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Increased Expression of NKX3.1 in Benign Prostatic Hyperplasia
Basic and Translational Science Increased Expression of NKX3.1 in Benign Prostatic Hyperplasia Bora Irer, Asli Toylu, Guven Aslan, Ilhan Celebi, Kutsal Yorukoglu, and Nese Atabey OBJECTIVES
METHODS
RESULTS
CONCLUSIONS
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. 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. 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. NKX3.1 expression is elevated in BPH tissues when compared with normal tissues, which may be important in the development of BPH. UROLOGY 73: 1140 –1144, 2009. © 2009 Elsevier Inc.
enign prostatic hyperplasia (BPH) is the most common benign neoplasm among aging men.1 Benign prostatic hyperplasia is a disease characterized by prostatic enlargement, and the prevalence of histologically identifiable BPH is greater than 50% for 60-year-old men and approximately 90% by age 85 years.1 The development of BPH is multifactorial, but two of the primary conditions required are the effect of androgens and aging.2,3 Androgens play an important role in BPH and prostate cancer by regulating the growth and function of prostate stromal and epithelial cells.4,5 In the target organs, such as prostate, androgens can stimulate or suppress the expression of certain genes.6 Modifications in the expression of these androgen-dependent genes are known to result in prostate cancer and BPH. Homeodomain proteins are encoded by homeobox genes. These proteins regulate transcription and are critical for normal organogenesis and maintenance of differentiated cellular function.7 NKX3.1 is an androgen-regulated and prostate- and testis-specific gene that belongs to the homeobox gene family.8 NKX3.1 maps to human
B
chromosome 8p21, and deletion of NKX3.1 in mice results in prostatic hyperplasia and dysplasia.9,10 NKX3.1 is known to be required for prostatic epithelial differentiation, but the specific function of NKX3.1 in the human prostate is not yet well defined. In the literature, numerous studies of NKX3.1 expression in human prostate cancer reached conflicting conclusions. One study suggests that NKX3.1 messenger ribonucleic acid (mRNA) expression is increased in prostate cancer, whereas others ascertained reduced expression or no significant change.11,12 In another study, loss of expression of NKX3.1 was reported to be strongly associated with prostate cancer progression and advanced stage.13 We previously reported that NKX3.1 expression was significantly decreased in prostate cancer patients when compared with BPH and NKX3.1 expression that was not correlated with prostate cancer progression.14 However, there is no study regarding the NKX3.1 expression profile in human BPH tissue compared with normal prostate tissue. In this study we evaluated the expression of NKX3.1 in mRNA and protein levels in normal prostate and BPH tissues.
Supported by the Dokuz Eylul University Research Foundation (grant no. 04.KB.SAG.067). From the Departments of Urology, Medical Biology and Genetics, and Pathology, Dokuz Eylul University, Inciralti, Izmir, Turkey Reprint requests: Guven Aslan, M.D., Department of Urology, Dokuz Eylul University School of Medicine, 35340 Inciralti, Izmir, Turkey. E-mail: aslang@ deu.edu.tr Submitted: October 4, 2007, accepted (with revisions): February 15, 2008
MATERIAL AND METHODS
1140
© 2009 Elsevier Inc. All Rights Reserved
Tissue Samples The ethics committee at Dokuz Eylul University approved this study, and all subjects provided informed consent. Tissue samples were obtained from patients who underwent transurethral prostatectomy for BPH (n ⫽ 12). No patient received any 0090-4295/09/$34.00 doi:10.1016/j.urology.2008.02.039
androgen ablation therapy before prostatectomy other than alpha-blockade therapy. Normal prostate tissue samples (n ⫽ 4) were collected from patients less than 40 years of age who underwent cystoscopy for microscopic hematuria. Freshly acquired tissues were separated into two portions, first for immunohistochemistry and pathologic assessment and then for RNA and protein extraction. Pathologic assessment of post-transurethral prostatectomy patients showed stromal and glandular hyperplasia of prostate. Total RNA and protein were extracted from snap-frozen tissues and stored at ⫺80°C until use.
Cell Culture The human prostate cancer cell lines LNCaP and DU145 were obtained from the American Type Culture Collection (Manassas, Va). The LNCaP cells were routinely maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum, 5 mg/mL penicillin/ streptomycin, and 2 mmol/L L-glutamine. The passage number of LNCaP cells was 12 to 18. Untreated LNCaP cells and androgen-unresponsive DU145 cells were used as negative control, whereas 24-hour androgen-treated LNCaP cells were used as positive control for NKX3.1 expression. Before androgen induction, LNCaP cells were grown for 48 hours in RPMI containing 2% fetal calf serum that had been charcoal treated to remove steroids, followed by an additional 24 hours in RPMI containing 0.5% charcoal-treated fetal calf serum. The synthetic androgen R1881 (10⫺8M) was then added, and cells were collected at the indicated time points for total RNA and protein extraction.
nyl fluoride, 1 mmol/L Na3VO4, 1 mmol/L NaF, 1 g/mL aprotinin, 1 g/mL leupeptin, and 1 g/mL pepstatin). After extraction, protein concentrations were determined by the BCA Protein Assay (Pierce, Rockford, Ill). Equal amount of protein samples were electrophoresed on 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. The proteins were then transferred to a polyvinylidene diflouride membrane (Millipore, Bedford, Ma) and blotted with NKX3.1-specific rabbit polyclonal antibody and peroxidase-conjugated secondary antibodies. Alpha-tubulin was used as a loading control (sc-5286; Santa Cruz Biotechnology, Santa Cruz, Calif). Detection was performed with an enhanced chemiluminescence kit (Amersham Pharmacia, Piscataway, NJ) according to the manufacturer’s recommendations.
