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The Loss of 5-Reductase Type I and Type II mRNA Expression in Metastatic Prostate Cancer to Bone and Lymph Node Metastasis

Prostate Research Group, University Departments of Oncology [F. K. H., M. R., C. W. B.], Medical Sciences [K. C.], Pathology [K. G.], and Urology [P. B.], Western General Hospital, Edinburgh, Scotland EH4 2XU, United Kingdom

	ABSTRACT

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Purpose: Considerable evidence has accumulated demonstrating that the 5-reduction of testosterone to dihydrotestosterone occurs more efficiently in the normal and benign hyperplastic prostate than in prostate cancer tissues. Efforts have also been channeled into investigating the distribution of 5-reductase isoenzymes in primary prostate tissues and in "in vitro" cell models of the human prostate. However, no one has, thus far, examined the expression of these isoenzymes in prostate cancer metastasis, although such studies might shed some light on the mechanism(s) responsible for the loss of hormone sensitivity in those tumors. The present report addresses this issue in the hope that this might help to identify the steps leading to the development of prostate cancer metastasis.

Experimental Design: In the present study we used in situ mRNA hybridization of sections from archival paraffin-embedded material to investigate the expression of 5-reductase type I (5R-I) and type II (5R-II) mRNAs in prostate cancer bony (n = 9) and lymph node (n = 13) metastasis, and compared the mRNA distributions with those observed in sections from primary prostate tumors (n = 12). In parallel, sections were investigated for androgen receptor (AR) mRNA expression, and immunostained for AR and prostate-specific antigen.

Results: Neither 5R-I nor 5R-II mRNA expression was detected in any of the prostate metastatic lesions, although the same metastatic sites expressed AR mRNA, and stained for cytoplasmic prostate-specific antigen and nuclear AR. In contrast, primary prostate tumors displayed intense staining for 5R-I and 5R-II.

Conclusion: These findings suggest that the loss of 5-reductase mRNA expression in bone and lymph node metastasis may be associated, in part, with the progression of these tumors to androgen insensitivity.

	INTRODUCTION

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Prostate cancer is the second leading cause of cancer-related death in men in the Western world, and in the United States alone 30,200 men will die of this disease in 2002 (1) . Prostate cancer has been well characterized at the histopathological level, and can be classified into different stages based on the location and size of the tumor, and into different grades dependent on the degree of differentiation of the epithelial cells in the cancer (2) . Additionally, the use, over the last 2 decades, of improved methods of detection and staging including PSA² testing has considerably facilitated the stratification and management of localized prostate cancer (3) . Furthermore, several molecular markers have been proposed as predictors in prostate carcinoma; However, most of these are severely limited by their heterogeneity of expression in prostate cancer (4, 5, 6) , and the molecular mechanisms underlying the development of the tumor and the progression of the disease still remain, for the most part, unclear.

Attention has focused recently on the enzymes responsible for the conversion of testosterone to the biologically more active DHT as a means of controlling the abnormal growth of the prostate. The formation of DHT is mediated through the action of 5R, of which 2 isoenzymes exist, 5R-I and 5R-II, encoded by separate genes (7, 8, 9) . Although the respective roles of 5R-I and 5R-II in the DHT-forming process are not fully understood, there is now general consensus that both isoenzymes are present in the human prostate and are expressed in a variety of cell types (10, 11, 12, 13, 14, 15, 16, 17) . Furthermore, lower 5-reductase activity (measured by monitoring the conversion of testosterone to DHT) was demonstrated in primary prostate cancers compared with benign tissues (18, 19, 20) ; the loss in activity correlated with the histological grade of the tumor (18) and mirrored the reduced hormone sensitivity of the cancer (21) . However no one has, to date, measured the expression of 5R-I and 5R-II in lymph node and bony lesions of human prostate cancer metastasis.

Therefore, the present study was undertaken to evaluate 5-reductase isoenzyme expression in prostate cancer metastasis. The presence of metastatic lesions secondary to prostate cancer was confirmed by immunostaining for PSA. The distribution of 5R-I and 5R-II mRNAs in bony and lymph node metastasis was studied and compared with the patterns found in primary prostate cancer tumors. In addition we examined, in parallel sections, the coexpression of AR mRNA.

