon binding to its cognate ligand CXCL12, CXCR4 couples to guanine nucleotide-binding protein αi (Gαi) and also recruits β-arrestin 1/2 to the receptor, leading to termination of G protein-mediated signaling, receptor internalization and activation of G protein-independent signaling pathways6. Several lines of evidence suggest that CXCR4 can form hetero-oligomeric complexes with other GPCRs, such as chemokine (C-C motif) receptor (CCR)2, CCR5, CXCR3, atypical chemokine receptor (ACKR) 3, chemerin receptor 23, β2-adrenergic receptor (AR), δ-opioid receptor or cannabinoid receptor 2, which is thought to alter the pharmacological properties of the interacting receptor partners14. Recently, we provided evidence that hetero-oligomeric complexes between CXCR4, ACKR3 and α1-AR are constitutively expressed on the cell surface of human vascular smooth muscle cells (hVSMC), which appear to be essential for normal α1-AR function and through which the chemokine receptors upon agonist stimulation diametrically regulate α1-AR signaling and function17. The effects of such hetero-oligomerization on CXCR4-mediated signaling and function, however, remain unknown. Here, we provide evidence for asymmetrical ligand-induced cross-regulation of CXCR4 by α1-AR within heteromeric CXCR4:α1-AR complexes in hVSMC.Chemokine (C-X-C motif) receptor (CXCR) 4 fulfills pleiotropic roles in the immune system and is regarded as an important regulator of directed cell migration1b/d-AR form heteromeric complexes, we co-transfected HEK293T cells with plasmids encoding human influenza hemagglutinin (HA)-tagged CXCR4 (HA-CXCR4) and with plasmids encoding FLAG-tagged α1b-AR (FLAG-α1b-AR) or FLAG-α1d-AR. We then performed proximity ligation assays (PLA) to visualize individual receptors and receptor-receptor interactions at single molecule resolution with anti-HA and anti-FLAG antibodies. Consistent with our previous observations17, we observed positive signals for interactions between HA-CXCR4 and FLAG-α1b/d-AR . As shown in Fig. 50 for PE-induced β-arrestin 2 recruitment to α1b-AR-Tango was 172 μM : 143–202 μM) and the top plateau 90 ± 2 relative luminescence units (RLU)% in cells transfected with α1b-AR/pcDNA3. In cells co-transfected with α1b-AR-Tango/CXCR4, potency and efficacy of PE to recruit β-arrestin 2 to α1b-AR were significantly reduced, as compared to cells co-transfected with α1b-AR/pcDNA3 (EC50: 475 (95%CI: 290–807) μM, p = 0.0001; top plateau: 23 ± 3 RLU, p < 0.0001). Because there were no differences in PE-induced β-arrestin 2 recruitment to α1d-AR in cells co-transfected with α1d-AR-Tango and pcDNA3 or CXCR4  218 (130–276) μM, top plateau: 110 ± 7 RLU%; α1b-AR-Tango/CXCR4-Tango: EC50 (95%CI) 22 (9–53) μM - p = 0.0027, top plateau: 178 ± 11 RLU% - p = 0.03)  Fig. .Figure 31b-AR recruits β-arrestin 2 to α1b-AR and CXCR4, we then expressed CXCR4-Tango in combination with α1b-AR or pcDNA, confirmed comparable CXCR4-Tango expression by flow cytometry  μM) was ∼10-fold higher than the potency of PE for β-arrestin 2 recruitment to α1b-AR-Tango co-transfected with pcDNA3 and ∼20-fold higher than the potency of PE for β-arrestin 2 recruitment to α1b-AR-Tango co-transfected with CXCR4. Similarly, the efficacy of PE to recruit β-arrestin 2 to CXCR4-Tango under these conditions was 2-fold higher than the efficacy of the cognate CXCR4 agonist CXCL12 . Thus, we tested whether the TM2 peptide analogue would also disrupt recombinant CXCR4:α1b-AR heteromeric complexes and affect PE-induced β-arrestin 2 cross-recruitment to CXCR4. We used a TM4 peptide analogue of CXCR4 as a control peptide because we observed previously that this peptide analogue did not disrupt heteromerization between CXCR4:α1B-AR17. As shown in Fig. 1b-AR interactions, as compared with vehicle-treated cells and cells treated with the TM4 peptide analogue. The TM4 peptide analogue did not significantly reduce PLA signals for CXCR4-Tango:α1b-AR interactions. In parallel experiments with cells co-expressing CXCR4-Tango and α1b-AR, we detected that the TM2 peptide reduced the potency of PE for β-arrestin 2 recruitment to CXCR4 3-fold, as compared with cells treated with vehicle (EC50 (95%CI): vehicle − 2228 μM; TM2 − 66 (47–93) μM, p < 0.001)   of GPCRs have previously been shown to interfere with receptor heteromerization and function and we reported that a peptide analogue of transmembrane domain 2 (TM2) of CXCR4 selectively interferes with CXCR4:α01) Fig. . In cont01) Fig. . While t01) Fig. . These f1b-AR would also result in the internalization of CXCR4 in cells co-expressing both recombinant