Immunohistochemistry
Total RNA isolation was performed by using RNAtidyG reagent (AppliChem, Darmstadt, Germany) according to the manufacturer’s instructions. Stability of the RNA samples was controlled by denaturing formamide-agarose gel electrophoresis. To analyze gene expression at the mRNA level, 2 g of total RNA from each sample was used as a template for complementary deoxyribonucleic acid (cDNA) synthesis. First-strand cDNA was synthesized in an oligo (dT)-primed polymerization with RevertAid M-MuLV reverse transcriptase (MBI Fermentas, Glen Burnie, Md). The cDNA was then used as template in quantitative polymerase chain reaction, using LightCycler-FastStart DNA Master SYBR Green I (Roche Diagnostics, Basel, Switzerland) to evaluate the mRNA expression levels of NKX3.1 (forward primer: 5-AGA AAG GCA CTT GGG GTC TT-3; reverse primer: 5-TAT GAG ACA CCC TGG GGA AG-3) and beta-actin control transcript. All samples were analyzed simultaneously, and the experiment was repeated at least three times. Expression values of NKX3.1 were normalized using the corresponding beta-actin value of each sample.
Immunohistochemical staining for NKX3.1 was performed according to a standard immunoperoxidase method. In brief, the paraffin-embedded prostatic tissue sections were first deparaffinized by xylene and serial ethanol dilutions. Antigen retrieval was performed by autoclaving at 121°C for 5 minutes in 0.01 mol/L citrate buffer (pH 6.4). Endogenous peroxidase activity was blocked with aqueous H2O2 (30 mL/L) for 15 minutes. Novostain Universal Detection Kit (NCL-RTU-D; Novocastra Laboratories., Newcastle upon Tyne, United Kingdom) was used for antigen detection. Sections were equilibrated in Trisbuffered saline (TBS)-Tween (0.05 mol/L Tris [pH 7.5], 0.3 mol/L NaCl, 0.1% Tween 20) for 10 minutes, followed by blocking with 1% bovine serum albumin in TBS. Rabbit antiNKX3.1 antibody was applied at a 1:200 dilution for 1 hour at room temperature in the blocking solution. After incubation for 30 minutes at room temperature, sections were washed with TBS-Tween three times, and biotinylated anti-rabbit antibody was added after being diluted fourfold in antibody dilution buffer. Sections were washed again after 30-minute incubation with the biotinylated anti-rabbit antibody at room temperature. After incubation and washing, streptavidin-tagged horseradish peroxidase was added for 30 minutes at room temperature. The sections were washed again, and detection was achieved by reaction with 3,3=-diaminobenzidine (Sigma Chemical, St. Louis, Mo) for 5 to 10 minutes. The slides were counterstained with hematoxylin and covered with coverslips. By immunohistochemistry, the expression of NKX3.1 was evaluated in BPH tissues. Tissue samples were examined by the same pathologist blind to the clinical data of the patients. Staining patterns were scored as 0 to 2 according to the intensity and uniformity of nuclear staining in malignant cells, as described in the literature (14). The staining patterns observed were scored as 0 for no staining, 1 for heterogeneous staining, and 2 for diffuse staining.
Rabbit Polyclonal Antiserum
Statistical Analysis
NKX3.1 antiserum was raised in rabbits using a recombinant NKX3.1 protein spanning the whole coding region produced in Escherichia coli.12 The antiserum was purified on immunoglobulin G affinity columns before use.
Differences between the groups were analyzed by Mann-Whitney rank sum test or chi-square with SigmaStat 2.03 (Systat Software, Chicago, Ill), with P ⬍0.05 being considered statistically significant.
RNA Isolation and RT-PCR
Protein Extraction and Western Analysis The whole cell extract and homogenate of tissue samples were prepared and resuspended in 200 L of lysis buffer (50 mmol/L Tris-HCL [pH 7.4], 150 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid, 1% NP-40, 1 mmol/L phenylmethylsulfoUROLOGY 73 (5), 2009
RESULTS The mean (⫾ standard deviation) age of patients with BPH and normal patients was 64.42 ⫾ 6.39 and 30.75 ⫾ 6.34 years, respectively. In the BPH group, mean pros1141
tate volume. The control group showed similar staining patterns with reduced staining intensities.
COMMENT
Figure 1. NKX3.1 expression in BPH versus normal prostate tissue. Total RNA was isolated from androgen-treated and untreated LNCaP cells, BPH tissues, and normal prostate tissues and used for cDNA synthesis and quantitative RT-PCR analysis with NKX3.1-specific primers. NKX3.1 expression levels were determined relative to the housekeeping gene beta-actin. Horizontal bars indicate fold differences of the mean values. The results were analyzed by Mann-Whitney rank sum test (P ⫽ 0.004).
