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Ha S, Ji C, Yang J, Xiao M, Xu Z, Pan WW, Xiang H, Luo G. Discovery of a highly potent, N-terminal domain-targeting degrader of AR-FL/AR-V7 for the treatment of prostate cancer. Eur J Med Chem 2025; 282:117079. [PMID: 39577229 DOI: 10.1016/j.ejmech.2024.117079] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2024] [Revised: 11/11/2024] [Accepted: 11/16/2024] [Indexed: 11/24/2024]
Abstract
The clinical development of PROTACs targeting the androgen receptor (AR) for degradation has made significant progress. However, effective treatments for metastatic prostate cancers containing the androgen receptor splice variant 7 (AR-V7), a constitutively active mutant without the ligand-binding domain (LBD), are still lacking. Here, we reported the identification of a highly potent, noncovalent PROTAC targeting the N-terminal domain (NTD) of AR, NP18, which is developed from the covalent AR-NTD antagonist EPI-002, and effectively degrades both AR-FL and AR-V7 in 22Rv1 cells (DC50: 18 and 26 nM respectively). Mechanistically, NP18 interacts with the N-terminal domain (NTD) of both full-length AR (AR-FL) and splice variant 7 (AR-V7), leading to their selective and proteasomal degradation. Importantly, NP18 exhibited remarkably superior antitumor activity in both 22Rv1 xenograft and patient-derived xenograft (PDX) models than EPI-002. Taken together, these findings highlight NP18 as a promising candidate to counteract AR splice variant-driven resistance.
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Affiliation(s)
- Si Ha
- State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 211198, China
| | - Chenxuan Ji
- State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 211198, China
| | - Jiaqi Yang
- State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 211198, China
| | - Maoxu Xiao
- State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 211198, China
| | - Ziyi Xu
- Department of Chemistry, Boston University, Boston, MA, 02215, United States
| | - Wei-Wei Pan
- Department of Cell Biology, College of Medicine, Jiaxing University, Jiaxing, 314001, China
| | - Hua Xiang
- State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 211198, China.
| | - Guoshun Luo
- State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 211198, China.
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Guzman J, Weigelt K, Neumann A, Tripal P, Schmid B, Winter Z, Palmisano R, Culig Z, Cronauer MV, Muschler P, Wullich B, Taubert H, Wach S. NanoLuc Binary Technology as a methodological approach: an important new tool for studying the localization of androgen receptor and androgen receptor splice variant V7 homo and heterodimers. BMC Cancer 2024; 24:346. [PMID: 38500100 PMCID: PMC10949640 DOI: 10.1186/s12885-024-12110-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2023] [Accepted: 03/12/2024] [Indexed: 03/20/2024] Open
Abstract
BACKGROUND The androgen/androgen receptor (AR)-signaling axis plays a central role in prostate cancer (PCa). Upon androgen-binding the AR dimerizes with another AR, and translocates into the nucleus where the AR-dimer activates/inactivates androgen-dependent genes. Consequently, treatments for PCa are commonly based on androgen deprivation therapy (ADT). The clinical benefits of ADT are only transitory and most tumors develop mechanisms allowing the AR to bypass its need for physiological levels of circulating androgens. Clinical failure of ADT is often characterized by the synthesis of a constitutively active AR splice variant, termed AR-V7. AR-V7 mRNA expression is considered as a resistance mechanism following ADT. AR-V7 no longer needs androgenic stimuli for nuclear entry and/or dimerization. METHODS Our goal was to mechanistically decipher the interaction between full-length AR (AR-FL) and AR-V7 in AR-null HEK-293 cells using the NanoLuc Binary Technology under androgen stimulation and deprivation conditions. RESULTS Our data point toward a hypothesis that AR-FL/AR-FL homodimers form in the cytoplasm, whereas AR-V7/AR-V7 homodimers localize in the nucleus. However, after androgen stimulation, all the AR-FL/AR-FL, AR-FL/AR-V7 and AR-V7/AR-V7 dimers were localized in the nucleus. CONCLUSIONS We showed that AR-FL and AR-V7 form heterodimers that localize to the nucleus, whereas AR-V7/AR-V7 dimers were found to localize in the absence of androgens in the nucleus.
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Affiliation(s)
- Juan Guzman
- Department of Urology and Pediatric Urology, Uniklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, 91054, Germany
- Comprehensive Cancer Center Erlangen-EMN (CCC ER-EMN), Erlangen, 91054, Germany
| | - Katrin Weigelt
- Department of Urology and Pediatric Urology, Uniklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, 91054, Germany
- Comprehensive Cancer Center Erlangen-EMN (CCC ER-EMN), Erlangen, 91054, Germany
| | - Angela Neumann
- Department of Urology and Pediatric Urology, Uniklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, 91054, Germany
- Comprehensive Cancer Center Erlangen-EMN (CCC ER-EMN), Erlangen, 91054, Germany
| | - Philipp Tripal
- Optical Imaging Centre Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, 91054, Germany
| | - Benjamin Schmid
- Optical Imaging Centre Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, 91054, Germany
| | - Zoltán Winter
- Optical Imaging Centre Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, 91054, Germany
| | - Ralph Palmisano
- Optical Imaging Centre Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, 91054, Germany
| | - Zoran Culig
- Department of Urology, Division of Experimental Urology, Medical University of Innsbruck, Innsbruck, 6020, Austria
| | - Marcus V Cronauer
- Institute of Pathology, Universitätsklinikum Bonn, Universität Bonn, Bonn, 53127, Germany
| | | | - Bernd Wullich
- Department of Urology and Pediatric Urology, Uniklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, 91054, Germany
- Comprehensive Cancer Center Erlangen-EMN (CCC ER-EMN), Erlangen, 91054, Germany
| | - Helge Taubert
- Department of Urology and Pediatric Urology, Uniklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, 91054, Germany.
- Comprehensive Cancer Center Erlangen-EMN (CCC ER-EMN), Erlangen, 91054, Germany.
| | - Sven Wach
- Department of Urology and Pediatric Urology, Uniklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, 91054, Germany
- Comprehensive Cancer Center Erlangen-EMN (CCC ER-EMN), Erlangen, 91054, Germany
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Ha S, Luo G, Xiang H. A Comprehensive Overview of Small-Molecule Androgen Receptor Degraders: Recent Progress and Future Perspectives. J Med Chem 2022; 65:16128-16154. [PMID: 36459083 DOI: 10.1021/acs.jmedchem.2c01487] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/03/2022]
Abstract
Prostate cancer (PC), the second most prevalent malignancy in men worldwide, has been proven to depend on the aberrant activation of androgen receptor (AR) signaling. Long-term androgen deprivation for the treatment of PC inevitably leads to castration-resistant prostate cancer (CRPC) in which AR remains a crucial oncogenic driver. Thus, there is an urgent need to develop new strategies to address this unmet medical need. Targeting AR for degradation has recently been in a vigorous development stage, and accumulating clinical studies have highlighted the benefits of AR degraders in CRPC patients. Herein, we provide a comprehensive summary of small-molecule AR degraders with diverse mechanisms of action including proteolysis-targeting chimeras (PROTACs), selective AR degraders (SARDs), hydrophobic tags (HyT), and other AR degraders with distinct mechanisms. Accordingly, their structure-activity relationships, biomedical applications, and therapeutic values are also dissected to provide insights into the future development of promising AR degradation-based therapeutics for CRPC.
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Affiliation(s)
- Si Ha
- State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 211198, P. R. China
| | - Guoshun Luo
- State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 211198, P. R. China
| | - Hua Xiang
- State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 211198, P. R. China
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Pandareesh MD, Kameshwar VH, Byrappa K. Prostate Carcinogenesis: Insights in Relation to Epigenetics and Inflammation. Endocr Metab Immune Disord Drug Targets 2021; 21:253-267. [PMID: 32682386 DOI: 10.2174/1871530320666200719020709] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/24/2020] [Revised: 04/17/2020] [Accepted: 04/29/2020] [Indexed: 12/24/2022]
Abstract
Prostate cancer is a multifactorial disease that mainly occurs due to the accumulation of somatic, genetic, and epigenetic changes, resulting in the inactivation of tumor-suppressor genes and activation of oncogenes. Mutations in genes, specifically those that control cell growth and division or the repair of damaged DNA, make the cells grow and divide uncontrollably to form a tumor. The risk of developing prostate cancer depends upon the gene that has undergone the mutation. Identifying such genetic risk factors for prostate cancer poses a challenge for the researchers. Besides genetic mutations, many epigenetic alterations, including DNA methylation, histone modifications (methylation, acetylation, ubiquitylation, sumoylation, and phosphorylation) nucleosomal remodeling, and chromosomal looping, have significantly contributed to the onset of prostate cancer as well as the prognosis, diagnosis, and treatment of prostate cancer. Chronic inflammation also plays a major role in the onset and progression of human cancer, via modifications in the tumor microenvironment by initiating epithelialmesenchymal transition and remodeling the extracellular matrix. In this article, the authors present a brief history of the mechanisms and potential links between the genetic aberrations, epigenetic changes, inflammation, and inflammasomes that are known to contribute to the prognosis of prostate cancer. Furthermore, the authors examine and discuss the clinical potential of prostate carcinogenesis in relation to epigenetics and inflammation for its diagnosis and treatment..
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Affiliation(s)
- Mirazkar D Pandareesh
- Center for Research and Innovation, BGSIT Campus, Adichunchanagiri University, B.G. Nagara, Mandya District, Karnataka 571448, India
| | - Vivek H Kameshwar
- Center for Research and Innovation, BGSIT Campus, Adichunchanagiri University, B.G. Nagara, Mandya District, Karnataka 571448, India
| | - Kullaiah Byrappa
- Center for Research and Innovation, BGSIT Campus, Adichunchanagiri University, B.G. Nagara, Mandya District, Karnataka 571448, India
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Sena LA, Wang H, Lim ScM SJ, Rifkind I, Ngomba N, Isaacs JT, Luo J, Pratz C, Sinibaldi V, Carducci MA, Paller CJ, Eisenberger MA, Markowski MC, Antonarakis ES, Denmeade SR. Bipolar androgen therapy sensitizes castration-resistant prostate cancer to subsequent androgen receptor ablative therapy. Eur J Cancer 2021; 144:302-309. [PMID: 33383350 PMCID: PMC9844588 DOI: 10.1016/j.ejca.2020.11.043] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2020] [Revised: 10/28/2020] [Accepted: 11/25/2020] [Indexed: 01/19/2023]
Abstract
BACKGROUND Cyclical, high-dose testosterone administration, termed bipolar androgen therapy (BAT), can induce clinical responses and restore sensitivity to androgen signalling inhibition in patients with previously treated castration-resistant prostate cancer (PCa) (CRPC). This trial evaluated whether BAT is a safe and effective first-line hormonal therapy for patients with CRPC. PATIENTS AND METHODS In cohort C of this single-centre, open-label, phase II, multi-cohort trial (RE-sensitizing with Supraphysiologic Testosterone to Overcome REsistance study), 29 patients with CRPC received first-line hormonal therapy with 400 mg of testosterone cypionate intramuscularly every 28 days concurrent with a luteinising hormone-releasing hormone agonist/antagonist. The primary end-point of the study was the PSA50 response rate to BAT treatment. RESULTS After treatment with BAT, four of 29 patients (14%; 95% confidence interval [CI]: 4-32%) experienced a PSA50 response. The median radiographic progression-free survival to BAT was 8.5 months (95% CI: 6.9-15.1) for patients with metastatic CRPC. After progression on BAT, 17 of 18 patients (94%; 95% CI: 73-100%) achieved a PSA50 response and 15 of 18 patients (83%; 95% CI: 59-96) achieved a PSA90 response on abiraterone or enzalutamide. Twelve of 15 patients (80%; 95% CI: 52-96) with metastatic CRPC remain on abiraterone or enzalutamide with a median duration of follow-up of 11.2 months. CONCLUSION As first-line hormonal treatment for CRPC, BAT was well tolerated and resulted in prolonged disease stabilisation. After progression on BAT, patients had favourable responses to second-generation androgen receptor-targeted therapy. TRIAL REGISTRATION ClinicalTrials.gov NCT02090114.
