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Steroid 5A-Reductase Isozymes I and II in Recurrent Prostate Cancer Mark A.Titus,4 Christopher W

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Steroid 5A-Reductase Isozymes I and II in Recurrent Prostate Cancer Mark A.Titus,4 Christopher W Imaging, Diagnosis, Prognosis Steroid 5A-Reductase Isozymes I and II in Recurrent Prostate Cancer Mark A.Titus,4 Christopher W. Gregory,1,4 O. Harris Ford III,4 Michael J. Schell, 3,4 Susan J. Maygarden,1andJames L. Mohler1,2,4,5,6 Abstract Purpose: Prostate cancer recurs during androgen deprivation therapy despite reduced circulating androgens. We showed that recurrent prostate cancer tissue has testosterone levels similar to androgen-stimulated benign prostate, whereas dihydrotestosterone levels were reduced 82% to 1.45 nmol/L, sufficient for androgen receptor activation.The altered testosterone/dihydrotestos- terone ratio in recurrent prostate cancer suggests loss of 5a-reducing capability. The aim of this study was to characterize steroid 5a-reductase isozymes I (S5aRI) and II (S5 aRII) in prostate tissues. Experimental Design: A tissue microarray was constructed from 22 recurrent prostate cancer specimens and matched pairs of androgen-stimulated benign prostate and androgen-stimulated prostate cancer from 23 radical prostatectomy specimens. Immunoblots were constructed from eight recurrent prostate cancers, eight androgen-stimulated benign prostate, and eight androgen- stimulated prostate cancer specimens. Isozyme expression was examined in microarray sections and immunoblots using S5aRI and S5 aRII polyclonal antibodies. Isozyme activities were mea- sured in 12 recurrent prostate cancer, 12 androgen-stimulated benign prostate, and 12 androgen- stimulated prostate cancer specimens. Results: Nuclear immunostaining exhibited higher S5aRI expression than S5aRII in recurrent prostate cancer, androgen-stimulated benign prostate, and androgen-stimulated prostate cancers (P < 0.0001); mean expression was125, 150, and 115 for S5aRI versus 10, 29, and 37 for S5aRII, respectively. Cytoplasmic immunostaining was moderate and similar for both isozymes in the three tissue types (P > 0.05). Immunoblots confirmed immunohistochemistry; S5aRI was expressed in recurrent prostate cancer specimens and S5aRII was not detected. The activity of S5aRI(114.4pmol/mgepithelialprotein/minute)was3.7-foldhigherthanS5aRII (30.7 pmol/ mg epithelial protein/minute) in recurrent prostate cancer specimens. Conclusions: Expression levels and isozyme activity shifts from S5aRII toward S5 aRI in recur- rent prostate cancer. Dual inhibition of S5aRI and S5aRIIshould reduce dihydrotestosterone bio- synthesis and may prevent or delay growth of recurrent prostate cancer. Androgen target cells in peripheral tissues use testosterone to metabolize testosterone to dihydrotestosterone. The adrenal activate androgen receptor, which interacts with androgen androgen androstenedione is also converted to 5a-reduced response elements in DNA to regulate gene transcription. androstanedione by steroid 5a-reductase in the prostate (2) Alternatively, intracellular testosterone acts as a prohormone and androstenedione has been implicated as a source of that is converted to dihydrotestosterone, a more potent dihydrotestosterone in prostate tissue after castration (3). androgen receptor ligand. In the prostate, an intracrine pathway Steroid 5a-reductase is a membrane-associated, NADPH- (1) uses the enzyme steroid 5a-reductase (EC 1.3.99.5) to dependent enzyme that catalyzes the irreversible stereospecific reduction of C19 3-keto-D4-5 steroid to 5a-reduced metabolites. Steroid 5a-reductase isozymes I (S5aRI) and II (S5aRII) in Authors’Affiliations: Departments of 1Pathology and Laboratory Medicine, humans are composed of 260 and 254 amino acids, respec- 2Surgery, 3Biostatistics, and 4UNC Lineberger Comprehensive Cancer Center, tively, with 47% sequence identity and distinct biochemical University of North Carolina School of Medicine, Chapel Hill, North Carolina, properties (4). S5aRI exhibits a broad neutral to basic optimum 5Department of Urologic Oncology, Roswell Park Cancer Institute, and 6Department of Urology, University at Buffalo School of Medicine and Biotechnology, Buffalo, pH range, whereas S5aRII displays a narrow acid pH optimum New York for maximum activity. Both isozymes contain an NH2-terminal Received 4/15/04; revised 3/9/05; accepted 3/17/05. steroid binding domain and a COOH-terminal NADPH Grant support: National Cancer Institute grant CA-77739. binding domain. S5aRI has higher turnover and decreased The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance substrate affinity, whereas S5aRII has lower turnover and with 18 U.S.C. Section 1734 solely to indicate this fact. increased substrate affinity. The apparent dissociation constant