Gnrh Antagonists Have Direct Inhibitory Effects on Castration-Resistant Prostate Cancer Via Intracrine Androgen and AR-V7 Expression Vito Cucchiara1, Joy C
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Published OnlineFirst July 24, 2019; DOI: 10.1158/1535-7163.MCT-18-1337 Small Molecule Therapeutics Molecular Cancer Therapeutics GnRH Antagonists Have Direct Inhibitory Effects On Castration-Resistant Prostate Cancer Via Intracrine Androgen and AR-V7 Expression Vito Cucchiara1, Joy C. Yang1, Chengfei Liu1, Hans H. Adomat2, Emma S. Tomlinson Guns2, Martin E. Gleave2, Allen C. Gao1,3, and Christopher P. Evans1,3 Abstract Hormone therapy is currently the mainstay in the manage- degarelix, leuprolide, or buffer alone for 4 weeks. Leuprolide ment of locally advanced and metastatic prostate cancer. slightly suppressed tumor growth compared with the vehicle Degarelix (Firmagon), a gonadotropin-releasing hormone control group (P > 0.05). Tumors in degarelix-treated mice (GnRH) receptor antagonist differs from luteinizing hor- were 67% of those in the leuprolide-treatment group but mone-releasing hormone (LHRH) agonists by avoiding "tes- 170% larger than in surgically castrated ones. Measurements tosterone flare" and lower follicle-stimulating hormone (FSH) of intratumoral steroids in serum, tumor samples, or treated levels. The direct effect of degarelix and leuprolide on human cell pellets by LC/MS confirmed that degarelix better decreased prostate cancer cells was evaluated. In LNCaP, C4-2BMDVR, the levels of testosterone and steroidogenesis pathway inter- and CWR22Rv1 cells, degarelix significantly reduced cell via- mediates, comparable to surgical castration, whereas leupro- bility compared with the controls (P 0.01). Leuprolide was lide had no inhibitory effect. Collectively, our results suggested stimulatory in the same cell lines. In C4-2B MDVR cells, a selective mechanism of action of degarelix against androgen degarelix alone or combined with abiraterone or enzaluta- steroidogenesis and AR-variants. This study provides addition- mide reduced the AR-V7 protein expression compared with al molecular insights regarding the mechanism of degarelix the control group. SCID mice bearing VCaP xenograft tumors compared with GnRH agonist therapy, which may have clin- were divided into 4 groups and treated with surgical castration, ical implications. Introduction one surge") are the most commonly used agents. GnRH agonists, after a desensitization of the GnRH receptor response, determine a Prostate cancer is the most common tumor and the second reduction in luteinizing hormone (LH), follicle-stimulating hor- cause of cancer death in men (1). For advanced and metastatic mone (FSH), and testosterone production (2, 3). GnRH antago- prostate cancer, androgen deprivation therapy (ADT) using lutei- nists, like degarelix, are also an approved form of ADT (4). GnRH nizing hormone-releasing hormone (LHRH) agonists or the antagonists, by blocking GnRH receptors, produce a more rapid gonadotropin-releasing hormone (GnRH) receptor antagonist, suppression of testosterone without testosterone surge or micro- alone or in combination with radiotherapy, is considered the best surge (4). Presently, international prostate cancer guidelines treatment option (2, 3). A new generation of androgen receptor recommend the use of either GnRH agonists or antagonists as signaling inhibitors, such as abiraterone or enzalutamide, have treatment options for ADT in patients with prostate cancer (2, 3). been approved as treatment options in castration-resistant pros- Although some studies have suggested differences in efficacy and tate cancer (CRPC; refs. 2, 3). Different ADT strategies have been disease-related outcomes (musculoskeletal and urinary events) tested to achieve castration levels of testosterone (<50 ng/dL). To with degarelix compared with LHRH agonists (4), a systematic date, long-acting depot formulations of GnRH agonists (in com- review of the literature did not support the superiority of antago- bination with antiandrogens for 4 weeks to avoid the "testoster- nists over agonists (5). All the available phase III studies included in the analysis have treatment bias, short-term follow-up and 1Department of Urologic Surgery, University of California at Davis, Sacramento, heterogeneous populations (5). California. 2Vancouver Prostate Centre, University of British Columbia, GnRH agonists and antagonists both induce castrate levels of Vancouver, British Columbia, Canada. 