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Affiliation(s)
- Laura A Sena
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Hao Wang
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Su J Lim ScM
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Irina Rifkind
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Nduku Ngomba
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - John T Isaacs
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Jun Luo
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Caroline Pratz
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Victoria Sinibaldi
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Michael A Carducci
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Channing J Paller
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Mario A Eisenberger
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Mark C Markowski
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Emmanuel S Antonarakis
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Samuel R Denmeade
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
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Pham T, Sadowski MC, Li H, Richard DJ, d'Emden MC, Richard K. Advances in hormonal therapies for hormone naïve and castration-resistant prostate cancers with or without previous chemotherapy. Exp Hematol Oncol 2016; 5:15. [PMID: 27340608 PMCID: PMC4918127 DOI: 10.1186/s40164-016-0046-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2016] [Accepted: 06/09/2016] [Indexed: 11/28/2022] Open
Abstract
Hormonal manipulation plays a significant role in the treatment of advanced hormone naïve prostate cancer and castration-resistant prostate cancer (CRPC) with or without previous chemotherapy. Combination of gonadotropin releasing hormone (GnRH) agonists and androgen receptor (AR) antagonists (combined androgen blockade; CAB) is the first line therapy for advanced hormone naïve prostate cancer, but current strategies are developing novel GnRH antagonists to overcome disadvantages associated with GnRH agonist monotherapy and CAB in the clinical setting. Abiraterone acetate and enzalutamide are hormonal agents currently available for patients with CRPC and are both shown to improve overall survival versus placebo. Recently, in clinical trials, testosterone has been administered in cycles with existing surgical and chemical androgen deprivation therapies (ADT) (intermittent therapy) to CRPC patients of different stages (low risk, metastatic) to abate symptoms of testosterone deficiency and reduce cost of treatment from current hormonal therapies for patients with CRPC. This review will provide an overview on the therapeutic roles of hormonal manipulation in advanced hormone naïve and castration-resistant prostate cancers, as well as the development of novel hormonal therapies currently in preclinical and clinical trials.
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Affiliation(s)
- Thy Pham
- Conjoint Endocrine Laboratory, Chemical Pathology, Pathology Queensland, Queensland Health, Level 9, Bancroft Centre, 300 Herston Road, Herston, QLD 4029 Australia
| | - Martin C Sadowski
- Australian Prostate Cancer Research Centre-Queensland, Institute of Health and Biomedical Innovation, Queensland University of Technology, Princess Alexandra Hospital, Translational Research Institute, Brisbane, QLD 4102 Australia
| | - Huika Li
- Conjoint Endocrine Laboratory, Chemical Pathology, Pathology Queensland, Queensland Health, Level 9, Bancroft Centre, 300 Herston Road, Herston, QLD 4029 Australia
| | - Derek J Richard
- School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD 4000 Australia
| | - Michael C d'Emden
- Conjoint Endocrine Laboratory, Chemical Pathology, Pathology Queensland, Queensland Health, Level 9, Bancroft Centre, 300 Herston Road, Herston, QLD 4029 Australia ; Department of Endocrinology and Diabetes, Royal Brisbane and Women's Hospital, Herston, QLD 4029 Australia
| | - Kerry Richard
- Conjoint Endocrine Laboratory, Chemical Pathology, Pathology Queensland, Queensland Health, Level 9, Bancroft Centre, 300 Herston Road, Herston, QLD 4029 Australia ; School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD 4000 Australia
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Recine F, Sternberg CN. Hormonal therapy and chemotherapy in hormone-naive and castration resistant prostate cancer. Transl Androl Urol 2016; 4:355-64. [PMID: 26816835 PMCID: PMC4708230 DOI: 10.3978/j.issn.2223-4683.2015.04.11] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
The management of advanced castration resistant prostate cancer (CRPC) has been rapidly changing and is still evolving. In the last years, there has been an increasing knowledge of prostate cancer biology. New therapeutic agents and approaches have been evaluated demonstrating benefits in survival and quality of life in patients with metastatic prostate cancer.
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Affiliation(s)
- Federica Recine
- Department of Medical Oncology, San Camillo and Forlanini Hospitals, Rome, Italy
| | - Cora N Sternberg
- Department of Medical Oncology, San Camillo and Forlanini Hospitals, Rome, Italy
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Dichotomy in the Epigenetic Mark Lysine Acetylation is Critical for the Proliferation of Prostate Cancer Cells. Cancers (Basel) 2015; 7:1622-42. [PMID: 26295410 PMCID: PMC4586787 DOI: 10.3390/cancers7030854] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2015] [Revised: 08/07/2015] [Accepted: 08/11/2015] [Indexed: 01/02/2023] Open
Abstract
The dynamics of lysine acetylation serve as a major epigenetic mark, which regulates cellular response to inflammation, DNA damage and hormonal changes. Microarray assays reveal changes in gene expression, but cannot predict regulation of a protein function by epigenetic modifications. The present study employs computational tools to inclusively analyze microarray data to understand the potential role of acetylation during development of androgen-independent PCa. The data revealed that the androgen receptor interacts with 333 proteins, out of which at least 92 proteins were acetylated. Notably, the number of cellular proteins undergoing acetylation in the androgen-dependent PCa was more as compared to the androgen-independent PCa. Specifically, the 32 lysine-acetylated proteins in the cellular models of androgen-dependent PCa were mainly involved in regulating stability as well as pre- and post-processing of mRNA. Collectively, the data demonstrate that protein lysine acetylation plays a crucial role during the transition of androgen-dependent to -independent PCa, which importantly, could also serve as a functional axis to unravel new therapeutic targets.
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Accumulation of trans-1-amino-3-[(18)F]fluorocyclobutanecarboxylic acid in prostate cancer due to androgen-induced expression of amino acid transporters. Mol Imaging Biol 2015; 16:756-64. [PMID: 24943499 DOI: 10.1007/s11307-014-0756-x] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
PURPOSE Androgens play a crucial role in prostate cancer progression, and trans-1-amino-3-[(18)F]fluorocyclobutanecarboxylic acid (anti-[(18) F]FACBC) are used for visualization of prostate cancer. We examined the effect of androgen on the expression of amino acid transporters related to anti-[(18)F]FACBC transport and uptake of trans-1-amino-3-fluoro-[1-(14)C]cyclobutanecarboxylic acid (anti-[(14)C]FACBC). PROCEDURES Expression of amino acid transporters and uptake of anti-[(14)C]FACBC in androgen receptor (AR)-positive LNCaP and AR-negative DU145 human prostate cancer cells cultured with/without 5α-dihydrotestosterone (DHT) and the effect of bicalutamide, an AR antagonist, on DHT-associated changes were investigated. RESULTS DHT stimulated the expression of amino acid transporters ASCT2, SNAT5, 4F2 heavy chain, and LAT3 in LNCaP but not in DU145 cells. Anti-[(14)C]FACBC uptake was enhanced, in a DHT-dependent manner, in LNCaP cells only. CONCLUSIONS DHT enhanced the expression of ASCT2, the transporter responsible for anti-[(18)F]FACBC uptake, thereby increasing anti-[(14)C]FACBC uptake in AR-positive LNCaP cells. Androgen-mediated induction may contribute to the distinct anti-[(18)F]FACBC accumulation pattern in prostate cancer.
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Zhu Y, Liu C, Armstrong C, Lou W, Sandher A, Gao AC. Antiandrogens Inhibit ABCB1 Efflux and ATPase Activity and Reverse Docetaxel Resistance in Advanced Prostate Cancer. Clin Cancer Res 2015; 21:4133-42. [PMID: 25995342 DOI: 10.1158/1078-0432.ccr-15-0269] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2015] [Accepted: 05/10/2015] [Indexed: 11/16/2022]
Abstract
PURPOSE Previous studies show that inhibition of ABCB1 expression overcomes acquired docetaxel resistance in C4-2B-TaxR cells. In this study, we examined whether antiandrogens, such as bicalutamide and enzalutamide, could inhibit ABCB1 activity and overcome resistance to docetaxel. EXPERIMENTAL DESIGN ABCB1 efflux activity was determined using a rhodamine efflux assay. ABCB1 ATPase activity was determined by Pgp-Glo assay systems. The effects of the antiandrogens bicalutamide and enzalutamide on docetaxel sensitivity were determined by cell growth assays and tumor growth in vivo. RESULTS We found that bicalutamide and enzalutamide inhibit ABCB1 ATP-binding cassette transporter activity through blocking ABCB1 efflux activity. Bicalutamide inhibited ABCB1 efflux activity by 40%, whereas enzalutamide inhibited ABCB1 efflux activity by approximately 60%. Both bicalutamide and enzalutamide inhibit ABCB1 ATPase activity. In addition, bicalutamide and enzalutamide inhibit ABCB1 efflux activity and desensitize docetaxel-resistant and androgen receptor (AR)-negative DU145 cells. Combination of bicalutamide with docetaxel had a significant antitumor effect in both AR-positive and AR-negative docetaxel-resistant xenograft models, suggesting that bicalutamide desensitizes docetaxel-resistant cells to docetaxel treatment independent of AR status. CONCLUSIONS We identified a novel mechanism of action for antiandrogens such as bicalutamide and enzalutamide as inhibitors of ABCB1 efflux and ATPase activity. Bicalutamide and enzalutamide desensitize docetaxel-resistant prostate cancer cells to docetaxel treatment independent of AR status. These studies may lead to the development of combinational therapies with bicalutamide/enzalutamide and docetaxel as effective regimens to treat advanced prostate cancer independent of AR status, and possibly other types of cancer.
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Affiliation(s)
- Yezi Zhu
- Department of Urology, University of California at Davis, Sacramento, California. Graduate Program in Pharmacology and Toxicology, University of California at Davis, Sacramento, California
| | - Chengfei Liu
- Department of Urology, University of California at Davis, Sacramento, California
| | - Cameron Armstrong
- Department of Urology, University of California at Davis, Sacramento, California
| | - Wei Lou
- Department of Urology, University of California at Davis, Sacramento, California
| | - Amandeep Sandher
- Department of Urology, University of California at Davis, Sacramento, California
| | - Allen C Gao
- Department of Urology, University of California at Davis, Sacramento, California. Graduate Program in Pharmacology and Toxicology, University of California at Davis, Sacramento, California. Comprehensive Cancer Center, University of California at Davis, Sacramento, California.