tate-specific antigen (PSA) level and prostate volume were 2.95 ⫾ 0.39 ng/mL, 50.58 ⫾ 17.41 mL, respectively. Total RNA was extracted from BPH and normal prostate tissue and androgen-treated LNCaP cells and DU145 cells. Complementary DNA was made and was then used in quantitative reverse transcriptase polymerase chain reaction (RT-PCR) with NKX3.1-specific primers. NKX3.1 expression levels were determined relative to the housekeeping gene beta-actin. The mean NKX3.1 mRNA transcript expressions were 19.17 ⫾ 3.05 versus 1.24 ⫾ 1.32 in BPH and normal tissues, respectively (P ⫽ 0.004). Mean NKX3.1 expression levels of BPH and normal tissues are presented in Figure 1. Our results showed that NKX3.1 expression was significantly higher in BPH compared with normal prostate tissue. We used Western blot analysis and immunohistochemistry to determine the expression level and profiling of NKX3.1 protein in BPH and normal prostate tissue. Total protein was extracted from BPH and normal prostate tissue and androgen-treated LNCaP cells and DU145 cells. Using immunohistochemistry, the expression of NKX3.1 was evaluated in the BPH group. Tissue samples were examined by the same pathologist blind to the clinical data of the patients. Western blot analysis demonstrated that the mean value for NKX3.1 protein expression in BPH tissues was 2.4-fold higher than in normal prostate tissues (4.23 ⫾ 1.8 and 1.78 ⫾ 0.95, respectively; P ⬍0.05); androgen treatment increased NKX3.1 protein expression in LNCaP cells, as expected (Fig. 2A). Immunohistochemistry results showed that all of the BPH tissues were stained with NKX3.1 (Fig. 2B,C) and that there were two types of immunostaining for NKX3.1 expression: heterogeneous staining and diffuse staining. The staining properties and distribution of staining scores are shown in Table 1. Mean age was significantly higher in the diffuse staining group compared with the heterogeneous staining group (P ⬍0.05), and there was no relationship with PSA level and pros1142
To date, studies focused on the relationship between NKX3.1 expression and prostate cancer have arrived at differing conclusions.11–13,15 Some investigators, including Bahatia-Gaur et al.,9 demonstrated that deletion of NKX3.1 in mice results in prostatic hyperplasia and dysplasia; and Ortner et al.16 showed that polymorphism of NKX3.1 gene has an effect on prostate size. However, the exact role of NKX3.1 in BPH pathogenesis has not yet been clearly defined. In this study we demonstrated that NKX3.1 expression is increased in BPH compared with normal prostate. To our knowledge, our study is the first to demonstrate this. The prevalence of histologic BPH is age dependent, with initial development usually after 40 years of age and reaching approximately 90% by age 85 years, with increasing morbidity with age.1 However, little is known about the molecular factors that contribute to the onset or progression of BPH. Recent studies suggest that hormonal changes alone may not explain BPH pathogenesis, and a number of growth factors have been implicated in the pathophysiology of BPH.4,17 Few regulatory genes are known to be expressed specifically during prostate development or to be required for prostate function, and several specific homeobox genes have been identified within developing prostate tissue and play a role in normal prostate development.18,19 These include Hoxb13, which belongs to the Hox gene family, necessary for normal differentiation and secretory function of the prostate20,21; and NKX3.1, a member of the NK gene family,22 which is primarily expressed in the prostate and testis23 and is required for normal prostate development in mice.9,22 Loss of function of NKX3.1 leads to defects in prostatic protein secretions and ductal morphogenesis.24 Deletion of NKX3.1 in mice results in prostatic hyperplasia and dysplasia.9 In light of these results we aimed to evaluate the NKX3.1 expression profile at the mRNA and protein level using quantitative RT-PCR and Western analyses in BPH and normal prostate tissue samples. We have shown that NKX3.1 expression increases in BPH and therefore may be involved in BPH pathogenesis. Androgens and estrogens have a critical role in the development and maintenance of the prostate and have roles both in prostate cancer and BPH.5 Regulation of NKX3.1 by androgens and 17beta-estradiol in prostate cancer cells suggests that it may have important regulatory roles during prostate cancer and BPH development.25 We previously reported that NKX3.1 expression was significantly decreased in prostate cancer patients compared with BPH; in contrast, NKX3.1 expression in prostate cancer patients already receiving antiandrogen therapy was similar to that seen in BPH.14 Our present results indicate that from normal prostate to developUROLOGY 73 (5), 2009
Figure 2. NKX3.1 expression profiling in BPH tissue. (A) LNCaP cells were untreated or treated with R1881 (10⫺8 mol/L) for 24 hours. Western blot analysis was performed with NKX 3.1 antiserum on LNCaP cells and normal and BPH prostate tissues. Values represent the mean of densitometrically analyzed NKX3.1 bands normalized to the amount of alpha tubulin detected in each sample. *Nonspecific bands. (B, C) NKX3.1 antiserum was used for immunohistochemistry on BPH tissue from human prostates. Immunohistochemistry results indicated that most of the BPH tissues stained diffusely. Original magnification, ⫻10 in B, ⫻20 in C. Table 1. Distribution of staining scores in BPH tissue according to age, PSA level, and prostate volume Immunohistochemistry Pattern Score 1 2 Mean (⫾SD) staining scores
n
%
Age (yr)
PSA (ng/mL)
Prostate Volume (cm3)
5 7 1.58 ⫾ 0.52
41.7 58.3
59.60 ⫾ 5.37* 67.86 ⫾ 4.74*
2.96 ⫾ 0.45 2.95 ⫾ 0.38
52.40 ⫾ 17.57 49.29 ⫾ 18.58
BPH ⫽ benign prostatic hyperplasia; PSA ⫽ prostate-specific antigen; SD ⫽ standard deviation. * P ⬍0.05, Mann-Whitney rank sum test.
ment of BPH there is an increase in NKX3.1 expression, whereas progression to prostate cancer is associated with a decrease in this regard. This reinforces the current understanding that development of BPH and prostate cancer must have different molecular signatures. We demonstrated that according to immunohistochemical analyses of BPH tissue there were two types of immunostaining for NKX3.1 expression, and we compared these staining groups with each other in terms of age, PSA level, and prostate volume. We found a relationship between age and NKX3.1 staining. Mean age was significantly higher in the diffuse staining group compared with the heterogeneous staining group. These results suggested that as a result of the increased expression of NKX3.1 with age, there was a relationship between NKX3.1, aging, and histologic BPH, but a larger UROLOGY 73 (5), 2009
population study must be planned to confirm this result. Taken together, our Western blot, quantitative RT-PCR, and immunohistochemical results demonstrated that NKX3.1 has a role in BPH pathophysiology as a regulatory gene and that NKX3.1 might be a target molecule for new treatment strategies for BPH. Most published studies used a benign area of pathologic specimens from patients more than 50 years of age undergoing radical prostatectomy as normal prostate to evaluate the molecular mechanism of NKX3.1 in pathophysiology of prostatic diseases.11–13,15 As a peculiarity compared with other studies, in our study we used prostate biopsy samples that were taken from patients less than 40 years of age during cystoscopy as normal prostate tissue, because histologic BPH is identifiable in 50% of men older than 50 years. For this reason our normal 1143
prostate tissue is more suitable than that in other studies that used radical prostatectomy specimens as normal prostate for the investigation of gene expression. One of the aspects of our study to be debated is the low number of normal prostate tissue samples used. Because of ethnic, cultural, and ethical reasons we were unable to collect a larger number of normal prostate tissue samples, but because we had homogeneous results in the specimens collected, we believe that our data are robust. Further studies with larger samples are of course desirable.
