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Endocrinology Vol. 140, No. 9 4056-4064
Copyright © 1999 by The Endocrine Society

ARTICLES

Regulation of Urokinase Production by Androgens in Human Prostate Cancer Cells: Effect on Tumor Growth and Metastases in Vivo1

Rosie Hongmei Xing and Shafaat A. Rabbani

Departments of Medicine, Physiology and Oncology, McGill University and Royal Victoria Hospital, Montréal, Québec Canada, H3A 1A1

Address all correspondence and requests for reprints to: Shafaat A. Rabbani, M.D., Royal Victoria Hospital, 687 Pine Avenue West, Room H4.72, Montréal, Québec, Canada H3A 1A1.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During the complex multistep process of tumor progression, prostate cancer is initiated as an androgen-sensitive, nonmetastatic cancer, followed by a gradual transition into a highly metastatic and androgen-insensitive variety that lacks the expression of functional androgen receptors (AR). Urokinase (uPA), a member of the serine protease family, has been implicated in the progression of various human malignancies, including prostate cancer. Although uPA production is regulated by various growth factors and cytokines, the role of sex steroids (androgens) in regulating uPA gene expression in prostate cancer is poorly understood. In the current study, we have examined the role of androgens in regulating uPA production and the invasive capacity of the androgen insensitive PC-3 cells transfected with the full-length human AR complementary DNA (PC-3T). Restoration of androgen responsiveness in PC-3T cells caused a marked decrease in cell doubling time. Treatment of PC-3T cells with dihydroxytestosterone (DHT) caused a dose-dependent decrease in uPA messenger RNA and protein production, resulting in their decreased ability to invade through the Matrigel. Nuclear runoff assays revealed that these effects were attributable to the ability of DHT to inhibit uPA gene transcription. AR antagonist flutamide (Flu) reversed the effect of DHT on proliferation and invasion of PC-3T cells. Both control (PC-3) and experimental (PC-3T) cells were injected into the right flank of male BALB/c nu/nu mice. Control animals developed palpable tumors and microscopic tumor metastases at lymph nodes, lungs, and liver at 6-week posttumor cell inoculation. In contrast to this, because of androgen sensitivity of PC-3T cells, palpable tumors were observed only at week 12, with occasional tumor metastases in lungs. Furthermore, inoculation of PC-3T cells into surgically castrated host animals resulted in the development of tumors at a much earlier time (week 10) and a high incidence of metastases, compared with regular animals receiving PC-3T cells. Collectively, these results demonstrate the ability of androgen to regulate uPA production, which may directly effect prostate cancer growth, invasion, and metastasis in vitro and in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADENOCARCINOMA of the prostate is a common hormone-dependent malignancy resulting in a high incidence of cancer-related morbidity and mortality (1, 2). Sex steroid androgens play key roles in the growth and differentiation of normal prostatic tissues to promote the initiation of malignant transformation and the progression of prostate cancer (3, 4). These effects of androgens are mediated via androgen receptors (AR), expressed in both the stromal and epithelial compartments of the prostate (5, 6, 7). During the complex multistep process of tumor progression, prostate cancer is initiated as a low-virulent and androgen-sensitive variety, which gradually transforms into a highly metastatic and hormone-insensitive variety caused by the outgrowth of AR-negative cells, resulting in the establishment of hormone resistance (2, 8). This hormone insensitivity is closely associated with a lack or mutation of the AR (9, 10, 11). Because of the close relationship between hormonal status and prostate cancer progression, treatment of early-stage prostate cancer consists of strategies aimed at eliminating the sources of circulating androgens via medical or surgical castration in combination with administration of antiandrogens (12). However, continued use of these therapeutic strategies, in treating late-stage prostate cancer, results in limited beneficial effects that may be attributed to the loss of functional AR in affected tumor cells. Additionally, various growth factors, hormones, and proteases are also implicated in prostate cancer progression.

The role of growth factors, steroids, and proteases in the acquisition of hormonal independence and the underlying molecular mechanism involved in this process remain poorly understood. The androgen-insensitive human prostate cancer cell line PC-3, which lacks a functional AR (13), has been used extensively as a model for androgen-independent prostate cancer (11, 14, 15).

Urokinase (uPA), a member of the serine protease family, is strongly implicated as a promoter of tumor progression in various human malignancies, including breast and prostate cancer (16, 17, 18). These effects are caused by the ability of uPA to break down various components of the extracellular matrix, including laminin, fibronectin, and collagen (19, 20). Although uPA is produced by normal and benign hyperplastic prostatic tissue, elevated levels of uPA are observed in patients with prostate cancer (21). In previous studies, we have demonstrated that overexpression of uPA by the rat prostate cancer cell line Dunning R3227 Mat Ly Lu results in increased tumor invasion and metastases in both skeletal and nonskeletal sites (17). These effects of uPA could be blocked by treating tumor-bearing animals with an active site inhibitor of uPA, which resulted in decreases in both tumor volume and tumor metastases (18). Although the expression of human uPA gene has been shown to be under the regulation of various growth factors and cytokines (22, 23, 24, 25), the role of sex steroids (estrogens and androgens) in regulating uPA production in hormone-dependent malignancies like breast and prostate cancer is poorly understood. Furthermore, among the currently available human prostate cancer cells, only hormone-insensitive (PC-3) cells express uPA, whereas androgen response Ln-CAP cells fail to produce any detectable levels of uPA (26).