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Figg WD, Chau CH, Price DK, Till C, Goodman PJ, Cho Y, Varella-Garcia M, Reichardt JKV, Tangen CM, Leach RJ, van Bokhoven A, Thompson IM, Lucia MS. Androgen receptor CAG repeat length and TMPRSS2:ETS prostate cancer risk: results From the Prostate Cancer Prevention Trial. Urology 2014; 84:127-31. [PMID: 24824408 DOI: 10.1016/j.urology.2014.03.015] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2014] [Revised: 03/14/2014] [Accepted: 03/17/2014] [Indexed: 11/18/2022]
Abstract
OBJECTIVE To investigate the association between the length of the polymorphic trinucleotide CAG microsatellite repeats in exon 1 of the AR gene and the risk of prostate cancer containing TMPRSS2:ETS fusion genes. METHODS This nested case-control study came from subjects enrolled in the Prostate Cancer Prevention Trial and included 195 biopsy-proven prostate cancer cases with a known TMPRSS2:ETS status and 1344 matched controls. RESULTS There was no association between the CAG repeat length and the risk of TMPRSS2:ETS-positive (odds ratio, 0.97; 95% confidence interval, 0.91-1.04) or TMPRSS2:ETS-negative prostate cancer (odds ratio, 1.04; 95% confidence interval, 0.97-1.11) and in patients with low- or high-grade disease. CONCLUSION Our findings suggested that AR CAG repeats are not associated with TMPRSS2:ETS formation in prostate cancer.
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Affiliation(s)
- William D Figg
- Genitourinary Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD.
| | - Cindy H Chau
- Genitourinary Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD
| | - Douglas K Price
- Genitourinary Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD
| | - Cathee Till
- SWOG Statistical Center, the Fred Hutchinson Cancer Research Center, Seattle, WA
| | - Phyllis J Goodman
- SWOG Statistical Center, the Fred Hutchinson Cancer Research Center, Seattle, WA
| | - Yonggon Cho
- Chonbuk National University Medical School, Jeonju, South Korea
| | | | - Juergen K V Reichardt
- School of Pharmacy and Molecular Sciences, James Cook University, Townsville, Queensland, Australia
| | - Catherine M Tangen
- SWOG Statistical Center, the Fred Hutchinson Cancer Research Center, Seattle, WA
| | - Robin J Leach
- Department of Urology and Cancer Therapy and Research Center, University of Texas Health Science Center at San Antonio, San Antonio, TX
| | - Adrie van Bokhoven
- Department of Pathology, University of Colorado Denver School of Medicine, Aurora, CO
| | - Ian M Thompson
- Department of Urology and Cancer Therapy and Research Center, University of Texas Health Science Center at San Antonio, San Antonio, TX
| | - M Scott Lucia
- Department of Pathology, University of Colorado Denver School of Medicine, Aurora, CO
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Al Sayed AA, Al Sulaiman MH, Mishriky A, Anil S. The role of androgen receptor gene in cyclosporine induced gingival overgrowth. J Periodontal Res 2013; 49:609-14. [PMID: 24206119 DOI: 10.1111/jre.12141] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/19/2013] [Indexed: 11/30/2022]
Abstract
BACKGROUND AND OBJECTIVE Gingival overgrowth is a prominent side effect of cyclosporine (CsA) therapy in renal transplant patients. Although the exact mechanism by which this drug induces gingival overgrowth is uncertain, marked variations in individual susceptibility to this drug suggest a genetic predisposition. Studies have shown that genetic variation (polymorphism) in the trinucleotide cytosine-adenine- guanine (CAG) sequence in exon 1 of the androgen receptor (AR) gene is related to altered activity of the AR as a transcription factor. However, the relationship between the length of the CAG repeat and gingival overgrowth has not yet been studied. The present study was carried out to determine whether there is an association between CsA-induced gingival overgrowth and the length of the CAG repeats in the AR gene. MATERIAL AND METHODS Genomic DNA samples were prepared from the blood of 50 renal transplant patients with CsA-induced gingival overgrowth and from the blood of 100 renal transplant patients on CsA with no gingival overgrowth. RESULTS The difference in allele distribution among the subjects with gingival overgrowth and control samples was statistically significant (p = 0.001). CONCLUSION The findings suggest a link between CsA7induced gingival overgrowth and a smaller size of CAG repeat in the AR gene.
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Affiliation(s)
- A A Al Sayed
- Prince Sultan Military Medical City, Ministry of Defense, Riyadh, Saudi Arabia
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13
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Zhu Y, Liu C, Tummala R, Nadiminty N, Lou W, Gao AC. RhoGDIα downregulates androgen receptor signaling in prostate cancer cells. Prostate 2013; 73:1614-22. [PMID: 23922223 PMCID: PMC3941975 DOI: 10.1002/pros.22615] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/05/2012] [Accepted: 10/15/2012] [Indexed: 12/11/2022]
Abstract
INTRODUCTION Treatment of primary prostate cancer (CaP) is the withdrawal of androgens. However, CaP eventually progresses to grow in a castration-resistant state due to aberrant activation of androgen receptor (AR). Understanding the mechanisms leading to the aberrant activation of AR is critical to develop effective therapy. We have previously identified Rho GDP Dissociation Inhibitor alpha (GDIα) as a novel suppressor in prostate cancer. In this study, we examine the effect of GDIα on AR signaling in prostate cancer cells. METHODS GDIα was transiently or stably transfected into several prostate cancer cell lines including LNCaP, C4-2, CWR22Rv1, and DU145. The regulation of AR expression by GDIα was analyzed by qRT-PCR and Western blot. AR activity was measured by luciferase reporter assays and electrophoretic mobility shift analysis (EMSA). Immunofluorescence assay was performed to study AR nuclear translocation. The interaction between GDIα and AR was examined by co-immunoprecipitation assays. RESULTS In this study, we have identified GDIα as a negative regulator of AR signaling pathway. Overexpression of GDIα downregulates AR expression at both mRNA and protein levels. Overexpression of GDIα is able to prevent AR nuclear translocation and inhibit transactivation of AR target genes. Co-immunoprecipitation assays showed that GDIα physically interacts with the N-terminal domain of AR. CONCLUSIONS GDIα suppresses AR signaling through inhibition of AR expression, nuclear translocation, and recruitment to androgen-responsive genes. GDIα regulatory pathway may play a critical role in regulating AR signaling and prostate cancer growth and progression.
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Affiliation(s)
- Yezi Zhu
- Department of Urology and Cancer Center, University of California at Davis, Sacramento, California
- Graduate Program of Pharmacology and Toxicology, University of California at Davis, Sacramento, California
| | - Chengfei Liu
- Department of Urology and Cancer Center, University of California at Davis, Sacramento, California
| | - Ramakumar Tummala
- Department of Urology and Cancer Center, University of California at Davis, Sacramento, California
| | - Nagalakshmi Nadiminty
- Department of Urology and Cancer Center, University of California at Davis, Sacramento, California
| | - Wei Lou
- Department of Urology and Cancer Center, University of California at Davis, Sacramento, California
| | - Allen C. Gao
- Department of Urology and Cancer Center, University of California at Davis, Sacramento, California
- Graduate Program of Pharmacology and Toxicology, University of California at Davis, Sacramento, California
- Correspondence to: Allen C. Gao, Department of Urology and Cancer Center, University of California Davis Medical Center, 4645 2nd Ave, Research III, Suite 1300, Sacramento, CA 95817.
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Abstract
Mel Greaves discusses the mechanisms underlying transformation in B cell chronic lymphocytic leukemia, including a new study suggesting a role for BCR ligation driven by recognition of an antigenic component of common yeast and fungi. Relatively few cancers arise in mature, differentiated cells. The propensity of mature B cells to transform has been linked to their longevity and proliferative potential, and stimulation of the B cell receptor (BCR) by cognate antigen may promote the transformation process. A study in this issue (Hoogeboom et al.) lends support to this notion, showing that cancer cells from a subset of patients with chronic lymphocytic leukemia (CLL) express a BCR specific for a sugar expressed by commensal yeast species. Another study, in contrast, suggests that B-CLL cells uniquely acquire the ability to signal in the complete absence of ligand.
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Affiliation(s)
- Mel Greaves
- Division of Molecular Pathology, Institute of Cancer Research, Sutton, Surrey SM2 5NG, England, UK.
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15
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Involvement of interleukin-1β mediated nuclear factor κB signalling pathways to down-regulate prostate-specific antigen and cell proliferation in LNCaP prostate cancer cells. Cell Biol Int 2012; 36:449-54. [PMID: 22103356 DOI: 10.1042/cbi20100922] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Involvement of NF-κB (nuclear factor κB) mediated by IL-1β (interleukin-1β) on cell proliferation and PSA (prostate-specific antigen) production of LNCaP prostate cell lines and the possible cross-talk with Akt (also known as protein kinase B) signalling pathway has been investigated. NF-κB and Akt were analysed by Western blotting from LNCaP cells treated by IL-1β before proliferation and PSA production were measured. IL-1β inhibited proliferation and decreased PSA production. The Akt pathway was not sensitive, whereas NF-κB phosphorylation occurred as a result of treatment. PSA production and proliferation of LNCaP cells were down-regulated by NF-κB mediated by IL-1β promoting anti-apoptotic signalling and co-suppressor factors of PSA expression. IL-1β through NF-κB activation provides a rationale for therapeutic approaches in the anticancer treatment of prostate.
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16
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Azevedo A, Cunha V, Teixeira AL, Medeiros R. IL-6/IL-6R as a potential key signaling pathway in prostate cancer development. World J Clin Oncol 2011; 2:384-96. [PMID: 22171281 PMCID: PMC3235657 DOI: 10.5306/wjco.v2.i12.384] [Citation(s) in RCA: 92] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/09/2011] [Revised: 11/08/2011] [Accepted: 11/15/2011] [Indexed: 02/06/2023] Open
Abstract
Interleukin-6 (IL-6) is a pleiotropic cytokine involved in prostate regulation and in prostate cancer (PC) development/progression. IL-6 acts as a paracrine and autocrine growth stimulator in benign and tumor prostate cells. The levels of IL-6 and respective receptors are increased during prostate carcinogenesis and tumor progression. Several studies reported that increased serum and plasma IL-6 and soluble interleukin-6 receptor levels are associated with aggressiveness of the disease and are associated with a poor prognosis in PC patients. In PC treatment, patients diagnosed with advanced stages are frequently submitted to hormonal castration, although most patients will eventually fail this therapy and die from recurrent castration-resistant prostate cancer (CRPC). Therefore, it is important to understand the mechanisms involved in CRPC. Several pathways have been proposed to be involved in CRPC development, and their understanding will improve the way to more effective therapies. In fact, the prostate is known to be dependent, not exclusively, on androgens, but also on growth factors and cytokines. The signaling pathway mediated by IL-6 may be an alternative pathway in the CRPC phenotype acquisition and cancer progression, under androgen deprivation conditions. The principal goal of this review is to evaluate the role of IL-6 pathway signaling in human PC development and progression and discuss the interaction of this pathway with the androgen recepto pathway. Furthermore, we intend to evaluate the inclusion of IL-6 and its receptor levels as a putative new class of tumor biomarkers.The IL-6/IL-6R signaling pathway may be included as a putative molecular marker for aggressiveness in PC and it may be able to maintain tumor growth through the AR pathway under androgen-deprivation conditions. The importance of the IL-6/IL-6R pathway in regulation of PC cells makes it a good candidate for targeted therapy.
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Affiliation(s)
- Andreia Azevedo
- Andreia Azevedo, Virginia Cunha, Ana Luisa Teixeira, Rui Medeiros, Molecular Oncology and Virology, Portuguese Institute of Oncology, 4200-072 Porto, Portugal
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17
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Jiang WG, Ablin RJ. Prostate transglutaminase: a unique transglutaminase and its role in prostate cancer. Biomark Med 2011; 5:285-91. [DOI: 10.2217/bmm.11.36] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
Prostate transglutaminase-4, also known as TGM4 or transglutaminase P, belongs to the prostate transglutaminase protein family, but is almost uniquely distributed in the prostate gland. Recent years have seen an expansion of interest in this enzyme, which is intriguingly expressed in prostate tissues and prostate cancer. In recent studies, the molecule has been found to have a diverse impact on prostate cancer cell growth, migration and invasiveness, and to be involved in the tumor–endothelial interaction and epithelial–mesenchymal transition, and has a wide interaction with other molecular complexes implicating it as a possible biomarker of aggressive versus nonaggressive cancer, as well as a therapeutic factor. This article reviews the recent progress and discusses the controversies and future directions in this exciting area of prostate cancer research.