receptors. After transfection with HA-CXCR4 plus pcDNA3 or α1b-AR, HEK293T cells were labeled with FITC-conjugated anti-HA at 4 °C, followed by stimulation with vehicle, CXCL12 or PE at room temperature. In cells co-expressing HA-CXCR4 and pcDNA3  and TM4 of CXCR4 were as previously described18, was generously provided by the laboratory of Dr. Bryan Roth and maintained in high glucose Dulbecco’s Modified’s Eagle Medium supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 µg/mL hygromycin B, and 2 µg/mL puromycin. All cells were cultured in a humidified environment at 37 °C, 5% CO2.Human vascular smooth muscle cells  and HEK 293 T cells (ATCC-CRL-11268) were purchased from American Type Culture Collection. hVSMC were cultured in vascular basal media (ATCC PCS-100-030) supplemented with the vascular smooth muscle growth kit (ATCC PCS-100-042), 100 U/mL penicillin, 100 μg/mL streptomycin. hVSMCs were used between passages 2–5. HEK 293 T cells were cultured in high-glucose Dulbecco’s Modified’s Eagle Medium containing 10 mg/mL sodium pyruvate, 2 mM L-glutamine, 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The HTLA cell line, a HEK293 cell line stably expressing a tTA-dependent luciferase reporter and a β-arrestin2-TEV fusion gene31. At least 10,000 cells/sample were recorded and analyzed with the FlowJo software (Tree Star).Flow cytometry after labeling cells with Phycoerythrin-conjugated anti-FLAG antibody , Alexa 647-conjugated anti-FLAG antibody , Alexa 488-conjugated anti-Myc antibody  or FITC-conjugated anti-HA antibody  was used to quantify recombinant receptor expression levels in HEK293T cells, as described16. To visualize individual receptors, slides were incubated with mouse anti-FLAG M2 , rabbit anti-HA , rabbit anti-α1B-AR (Abcam Ab169523) or goat anti-CXCR4 (Abcam Ab1670) at 37 °C for 105 min in a humidifying chamber at 37 °C. Anti-α1B-AR and anti-CXCR4 have been validated for sufficient selectivity previously31. Slides were then washed and incubated  with secondary species specific (rabbit/mouse/goat) antibodies conjugated with plus and minus Duolink II PLA probes (1:5), as appropriate. To visualize receptor-receptor interactions, slides were incubated with a combination of rabbit anti-α1B-AR (Abcam Ab169523) and goat anti-CXCR4 (Abcam Ab1670) or a combination of mouse anti-FLAG M2 and rabbit anti-HA. All antibodies, except anti-HA (dilution 1:1000), were used in dilutions of 1:500. Slides were then washed and incubated with secondary species specific antibodies conjugated with plus and minus Duolink II PLA probes (1:5) corresponding to the two primary antibodies. PLA signals were quantified using the Duolink Image Tool software (Sigma Aldrich). Comparisons and statistical analyses were performed only when PLA assays were performed on the same day in parallel experiments and fluorescence microscopy was performed with the identical settings. For each experiment and condition, 10 randomly selected non-overlapping vision fields were analyzed.PLA were performed as described in detail previously1b-AR-TANGO, #66214; α1d-TANGO, #66215) were from Addgene deposited by the laboratory of Dr. Bryan Roth. Myc-tagged TANGO plasmids were generated by replacing the original FLAG-tag plasmids with Myc tag after PCR amplification with primers carrying the Myc tag sequence. HA- or FLAG-tagged CXCR4, α1b-AR or α1d-AR were generated by PCR amplification using corresponding TANGO plasmids as cDNAs with primers carrying Xho I and Xba I sites and inserted in pcDNA3 with an N-terminal FLAG or HA tag. All plasmids were verified by sequencing. HEK293T cells were transiently transfected with a single DNA or two DNAs that encode HA- or FLAG-tagged CXCR4, α1b-AR or α1d-AR or TANGO plasmids at the amounts indicated, using Lipofectamine 3000 (Thermo Scientific) as per manufacturer’s protocol. For single GPCR-encoding DNA transfection, empty vector pcDNA3 was added to maintain the total DNA amount per transfection constant.FLAG-tagged Tango plasmids  were seeded in a 6-well plate and transfected with 750 ng of each of the Tango plasmids using Lipofectamine 3000 (Thermo Scientific). The following day, transfected HTLA cells  were plated onto Poly-L-Lysine pre-coated 96-well microplates and allowed to attach to the plate surface for at least 4 hours prior to treatment. Cells were treated with receptor agonists for 2 h and then replaced with fresh full medium and incubated overnight at 37 °C, 5% CO2 in a humidified environment. To test the effects of TM peptides (50 μM), AMD3100 (10 μM) or phentolamine (100 μM), cells were pre-incubated with