	MATERIALS AND METHODS

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Archival Material and Tissue Samples.
Sections from archival paraffin-embedded primary prostate cancers (n = 12) and prostate cancer metastases (n = 22) were supplied by the Department of Pathology, Western General Hospital. The metastatic lesions were located in bone (9 cases) and lymph node (13 cases). All of the primary tumor cases (M₀, T₃-T₄; PSA = >10) and all of the lymph node metastatic specimens were from newly diagnosed patients who had not been treated with endocrine therapy. Six patients with bone metastasis had also not received endocrine therapy, whereas the remaining 3 patients in that group were on endocrine therapy but had not been subjected to radical prostatectomy. Serial 5-µm sections were cut, and representative sections from each block of archival tissue were stained with H&E for histopathological evaluation. Suitable blocks for examination were selected by an experienced uropathologist (K. G.), and only sections, which were decalcified using EDTA, were used. Preliminary experiments had demonstrated that decalcification with formic acid inhibited ISH, whereas EDTA treatment had no effect.

Antibodies.
A commercial monoclonal anti-AR antibody (NCL-2F-AR; Novacastra, Newcastle, United Kingdom) was used. This antibody does not cross-react with estrogen, progesterone, or glucocorticoid receptors. The secondary antibody applied for AR detection was biotinylated goat antimouse IgG (Boehringer Mannheim, Lewes, Sussex, United Kingdom). The PSA staining was detected with a commercial monoclonal anti-PSA antibody (Dako, High Wycombe, United Kingdom) and a biotinylated-labeled goat antimouse secondary antibody (Boehringer Mannheim).

Immunohistochemistry.
Human AR immunohistochemistry was performed as described previously (22) . Briefly, the archival sections were deparaffinized and dehydrated in xylene and graded alcohols. Before the application of primary antisera, heat-mediated antigen retrieval was performed by boiling slides in 0.01 M citrate buffer in a microwave oven.

Endogenous peroxidase was blocked with 3.0% H₂0₂ for 10 min, and this was followed by incubation with 10% nonimmune normal goat serum in Tris-buffered saline. Human AR primary antibody (1:25 dilution) was applied to the tissue sections at room temperature for 60 min. After two washes in Tris-buffered saline, the biotinylated secondary antibody (1:500 dilution) was applied at room temperature for 30 min followed by peroxidase-labeled streptavidin-biotin complex (1:800 dilution) for 30 min. For PSA immunohistochemistry, no microwave pretreatment was performed. The primary anti-PSA antibody (1:2500 dilution) was applied for 60 min at room temperature followed by biotinylated secondary antibody as described above. To visualize the immunoreaction a freshly prepared solution of 3',3' diaminobenzidine tetrahydrochloride (0.5 mg/ml; Sigma, Poole, Dorset, United Kingdom) was added for 5 min. Sections were then counterstained, dehydrated, cleared, and mounted. LNCaP cells, which contain high levels of AR protein and PSA, were used as positive control, whereas PC3 and DU145 cells served as negative controls; all of these cells were cultured in the laboratory as described elsewhere (23) .

ISH.
Expression of mRNAs encoding 5R-1 and 5R-II was investigated using nonisotopic mRNA ISH as described previously (15) . Briefly, archival sections were dewaxed in xylene, refixed, and permealized by covering the sections with 100 µl 50 mM Tris-HCl (pH 7.6) containing 20 µg proteinase K (Sigma) and 5 mM EDTA for 7.5 min at room temperature. The DIG-labeled cRNA probe used to detect 5R-I mRNA was synthesized from a plasmid encoding a 780-bp fragment of human 5R-I cDNA (7) subcloned into pBSII SK. For the cRNA (or "antisense") probe, the plasmid was linearized with Kpnl and transcription carried out using T3 RNA polymerase. For the "sense" probe, NotI cleaved plasmid was transcribed using T7 RNA polymerase. The DIG-labeled cRNA probe used to detect 5R-II mRNA was synthesized in a similar fashion, except that a 365-bp cDNA encoding 5R-II (9) complementary to the 5R-II mRNA was used as detailed in our earlier study (15) . The specificity of the 5R-I and -II antisense probes was checked by ISH of Chinese hamster ovary cells, which had been transfected with either the 5R-I or -II gene. The results showed no cross-reactivity between the probes (15) . Hybridization to DIG-UTP-labeled RNA probes (1.6 µg/80 µl) was carried out in a hybridization mixture containing 40 µl formamide, 9.6 µl 5 M NaCl, 1.6 µl 50x Denhardt’s solution, 0.8 µl 1 M DTT, 1.6 µl tRNA (10 µg/ml), and 16 µl 50% dextran sulfate, and proceeded overnight at 50°C in a moist chamber under sealed silane-treated coverslips. Posthybridization washes (30 min each) included 2x SSC (300 mM sodium chloride and 30 mM sodium citrate) at 37°C followed by a single wash in 2x SSC at room temperature before RNase treatment (30 min at 37°C in 2x SSC containing 10 µg/ml RNase-A) then washed in 2x SSC for 20 min, 2x SSC containing 50% formamide at 65°C for 20 min, 2x SSC at 65°C for 20 min, 0.2x SSC at room temperature for 10 min, and finally in 0.1x SSC at 42°C for 20 min. Visualization of the DIG-labeled probes was effected by immersing the sections in DIG-alkaline phosphatase-conjugated antibody (Boehringer Mannheim) followed by overnight immersion in a developing agent (Boehringer Mannheim) to the instruction of the manufacturers. For negative control, hybridization with sense-strand riboprobes was carried out. In addition to establishing the viability of the metastatic specimens for ISH, AR mRNA expression was used as a positive control; earlier studies had demonstrated that distant metastases from prostatic carcinoma expressed ARs (24) . The AR probe was made by subcloning an EcoRI generated fragment of 465 bp from the expression vector, pSVAR0 (kindly donated by Dr. A. Brinkman, Rotterdam, Holland), into pGem, which has T7 and SP6 promoter sites producing the antisense and sense AR probes, respectively. The viability of the fragment was established after digestion with SphI restriction enzyme to yield 120- and 345-bp fragments.