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Affiliation(s)
- Wen G Jiang
- Metastasis & Angiogenesis Research Group, Cardiff University School of Medicine, Heath Park, Cardiff, CF14 4XN, UK
| | - Richard J Ablin
- Department of Pathology, Health Sciences Center, University of Arizona College of Medicine, Arizona Cancer Center & BIO5 Institute, 1501 North Campbell Avenue, PO Box 245043, Tucson, AZ 85724-5043, USA
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18
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Loiarro M, Campo S, Arseni B, Rossi S, D'Alessio V, De Santis R, Sette C, Ruggiero V. Anti-proliferative effect of a triazole derivative (ST1959) on LNCaP human prostate cancer cells through down-regulation of cyclin and androgen receptor expression. Prostate 2011; 71:32-41. [PMID: 20607765 DOI: 10.1002/pros.21219] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
BACKGROUND Previous studies demonstrated that ST1959, a triazole derivative endowed with immunomodulatory activities, also exerts inhibitory effects on proliferation and survival of a panel of tumor cells. In this study, we sought to ascertain the effects of ST1959 on the growth of androgen-dependent and androgen-independent prostate cancer (PCa) cells. METHODS The growth of androgen-dependent (LNCaP) and androgen-independent (PC3, DU-145) cells was analyzed in vitro both in the presence and absence of ST1959. Modulation of cyclin and androgen receptor (AR) expression following treatment with ST1959 was analyzed by Western blot and cytofluorimetric analysis. RESULTS We observed that ST1959 causes a significant growth inhibition of LNCaP cells without affecting proliferation of androgen-insensitive DU-145 and PC3 cell lines. These effects were associated with G0/G1 cell cycle arrest and down-regulation of cyclin D1, A and B and AR expression. CONCLUSIONS Our present findings indicate that the anti-proliferative activity of ST1959 on cell growth of androgen-dependent LNCaP PCa cells may be brought about by decreasing expression of functional AR and selected cyclins, ultimately leading to cell growth inhibition.
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Affiliation(s)
- Maria Loiarro
- Department of Public Health and Cell Biology, University of Rome Tor Vergata, Rome, Italy
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19
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Price DK, Chau CH, Till C, Goodman PJ, Baum CE, Ockers SB, English BC, Minasian L, Parnes HL, Hsing AW, Reichardt JKV, Hoque A, Tangen CM, Kristal AR, Thompson IM, Figg WD. Androgen receptor CAG repeat length and association with prostate cancer risk: results from the prostate cancer prevention trial. J Urol 2010; 184:2297-302. [PMID: 20952028 DOI: 10.1016/j.juro.2010.08.005] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2010] [Indexed: 12/17/2022]
Abstract
PURPOSE We investigated the association between the length of the polymorphic trinucleotide CAG microsatellite repeats in exon 1 of the AR gene and the risk of prostate cancer. MATERIALS AND METHODS This is a nested case-control study of 1,159 cases and 1,353 controls from the Prostate Cancer Prevention Trial, a randomized, placebo controlled trial testing whether the 5α-reductase inhibitor finasteride could decrease the 7-year prevalence of prostate cancer. During the course of the trial men underwent annual digital rectal examination and prostate specific antigen measurement. Prostate biopsy was recommended in all men with abnormal digital rectal examination or finasteride adjusted prostate specific antigen greater than 4.0 ng/ml. Cases were drawn from men with biopsy determined prostate cancer identified by for cause or end of study biopsy. Controls were selected from men who completed the end of study biopsy. RESULTS Mean CAG repeat length did not differ between cases and controls. The frequency distribution of cases and controls for the AR CAG repeat length was similar. There were no significant associations of CAG repeat length with prostate cancer risk when stratified by treatment arm (finasteride or placebo), or when combined. There was also no significant association between CAG repeat length and the risk of low or high grade prostate cancer. CONCLUSIONS There is no association of AR CAG repeat length with prostate cancer risk. Knowledge of AR CAG repeat length provides no clinically useful information to predict prostate cancer risk.
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Affiliation(s)
- Douglas K Price
- Medical Oncology Branch, National Cancer Institute, Bethesda, Maryland 20892, USA
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20
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Abstract
Hormone-refractory prostate cancer is the result of regrowth of prostate cancer cells that have adapted to the hormone-deprived environment of the prostate. The process by which castration-resistant prostate cancer (CRPC) cells are generated appears to be varied. The complex mechanism of hormone resistance has been the topic of research in most laboratories that have analyzed the process from different angles. This review compiles research findings that explain the methods of development of hormone resistance in prostate cancer. Research data show many different processes to be involved in the acquisition of hormone resistance. Interestingly, one observes interdependence between these processes, indicating a complex network at play in the development of hormone resistance. Cytokines such as IL-6 have been shown to initiate an alternative signaling pathway, compared with the androgen receptor signaling pathway, in CRPC. IL-6 has been proposed to be the effector of the intracrine signaling pathway by influencing the levels of metabolic enzymes. Neuroendocrine cells are present at low levels in normal prostate, and signify the transitory phase of normal hormone-sensitive cells to hormone-refractory cells. IL-6 induces growth of neuroendocrine cells or neuroendocrine-like features in cells in CRPC. The increased presence of neuroendocrine cells in CRPC signifies a change in the prostate cell microenvironment. The stromal microenvironment also influences the development of CRPC in the hormone-refractory stage. In addition, intracrine androgen metabolic enzymes play a significant role in the development of the hormone refractory process. Despite hormone ablation, there is a residual level of hormones in cells due to active intracrine metabolic pathways. It is acknowledged that the androgen receptor plays the most influential role in development of prostate cancer. In addition to mutation and amplification, the androgen receptor has been characterized and shown to differ in sequence in CRPC compared with the androgen-sensitive prostate cancer cells. These variants of the androgen receptor through sequence changes may preserve the basic function of the molecule, but have far-reaching consequences on the cell as a whole. A multicombinatorial drug treatment approach has been suggested to target these multiple pathways in an effort to reduce the possibility of recurrence of CRPC.
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Affiliation(s)
- Smitha S Dutt
- University of California School of Medicine at Davis, CA, USA
| | - Allen C Gao
- Department of Urology and Cancer Center, Research III Bldg, Suite 1300, University of California School of Medicine at Davis, 4645 2nd Ave, Sacramento, CA 95817, USA, Tel.: +1 916 734 8718, Fax: +1 916 734 8714
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21
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The Profile of Prostate Epithelial Cytokines and its Impact on Sera Prostate Specific Antigen Levels. Inflammation 2009; 32:202-10. [DOI: 10.1007/s10753-009-9121-7] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2009] [Accepted: 04/06/2009] [Indexed: 10/20/2022]
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22
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van Royen ME, Dinant C, Farla P, Trapman J, Houtsmuller AB. FRAP and FRET methods to study nuclear receptors in living cells. Methods Mol Biol 2009; 505:69-96. [PMID: 19117140 DOI: 10.1007/978-1-60327-575-0_5] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/27/2023]
Abstract
Quantitative imaging techniques of fluorescently-tagged proteins have been instrumental in the study of the behavior of nuclear receptors (NRs) and coregulators in living cells. Ligand-activated NRs exert their function in transcription regulation by binding to specific response elements in promotor and enhancer sequences of genes. Fluorescence recovery after photobleaching (FRAP) has proven to be a powerful tool to study the mobility of fluorescently-labeled molecules in living cells. Since binding to DNA leads to the immobilization of DNA-interacting proteins like NRs, FRAP is especially useful for determining DNA-binding kinetics of these proteins. The coordinated interaction of NRs with promoters/enhancers and subsequent transcription activation is not only regulated by ligand but also by interactions with sets of cofactors and, at least in the case of the androgen receptor (AR), by dimerization and interdomain interactions. In living cells, these interactions can be studied by fluorescence resonance energy transfer (FRET). Here we provide and discuss detailed protocols for FRAP and FRET procedures to study the behavior of nuclear receptors in living cells. On the basis of our studies of the AR, we provide protocols for two different FRAP methods (strip-FRAP and FLIP-FRAP) to quantitatively investigate DNA-interactions and for two different FRET approaches, ratio imaging, and acceptor photobleaching FRET to study AR domain interactions and interactions with cofactor motifs. Finally, we provide a protocol of a technique where FRAP and acceptor photobleaching FRET are combined to study the dynamics of interacting ARs.
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Affiliation(s)
- Martin E van Royen
- Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands
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23
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Irer B, Toylu A, Aslan G, Celebi I, Yorukoglu K, Atabey N. Increased expression of NKX3.1 in benign prostatic hyperplasia. Urology 2008; 73:1140-4. [PMID: 18597829 DOI: 10.1016/j.urology.2008.02.039] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2007] [Revised: 02/10/2008] [Accepted: 02/15/2008] [Indexed: 11/24/2022]
Abstract
OBJECTIVES To establish the role of the NKX3.1 gene in the development of benign prostatic hyperplasia by comparing the expression of NKX3.1 in messenger ribonucleic acid (mRNA) and protein levels in young adult prostate and BPH tissues. METHODS Normal prostate tissue samples (n = 4) were obtained from prostate biopsies of patients less than 40 years of age who underwent diagnostic cystoscopy for microscopic hematuria. Benign prostatic hyperplasia tissues (n = 12) were obtained from patients who underwent transurethral prostate resection for bladder outlet obstruction. The RNAs isolated from these tissue samples were analyzed with quantitative reverse transcriptase polymerase chain reaction; the proteins were analyzed with Western blotting and immunohistochemistry. RESULTS The mean NKX3.1 mRNA transcript expression was 19.17 +/- 3.05 vs 1.24 +/- 1.32 in BPH and normal tissues, respectively, and NKX3.1 protein expression of BPH was approximately 2.4-fold higher than in normal prostate tissue. Reverse transcriptase polymerase chain reaction and Western blot analyses revealed that NKX3.1 gene expression in BPH patient tissues were higher compared with normal prostate tissues. Immunohistochemistry results indicated that most of the BPH tissues stained diffusely, and there was no BPH tissue that lacked NKX3.1 expression. CONCLUSIONS NKX3.1 expression is elevated in BPH tissues when compared with normal tissues, which may be important in the development of BPH.
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Affiliation(s)
- Bora Irer
- Department of Urology, Dokuz Eylul University, Inciralti, Izmir, Turkey
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24
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Lai CL, van den Ham R, Mol J, Teske E. Immunostaining of the androgen receptor and sequence analysis of its DNA-binding domain in canine prostate cancer. Vet J 2008; 181:256-60. [PMID: 18583166 DOI: 10.1016/j.tvjl.2008.04.009] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2008] [Revised: 03/29/2008] [Accepted: 04/13/2008] [Indexed: 11/29/2022]
Abstract
Prostate cancer in the dog (cPC) has many features in common with hormone refractory human prostate cancer. As cPC is seen more often in castrated dogs, the contribution of the androgen receptor (AR) to the development of prostate cancer remains questionable. The aim of the present study was to evaluate the presence of the AR by immunohistochemistry in cPC. AR staining was observed in most tumors from intact and castrated dogs, but the proportion of positive cells and the staining intensity were much lower than in the prostate of healthy, non-castrated dogs. Most of the positive staining was seen in the cytoplasm rather than in the nuclei of the tumor cells. The predominant cytoplasmic localization was not related to mutations in exon 3 of the DNA-binding domain of the AR, as shown by sequence analysis of microdissected AR positive tumor cells. Other mechanisms that lead to an impaired androgen-AR signaling or a basal/stem cell like origin may explain the low cytoplasmic AR staining in cPC.