CONCLUSIONS Elevated expression of NKX3.1 in BPH tissues suggests that it may contribute to BPH development during aging and may therefore be a therapeutic target for the treatment of BPH. However, further work is needed to confirm the role of NKX3.1 in transformation of normal prostate gland to BPH and prostate cancer and the relationship between NKX3.1 expression and the pathogenesis of prostatic diseases. Acknowledgment. To Fahri Saatcioglu for the generous gift of the NKX3.1 antiserum. References 1. Ziada A, Rosenblum M, and Crawford ED: Benign prostatic hyperplasia: an overview. Urology 53: 1-6, 1999. 2. Eaton CL. Aetiology and pathogenesis of benign prostatic hyperplasia. Curr Opin Urol 13: 7-10, 2003. 3. Berry SJ, Coffey DS, Walsh PC, et al: The development of human benign prostatic hyperplasia with age. J Urol 132: 474-479, 1984. 4. Mullan RJ, Bergstralh EJ, Farmer SA, et al: Growth factor, cytokine, and vitamin D receptor polymorphisms and risk of benign prostatic hyperplasia in a community-based cohort of men. Urology 67: 300-305, 2006. 5. Marcelli M, and Cunningham GR: Hormonal signaling in prostatic hyperplasia and neoplasia. J Clin Endocrinol Metab 84: 34633468, 1999. 6. Trapman J, and Cleutjens KB: Androgen-regulated gene expression in prostate cancer. Semin Cancer Biol 8: 29-36, 1997. 7. Kim Y, and Nirenberg M: Drosophila NK-homeobox genes (NK-1, NK-2, NK-3, and NK-4 DNA clones/chromosome locations of genes). Proc Natl Acad Sci U S A 86: 7716-7720, 1989. 8. He WW, Sciavolino PJ, Wing J, et al: A novel human prostatespecific, androgen-regulated homeobox gene (NKX3.1) that maps to 8p21, a region frequently deleted in prostate cancer. Genomics 43: 69-77, 1997.
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9. Bhatia-Gaur R, Donjacour AA, Sciavolino PJ, et al: Roles for Nkx3.1 in prostate development and cancer. Genes Dev 13: 966977, 1999. 10. Voeller HJ, Augustus M, Madike V, et al: Coding region of NKX3.1, a prostate-specific homeobox gene on 8p21, is not mutated in human prostate cancers. Cancer Res 57: 4455-4459, 1997. 11. Ornstein DK, Cinquanta M, Weiler S, et al: Expression studies and mutational analysis of the androgen regulated homeobox gene NKX3.1 in benign and malignant prostate epithelium. J Urol 165: 1329-1334, 2001. 12. Korkmaz CG, Korkmaz KS, Onody T, et al: Analysis of androgen regulated homeobox gene NKX3.1 during prostate carcinogenesis. J Urol 172: 1134-1139, 2004. 13. Bowen C, Bubendorf L, Voeller HJ, et al: Loss of NKX3.1 expression in human prostate cancers correlates with tumor progression. Cancer Res 60: 6111-6115, 2000. 14. Aslan G, Irer B, Tuna B, et al: Analysis of NKX3.1 expression in prostate cancer tissues and correlation with clinicopathologic features. Pathol Res Pract 202: 93-98, 2006. 15. Asatiani E, Huang WX, Wang A, et al: Deletion, methylation, and expression of the NKX3.1 suppressor gene in primary human prostate cancer. Cancer Res 65: 1164-1173, 2005. 16. Ortner ER, Hayes R, Weissfeld J, et al: Effect of homeodomain protein NKX3.1 R52C polymorphism on prostate gland size. Urology 67: 311-315, 2006. 17. Schwartz S Jr, Caceres C, Morote J, et al: Over-expression of epidermal growth factor receptor and c-erbB2/neu but not of int-2 genes in benign prostatic hyperplasia by means of semi-quantitative PCR. Int J Cancer 76: 464-467, 1998. 18. Tiniakos DG, Mitropoulos D, Kyroudi-Voulgari A, et al: Expression of c-jun oncogene in hyperplastic and carcinomatous human prostate. Urology 67: 204-208, 2006. 19. Luo J, Dunn T, Ewing C, et al: Gene expression signature of benign prostatic hyperplasia revealed by cDNA microarray analysis. Prostate 51: 189-200, 2002. 20. Warot X, Fromental-Ramain C, Fraulob V, et al: Gene dosagedependent effects of the Hoxa-13 and Hoxd-13 mutations on morphogenesis of the terminal parts of the digestive and urogenital tracts. Development 124: 4781-4791, 1997. 21. Economides KD, and Capecchi MR: Hoxb13 is required for normal differentiation and secretory function of the ventral prostate. Development 130: 2061-2069, 2003. 22. Bieberich CJ, Fujita K, He WW, et al: Prostate-specific and androgen dependent expression of a novel homeobox gene. J Biol Chem 271: 31779-31782, 1996. 23. Gelmann EP, Bowen C, and Bubendorf L: Expression of NKX3.1 in normal and malignant tissues. Prostate 55: 111-117, 2003. 24. Shen MM, and Abate-Shen C: Roles of the NKX3.1 homeobox gene in prostate organogenesis and carcinogenesis. Dev Dyn 228: 767-778, 2003. 25. Korkmaz KS, Korkmaz CG, Ragnhildstveit E, et al: Full-length cDNA sequence and genomic organization of human NKX3a— alternative forms and regulation by both androgens and estrogens. Gene 260: 25-36, 2000.