these reagents for 15 min at 37 °C before adding agonists. The following morning, medium was removed from cell culture plates and replaced with a 100 µL 1:5 mixture of Bright-Glo (Promega) and 1× HBSS, 20 mM HEPES solution. Plates were then incubated at room temperature for 20 min before measuring luminescence on a Biotek Synergy II plate reader. Relative luminescence (RLU) is expressed as % of luminescence upon stimulation with the highest concentration of the corresponding receptor agonist when the Tango receptor was co-expressed with pcDNA3 (=100%). When HTLA cells were not transfected with a Tango plasmid, no change in luminescence was detectable upon agonist treatment  assay was performed as recently described1b-AR at 375 ng each DNA/well of a 12-well plate. After 24 hours, cells were re-plated in poly D-lysine coated 8-well chamber slides. After overnight incubation, cells were chilled on ice and labeled with FITC-conjugated anti-HA  for 1 h at 4 °C. Subsequently, labeled cells were washed with Opti-MEM twice and then incubated with CXCL12 (200 nM) or PE (200 μM) for 1 h at room temperature. Thereafter, cells were washed with PBS and fixed with 4% paraformaldehyde. hVSMC were plated in 8-well chamber slides. The next day, slides were cooled on ice, washed once with Opti-MEM and then incubated with FITC-conjugated anti-CXCR4 antibody  or rabbit anti-CXCR4 antibody  at 4 °C for 1 h. Subsequently, slides were washed twice and then treated with CXCL12 (100 nM) or PE (10 μM) in Opti-MEM at 37 °C for 20 min. Cells were fixed with 4% paraformaldehyde/PBS at room temperature for 10 min. After three washes with PBS, cells incubated with non-conjugated anti-CXCR4 were permeabilized with 0.2% Triton X-100/PBS at room temperature for 10 min and then blocked with 1% BSA/PBS for 30 min, followed by incubation with Alexa 488-conjugated goat anti-rabbit antibody (1:250) at room temperature for 1 h. HEK293T cells and hVSMC Cells were mounted with mounting medium for fluorescence microscopy. Images were obtained under a florescence microscope (Carl Zeiss Axiovert 200 M) with a 40x objective lens equipped with Axio CamMRc5. For each well, 10 vision fields were imaged.Receptor internalization assays were performed with HEK293T cells and hVSMC. HEK293T cells were transfected with HA-CXCR4 plus pcDNA3 or FLAG- α32. The chemotactic index (CI) was calculated as the ratio of cells that transmigrated through the filter in the presence versus the absence (=PBS/control) of the test solutions.Cell migration was assessed using the ChemoTx 96-well cell migration system, as describedCell viability was assessed using the Trypan blue exclusion method. HTLA cells were incubated with various concentrations of phentolamine under the same conditions used in Presto-Tango assays. After staining cells with Trypan blue, at least 100 cells per condition and experiment were examined in a hemocytometer and the percentage of viable cells quantified.Data are expressed as mean ± standard error of the mean (SEM) from n independent experiments that were performed on different days. Data were analyzed using the GraphPad-Prism 7 software. Unpaired Student’s t test or one-way analyses of variance (ANOVA) with Dunnett’s multiple comparison post-hoc test for multiple comparisons were used, as appropriate. Dose response curves were generated using non-linear regression analyses. A two-tailed p < 0.05 was considered significant.The datasets generated during the current study are available from the corresponding author on reasonable request.Supplementary Information


The aims of this study were (1) to characterise the type of patient prescribed MP-AzeFlu  in real life in the UK and physicians' reasons for prescribing it and (2) to quantify the personal and societal burden of allergic rhinitis (AR) in the UK prior to MP-AzeFlu prescription.This multicentre, non-interventional study enrolled patients (n=193) with moderate-to-severe AR and acute symptoms who were eligible to receive treatment with MP-AzeFlu according to its licensed indications. Information was gathered on patient demographics, AR history and symptom severity, symptomatology and AR treatments in the previous calendar year (prior to MP-AzeFlu prescription). Physicians also recorded the number of previous AR visits, specific reasons for these visits and their reason for prescribing MP-AzeFlu.Most patients had seasonal AR either alone (10.4%) or in combination with perennial AR (35.2%), but many had AR of unknown origin (35.8%). Prior to MP-AzeFlu prescription, patients reported troublesome symptoms (78.2%) and sleep disturbance (64.8%), with congestion considered the most bothersome (54.4%) and ocular symptoms reported by 68.4% of patients. The most frequent reason for MP-AzeFlu prescription was that other therapies were not sufficient in the past (78.8%) or not sufficient to treat acute symptoms (16.1%). 