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Affiliation(s)
- Chen-Li Lai
- Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.154, 3508 TD Utrecht, The Netherlands
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Paliouras M, Diamandis EP. An AKT activity threshold regulates androgen-dependent and androgen-independent PSA expression in prostate cancer cell lines. Biol Chem 2008; 389:773-80. [DOI: 10.1515/bc.2008.072] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
AbstractThe androgen receptor (AR) plays an important role in early prostate cancer by activating transcription of a number of genes participating in cell proliferation and growth and cancer progression. However, as the cancer progresses, prostate cancer cells transform from an androgen-dependent to an androgen-independent state. Androgen-independent prostate cancer can manifest itself in several forms, including a percentage of cancers that show reduced levels of prostate-specific antigen (PSA) and can progress without the need for the ligand or active receptor. Therefore, our goal was to examine the role of intracellular signaling pathways in an androgen-independent prostate cancerin vitromodel. Using the cell line PC3(AR)2, we stimulated cells with 5-α-dihydrotestosterone (DHT) and epidermal growth factor (EGF) and then analyzed PSA expression. We observed lower PSA expression when cells were jointly stimulated with DHT and EGF, and this was associated with an increase in AKT activity. We examined the role of AKT in AR activity and PSA expression by creating stable PC3(AR)2cell lines transfected with a PI3K-Ras-effector loop mutant. These cell lines showed lower DHT-stimulated PSA expression that correlated to changes in the phosphorylated state of AR. Therefore, we propose anin vitroandrogen-independent model in which a PI3K/AKT activity threshold and subsequent AR transactivation regulate PSA expression.
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26
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Chen PH, Tsao YP, Wang CC, Chen SL. Nuclear receptor interaction protein, a coactivator of androgen receptors (AR), is regulated by AR and Sp1 to feed forward and activate its own gene expression through AR protein stability. Nucleic Acids Res 2007; 36:51-66. [PMID: 17984071 PMCID: PMC2248731 DOI: 10.1093/nar/gkm942] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Previously, we found a novel gene, nuclear receptor interaction protein (NRIP), a transcription cofactor that can enhance an AR-driven PSA promoter activity in a ligand-dependent manner in prostate cancer cells. Here, we investigated NRIP regulation. We cloned a 413-bp fragment from the transcription initiation site of the NRIP gene that had strong promoter activity, was TATA-less and GC-rich, and, based on DNA sequences, contained one androgen response element (ARE) and three Sp1-binding sites (Sp1-1, Sp1-2, Sp1-3). Transient promoter luciferase assays, chromatin immunoprecipitation and small RNA interference analyses mapped ARE and Sp1-2-binding sites involved in NRIP promoter activation, implying that NRIP is a target gene for AR or Sp1. AR associates with the NRIP promoter through ARE and indirectly through Sp1-binding site via AR–Sp1 complex formation. Thus both ARE and Sp1-binding site within the NRIP promoter can respond to androgen induction. More intriguingly, NRIP plays a feed-forward role enhancing AR-driven NRIP promoter activity via NRIP forming a complex with AR to protect AR protein from proteasome degradation. This is the first demonstration that NRIP is a novel AR-target gene and that NRIP expression feeds forward and activates its own expression through AR protein stability.
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Affiliation(s)
- Pei-Hong Chen
- Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan
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27
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Park SY, Yu X, Ip C, Mohler JL, Bogner PN, Park YM. Peroxiredoxin 1 interacts with androgen receptor and enhances its transactivation. Cancer Res 2007; 67:9294-303. [PMID: 17909037 DOI: 10.1158/0008-5472.can-07-0651] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Although hypoxia is accepted as an important microenvironmental factor influencing tumor progression and treatment response, it is usually regarded as a static global phenomenon. Consequently, less attention is given to the impact of dynamic changes in tumor oxygenation in regulating the behavior of cancer cells. Androgen receptor (AR) signaling plays a critical role in prostate cancer. We previously reported that hypoxia/reoxygenation, an in vitro condition used to mimic an unstable oxygenation climate in a tumor, stimulates AR activation. In the present study, we showed that peroxiredoxin 1 (Prx1), a member of the peroxiredoxin protein family, acts as a key mediator in this process. We found that the aggressive LN3, C4-2, and C4-2B prostate cancer cell lines derived from LNCaP possess constitutively elevated Prx1 compared with parental cells, and display greater AR activation in response to hypoxia/reoxygenation. Although the cell survival-enhancing property of Prx1 has traditionally been attributed to its antioxidant activity, the reactive oxygen species-scavenging activity of Prx1 was not essential for AR stimulation because Prx1 itself was oxidized and inactivated by hypoxia/reoxygenation. Increased AR transactivation was observed when wild-type Prx1 or mutant Prx1 (C52S) lacking antioxidant activity was introduced into LNCaP cells. Reciprocal immunoprecipitation, chromatin immunoprecipitation, and in vitro pull-down assays corroborated that Prx1 interacts with AR and enhances its transactivation. We also show that Prx1 is capable of sensitizing a ligand-stimulated AR. Based on the above information, we suggest that disrupting the interaction between Prx1 and AR may serve as a fruitful new target in the management of prostate cancer.
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Affiliation(s)
- Soo-Yeon Park
- Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA
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28
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Cai C, Hsieh CL, Shemshedini L. c-Jun has multiple enhancing activities in the novel cross talk between the androgen receptor and Ets variant gene 1 in prostate cancer. Mol Cancer Res 2007; 5:725-35. [PMID: 17634427 DOI: 10.1158/1541-7786.mcr-06-0430] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
The multiple transcriptional roles of c-Jun are shown in a novel cross-talk between the androgen receptor (AR) and its new target gene, Ets variant gene 1 (ETV1). In this report, we show that c-Jun can mediate AR induction of ETV1 expression independent of c-Jun transactivation function. Interestingly, c-Jun can transactivate the cloned ETV1 promoter also in the absence of ligand-activated AR, suggesting two mechanisms by which c-Jun can induce ETV1 expression. In addition, both wild-type c-Jun and a transactivation-deficient mutant can enhance the transcriptional activity of ETV1, as measured by both reporter gene assay and endogenous expression of matrix metalloproteinase genes, well-known targets of Ets proteins. Overexpression of the c-Jun mutant protein also led to increased prostate cancer cell invasion. Immunoprecipitation and immunocytochemistry experiments showed copurification and colocalization of c-Jun with AR or ETV1, suggesting that c-Jun acts on AR or ETV1 via a physical association. Collectively, these results, together with a parallel overexpression of ETV1, c-Jun, and AR in prostate tumors, imply that c-Jun plays a pivotal role in the pathway that connects ligand-activated AR to elevated ETV1 expression, leading to enhanced expression of matrix metalloproteinases and prostate cancer cell invasion.
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Affiliation(s)
- Changmeng Cai
- Department of Biological Sciences, University of Toledo, Toledo, OH 43606, USA
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Cai C, Chen SY, Zheng Z, Omwancha J, Lin MF, Balk SP, Shemshedini L. Androgen regulation of soluble guanylyl cyclasealpha1 mediates prostate cancer cell proliferation. Oncogene 2006; 26:1606-15. [PMID: 16964290 DOI: 10.1038/sj.onc.1209956] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The growth and progression of prostate cancer are dependent on androgens and androgen receptor (AR), which act by modulating gene expression. Utilizing a gene microarray approach, we have identified the alpha1-subunit gene of soluble guanylyl cyclase (sGC) as a novel androgen-regulated gene. A heterodimeric cytoplasmic protein composed of one alpha and one beta subunit, sGC mediates the widespread cellular effects of nitric oxide (NO). We report here that, in prostate cancer cells, androgens stimulate the expression of sGCalpha1. A cloned human sGCalpha1 promoter is activated by androgen in an AR-dependent manner, suggesting that sGCalpha1 may be a direct AR target gene. Disruption of sGCalpha1 expression severely compromises the growth of both androgen-dependent and androgen-independent AR-positive prostate cancer cells. Overexpression of sGCalpha1 alone is sufficient for stimulating prostate cancer cell proliferation. Interestingly, the major growth effect of sGCalpha1 is independent of NO and cyclic guanosine monophosphate, a major mediator of the sGC enzyme. These data strongly suggest that sGCalpha1 acts in prostate cancer via a novel pathway that does not depend on sGCbeta1. Tissue studies show that sGCalpha1 expression is significantly elevated in advanced prostate cancer. Thus, sGCalpha1 may be an important mediator of the procarcinogenic effects of androgens.
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Affiliation(s)
- C Cai
- Department of Biological Sciences, University of Toledo, Toledo, OH 43606, USA
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30
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Park SY, Kim YJ, Gao AC, Mohler JL, Onate SA, Hidalgo AA, Ip C, Park EM, Yoon SY, Park YM. Hypoxia increases androgen receptor activity in prostate cancer cells. Cancer Res 2006; 66:5121-9. [PMID: 16707435 DOI: 10.1158/0008-5472.can-05-1341] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Recent studies show that prostate cancer cells are able to survive in a hypoxic tumor environment, and the extent of tumor hypoxia correlates with poor clinical outcome. Androgen deprivation, the most common form of prostate cancer therapy, was itself shown to induce a state of transient hypoxia at the microenvironmental level. Because androgen receptor (AR) signaling plays a critical role in prostate cancer, we investigated the effect of hypoxia in regulating AR function. We found that in LNCaP prostate cancer cells, AR binding to the androgen-responsive element (ARE), prostate-specific antigen accumulation, and ARE-reporter gene activity were increased after hypoxia treatment. Hypoxia-enhanced AR function was also observed when AR was exogenously introduced into AR-null DU145 cells. Confocal microscopy and chromatin immunoprecipitation assays showed that AR translocation to the nucleus and AR recruitment to the prostate-specific antigen promoter were facilitated after hypoxia treatment. The AR stimulatory effect seemed to be ligand-dependent because it was abrogated when cells were cultured in an androgen-depleted medium, but was restored with the addition of R1881, a synthetic androgen. The sensitivity of AR activation to R1881 was also increased after hypoxia treatment. Although concentrations of <1 nmol/L R1881 did not induce ARE reporter activity under normoxic conditions, exposure to hypoxia greatly potentiated the AR response to low levels of R1881. Collectively, our results provide compelling evidence that changes in hypoxia/reoxygenation stimulate AR trans-activation and sensitization. The AR-stimulatory effect of an unstable tissue oxygenation milieu of a tumor is likely to contribute to treatment resistance and the emergence of recurrent prostate cancer.
