The Prostate 76:491–511 (2016) NF-kB and Androgen Receptor Variant Expression Correlate With Human BPH Progression David C. Austin,1 Douglas W. Strand,2* Harold L. Love,2 Omar E. Franco,3 Alex Jang,2 Magdalena M. Grabowska,2 Nicole L. Miller,2 Omar Hameed,4 Peter E. Clark,2,5 Jay H. Fowke,2,6 Robert J. Matusik,1,5 Ren J. Jin,2 and Simon W. Hayward2,3* 1 Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, Tennessee Department of Urologic Surgery, Vanderbilt University Medical Center, Nashville, Tennessee 3 Department of Surgery, NorthShore University HealthSystem Research Institute, Evanston, Illinois 4 Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee 5 Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee 6 Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 2 BACKGROUND. Benign prostatic hyperplasia (BPH) is a common, chronic progressive disease. Inflammation is associated with prostatic enlargement and resistance to 5a-reductase inhibitor (5ARI) therapy. Activation of the nuclear factor-kappa B (NF-kB) pathway is linked to both inflammation and ligand-independent prostate cancer progression. METHODS. NF-kB activation and androgen receptor variant (AR-V) expression were quantified in transition zone tissue samples from patients with a wide range of AUASS from incidental BPH in patients treated for low grade, localized peripheral zone prostate cancer to advanced disease requiring surgical intervention. To further investigate these pathways, human prostatic stromal and epithelial cell lines were transduced with constitutively active or kinase dead forms of IKK2 to regulate canonical NF-kB activity. The effects on AR full length (AR-FL) and androgen-independent AR-V expression as well as cellular growth and differentiation were assessed. RESULTS. Canonical NF-kB signaling was found to be upregulated in late versus early stage BPH, and to be strongly associated with non-insulin dependent diabetes mellitus. Elevated expression of AR-variant 7 (AR-V7), but not other AR variants, was found in advanced BPH samples. Expression of AR-V7 significantly correlated with the patient AUASS and TRUS volume. Forced activation of canonical NF-kB in human prostatic epithelial and stromal cells resulted in elevated expression of both AR-FL and AR-V7, with concomitant ligand-independent activation of AR reporters. Activation of NF-kB and over expression of AR-V7 in human prostatic epithelial cells maintained cell viability in the face of 5ARI treatment. Grant sponsor: National Institutes of Health; Grant numbers: DK098277; R01-CA076142; R01-DK055748; DK103483; DK067049; DK097782; DK104280; Grant sponsor: National Cancer Institute; Grant number: P30 CA068485. Present address of Douglas W. Strand is Department of Urology, University of Texas Southwestern Medical Center, Dallas, TX. Disclosure Statement: The authors have nothing to disclose.
Correspondence to: Simon W. Hayward, Department of Surgery, NorthShore University HealthSystem Research Institute, 1001 University Place, Evanston, IL 60201. E-mail: shayward@northshore.org Received 26 June 2015; Accepted 1 December 2015 DOI 10.1002/pros.23140 Published online 28 December 2015 in Wiley Online Library (wileyonlinelibrary.com). ß 2015 Wiley Periodicals, Inc. 492 Austin et al. CONCLUSION. Activation of NF-kB and AR-V7 in the prostate is associated with increased disease severity. AR-V7 expression is inducible in human prostate cells by forced activation of NF-kB resulting in resistance to 5ARI treatment, suggesting a potential mechanism by which patients may become resistant to 5ARI therapy. Prostate 76:491–511, 2016. # 2015 Wiley Periodicals, Inc. KEY WORDS: NF-kB; Inflammation; BPH; androgen receptor; androgen receptor variant 7 INTRODUCTION Benign prostatic hyperplasia (BPH) is the most common urologic disease in men over the age of 50 [1]. Comorbidities of BPH include age, systemic inflammation, autoimmune and inflammatory disease, and individual components of metabolic syndrome [2–5]. There are many potential etiological factors contributing to BPH pathogenesis such as disruption of growth factor and hormone signaling, inflammation, fibrosis, and sympathetic nerve activity [6–9]. Consistent with concepts discussed by other investigators [10,11] we have recently demonstrated that gene expression profiles of advanced BPH show marked similarities with conditions such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease suggesting the possibility of an autoimmune/inflammatory component to the disease process [12]. There are two major medical approaches for patients presenting with symptoms suggestive of BPH: alpha-adrenergic receptor antagonists (ablockers) that decrease smooth muscle tone [13] and 5a-reductase inhibitors (5ARI) that reduce the enzymatic conversion of testosterone to dihydrotestosterone, resulting in apoptosis and a decrease of around 25% in total prostate volume [14]. Combination treatment with these therapies was shown to provide a significant reduction in the risk of symptomatic progression. However, nearly 20% of patients display serious adverse complications to these medications and many patients either fail to respond or become resistant over time, with 5–7% progressing to surgical intervention [15]. Given the age and comorbidity profile of this population, many of these patients are not good candidates for surgery [16]. The variability in clinical responses to existing BPH therapies highlights the need to better understand the molecular basis of BPH progression with a view to developing new therapies appropriately targeted to specific patient groups [17]. The eukaryotic nuclear factor-kappa B (NF-kB) transcription factor family regulates the expression of a large variety of genes involved in inflammatory and immune responses as well as cellular growth and development [18]. NF-kB transcription factors are activated as a response to a variety of stress signals, The Prostate including cytokines and pathogens. Activation of NF-kB proteins is tightly regulated, and inappropriate activation of these signaling pathways has been linked to autoimmunity, chronic inflammation, and various cancers [19,20]. NF-kB signaling occurs through canonical (p50/RelA) and non-canonical (p52/RelB) pathways resulting in activation of overlapping sets of downstream genes. The androgen receptor (AR) has been shown to be expressed in the form of C-terminal truncated variants (AR-V) in prostate cancer [21–27]. These AR-Vs lack the ligand-binding domain of full-length AR. AR-V can be constitutively active, driving ARregulated transcription and promoting tumor progression, even under castrate conditions [23,28–30]. Expression of AR variant 7 (AR-V7) in circulating prostate tumor cells has been shown to be predictive of resistance to enzalutamide (which interacts with the ligand binding domain of the AR) and abiraterone (which depletes androgen levels) [28]. Gao et al. have reported that non-canonical NF-kB signaling induces the expression of AR-V in prostate cancer [31]. We have reported that AR-Vs are also induced by canonical NF-kB signaling resulting in castrate-resistant prostate cancer (CRPC) [32]. Inhibition of NF-kB expression results in AR-V down-regulation and restores sensitivity of CRPC to anti-androgens [32]. Baseline prostate volume is the most reliable predictor of therapeutic failure of BPH and lower urinary tract symptom (LUTS) progression [33] and is most commonly targeted by 5ARI therapy; therefore, our goal is to understand the potential mechanisms of 5ARI resistance. Currently, there is not an established link between AR-V expression and resistance to 5ARI therapy in BPH. In this study, we investigated the activation of NF-kB and AR-V7 in BPH. We compared human tissue samples from patients who underwent surgery for moderate-to-severe BPH/LUTS to a cohort of patients with, mildly symptomatic BPH incidental to radical prostatectomy for prostate cancer. We utilized benign human prostate epithelial and stromal cells, to test the presence and consequences of NF-kB activation on AR and AR-V7. To our knowledge, this is the first study to link chronic activation of NF-kB NK-kB Stimulates Androgen Receptor Variant Expression signaling in BPH to increased AR-V7 expression. This provides the basis for a mechanism that could explain why certain patients with BPH fail 5ARI therapy. Our previous study with CRPC demonstrated that inhibition of NF-kB and AR-V7 expression restores responsiveness to medical therapy [34], suggesting that targeting both NF-kB and AR could have an impact in reducing failure of treatment for BPH. MATERIALS AND METHODS Cells, Reagents, and Antibodies BHPrS1 human prostate stromal cells [35] were cultured in RPMI 1640 (Gibco, Grand Island, NY), 5% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA), and 1% penicillin/streptomycin (Gibco). NHPrE1 prostate epithelial cells [36] were cultured in DMEM/F12 (Gibco) containing either 5% complete fetal bovine serum (FBS) or 5% dextran-coated charcoalstripped FBS (CS-FBS) (Atlanta Biologicals), 1% penicillin/streptomycin (Gibco), 10 ng/ml epidermal growth factor (Sigma–Aldrich, St. Louis, MO), 1% insulintransferrin-selenium (ITS) (Gibco), and 0.4% bovine pituitary extract (Atlanta Biologicals). Primary