	RESULTS

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None of the metastatic prostate cancer deposits in bone and lymph node entered in this study showed any evidence of either 5R-I or 5R-II mRNA expression (data not shown). This contrasted with the strong expression of 5R-I and 5R-II mRNAs detected mainly in the glandular lesions of all 12 of the primary prostate tumors examined (Fig. 1, A and B) . However, the mRNA expression of 5R-I and 5R-II in primary prostate cancers was variable across the tumor sections, and the intensity of the staining did not correlate with the degree of differentiation of the tumors (Gleason score). mRNAs encoding both isoenzymes were also found in the stroma/fibroblast component of the primary tumors (Fig. 1, A and B) , but the staining was significantly weaker than in the corresponding epithelial areas.

View larger version (74K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Representative photomicrographs of ISH studies for 5R-I and 5R-II in primary prostate cancers (A and B). Hybridization was carried out with DIG-UTP-labeled 5R-I (A) and 5R-II (B) mRNA antisense probes, and visualized by immersion in the DIG-alkaline phosphatase-conjugated antibody. The patterns showed strong staining in the epithelial as well as the stroma/fibroblast for both the 5R-I (A) and 5R-II (B). Magnification: x250.

View larger version (58K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Photomicrographs demonstrating AR mRNA expression by ISH in sections of prostate cancer bony (A) and lymph node (B) metastasis. Hybridization was carried out with DIG-UTP-labeled AR mRNA antisense probes and visualized by immersion in the DIG-alkaline phosphatase-conjugated antibody. Positive labeling for AR mRNA in the prostate deposits of the metastatic lesions (A and B) was noted. Magnification: x250.

View larger version (121K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Photomicrographs of immunohistochemical staining for AR in sections of prostate cancer bony (A) and lymph node (B) metastasis. The sections show strong nuclear staining for AR in the prostate metastatic lesions (A and B). Magnification: x250.

View larger version (133K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Photomicrographs of immunohistochemical staining for PSA in sections of prostate cancer bony (A) and lymph node (B) metastasis. The sections show strong PSA staining confined to the prostate epithelial cells (A and B) with no staining detected out with the prostatic lesions of the bone (A) and lymph node (B) metastasis. Magnification: x250.

	DISCUSSION

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This is the first study to examine 5-reductase expression in prostate cancer metastases. Our findings demonstrated the complete absence of 5R-I and 5R-II mRNA expression in bony and lymph node metastases, although these lesions contained substantial prostate deposits, which expressed AR mRNA and were immunoreactive to both the AR and PSA antibodies.

The absence of 5R isoenzyme mRNA expression in the metastatic lesions contrasted with primary prostate cancers where mRNAs encoding both isoenzymes were present predominantly in the glandular regions but DIG-labeling was also detected in the stroma/fibroblast component of the tumors. These findings confirm earlier reports describing the presence of 5-reductase enzyme activity in primary prostate cancer (16, 17, 18, 19, 20, 21 , 25 , 26) , but unlike the previous studies we were able to identify which isoenzyme was involved. The difference in outcome between the various studies merely reflects the approaches used for detecting 5-reductase. Although earlier studies (16 , 18, 19, 20 , 25 , 26) provided quantitative information on 5R expression, they were unable to distinguish between the two isoenzymes (16 , 18, 19, 20, 21 , 25 , 26) , whereas in the present report and in an earlier study (15) we have demonstrated mRNA expression of both 5R-1 and -II, and identified the cellular distribution of both isoenzymes.