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Affiliation(s)
- Soo-Yeon Park
- Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA
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Chun JY, Nadiminty N, Lee SO, Onate SA, Lou W, Gao AC. Mechanisms of selenium down-regulation of androgen receptor signaling in prostate cancer. Mol Cancer Ther 2006; 5:913-8. [PMID: 16648561 DOI: 10.1158/1535-7163.mct-05-0389] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Prevention trials showed that selenium reduced prostate cancer incidence by 50%, establishing selenium as a promising chemopreventive agent for prostate cancer. Selenium inhibited human prostate cancer cell growth, blocked cell cycle progression at multiple transition points, and induced apoptotic cell death. Previous studies showed a novel mechanism of selenium anticancer action in which selenium markedly reduces androgen signaling and androgen receptor (AR)-mediated gene expression, including prostate-specific antigen (PSA), in human prostate cancer cells. The molecular mechanisms of selenium-mediated down-regulation of AR signaling are not clear. In this study, a systemic approach was taken to examine the modification of androgen signaling by selenium in human prostate cancer cells. In addition to reduced AR mRNA expression, selenium was found to initially increase the stability of AR mRNA within 6 hours while decreasing the stability of AR mRNA after 8 hours. Selenium increased AR protein degradation and reduced AR nuclear localization. Scatchard analysis indicated that selenium did not affect ligand binding to AR in LNCaP cells. Chromatin immunoprecipitation analyses showed that DHT increased the recruitment of AR and coactivators, such as SRC-1 and TIF-2, to the promoter of the PSA gene, and that recruitment was greatly diminished in the presence of 5 micromol/L selenium. On the other hand, selenium enhanced the recruitment of corepressors, such as SMRT, to the promoter of the PSA gene. Taken together, these results suggest that selenium disrupts AR signaling at multiple stages, including AR mRNA expression, mRNA stability, protein degradation, nuclear translocation, and recruitment of coregulators.
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Affiliation(s)
- Jae Yeon Chun
- Department of Medicine, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA
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Abdel-Wahab M, Berkey BA, Krishan A, O'Brien T, Hammond E, Roach M, Lawton C, Pilepich M, Markoe A, Pollack A. Influence of Number of CAG Repeats on Local Control in the RTOG 86-10 Protocol. Am J Clin Oncol 2006; 29:14-20. [PMID: 16462497 DOI: 10.1097/01.coc.0000195085.34162.88] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
OBJECTIVES The number of CAG repeats on the androgen receptor (AR) gene is inversely proportional to transcriptional activity. The purpose of this study was to determine if short-term androgen deprivation therapy (RT + HT) can improve outcome in patients with tumors with short CAG repeats (<19). MATERIALS AND METHODS Prostate cancer patients were randomized to receive either radiotherapy (RT) alone or (RT + HT) in the RTOG 86-10 study. CAG repeats were measured in 94 tumor specimens (21%; test cohort) of the 456 (parent cohort) analyzable cases. AR flow cytometry measurements were done on 13 patients. The effect on local failure (LF), distant metastases (DM), prostate cancer survival (PSS), and overall survival (OS) was studied. RESULTS Pretreatment characteristics and assigned treatment arm were not significantly different between the parent and test groups except for a significantly higher risk of death (P = 0.049) in the test group. The median CAG repeat was 19. There were no significant differences in stage, or Gleason score between high (19 or greater) and low CAG (<19) patients within each treatment group. Number of CAG repeats alone did not significantly influence LF, DM, PSS, and OS. However, when the CAG repeat outcome was studied in conjunction with androgen deprivation therapy, patients with CAG <19 who received H + RT had improved local control as compared with patients who received RT alone (P = 0.026, 5-year rates 4.6% versus 36.4%) and improved local control over patients with CAG > or =19 that received H + RT (P = 0.028). CONCLUSIONS Patients with short CAG repeats show a local control benefit with short-term androgen deprivation therapy, but no improvement in survival.
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Affiliation(s)
- May Abdel-Wahab
- Department of Radiation Oncology, University of Miami School of Medicine, Miami, FL 33136, USA.
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Shin T, Sumiyoshi H, Matsuo N, Satoh F, Nomura Y, Mimata H, Yoshioka H. Sp1 and Sp3 transcription factors upregulate the proximal promoter of the human prostate-specific antigen gene in prostate cancer cells. Arch Biochem Biophys 2005; 435:291-302. [PMID: 15708372 DOI: 10.1016/j.abb.2005.01.002] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2004] [Revised: 12/31/2004] [Indexed: 11/18/2022]
Abstract
The serum level of prostate-specific antigen (PSA) is useful as a clinical marker for diagnosis and assessment of the progression of prostate cancer, and in evaluating the effectiveness of treatment. We characterized four Sp1/Sp3 binding sites in the proximal promoter of the PSA gene. In a luciferase assay, these sites contributed to the basal promoter activity in prostate cancer cells. In an electrophoretic mobility shift assay and chromatin immunoprecipitation assay, we confirmed that Sp1 and Sp3 bind to these sites. Overexpression of wild-type Sp1 and Sp3 further upregulated the promoter activity, whereas overexpression of the Sp1 dominant-negative form or addition of mithramycin A significantly reduced the promoter activity and the endogenous mRNA level of PSA. Among the four binding sites, a GC box located at nucleotides -53 to -48 was especially critical for basal promoter activity. These results indicate that Sp1 and Sp3 are involved in the basal expression of PSA in prostate cancer cells.
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Affiliation(s)
- Toshitaka Shin
- Department of Anatomy, Biology, and Medicine, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Oita 879-5593, Japan.
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Yousef GM, Obiezu CV, Luo LY, Magklara A, Borgoño CA, Kishi T, Memari N, Michael LP, Sidiropoulos M, Kurlender L, Economopolou K, Kapadia C, Komatsu N, Petraki C, Elliott M, Scorilas A, Katsaros D, Levesque MA, Diamandis EP. Human Tissue Kallikreins: From Gene Structure to Function and Clinical Applications. Adv Clin Chem 2005; 39:11-79. [PMID: 16013667 DOI: 10.1016/s0065-2423(04)39002-5] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Affiliation(s)
- George M Yousef
- Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
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Cinar B, Yeung F, Konaka H, Mayo MW, Freeman MR, Zhau HE, Chung LWK. Identification of a negative regulatory cis-element in the enhancer core region of the prostate-specific antigen promoter: implications for intersection of androgen receptor and nuclear factor-kappaB signalling in prostate cancer cells. Biochem J 2004; 379:421-31. [PMID: 14715080 PMCID: PMC1224078 DOI: 10.1042/bj20031661] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2003] [Revised: 01/07/2004] [Accepted: 01/09/2004] [Indexed: 11/17/2022]
Abstract
The NF-kappaB (nuclear factor-kappaB) transcription factors mediate activation of a large number of gene promoters containing diverse kappaB-site sequences. Here, PSA (prostate-specific antigen) was used as an AR (androgen receptor)-responsive gene to examine the underlying mechanism by which the NF-kappaB p65 transcription factor down-regulates the transcriptional activity of AR in cells. We observed that activation of NF-kappaB by TNFalpha (tumour necrosis factor alpha) inhibited both basal and androgen-stimulated PSA expression, and that this down-regulation occurred at the promoter level, as confirmed by the super-repressor IkappaBalpha (S32A/S36A), a dominant negative inhibitor of NF-kappaB. Using a linker-scanning mutagenesis approach, we identified a cis -element, designated XBE (X-factor-binding element), in the AREc (androgen response element enhancer core) of the PSA promoter, which negatively regulated several AR-responsive promoters, including that of PSA. When three copies of XBE in tandem were juxtaposed to GRE4 (glucocorticoid response element 4), a 4-6-fold reduction of inducible GRE4 activity was detected in three different cell lines, LNCaP, ARCaP-AR and PC3-AR. Bioinformatics and molecular biochemical studies indicated that XBE is a kappaB-like element that binds specifically to the NF-kappaB p65 subunit; consistent with these observations, only NF-kappaB p65, but not the NF-kappaB p50 subunit, was capable of inhibiting AR-mediated PSA promoter transactivation in LNCaP cells. In addition, our data also showed that AR binds to XBE, as well as to the kappaB consensus site, and that the transfection of AR inhibits the kappaB-responsive promoter in transient co-transfection assays. Collectively, these data indicate that cross-modulation between AR and NF-kappaB p65 transcription factors may occur by a novel mechanism involving binding to a common cis -DNA element.
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Affiliation(s)
- Bekir Cinar
- Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA
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Koochekpour S, Maresh GA, Katner A, Parker-Johnson K, Lee TJ, Hebert FE, Kao YS, Skinner J, Rayford W. Establishment and characterization of a primary androgen-responsive African-American prostate cancer cell line, E006AA. Prostate 2004; 60:141-52. [PMID: 15162380 DOI: 10.1002/pros.20053] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
BACKGROUND Establishment of human prostate cancer cell lines is essential to advance our understanding of complex processes associated with the initiation and progression of the disease. In the present study, we report the establishment of a primary African-American prostate cancer cell line (E006AA) as well as its associated stromal cells (S006AA). METHODS E006AA cell line was established as a spontaneously immortalized cells from a patient with a clinically localized prostate cancer. Extensive characterization of the cells was accomplished using androgen-dependent growth and sensitivity assays, Western analyses, RT-PCR/real-time PCR, cytogenetic analyses, and tumorigenicity in nude mice. RESULTS E006AA cell line shows androgen-dependent growth, expresses PSA and the androgen receptor (AR) with 26 CAG repeats in exon 1 of AR. Cytogenetic analyses revealed a hypertriploid karyotype with additional numerical gains in chromosomes 5, 6, 8, 10, 17, 20, 21 and a marker chromosome of unknown origin as well as structural abnormalities in chromosomes 4, 5, 8, 9, 11-14, 18, and 20. This cell line is not tumorigenic in nude mice. S006AA cell line, isolated from the same tumor specimen, also expresses AR and shows the morphological characteristics of smooth muscle cells of prostatic stroma. CONCLUSIONS These cell lines are the first available primary epithelial and stromal cells derived from an African-American patient with organ-confined prostate cancer and in conjunction with other established cell lines, could provide an in vitro model system to investigate early transforming events in prostate cancer.
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Affiliation(s)
- Shahriar Koochekpour
- Department of Biochemistry and Molecular Biology, Louisiana State University-Health Sciences Center, New Orleans, 70112, USA
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Borgoño CA, Michael IP, Diamandis EP. Human Tissue Kallikreins: Physiologic Roles and Applications in Cancer. Mol Cancer Res 2004. [DOI: 10.1158/1541-7786.257.2.5] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
Abstract
Tissue kallikreins are members of the S1 family (clan SA) of trypsin-like serine proteases and are present in at least six mammalian orders. In humans, tissue kallikreins (hK) are encoded by 15 structurally similar, steroid hormone–regulated genes (KLK) that colocalize to chromosome 19q13.4, representing the largest cluster of contiguous protease genes in the entire genome. hKs are widely expressed in diverse tissues and implicated in a range of normal physiologic functions from the regulation of blood pressure and electrolyte balance to tissue remodeling, prohormone processing, neural plasticity, and skin desquamation. Several lines of evidence suggest that hKs may be involved in cascade reactions and that cross-talk may exist with proteases of other catalytic classes. The proteolytic activity of hKs is regulated in several ways including zymogen activation, endogenous inhibitors, such as serpins, and via internal (auto)cleavage leading to inactivation. Dysregulated hK expression is associated with multiple diseases, primarily cancer. As a consequence, many kallikreins, in addition to hK3/PSA, have been identified as promising diagnostic and/or prognostic biomarkers for several cancer types, including ovarian, breast, and prostate. Recent data also suggest that hKs may be causally involved in carcinogenesis, particularly in tumor metastasis and invasion, and, thus, may represent attractive drug targets to consider for therapeutic intervention.
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Affiliation(s)
- Carla A. Borgoño
- Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
| | - Iacovos P. Michael
- Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
| | - Eleftherios P. Diamandis
- Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
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Abstract
Multiple factors contribute to the high incidence and prevalence of prostate cancer including race, ethnicity, diet, environment, widespread awareness through prostate-specific antigen screening and genetics. Linkage analysis has identified several candidate sites for hereditary prostate cancer gene loci. Molecular studies have also identified genes that are frequently altered in sporadic prostate cancer. It appears that due to the heterogeneity of prostate cancer, multiple genes may be involved in the neoplastic process.