UROLOGY 73 (5), 2009
METHODS
RESULTS
CONCLUSIONS
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. 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. 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. NKX3.1 expression is elevated in BPH tissues when compared with normal tissues, which may be important in the development of BPH. UROLOGY 73: 1140 –1144, 2009. © 2009 Elsevier Inc.
enign prostatic hyperplasia (BPH) is the most common benign neoplasm among aging men.1 Benign prostatic hyperplasia is a disease characterized by prostatic enlargement, and the prevalence of histologically identifiable BPH is greater than 50% for 60-year-old men and approximately 90% by age 85 years.1 The development of BPH is multifactorial, but two of the primary conditions required are the effect of androgens and aging.2,3 Androgens play an important role in BPH and prostate cancer by regulating the growth and function of prostate stromal and epithelial cells.4,5 In the target organs, such as prostate, androgens can stimulate or suppress the expression of certain genes.6 Modifications in the expression of these androgen-dependent genes are known to result in prostate cancer and BPH. Homeodomain proteins are encoded by homeobox genes. These proteins regulate transcription and are critical for normal organogenesis and maintenance of differentiated cellular function.7 NKX3.1 is an androgen-regulated and prostate- and testis-specific gene that belongs to the homeobox gene family.8 NKX3.1 maps to human
B
chromosome 8p21, and deletion of NKX3.1 in mice results in prostatic hyperplasia and dysplasia.9,10 NKX3.1 is known to be required for prostatic epithelial differentiation, but the specific function of NKX3.1 in the human prostate is not yet well defined. In the literature, numerous studies of NKX3.1 expression in human prostate cancer reached conflicting conclusions. One study suggests that NKX3.1 messenger ribonucleic acid (mRNA) expression is increased in prostate cancer, whereas others ascertained reduced expression or no significant change.11,12 In another study, loss of expression of NKX3.1 was reported to be strongly associated with prostate cancer progression and advanced stage.13 We previously reported that NKX3.1 expression was significantly decreased in prostate cancer patients when compared with BPH and NKX3.1 expression that was not correlated with prostate cancer progression.14 However, there is no study regarding the NKX3.1 expression profile in human BPH tissue compared with normal prostate tissue. In this study we evaluated the expression of NKX3.1 in mRNA and protein levels in normal prostate and BPH tissues.
Supported by the Dokuz Eylul University Research Foundation (grant no. 04.KB.SAG.067). From the Departments of Urology, Medical Biology and Genetics, and Pathology, Dokuz Eylul University, Inciralti, Izmir, Turkey Reprint requests: Guven Aslan, M.D., Department of Urology, Dokuz Eylul University School of Medicine, 35340 Inciralti, Izmir, Turkey. E-mail: aslang@ deu.edu.tr Submitted: October 4, 2007, accepted (with revisions): February 15, 2008
MATERIAL AND METHODS
1140
© 2009 Elsevier Inc. All Rights Reserved
Tissue Samples The ethics committee at Dokuz Eylul University approved this study, and all subjects provided informed consent. Tissue samples were obtained from patients who underwent transurethral prostatectomy for BPH (n ⫽ 12). No patient received any 0090-4295/09/$34.00 doi:10.1016/j.urology.2008.02.039
androgen ablation therapy before prostatectomy other than alpha-blockade therapy. Normal prostate tissue samples (n ⫽ 4) were collected from patients less than 40 years of age who underwent cystoscopy for microscopic hematuria. Freshly acquired tissues were separated into two portions, first for immunohistochemistry and pathologic assessment and then for RNA and protein extraction. Pathologic assessment of post-transurethral prostatectomy patients showed stromal and glandular hyperplasia of prostate. Total RNA and protein were extracted from snap-frozen tissues and stored at ⫺80°C until use.
Cell Culture The human prostate cancer cell lines LNCaP and DU145 were obtained from the American Type Culture Collection (Manassas, Va). The LNCaP cells were routinely maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum, 5 mg/mL penicillin/ streptomycin, and 2 mmol/L L-glutamine. The passage number of LNCaP cells was 12 to 18. Untreated LNCaP cells and androgen-unresponsive DU145 cells were used as negative control, whereas 24-hour androgen-treated LNCaP cells were used as positive control for NKX3.1 expression. Before androgen induction, LNCaP cells were grown for 48 hours in RPMI containing 2% fetal calf serum that had been charcoal treated to remove steroids, followed by an additional 24 hours in RPMI containing 0.5% charcoal-treated fetal calf serum. The synthetic androgen R1881 (10⫺8M) was then added, and cells were collected at the indicated time points for total RNA and protein extraction.
nyl fluoride, 1 mmol/L Na3VO4, 1 mmol/L NaF, 1 g/mL aprotinin, 1 g/mL leupeptin, and 1 g/mL pepstatin). After extraction, protein concentrations were determined by the BCA Protein Assay (Pierce, Rockford, Ill). Equal amount of protein samples were electrophoresed on 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. The proteins were then transferred to a polyvinylidene diflouride membrane (Millipore, Bedford, Ma) and blotted with NKX3.1-specific rabbit polyclonal antibody and peroxidase-conjugated secondary antibodies. Alpha-tubulin was used as a loading control (sc-5286; Santa Cruz Biotechnology, Santa Cruz, Calif). Detection was performed with an enhanced chemiluminescence kit (Amersham Pharmacia, Piscataway, NJ) according to the manufacturer’s recommendations.