The loss of 5-reductase expression in the secondary sites is striking and in marked contrast to the maintenance of expression in the primary tumors. However, prostate metastatic sites in bone and lymph node do maintain their capacity to express AR as demonstrated by ISH and immunostaining, and the presence of PSA, an AR dependent gene, was detected in all of the specimens, thus confirming the prostate nature of these metastatic lesions. The lack of 5-reductase mRNA in the secondary sites cannot be attributed to a methodological problem, because the positive ISH controls (AR mRNA) testified to the viability of the metastatic specimens, whereas the expression of the 5R isoforms by primary tumors confirmed that the in situ techniques were working. Furthermore, our earlier report (15) on the distribution of 5R isoenzymes in benign prostatic hyperplasia testified to the reliability, sensitivity, and isoform specificity of the ISH approach. Therefore, the absence of 5R mRNA expression in the metastatic lesions must reflect a genuine change in the molecular structure of the invading prostate cancer cells. Significantly, this loss of expression is manifested at a time when the cancer has progressed from a well-differentiated primary tumor to a metastatic lesion, and this transition parallels the development of hormone refractiveness in those tumors. Earlier studies had attributed the development of hormone resistance to an amplification of the AR gene (27), which allows the cells to resume hormone-dependent growth even in the presence of low concentrations of androgen (28) . The high levels of AR immunoreactivity detected in the metastatic sites as reported herein are compatible with those from previous immunohistochemical studies (24 , 28) and provide a basis for the AR gene amplification theory. Whether the amplification of AR is secondary to the loss of 5R mRNA expression and to the depletion of the DHT concentrations in the metastasizing cells is not evident. However, the observations made in this report lend support to the view that the loss of DHT-forming 5R isoenzymes may be of crucial pathogenic relevance to the development of androgen-insensitive growth in prostate cancer.

Thus far, we have been unable to identify the causes for the loss of 5R expression in prostate cancer metastases. However, mutations of the gene encoding 5R-II have been described. Lack of 5R-II causes pseudohermaphroditism (7) in which the prostate remains highly underdeveloped and DHT levels are low. However, these mutations do not appear to be involved in prostatic diseases in adult. More recently, other reports have demonstrated that those genetic variants in 5R-II correlate with androgen metabolism and prostatic cancer risk. The 5R gene harbors two frequent polymorphic sites, one in the coding region at codon 89 of exon 1, where valine is substituted by leucine (V89L; Refs. 29, 30, 31 ), and the other in the 3' untranslated region, where a variable number of dinucleotide TA repeat lengths exist (31) . Both polymorphisms are known to increase the activity of this enzyme and may also play a role in predisposition to prostate cancer (29, 30, 31) . Thus, it is unlikely that any of these genetic variants are responsible for the loss of 5R-II mRNA expression as seen in the metastatic process. It is more likely that the absence of 5R-II mRNA expression in prostate bony and lymph node metastasis reflects the loss of a factor in the metastatic lesions, which is essential for 5R-II expression. A number of studies have recently implicated smooth muscle in the development of prostate function and growth (32, 33, 34) , and we have demonstrated that human prostate epithelial cells cocultured with prostate mesenchymal cells maintain their differentiated phenotype, including expression of 5R-II (34) . However, epithelial cells grown in isolation rapidly lose their differentiated features including the expression of 5R-I and 5R-II (34) . We have additionally demonstrated that a soluble paracrine factor of mesenchymal origin is likely to be responsible for maintaining the differentiated state of the epithelial cell (34) . Once prostate cancer epithelial cells invade secondary organs, they lose contact/proximity to prostate stroma, and, as a result, lose their differentiated phenotypic characteristics and fail to express 5-reductase. We are at present examining this hypothesis using a new model for prostate cancer bony metastasis.

	FOOTNOTES

 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at Prostate Research Group, University Department of Oncology, Western General Hospital, Edinburgh, EH4 2XU Scotland, United Kingdom. Phone: 44-131-537-1568; Fax: 44-131-343-6529; E-mail: f.k.habib{at}ed.ac.uk

² The abbreviations used are: PSA, prostate-specific antigen; DHT, dihydrotestosterone; 5R, 5-reductase; AR, androgen receptor; TNM, Tumor-Node-Metastasis; ISH, in situ hybridization; DIG, digoxigenin.

Received 2/25/02; revised 12/ 3/02; accepted 12/30/02.

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