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Affiliation(s)
- Mark A Rubin
- Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115, USA.
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Bostwick DG, Burke HB, Djakiew D, Euling S, Ho SM, Landolph J, Morrison H, Sonawane B, Shifflett T, Waters DJ, Timms B. Human prostate cancer risk factors. Cancer 2004; 101:2371-490. [PMID: 15495199 DOI: 10.1002/cncr.20408] [Citation(s) in RCA: 401] [Impact Index Per Article: 19.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Prostate cancer has the highest prevalence of any nonskin cancer in the human body, with similar likelihood of neoplastic foci found within the prostates of men around the world regardless of diet, occupation, lifestyle, or other factors. Essentially all men with circulating androgens will develop microscopic prostate cancer if they live long enough. This review is a contemporary and comprehensive, literature-based analysis of the putative risk factors for human prostate cancer, and the results were presented at a multidisciplinary consensus conference held in Crystal City, Virginia, in the fall of 2002. The objectives were to evaluate known environmental factors and mechanisms of prostatic carcinogenesis and to identify existing data gaps and future research needs. The review is divided into four sections, including 1) epidemiology (endogenous factors [family history, hormones, race, aging and oxidative stress] and exogenous factors [diet, environmental agents, occupation and other factors, including lifestyle factors]); 2) animal and cell culture models for prediction of human risk (rodent models, transgenic models, mouse reconstitution models, severe combined immunodeficiency syndrome mouse models, canine models, xenograft models, and cell culture models); 3) biomarkers in prostate cancer, most of which have been tested only as predictive factors for patient outcome after treatment rather than as risk factors; and 4) genotoxic and nongenotoxic mechanisms of carcinogenesis. The authors conclude that most of the data regarding risk relies, of necessity, on epidemiologic studies, but animal and cell culture models offer promise in confirming some important findings. The current understanding of biomarkers of disease and risk factors is limited. An understanding of the risk factors for prostate cancer has practical importance for public health research and policy, genetic and nutritional education and chemoprevention, and prevention strategies.
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Lee SO, Lou W, Hou M, Onate SA, Gao AC. Interleukin-4 enhances prostate-specific antigen expression by activation of the androgen receptor and Akt pathway. Oncogene 2003; 22:7981-8. [PMID: 12970746 DOI: 10.1038/sj.onc.1206735] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Androgen receptor (AR) plays an important role in the development and progression of prostate cancer upon the action of androgen through the binding of the androgen-responsive elements (AREs) on the target genes. Abnormal activation of the AR by nonandrogen has been implicated in the progression of androgen-independent prostate cancer. The levels of interleukin-4 (IL-4) are significantly elevated in sera of patients with hormone refractory prostate cancer. The potential role of IL-4 on the activation of AR was investigated in prostate cancer cells. IL-4 enhances AR-mediated prostate-specific antigen (PSA) expression and ARE-containing gene activity through activation of the AR in the androgen ablation condition in human prostate cancer cells. The AR can also be sensitized by IL-4 and activated by significantly lower levels of androgen (10 pM of R1881) in prostate cancer cells. IL-4 enhances nuclear translocation of AR and increases binding of the AR to the ARE in LNCaP prostate cancer cells. Blocking of the Akt pathway by an Akt-specific inhibitor LY294002 abrogates IL-4-induced PSA expression and AR signaling. These results demonstrate that IL-4 enhances PSA expression through activation of the AR and Akt signaling pathways in LNCaP prostate cancer cells. Understanding IL-4-induced signaling leading to abnormal activation of AR will provide insights into the molecular mechanisms of androgen-independent progression of prostate cancer cells.
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Affiliation(s)
- Soo Ok Lee
- Department of Medicine and Pharmacology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
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Franco OE, Onishi T, Yamakawa K, Arima K, Yanagawa M, Sugimura Y, Kawamura J. Mitogen-activated protein kinase pathway is involved in androgen-independent PSA gene expression in LNCaP cells. Prostate 2003; 56:319-25. [PMID: 12858361 DOI: 10.1002/pros.10258] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
BACKGROUND Prostate specific antigen (PSA) is regulated by growth factors and hormones through functional androgen responsive elements in the promoter region of the PSA gene. However, the molecular basis for androgen independent PSA elevation in hormone refractory prostate cancer is unknown. The purpose of this study was to investigate the role of MAP kinase activation in androgen independent regulation of PSA expression. METHODS LNCaP cells transfected with MEK1 expression vector with or without the MAP kinase inhibitor U0126 under low androgen conditions were analyzed by luciferase assay and electrophoretic mobility shift assay (EMSA). RESULTS Transfection experiments of the proximal PSA promoter linked to Luc-reporter identified one region designated as "B" motif centered at -60 bp to be essential for basal activation. Co-transfection with the MEK1 activated vector enhanced PSA expression, while mutation of the "B" motif totally abrogated this induction. EMSA showed a specific DNA-protein complex, but Sp1 family members and AR do not interact with the "B" region by supershift analysis. CONCLUSIONS Our data suggest that enhanced androgen-independent PSA gene expression in MAP kinase-induced LNCaP cells is mediated, at least in part, by the "B" motif of the PSA promoter.
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Affiliation(s)
- Omar E Franco
- Department of Urology, Mie University School of Medicine, Tsu, Mie, Japan
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Edwards J, Krishna NS, Grigor KM, Bartlett JMS. Androgen receptor gene amplification and protein expression in hormone refractory prostate cancer. Br J Cancer 2003; 89:552-6. [PMID: 12888829 PMCID: PMC2394367 DOI: 10.1038/sj.bjc.6601127] [Citation(s) in RCA: 263] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
This study examined androgen receptor (AR) gene amplification and protein expression in 102 matched paired hormone sensitive and resistant tumours from 51 patients. AR gene amplification and X chromosome copy number were assessed by fluorescent in situ hybridisation, and protein expression was assessed by immunohistochemistry. All tumours were stained for PSA protein expression. Significantly more tumours exhibited AR amplification following the development of hormone resistance (20%, 10 out of 49) compared to matched hormone-sensitive tumours from the same patient (2%, one out of 48) (P=0.0085). The level of AR expression was significantly higher in hormone-resistant tumours compared to matched hormone-sensitive tumours from the same patient (130, interquartile range, 55-167 vs 94.5 interquartile range, 55-120, P=0.019). AR expression levels in hormone-resistant tumours with and without AR amplification were not significantly different. However, an increase in AR expression was seen with the development of AR amplification in paired tumours. The rate of AR gene amplification and/or an increase in AR protein expression during androgen resistant is too low to wholly explain the development of androgen resistance. Alternative mechanisms for modulating the function of the AR, or other signalling pathways, must be considered as key factors in the development of hormone-resistant prostate.
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Affiliation(s)
- J Edwards
- Endocrine Cancer Group, Surgical and Translational research section, Division of Cancer Sciences and Molecular Pathology, Glasgow Royal Infirmary, Glasgow G31 2ER, Scotland
| | - N S Krishna
- Endocrine Cancer Group, Surgical and Translational research section, Division of Cancer Sciences and Molecular Pathology, Glasgow Royal Infirmary, Glasgow G31 2ER, Scotland
| | - K M Grigor
- University Department of Pathology, Edinburgh Royal Infirmary, Edinburgh EH8 9AG, Scotland
| | - J M S Bartlett
- Endocrine Cancer Group, Surgical and Translational research section, Division of Cancer Sciences and Molecular Pathology, Glasgow Royal Infirmary, Glasgow G31 2ER, Scotland
- Endocrine Cancer Group, Surgical and Translational Research Section, Division of Cancer Sciences and Molecular Pathology, University Department of Surgery, Level II, Queen Elizabeth Building, Glasgow Royal Infirmary, Glasgow, G31 2ER, Scotland. E-mail:
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Cheng WS, Giandomenico V, Pastan I, Essand M. Characterization of the androgen-regulated prostate-specific T cell receptor gamma-chain alternate reading frame protein (TARP) promoter. Endocrinology 2003; 144:3433-40. [PMID: 12865322 DOI: 10.1210/en.2003-0121] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
TARP (T cell receptor gamma-chain alternate reading frame protein) is uniquely expressed in males in prostate epithelial cells and prostate cancer cells. Here we demonstrate that TARP expression is regulated by testosterone at the transcriptional level through specific binding of androgen receptor to an androgen response element in the proximal TARP promoter. We further demonstrate that the promoter specifically initiates reporter gene expression in TARP-positive prostate cancer cell lines. To develop a regulatory sequence for prostate-specific gene expression, we constructed a chimeric sequence consisting of the TARP promoter and the prostate-specific antigen (PSA) enhancer. We found that in the prostatic adenocarcinoma cell line LNCaP, the transcriptional activity of the regulatory sequence consisting of a TARP promoter and PSA enhancer is 20 times higher than the activity of a regulatory sequence consisting of the PSA promoter and PSA enhancer. Thus, our studies define a regulatory sequence that may be used to restrict expression of therapeutic genes to prostate cancer cells and may therefore play a role in prostate cancer gene therapy.
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Affiliation(s)
- Wing-Shing Cheng
- Clinical Immunology, Rudbeck Laboratory, Uppsala University, SE-75185 Uppsala, Sweden
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44
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Steketee K, Timmerman L, Ziel-van der Made ACJ, Doesburg P, Brinkmann AO, Trapman J. Broadened ligand responsiveness of androgen receptor mutants obtained by random amino acid substitution of H874 and mutation hot spot T877 in prostate cancer. Int J Cancer 2002; 100:309-17. [PMID: 12115546 DOI: 10.1002/ijc.10495] [Citation(s) in RCA: 104] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
In a subset of endocrine therapy-resistant prostate cancers, amino acid substitutions H874Y, T877A and T877S, which broaden ligand specificity of the ligand binding domain (LBD) of the androgen receptor (AR), have been detected. To increase our knowledge of the role of amino acid substitutions at these specific positions in prostate cancer, codons 874 and 877 were subjected to random mutagenesis. AR mutants were screened in a yeast readout system for responsiveness to 5 alpha-dihydrotestosterone, progesterone and dehydroepiandrosterone. At position 874, only the histidine to tyrosine substitution could broaden AR ligand specificity. At position 877, 4 ligand specificity broadening substitutions were found: T877A, T877S, T877C and T877G. The latter 2 were not found in prostate cancer. The AR mutants were tested in mammalian (Hep3B) cells for responsiveness to 13 different ligands. All mutants displayed their own ligand specificity spectrum. Importantly, AR(H874Y) and AR(T877A) could be activated by cortisol. According to the 3-dimensional structure of the AR LBD, T877 interacts directly with the 17 beta-hydroxyl group of androgens. All amino acid substitutions identified at position 877 had smaller side chains than the threonine in the wild-type receptor, indicating that increased space in the ligand binding pocket is important in broadened ligand specificity. Because H874 does not interact directly with the ligand, its substitution by a tyrosine is expected to change the ligand binding pocket conformation indirectly. For T877C and T877G substitutions, 2-point mutations are required, and for H874Y, T877A and T877S substitutions, only a 1-point mutation is sufficient. This most likely explains that the latter 3 have been found in prostate cancer.