Immunohistochemistry
Total RNA isolation was performed by using RNAtidyG reagent (AppliChem, Darmstadt, Germany) according to the manufacturer’s instructions. Stability of the RNA samples was controlled by denaturing formamide-agarose gel electrophoresis. To analyze gene expression at the mRNA level, 2 g of total RNA from each sample was used as a template for complementary deoxyribonucleic acid (cDNA) synthesis. First-strand cDNA was synthesized in an oligo (dT)-primed polymerization with RevertAid M-MuLV reverse transcriptase (MBI Fermentas, Glen Burnie, Md). The cDNA was then used as template in quantitative polymerase chain reaction, using LightCycler-FastStart DNA Master SYBR Green I (Roche Diagnostics, Basel, Switzerland) to evaluate the mRNA expression levels of NKX3.1 (forward primer: 5-AGA AAG GCA CTT GGG GTC TT-3; reverse primer: 5-TAT GAG ACA CCC TGG GGA AG-3) and beta-actin control transcript. All samples were analyzed simultaneously, and the experiment was repeated at least three times. Expression values of NKX3.1 were normalized using the corresponding beta-actin value of each sample.
Immunohistochemical staining for NKX3.1 was performed according to a standard immunoperoxidase method. In brief, the paraffin-embedded prostatic tissue sections were first deparaffinized by xylene and serial ethanol dilutions. Antigen retrieval was performed by autoclaving at 121°C for 5 minutes in 0.01 mol/L citrate buffer (pH 6.4). Endogenous peroxidase activity was blocked with aqueous H2O2 (30 mL/L) for 15 minutes. Novostain Universal Detection Kit (NCL-RTU-D; Novocastra Laboratories., Newcastle upon Tyne, United Kingdom) was used for antigen detection. Sections were equilibrated in Trisbuffered saline (TBS)-Tween (0.05 mol/L Tris [pH 7.5], 0.3 mol/L NaCl, 0.1% Tween 20) for 10 minutes, followed by blocking with 1% bovine serum albumin in TBS. Rabbit antiNKX3.1 antibody was applied at a 1:200 dilution for 1 hour at room temperature in the blocking solution. After incubation for 30 minutes at room temperature, sections were washed with TBS-Tween three times, and biotinylated anti-rabbit antibody was added after being diluted fourfold in antibody dilution buffer. Sections were washed again after 30-minute incubation with the biotinylated anti-rabbit antibody at room temperature. After incubation and washing, streptavidin-tagged horseradish peroxidase was added for 30 minutes at room temperature. The sections were washed again, and detection was achieved by reaction with 3,3=-diaminobenzidine (Sigma Chemical, St. Louis, Mo) for 5 to 10 minutes. The slides were counterstained with hematoxylin and covered with coverslips. By immunohistochemistry, the expression of NKX3.1 was evaluated in BPH tissues. Tissue samples were examined by the same pathologist blind to the clinical data of the patients. Staining patterns were scored as 0 to 2 according to the intensity and uniformity of nuclear staining in malignant cells, as described in the literature (14). The staining patterns observed were scored as 0 for no staining, 1 for heterogeneous staining, and 2 for diffuse staining.
Rabbit Polyclonal Antiserum
Statistical Analysis
NKX3.1 antiserum was raised in rabbits using a recombinant NKX3.1 protein spanning the whole coding region produced in Escherichia coli.12 The antiserum was purified on immunoglobulin G affinity columns before use.
Differences between the groups were analyzed by Mann-Whitney rank sum test or chi-square with SigmaStat 2.03 (Systat Software, Chicago, Ill), with P ⬍0.05 being considered statistically significant.
RNA Isolation and RT-PCR
Protein Extraction and Western Analysis The whole cell extract and homogenate of tissue samples were prepared and resuspended in 200 L of lysis buffer (50 mmol/L Tris-HCL [pH 7.4], 150 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid, 1% NP-40, 1 mmol/L phenylmethylsulfoUROLOGY 73 (5), 2009
RESULTS The mean (⫾ standard deviation) age of patients with BPH and normal patients was 64.42 ⫾ 6.39 and 30.75 ⫾ 6.34 years, respectively. In the BPH group, mean pros1141
tate volume. The control group showed similar staining patterns with reduced staining intensities.
COMMENT
Figure 1. NKX3.1 expression in BPH versus normal prostate tissue. Total RNA was isolated from androgen-treated and untreated LNCaP cells, BPH tissues, and normal prostate tissues and used for cDNA synthesis and quantitative RT-PCR analysis with NKX3.1-specific primers. NKX3.1 expression levels were determined relative to the housekeeping gene beta-actin. Horizontal bars indicate fold differences of the mean values. The results were analyzed by Mann-Whitney rank sum test (P ⫽ 0.004).