3UC Davis Comprehensive Cancer Center, testosterone by altering the intracellular signaling of pituitary University of California at Davis, Sacramento, California. cells, but several attempts have been made to elucidate the Note: Supplementary data for this article are available at Molecular Cancer effect of these agents on other cells that express GNRH Therapeutics Online (http://mct.aacrjournals.org/). receptor (GnRH-R; refs. 6, 7). These studies revealed that extra- V. Cucchiara and J.C. Yang contributed equally to this article. pituitary tissues are affected by compounds directed toward GnRH-R (8–11). In prostate cells, GnRH-R manipulation may Corresponding Author: Christopher P. Evans, University of California at Davis, influence several biological processes such as cell growth, apo- 4860 Y St., Suite 3500, Sacramento, CA 95817. Phone: 916-734-7520; Fax: 916- – 734-8904; E-mail: [email protected] ptosis, angiogenesis, and cell adhesion (8 11). We hypothesized that, unlike agonists, GnRH antagonists may Mol Cancer Ther 2019;18:1811–21 have a direct mechanism of action on prostate cancer cells growth, doi: 10.1158/1535-7163.MCT-18-1337 by affecting the AR signaling pathway. Specifically, we investigat- Ó2019 American Association for Cancer Research. ed the role of GnRH agonists and antagonists in castration www.aacrjournals.org 1811 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst July 24, 2019; DOI: 10.1158/1535-7163.MCT-18-1337 Cucchiara et al. sensitive and castration resistant prostate cancer cell lines and group was treated with 10 nmol/L of R1881, one cotransfected xenograft models and their possible interaction with AR and AR with an AR-V7 expression vector, the last with DMSO as control. splice variants (AR-Vs such as AR-V7). It is already know that All were treated with 10 to 20 mmol/L of leuprolide or degarelix. aberrant AR signaling and AR-Vs are able to promote the devel- Cells were harvested 48 hours after transfection and transactiva- opment of CRPC and may drive drug resistance (8–11). Preclin- tion was examined by the dual-luciferase assay (Promega). ical and clinical trials have described a direct correlation between AR-V7 expression, one of the most intensively studied AR-variant, Proliferation assay and resistance to enzalutamide and abiraterone. Moreover, AR-V7 LNCaP, C4-2B, CWR22Rv1, VCaP, and C4-2B MDVR cells were detection has been independently associated with poor prognosis seeded on 12-well plates at a density of 0.5 Â 105 cells/well in in CRPC (12, 13). AR-V7 has been proposed as a prognostic media containing 10% FBS. When treated with leuprolide marker of PSA response, progression-free survival (PFS), and (20 mmol/L) and degarelix (20 mmol/L) alone or in combination overall survival (OS) among CRPC patients treated with AR- with enzalutamide (20 mmol/L) and abiraterone acetate targeted agents (abiraterone and enzalutamide) or chemothera- (5 mmol/L), cells were maintained in complete medium and py (14, 15), but there are no studies that have investigated the harvested after 3 or 6 days of treatment for cell counting. A total relationship between the type of ADT (GnRH agonist or antag- of 20 mmol/L of leuprolide and degarelix are equivalent to 24.2 onist) and the expression of AR-V7. and 32.6 mg/kg if using density of water 1 g/mL. In this study, we showed that different prostate cancer cell lines are sensitive to the antiproliferative effect of the GnRH antagonist Real-time qRT-PCR degarelix. Furthermore, the use of degarelix, alone or in combi- Total RNAs was extracted from LNCaP, C4-2B, CWR22Rv1, nation with enzalutamide or abiraterone, affected the expression VcaP, and C4-2B MDVR cells using the Qiagen Rneasy Kit. qPCR of AR-V7. In particular, we observed a reduction of AR-V7 at both analysis was performed in SsoFast EvaGreen Supermix (Bio-Rad) the protein and transcription levels. These insights suggest extra- with specific primers for AR-FL, AR-V7, or PSA and analyzed with pituitary activity of GnRH-R in prostate cancer tumors and may Bio-Rad CFX96 Real-Time PCR system (Bio-Rad). Each reaction have implications regarding resistance to second generation AR was normalized by co-amplification of b-action and triplicate pathway inhibitors. runs. The experiments were repeated 2 to 3 times for statistical analysis. Primers used for real-time-PCR were: AR-full length: 50-AAG Materials and Methods CCA GAG CTG TGC AGA TGA, 30-TGT CCT GCA GCC ACT GGT Reagents and cell culture TC; AR-V7: 50-AAC AGA AGT ACC TGT GCG CC, 30-TCA GGG TCT LNCaP, VCaP, and CWR22Rv1 cells were obtained from the GGT CAT TTT GA; actin: 50-AGA ACT GGC CCT TCT TGG AGG, 30- ATCC. All experiments with cell lines were performed within GTT TTT ATG TTC CTC TAT GGG; PSA: 5-GAT GAA ACA GGC TGT 6 months of receipt from ATCC or resuscitation after cryopreser- GCC G, 30-CCT CAC AGC TAC CCA CTG CA; GnRH-R subtype 1: vation. The cells were maintained in RPMI1640 supplemented 50-CAC CCT GAC ACG GGT CCT; 30-TTT ACT GGG TCT GAC AAC with 10% FBS, 100 units/mL penicillin, and 0.1 mg/mL strepto- C; GnRH-R subtype 2: 50-GTT TCT CTC CAG GCC ACC AT, 30-CAT mycin. VCaP cells were maintained in DMEM supplemented with CAG TGT CCG ACA TGC GA. 10% FBS, 100 units/mL penicillin, and 0.1 mg/mL streptomycin. C4-2B MDVR (C4-2B enzalutamide resistant; ref. 16) cells were In vivo tumorigenesis assay maintained in 20 mmol/L enzalutamide containing medium. All Animal studies were performed based on the protocols of the cells were maintained at 37C in a humidified incubator with 5% Institutional Animal Care and Use Committee of the University of carbon dioxide. California at Davis (Sacramento, CA).