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Affiliation(s)
- Karine Steketee
- Department of Pathology, Josephine Nefkens Institute, Erasmus University, Rotterdam, The Netherlands
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45
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Abstract
Serine proteases are proteolytic enzymes with an active serine residue in their catalytic site. Kallikreins are a subgroup of the serine protease family which is known to have diverse physiological functions. The human kallikrein gene family has now been fully characterized and includes 15 members tandemly located on chromosome 19q13.4. Here we discuss the common structural features of kallikreins at the DNA, mRNA and protein levels and summarize their tissue expression and hormonal regulation patterns. Kallikreins are expressed in many tissues including the salivary gland, endocrine tissues such as testis, prostate, breast and endometrium, and in the central nervous system. Most genes appear to be under steroid hormone regulation. The occurrence of several splice variants is common among kallikreins, and some of the splice variants seem to be tissue-specific and might be related to certain pathological conditions. Kallikreins are secreted in an inactive 'zymogen' form which is activated by cleavage of an N-terminal peptide. Some kalikreins can undergo autoactivation while others may be activated by other kallikreins or other proteases. Most kallikreins are predicted to have trypsin-like enzymatic activity except three which are probably chymotrypsin-like. New, but mainly circumstantial evidence, suggests that at least some kallikreins may be part of a novel enzymatic cascade pathway which is turned-on in aggressive forms of ovarian and probably other cancers.
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Affiliation(s)
- George M Yousef
- Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada
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46
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Gurova KV, Roklin OW, Krivokrysenko VI, Chumakov PM, Cohen MB, Feinstein E, Gudkov AV. Expression of prostate specific antigen (PSA) is negatively regulated by p53. Oncogene 2002; 21:153-7. [PMID: 11791186 DOI: 10.1038/sj.onc.1205001] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2001] [Revised: 09/17/2001] [Accepted: 09/20/2001] [Indexed: 11/09/2022]
Abstract
Although prostate-specific antigen (PSA) is considered a uniquely important tumor marker and is broadly used for early detection of prostate cancer, the molecular mechanisms underlying its elevated expression in tumors have been unknown. By using cDNA microarray gene expression profiling, we found a fourfold increase in the PSA mRNA level in prostatic carcinoma cell line LNCaP, in which the p53 pathway was suppressed by a dominant negative p53 mutant. Consistently, p53 suppression caused a 4-8-fold increase in secretion of PSA protein in culture medium, suggesting that PSA gene expression is under negative control of p53. While wild type p53 strongly repressed, dominant negative p53 mutants stimulated PSA promoter-driven transcription and secretion of PSA in transient transfection experiments. The inhibitory effect of wild type p53 was undetectable in the presence of trichostatin A, suggesting the involvement of histone deacetylation in negative regulation of PSA promoter activity. Thus, PSA is likely to be a tissue specific indicator of transformation-associated p53 suppression in prostate cells. This finding provides a plausible explanation for a frequent increase of PSA levels in advanced prostate cancer.
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MESH Headings
- Acetylation
- Adenocarcinoma/genetics
- Adenocarcinoma/metabolism
- Adenocarcinoma/pathology
- Chloramphenicol O-Acetyltransferase/biosynthesis
- Chloramphenicol O-Acetyltransferase/genetics
- Culture Media, Conditioned
- DNA, Complementary/genetics
- Gene Expression Regulation, Neoplastic/physiology
- Genes, Dominant
- Genes, Reporter
- Genes, p53
- Humans
- Hydroxamic Acids/pharmacology
- Male
- Neoplasm Proteins/deficiency
- Neoplasm Proteins/genetics
- Neoplasm Proteins/metabolism
- Neoplasm Proteins/physiology
- Oligonucleotide Array Sequence Analysis
- Promoter Regions, Genetic/genetics
- Prostate-Specific Antigen/biosynthesis
- Prostate-Specific Antigen/genetics
- Prostate-Specific Antigen/metabolism
- Prostatic Neoplasms/genetics
- Prostatic Neoplasms/metabolism
- Prostatic Neoplasms/pathology
- Protein Processing, Post-Translational
- RNA, Messenger/biosynthesis
- RNA, Messenger/genetics
- RNA, Neoplasm/biosynthesis
- RNA, Neoplasm/genetics
- Recombinant Fusion Proteins/biosynthesis
- Transcription, Genetic
- Tumor Cells, Cultured/metabolism
- Tumor Suppressor Protein p53/deficiency
- Tumor Suppressor Protein p53/physiology
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Affiliation(s)
- Katerina V Gurova
- Department of Molecular Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio, OH 44195, USA
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47
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Clegg N, Eroglu B, Ferguson C, Arnold H, Moorman A, Nelson PS. Digital expression profiles of the prostate androgen-response program. J Steroid Biochem Mol Biol 2002; 80:13-23. [PMID: 11867260 DOI: 10.1016/s0960-0760(01)00167-4] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
The androgen receptor (AR) and cognate ligands regulate vital aspects of prostate cellular growth and function including proliferation, differentiation, apoptosis, lipid metabolism, and secretory action. In addition, the AR pathway also influences pathological processes of the prostate such as benign prostatic hypertrophy and prostate carcinogenesis. The pivotal role of androgens and the AR in prostate biology prompted this study with the objective of identifying molecular mediators of androgen action. Our approach was designed to compare transcriptomes of the LNCaP prostate cancer cell line under conditions of androgen depletion and androgen stimulation by generating and comparing collections of expressed sequence tags (ESTs). A total of 4400 ESTs were produced from LNCaP cDNA libraries and these ESTs assembled into 2486 distinct transcripts. Rigorous statistical analysis of the expression profiles indicated that 17 genes exhibited a high probability (P>0.9) of androgen-regulated expression. Northern analysis confirmed that the expression of KLK3/PSA, FKBP5, KRT18, DKFZP564K247, DDX15, and HSP90 is regulated by androgen exposure. Of these, only KLK3/PSA is known to be androgen-regulated while the other genes represent new members of the androgen-response program in prostate epithelium. LNCaP gene expression profiles defined by two independent experiments using the serial analysis of gene expression (SAGE) method were compared with the EST profiles. Distinctly different expression patterns were produced from each dataset. These results are indicative of the sensitivity of the methods to experimental conditions and demonstrate the power and the statistical limitations of digital expression analyses.
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Affiliation(s)
- Nigel Clegg
- Division of Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA
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48
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Fillmore RA, Dean DA, Zimmer WE. The smooth muscle gamma-actin gene is androgen responsive in prostate epithelia. Gene Expr 2002; 10:201-11. [PMID: 12450213 PMCID: PMC5977519 DOI: 10.3727/000000002783992424] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/01/2002] [Indexed: 11/24/2022]
Abstract
Nkx 3.1 is an evolutionarily conserved vertebrate homolog of the Drosophila Nk-3 homeodomain gene bagpipe that is expressed by a variety of cells during early mammalian development and has been shown to be a critical factor for prostate development and function. Previous studies utilizing a heterologous cell transfection strategy from our laboratory identified the smooth muscle gamma-actin (SMGA) gene as a novel molecular target of Nkx 3.1 regulatory activity. In the studies presented here, SMGA gene activity and regulation were evaluated in normal and cancerous prostate epithelial cells. SMGA transcripts were demonstrated in prostate epithelia and SMGA mRNA levels were increased in androgen-responsive LNCaP cancer and normal prostate epithelial cells. SMGA gene transcriptional activity was androgen responsive in these cells and required a segment of the human SMGA promoter containing NKE and SRF (serum response factor) binding elements. This region of the human SMGA proximal promoter is well conserved across species and is synergistically activated by coexpression of Nkx 3.1 and SRF in heterologous CV-1 cells. SMGA transcription was not responsive to steroid in PC-3 prostate epithelial cancer cells, which do not express Nkx 3.1. However, SMGA transcription was influenced by expression of androgen receptor in these cells, a situation that allows the androgen-dependent expression of Nkx 3.1. Furthermore, SMGA gene activity was influenced by direct Nkx 3.1 expression in the PC-3 cells. Thus, SMGA gene activity in prostate epithelia is due, in part, to the androgen-dependent expression of Nkx 3.1. As such, our studies provide the initial description of Nkx 3.1 target gene regulatory activity in the prostate.
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Affiliation(s)
- R. A. Fillmore
- *Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, AL 36688
| | - D. A. Dean
- †Department of Microbiology and Immunology, University of South Alabama, Mobile, AL 36688
| | - W. E. Zimmer
- *Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, AL 36688
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49
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Gross M, Liu B, Tan J, French FS, Carey M, Shuai K. Distinct effects of PIAS proteins on androgen-mediated gene activation in prostate cancer cells. Oncogene 2001; 20:3880-7. [PMID: 11439351 DOI: 10.1038/sj.onc.1204489] [Citation(s) in RCA: 133] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2001] [Revised: 03/20/2001] [Accepted: 03/26/2001] [Indexed: 11/09/2022]
Abstract
Androgen signaling influences the development and growth of prostate carcinoma. The transcriptional activity of androgen receptor (AR) is regulated by positive or negative transcriptional cofactors. We report here that PIAS1, PIAS3, and PIASy of the protein inhibitor of activated STAT (PIAS) family, which are expressed in human prostate, display distinct effects on AR-mediated gene activation in prostate cancer cells. While PIAS1 and PIAS3 enhance the transcriptional activity of AR, PIASy acts as a potent inhibitor of AR in prostate cancer cells. The effects of PIAS proteins on AR are competitive. PIASy binds to AR but does not affect the DNA binding activity of AR. An NH2-terminal LXXLL signature motif of PIASy, although not required for PIASy-AR interaction, is essential for the transrepression activity of PIASy. Our results identify PIASy as a transcriptional corepressor of AR and suggest that different PIAS proteins have distinct effects on AR signaling in prostate cancer cells.
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Affiliation(s)
- M Gross
- Division of Hematology-Oncology, Department of Medicine, University of California, Los Angeles, California, CA 90095, USA
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50
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Xu LL, Su YP, Labiche R, Segawa T, Shanmugam N, McLeod DG, Moul JW, Srivastava S. Quantitative expression profile of androgen-regulated genes in prostate cancer cells and identification of prostate-specific genes. Int J Cancer 2001; 92:322-8. [PMID: 11291065 DOI: 10.1002/ijc.1196] [Citation(s) in RCA: 66] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Quantitative expression profile of androgen-regulated genes (ARGs) was evaluated in the hormone-responsive prostate cancer cell line LNCaP by serial analysis of gene expression (SAGE). A total of 83,489 SAGE tags representing 23,448 known genes or expressed sequence tags (ESTs) and 1,655 potentially novel sequences have unraveled the transcriptome of LNCaP cells, the most common cell line used in prostate cancer research. Comparison of transcripts between control and R1881-treated LNCaP cells revealed the induction of 136 genes and repression of 215 genes in response to androgen (p < 0.05). Strikingly, a high fraction ( approximately 90%) of ARGs identified in our study has not been described as ARGs previously. A number of prostate-specific transcription factors were among the ARGs identified here. Classification of the ARGs on the basis of biochemical functions revealed that a great majority of ARGs identified in our experimental system appear to be involved in regulation of transcription, splicing, ribosomal biogenesis, mitogenesis, bioenergetics and redox processes. One of the novel aspects of androgen signaling included androgen regulation of genes involved in DNA repair/recombination process. By comparing our LNCaP-C and LNCaP-T SAGE libraries with SAGE tag libraries available at the NCBI-SAGE website, we have identified >200 potential prostate specific/abundant transcripts. The discovery of new prostate-specific genes and ARGs provides a unique opportunity to determine the role of these genes in prostate cell growth, differentiation and tumorigenesis.
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Affiliation(s)
- L L Xu
- Center for Prostate Disease Research (CPDR), Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA
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