tate-specific antigen (PSA) level and prostate volume were 2.95 ⫾ 0.39 ng/mL, 50.58 ⫾ 17.41 mL, respectively. Total RNA was extracted from BPH and normal prostate tissue and androgen-treated LNCaP cells and DU145 cells. Complementary DNA was made and was then used in quantitative reverse transcriptase polymerase chain reaction (RT-PCR) with NKX3.1-specific primers. NKX3.1 expression levels were determined relative to the housekeeping gene beta-actin. The mean NKX3.1 mRNA transcript expressions were 19.17 ⫾ 3.05 versus 1.24 ⫾ 1.32 in BPH and normal tissues, respectively (P ⫽ 0.004). Mean NKX3.1 expression levels of BPH and normal tissues are presented in Figure 1. Our results showed that NKX3.1 expression was significantly higher in BPH compared with normal prostate tissue. We used Western blot analysis and immunohistochemistry to determine the expression level and profiling of NKX3.1 protein in BPH and normal prostate tissue. Total protein was extracted from BPH and normal prostate tissue and androgen-treated LNCaP cells and DU145 cells. Using immunohistochemistry, the expression of NKX3.1 was evaluated in the BPH group. Tissue samples were examined by the same pathologist blind to the clinical data of the patients. Western blot analysis demonstrated that the mean value for NKX3.1 protein expression in BPH tissues was 2.4-fold higher than in normal prostate tissues (4.23 ⫾ 1.8 and 1.78 ⫾ 0.95, respectively; P ⬍0.05); androgen treatment increased NKX3.1 protein expression in LNCaP cells, as expected (Fig. 2A). Immunohistochemistry results showed that all of the BPH tissues were stained with NKX3.1 (Fig. 2B,C) and that there were two types of immunostaining for NKX3.1 expression: heterogeneous staining and diffuse staining. The staining properties and distribution of staining scores are shown in Table 1. Mean age was significantly higher in the diffuse staining group compared with the heterogeneous staining group (P ⬍0.05), and there was no relationship with PSA level and pros1142
To date, studies focused on the relationship between NKX3.1 expression and prostate cancer have arrived at differing conclusions.11–13,15 Some investigators, including Bahatia-Gaur et al.,9 demonstrated that deletion of NKX3.1 in mice results in prostatic hyperplasia and dysplasia; and Ortner et al.16 showed that polymorphism of NKX3.1 gene has an effect on prostate size. However, the exact role of NKX3.1 in BPH pathogenesis has not yet been clearly defined. In this study we demonstrated that NKX3.1 expression is increased in BPH compared with normal prostate. To our knowledge, our study is the first to demonstrate this. The prevalence of histologic BPH is age dependent, with initial development usually after 40 years of age and reaching approximately 90% by age 85 years, with increasing morbidity with age.1 However, little is known about the molecular factors that contribute to the onset or progression of BPH. Recent studies suggest that hormonal changes alone may not explain BPH pathogenesis, and a number of growth factors have been implicated in the pathophysiology of BPH.4,17 Few regulatory genes are known to be expressed specifically during prostate development or to be required for prostate function, and several specific homeobox genes have been identified within developing prostate tissue and play a role in normal prostate development.18,19 These include Hoxb13, which belongs to the Hox gene family, necessary for normal differentiation and secretory function of the prostate20,21; and NKX3.1, a member of the NK gene family,22 which is primarily expressed in the prostate and testis23 and is required for normal prostate development in mice.9,22 Loss of function of NKX3.1 leads to defects in prostatic protein secretions and ductal morphogenesis.24 Deletion of NKX3.1 in mice results in prostatic hyperplasia and dysplasia.9 In light of these results we aimed to evaluate the NKX3.1 expression profile at the mRNA and protein level using quantitative RT-PCR and Western analyses in BPH and normal prostate tissue samples. We have shown that NKX3.1 expression increases in BPH and therefore may be involved in BPH pathogenesis. Androgens and estrogens have a critical role in the development and maintenance of the prostate and have roles both in prostate cancer and BPH.5 Regulation of NKX3.1 by androgens and 17beta-estradiol in prostate cancer cells suggests that it may have important regulatory roles during prostate cancer and BPH development.25 We previously reported that NKX3.1 expression was significantly decreased in prostate cancer patients compared with BPH; in contrast, NKX3.1 expression in prostate cancer patients already receiving antiandrogen therapy was similar to that seen in BPH.14 Our present results indicate that from normal prostate to developUROLOGY 73 (5), 2009
Figure 2. NKX3.1 expression profiling in BPH tissue. (A) LNCaP cells were untreated or treated with R1881 (10⫺8 mol/L) for 24 hours. Western blot analysis was performed with NKX 3.1 antiserum on LNCaP cells and normal and BPH prostate tissues. Values represent the mean of densitometrically analyzed NKX3.1 bands normalized to the amount of alpha tubulin detected in each sample. *Nonspecific bands. (B, C) NKX3.1 antiserum was used for immunohistochemistry on BPH tissue from human prostates. Immunohistochemistry results indicated that most of the BPH tissues stained diffusely. Original magnification, ⫻10 in B, ⫻20 in C. Table 1. Distribution of staining scores in BPH tissue according to age, PSA level, and prostate volume Immunohistochemistry Pattern Score 1 2 Mean (⫾SD) staining scores
n
%
Age (yr)
PSA (ng/mL)
Prostate Volume (cm3)
5 7 1.58 ⫾ 0.52
41.7 58.3
59.60 ⫾ 5.37* 67.86 ⫾ 4.74*
2.96 ⫾ 0.45 2.95 ⫾ 0.38
52.40 ⫾ 17.57 49.29 ⫾ 18.58
BPH ⫽ benign prostatic hyperplasia; PSA ⫽ prostate-specific antigen; SD ⫽ standard deviation. * P ⬍0.05, Mann-Whitney rank sum test.
ment of BPH there is an increase in NKX3.1 expression, whereas progression to prostate cancer is associated with a decrease in this regard. This reinforces the current understanding that development of BPH and prostate cancer must have different molecular signatures. We demonstrated that according to immunohistochemical analyses of BPH tissue there were two types of immunostaining for NKX3.1 expression, and we compared these staining groups with each other in terms of age, PSA level, and prostate volume. We found a relationship between age and NKX3.1 staining. Mean age was significantly higher in the diffuse staining group compared with the heterogeneous staining group. These results suggested that as a result of the increased expression of NKX3.1 with age, there was a relationship between NKX3.1, aging, and histologic BPH, but a larger UROLOGY 73 (5), 2009
population study must be planned to confirm this result. Taken together, our Western blot, quantitative RT-PCR, and immunohistochemical results demonstrated that NKX3.1 has a role in BPH pathophysiology as a regulatory gene and that NKX3.1 might be a target molecule for new treatment strategies for BPH. Most published studies used a benign area of pathologic specimens from patients more than 50 years of age undergoing radical prostatectomy as normal prostate to evaluate the molecular mechanism of NKX3.1 in pathophysiology of prostatic diseases.11–13,15 As a peculiarity compared with other studies, in our study we used prostate biopsy samples that were taken from patients less than 40 years of age during cystoscopy as normal prostate tissue, because histologic BPH is identifiable in 50% of men older than 50 years. For this reason our normal 1143
prostate tissue is more suitable than that in other studies that used radical prostatectomy specimens as normal prostate for the investigation of gene expression. One of the aspects of our study to be debated is the low number of normal prostate tissue samples used. Because of ethnic, cultural, and ethical reasons we were unable to collect a larger number of normal prostate tissue samples, but because we had homogeneous results in the specimens collected, we believe that our data are robust. Further studies with larger samples are of course desirable.