Note: C.W. Gregory is currently at Voyager Pharmaceutical Corporation, for NADPH cofactor is similar for both isozymes. Raleigh, NC. Prostate cancer is the second leading cause of death and the Requests for reprints: Mark A. Titus, UNC Lineberger Comprehensive Cancer Center, CB#7295, University of North Carolina, Chapel Hill, NC 27599-7295. most common non–skin cancer in American men. Current Phone: 919-966-9257; Fax: 919-966-3015; E-mail: [email protected]. estimates predict 232,090 new cases of prostate cancer in F 2005 American Association for Cancer Research. American men and 30,350 deaths from prostate cancer in 2004 www.aacrjournals.org 4365 Clin Cancer Res 2005;11(12) June 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. Imaging, Diagnosis, Prognosis (5). Men who fail curative therapy or present with advanced steroid enzymes. The patients with androgen-stimulated benign disease usually receive androgen deprivation therapy that causes prostate had a mean age of 63 years (range 50-75). Patients with regression of androgen-dependent prostate cancer through androgen-stimulated prostate cancer had a mean age of 54 years (range programmed cell death (6). However, androgen deprivation 39-76) and Gleason sums were 6 or 7. Patients with recurrent prostate cancer had a mean age of 71 years (range 60-86) and Gleason sums therapy is palliative in advanced prostate cancer (7) because ranged from 8 to 10. prostate cancer recurs and almost always causes death. A An immunoblot was constructed from frozen prostate tissue molecular role for androgen receptor in the transition from specimen lysates from 24 different men (19) that yielded eight samples androgen-stimulated prostate cancer to recurrent prostate cancer each of androgen-stimulated benign prostate, androgen-stimulated is supported by androgen receptor expression in recurrent prostate cancer, and recurrent prostate cancer. The androgen-stimulated prostate cancer (8–10) and expression of androgen-regulated benign prostate patients had a mean age of 65 years (range 58-71). genes (10–12). Potential mechanisms include androgen recep- Androgen-stimulated prostate cancer patients had a mean age of 59 tor amplification (13), androgen receptor gene mutations years (range 41-71) and Gleason sums were 6 or 7. Recurrent prostate leading to transactivation by low levels of dihydrotestosterone cancer patients had a mean age of 71 years (range 60-86) and Gleason (14) or promiscuous ligand binding (15) and posttranslational sums ranged from 8 to 10. modification of androgen receptor through peptide growth Microarray construction. A high-density tissue microarray was constructed using formalin-fixed, paraffin-embedded human prostate factor signaling (16, 17). In addition to these mechanisms, our specimens as previously described (20). The tissue microarrays were investigations have recently found tissue levels of testosterone constructed with matched pairs of androgen-stimulated benign prostate and dihydrotestosterone in recurrent prostate cancer that are and prostate cancer from radical prostatectomy specimens from 23 men sufficient for activation of even wild-type androgen receptor and recurrent prostate cancer from transurethral resection specimens (10). However, lack of efficacy of finasteride, a S5aRII-specific from 22 men using the Beecher Instruments (Silver Spring, MD) inhibitor, suggests that S5aRII does not contribute significantly manual tissue arrayer. Benign prostate obtained by transurethral to dihydrotestosterone production (18). resection, colon cancer, and mouse liver cores were included as internal We investigated the expression levels, subcellular location controls for standardization. and in vitro enzymatic activity of S5aR isozymes I and II in Six-micrometer sections were cut from donor paraffin blocks and recurrent prostate cancer compared with androgen-stimulated stained with H&E using routine methods. A pathologist (S.J. Maygarden) evaluated the tissue sections and identified benign prostate benign prostate and androgen-stimulated prostate cancer. and prostate cancer. The regions of interest were sampled by removing a a S5 RI and II expression were characterized using tissue micro- 1.5 mm tissue core. These cores were implanted into a recipient paraffin array immunohistochemistry and Western analysis, and enzy- block to create an array containing a total of 84 tissue cores that matic activity was assessed using pH-optimized assays for each includes 22 cores of recurrent prostate cancer, 23 cores of androgen- isozyme. stimulated benign prostate, 23 cores of androgen-stimulated prostate cancer, and 16 duplicate and control tissues. Materials and Methods Immunohistochemistry. Optimal conditions were defined for immunodetection of 5a-reductase isozymes (21). Tissue microarray Patient and
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