79.3% of patients reported using ≥2 AR therapies in the past year. An average of 1.6 (SD 1.9) doctor visits due to AR were reported prior to MP-AzeFlu prescription.In the UK, MP-AzeFlu was prescribed for individuals (≥12 years) with moderate/severe AR irrespective of (1) previous AR treatment (mono or multiple), (2) previous or likely treatment failure, (3) phenotype, (4) number of previous physician visits for AR and (5) for the relief of both acute symptoms and in anticipation of allergen exposure. Sufficiently large patient numbers to draw general conclusion on use of MP-AzeFlu in routine clinical practice in the UK.Low amount of missing data.Real-life data obtained from daily practice highlighting the burden of allergic rhinitis in the UK and the type of patient prescribed MP-AzeFlu.Relates type of patient prescribed MP-AzeFlu with the labelled indication, thus providing sound estimates for relevant subgroups from a socioeconomic perspective.For such single-arm epidemiological research, standard bias limiting methods  could not be applied.One in four individuals in the UK has allergic rhinitis (AR),10The burden of AR and the importance of achieving disease control has now been recognised both by the European Academy of Allergy and Clinical Immunology14MP-AzeFlu  comprises a novel formulation containing an intranasal antihistamine (azelastine hydrochloride), an intranasal corticosteroid (INS), fluticasone propionate (FP) and excipients delivered in a single spray. It has been approved for use in the UK since January 2013, indicated for the relief of symptoms of moderate-to-severe seasonal AR (SAR) and perennial AR (PAR) if monotherapy with either intranasal antihistamine or glucocorticoid is not considered sufficient.17Characteristics of patients who received MP-AzeFlu in an RCT setting are well established, due to strict inclusion/exclusion criteria applied to ensure a homogeneous patient population and low external variability.17This was a multicentre, non-interventional study conducted in the UK between October 2013 and May 2014, as part of a larger Europe-wide study of similar design. During the study, patients were treated with MP-AzeFlu (one spray in each nostril twice daily). MP-AzeFlu is indicated for the relief of symptoms of moderate-to-severe SAR and PAR if monotherapy with either intranasal antihistamine or glucocorticoid is not considered sufficient.Patients were eligible for inclusion into the study if they were aged ≥12 years, had a documented diagnosis of moderate-to-severe SAR or PAR and for whom monotherapy with either intranasal antihistamine or glucocorticoid were not considered sufficient.Patients were excluded if they had hypersensitivity to azelastine hydrochloride, FP or any MP-AzeFlu excipients. There were no restrictions regarding concomitant treatments, apart from ritonavir, which was to be avoided. Female patients were excluded if they were pregnant or breast feeding. All patients provided written informed consent prior to their participation in the study. If patients were below 16 years old, their caregiver also provided written informed consent.Physicians who were usually involved in AR management participated in this study and included secondary care ear-nose-and-throat (ENT) specialists, immunologists and allergists. These physicians were identified by an independent agency . Each physician could enrol up to 20 patients. The decision whether to include a patient in the study was made by the physician independently from and after the decision to prescribe MP-AzeFlu had been made.Information on patient demographics, clinical symptoms and AR treatments in the previous calendar year (prior to MP-AzeFlu prescription) was documented by physicians. Physicians also recorded information on type of AR, number of visits in the current calendar year due to AR, predominant symptoms and ARIA-defined AR severity. The reason for the patient's visit  and the reason for prescribing MP-AzeFlu  was documented by the physician. All data were recorded by physicians in a prespecified English language electronic case report from  based on the investigators' patient files and on information obtained at the patient visit.This study included 193 patients with AR, which was deemed sufficient to provide insight into use