CONCLUSIONS Elevated expression of NKX3.1 in BPH tissues suggests that it may contribute to BPH development during aging and may therefore be a therapeutic target for the treatment of BPH. However, further work is needed to confirm the role of NKX3.1 in transformation of normal prostate gland to BPH and prostate cancer and the relationship between NKX3.1 expression and the pathogenesis of prostatic diseases. Acknowledgment. To Fahri Saatcioglu for the generous gift of the NKX3.1 antiserum. References 1. Ziada A, Rosenblum M, and Crawford ED: Benign prostatic hyperplasia: an overview. Urology 53: 1-6, 1999. 2. Eaton CL. Aetiology and pathogenesis of benign prostatic hyperplasia. Curr Opin Urol 13: 7-10, 2003. 3. Berry SJ, Coffey DS, Walsh PC, et al: The development of human benign prostatic hyperplasia with age. J Urol 132: 474-479, 1984. 4. Mullan RJ, Bergstralh EJ, Farmer SA, et al: Growth factor, cytokine, and vitamin D receptor polymorphisms and risk of benign prostatic hyperplasia in a community-based cohort of men. Urology 67: 300-305, 2006. 5. Marcelli M, and Cunningham GR: Hormonal signaling in prostatic hyperplasia and neoplasia. J Clin Endocrinol Metab 84: 34633468, 1999. 6. Trapman J, and Cleutjens KB: Androgen-regulated gene expression in prostate cancer. Semin Cancer Biol 8: 29-36, 1997. 7. Kim Y, and Nirenberg M: Drosophila NK-homeobox genes (NK-1, NK-2, NK-3, and NK-4 DNA clones/chromosome locations of genes). Proc Natl Acad Sci U S A 86: 7716-7720, 1989. 8. He WW, Sciavolino PJ, Wing J, et al: A novel human prostatespecific, androgen-regulated homeobox gene (NKX3.1) that maps to 8p21, a region frequently deleted in prostate cancer. Genomics 43: 69-77, 1997.
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9. Bhatia-Gaur R, Donjacour AA, Sciavolino PJ, et al: Roles for Nkx3.1 in prostate development and cancer. Genes Dev 13: 966977, 1999. 10. Voeller HJ, Augustus M, Madike V, et al: Coding region of NKX3.1, a prostate-specific homeobox gene on 8p21, is not mutated in human prostate cancers. Cancer Res 57: 4455-4459, 1997. 11. Ornstein DK, Cinquanta M, Weiler S, et al: Expression studies and mutational analysis of the androgen regulated homeobox gene NKX3.1 in benign and malignant prostate epithelium. J Urol 165: 1329-1334, 2001. 12. Korkmaz CG, Korkmaz KS, Onody T, et al: Analysis of androgen regulated homeobox gene NKX3.1 during prostate carcinogenesis. J Urol 172: 1134-1139, 2004. 13. Bowen C, Bubendorf L, Voeller HJ, et al: Loss of NKX3.1 expression in human prostate cancers correlates with tumor progression. Cancer Res 60: 6111-6115, 2000. 14. Aslan G, Irer B, Tuna B, et al: Analysis of NKX3.1 expression in prostate cancer tissues and correlation with clinicopathologic features. Pathol Res Pract 202: 93-98, 2006. 15. Asatiani E, Huang WX, Wang A, et al: Deletion, methylation, and expression of the NKX3.1 suppressor gene in primary human prostate cancer. Cancer Res 65: 1164-1173, 2005. 16. Ortner ER, Hayes R, Weissfeld J, et al: Effect of homeodomain protein NKX3.1 R52C polymorphism on prostate gland size. Urology 67: 311-315, 2006. 17. Schwartz S Jr, Caceres C, Morote J, et al: Over-expression of epidermal growth factor receptor and c-erbB2/neu but not of int-2 genes in benign prostatic hyperplasia by means of semi-quantitative PCR. Int J Cancer 76: 464-467, 1998. 18. Tiniakos DG, Mitropoulos D, Kyroudi-Voulgari A, et al: Expression of c-jun oncogene in hyperplastic and carcinomatous human prostate. Urology 67: 204-208, 2006. 19. Luo J, Dunn T, Ewing C, et al: Gene expression signature of benign prostatic hyperplasia revealed by cDNA microarray analysis. Prostate 51: 189-200, 2002. 20. Warot X, Fromental-Ramain C, Fraulob V, et al: Gene dosagedependent effects of the Hoxa-13 and Hoxd-13 mutations on morphogenesis of the terminal parts of the digestive and urogenital tracts. Development 124: 4781-4791, 1997. 21. Economides KD, and Capecchi MR: Hoxb13 is required for normal differentiation and secretory function of the ventral prostate. Development 130: 2061-2069, 2003. 22. Bieberich CJ, Fujita K, He WW, et al: Prostate-specific and androgen dependent expression of a novel homeobox gene. J Biol Chem 271: 31779-31782, 1996. 23. Gelmann EP, Bowen C, and Bubendorf L: Expression of NKX3.1 in normal and malignant tissues. Prostate 55: 111-117, 2003. 24. Shen MM, and Abate-Shen C: Roles of the NKX3.1 homeobox gene in prostate organogenesis and carcinogenesis. Dev Dyn 228: 767-778, 2003. 25. Korkmaz KS, Korkmaz CG, Ragnhildstveit E, et al: Full-length cDNA sequence and genomic organization of human NKX3a— alternative forms and regulation by both androgens and estrogens. Gene 260: 25-36, 2000.
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