antibodies against p65, acetyl-p65 (K310) were purchased from Cell Signaling (Beverly, MA), AR (N-20 and C-19) from Santa Cruz Biotechnology (Santa Cruz, CA), AR-V7 from Precision Antibody (Western blotting) (Columbia, MD) and Abcam (IHC) (Cambridge, MA) and, and phospho-p65(S276) from Abcam. Secondary antibodies were purchased from GE Healthcare (Pittsburg, PA). Matrigel Culture Single cell suspensions of NHPrE1 cells in monolayer culture medium containing 2% growth factor reduced Matrigel (BD Bioscience, Oxford, UK) were seeded at 2,000 cells/well in 48 well plate containing 200 ml of medium containing 4% growth factor reduced Matrigel, 5% charcoal- stripped FBS (CS-FBS in DMEM/F12 medium with or without DHT (108 M). Cells were incubated at 37°C with replacement of the growth medium containing 4% growth factor reduced Matrigel with/without DHT at day 3. Medium was then changed every 2 days. NHPrE1 cells were extracted from the Matrigel using cell recovery solution (BD Biosciences). Extracted NHPrE1 cells were then dissociated to a single cell suspension using enzymatic disaggregation (0.25% Trypsin EDTA, Sigma). 493 complementary DNA inserts of IKK2-Empty Vector (EV), IKK2-constitutively active (EE), IKK2-kinase dead (KD) (gifts from Dr. Timothy Blackwell, Vanderbilt), AR-FL and V7 (gift from Dr. Ganesh Raj, University of Texas Southwestern) were used for infection of NHPrE1 and BHPrS1 prostatic epithelial and stromal cells as previously described [37–39]. Briefly, the amphotropic producer cell line fNX was transfected with 10 mg of the retroviral vectors by calcium phosphate precipitation. To select transfected producer cells, 0.5 mg/ml zeocin (IKK2) (Life Technologies, Carlsbad, CA) or blasticidin (AR-FL, V7) (Life Technologies) was added to the culture medium for 3 days to obtain >95% green fluorescent protein-positive producer cells. Cell culture supernatants containing viral particles were generated by incubation of producer cells with DMEM containing 10% FBS overnight. Following filtration at 45 mm (Millipore Billerica, MA), culture supernatant was added to NHPrE1 or BHPrS1 cells seeded in six-well plates 24 hr earlier in the presence of 1 mg/ml polybrene. After 5 days recovery of bulk-infected cultures, FACS analysis for green fluorescent protein expression and western blot analysis was performed on expanded polyclonal cells to confirm ectopic expression of the respective molecules. Cell lines used were the IKK2-empty vector (NHPrE1-EV and BHPrS1-EV), IKK2-kinase dead (NHPrE1-KD andBHPrS1-KD), or IKK2-constitutively active, (NHPrE1-EE and BHPrS1-EE). Transient Transfection Assay The NGL vector (a NF-kB responsive reporter vector that has luciferase and green fluorescent protein (GFP) reporter genes [40]) was a gift from Dr. Timothy Blackwell (Vanderbilt), ARR2PB-Luc vector (an AR-responsive reporter vector that does not respond directly to NF-kB [41]), and pGL3-PSA-PSAE1-Luc reporters [42] along with p65 and AR siRNA purchased from Cell Signaling, Life Technologies, and Santa Cruz Biotechnology, respectively. The transfection efficiency was determined by co-transfecting pRL-CMV containing the Renilla luciferase reporter gene (Promega, Madison, WI). Luciferase activity was determined using the Promega Corp luciferase assay system 24 hr after transfection. The values plotted represent the mean of at least three individual samples  SD. Cell Viability Assay Viral Transduction The pCFG5-IEGZ retroviral vector, gift from Martin Leverkus (Heidelberg, Germany) containing Cell viability assay was performed using CellTiterGlo (Promega) according to manufacturer’s instructions. NHPrE1 cells were seeded at 2,000 cells/well in a 96-well plate, grown over 5 and 7 days in the The Prostate 494 Austin et al. presence and absence of testosterone and Finasteride. CellTiter-Glo measurements were taken at several time points to track cell survival. holmium laser enucleation of the transition zone of the prostate (HoLEP) for symptomatic BPH with LUTS, referred to here as “Surgical BPH.” In the majority of cases, these patients had failed medical therapy with an a-blocker, a 5ARI, or both (see Table I). As a control, a pathologist resected transition zone nodules from 53 patients undergoing radical prostatectomy for small volume, low risk, clinically localized peripheral zone prostate cancer (referred to here as “Incidental BPH”) at Vanderbilt University Medical Center (Nashville, TN) as previously described [12]. Low risk prostate cancer was defined as clinical stage T1c, pathologic stage T2a, pre-operative PSA
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