of MP-AzeFlu in routine clinical practice in the UK. Analyses were conducted on the safety population, defined as all patients who were treated at least once with MP-AzeFlu and whose physician provided an electronic signature to confirm data accuracy. All data were reported with descriptive statistics. Categorical data were described in terms of frequency and as percentage of patients. Continuous data were described as mean and SD. Analyses were performed by the Contract Research Organisation, Syneed Medidata GmbH, with SAS V.9.1.3.The study was conducted in 15 study centres in the UK who enrolled 195 patients. Of these, 2 patients were excluded from data analysis due to unconfirmed data documentation, leaving 193 confirmed patients in the safety population.A summary of patient demographics and baseline clinical characteristics is presented in In the current calendar year, the mean (SD) number of physician visits due to AR was 1.6 (1.9). In total, 62.7% (n=121) of patients had visited their physician at least once before inclusion into the study in the current calendar year due to their AR ; with moMP-AzeFlu was prescribed according to its licensed indication. The most frequent reasons for the physician visit were ‘acute AR symptoms’ , ‘expected allergen exposure in the near future’  and ‘other’ . The most frequent reason for prescribing MP-AzeFlu was that ‘other therapies were not sufficient in the past’. For the remaining patients, other reasons were cited, including ‘other therapies were not considered sufficient to treat acute symptoms’ .The majority of patients had received or used at least one AR therapy, either with prescription or over the counter, prior to MP-AzeFlu prescription . The AR This study represents a valuable addition to the AR knowledge base for several reasons. First, it provides important epidemiological data on the type of patient with AR presenting to secondary care allergy clinics in the UK, as well as information on AR history and symptomatology of those who received MP-AzeFlu. Second, it confirms the enormous (and frequently underestimated)Physicians who took part in this non-interventional study  prescribed MP-AzeFlu to adolescents, adults and elderly patients with moderate/severe AR Although MACVIA ARIA clearly recommends MP-AzeFlu for the treatment of intermittent and persistent AR,22The results of this study also show that the most commonly prescribed AR treatments in the UK do not provide adequate symptom relief for many patients, further endorsing the need for MP-AzeFlu. Prescribing physicians considered that other therapies were not sufficient in the past for 66.4% of patients and would not provide adequate symptom relief for 34.5% of patients. Most patients prescribed MP-AzeFlu had previously been treated with INS and/or oral antihistamines. Inadequacy of previous AR treatments was also evident from the high symptomatic burden on treatment reported by patients, the high degree of polypharmacy noted (79.3%) and the high incidence of use of systemic corticosteroids (18.7%) and decongestants (45.6%). The former are rarely indicated for the management of rhinitis (except for severe obstruction or short-term rescue use),23Allergy Diary) to empower patients to take control of their own AR.The concept of achieving symptom relief  has been now superseded by the concept of achieving symptom control.The limitations of this study are those typically associated with non-interventional, observational data sets as the standard methods of blinding and randomisation cannot be applied. Thus, confounding can never safely be ruled out. Another limitation is that our study included no investigation of the effectiveness of MP-AzeFlu, and there was no direct head-to-head comparison of MP-AzeFlu with other AR treatments. However, the effectiveness of MP-AzeFlu has been demonstrated in real-life studies conducted in other countries.In conclusion, the symptomatic burden of AR remains high for many patients in the UK, who live with uncontrolled disease, despite treatment with monotherapy and multiple therapies and repeat doctor visits. In the UK, MP-AzeFlu was prescribed for individuals (≥12 years) with moderate/severe AR irrespective of (1) previous AR treatment (mono or multiple), (2) previous or likely treatment failure, (3) phenotype, (4) number of previous physician visits for AR and (5) for the relief of both acute symptoms and in anticipation of allergen exposure. A more effective treatment option, like MP-AzeFlu, with established superiority over INS and antihistamine monotherapy, should improve AR control.