In the current study, we have examined the regulation of uPA production by androgens in the androgen-insensitive human prostate cancer cell line PC-3, which lacks a functional AR, and in androgen-sensitive PC-3 cells, transfected with the full-length human AR complementary DNA (cDNA) (PC-3T). The effect of androgens on uPA production, tumor cell growth, invasion, and metastasis was evaluated both in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, and reagents
The human prostate cancer cell line PC-3 (11) was obtained from the American Type Culture Collection (Rockville, MD). PC-3 cells, transfected with a functional full-length human AR cDNA (PC-3T), were kindly provided by Dr. T. J. Brown (The Toronto Hospital Research Institute, Toronto, Canada) (27). PC-3 cells were maintained in F-12 (Life Technologies, Inc., Gaithersburg, MD), and the transfected PC-3T cells were maintained in RPMI 1640 (Life Technologies, Inc.) supplemented with 100 µg/ml hygromycin B (Sigma Chemical Co., St. Louis, MO). Wild-type PC-3 cells were also transfected with empty vector and used as control (27, 28). All culture media were supplemented with 10% FBS (Life Technologies, Inc.), 25 mM 4-(2-hydroxyethy)-1piperazineethanesulfonic acid, 26 mM sodium bicarbonate, 5000 U/ml penicillin G (Life Technologies, Inc.), and 5000 mg/ml streptomycin (Life Technologies, Inc.). Cells were incubated at 37 C in 5% CO2. Stripped FBS (SFBS; Life Technologies, Inc.), which is depleted of steroids, was used during androgen treatment. PC-3T cells were treated with different concentrations of androgen dihydroxytestosterone (DHT) and the AR antagonist flutamide (Sigma Chemical Co.).

Stable transfection of PC-3 and PC-3T cells with green fluorescent protein (GFP)
The expression vector containing the codon-optimized hGFP-S65T gene was obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA). PC-3 and PC-3T cells were transfected with hGFP-S65T using lipofectin reagent (Life Technologies, Inc.). Cells with stably integrated plasmids were selected for neomycin-resistant gene with G418 (28).

Cell proliferation
Cell growth was determined by cell proliferation assays. Ten thousand PC-3 and PC-3T cells were plated in 2 ml of medium in 6-well tissue culture plates. Where indicated, PC-3T cells were treated with either 10 nM DHT alone or with 10 nM DHT and 10 nM flutamide. Cell culture medium was replenished every third day. At 4 h and at the indicated time point, cells were trypsinized and counted using a Coulter Counter (model ZF, Coulter Electronics, Harpenden, Herts, UK). After 4 h of incubation, the number of cells in each well was determined, to establish that equal numbers of cells were present in all wells.

Northern blot analysis
Total cellular RNA was isolated from control and experimental PC-3 and PC-3T cells and tumors, as previously described (28). Briefly, 20 µg total cellular RNA was electrophoresed on a 1.1% agarose-formaldehyde gel and transferred to a nylon membrane (Nytran, Pharmacia, Montréal, Québec, Canada) by capillary blotting. All blots were hybridized with a 32P-labeled human uPA cDNA or with 18S cDNA as a control for the amount of RNA loaded (17, 28). All filters were incubated at 42 C for 24 h and successively washed in 1 x SSC (10 x SSC is 1.5 M NaCl, 0.5 M sodium citrate, pH 7.0), 1% SDS for 15 min at room temperature; 0.5 x SSC, 0.5% SDS for 15 min at room temperature; 0.1 x SSC, 0.1% SDS twice for 15 min at room temperature; and then once for 30 min at 55 C. Autoradiography of filters was carried out at -70 C using XAR film (Eastman Kodak Co., Rochester, NY) with two intensifying screens. The level of uPA messenger RNA (mRNA) expression was quantified by densitometric scanning.

Indirect immunofluorescence
To examine the cell surface expression of uPA by PC-3 and control and experimental PC-3T cells, 5 x 104 cells were plated in Lab-Tek tissue culture chambers (Nunc Inc., Naperville, IL) and allowed to grow to 70–80% confluence. Cells were then incubated with 30% goat serum (Sigma Chemical Co.) for 1 h at room temperature and washed with PBS containing 1% BSA. Cells were subsequently incubated with 100 µg/ml of rabbit antirat uPA IgG (American Diagnostica Inc. Greenwich, CT) and with goat-antirabbit IgG conjugated to fluorescein isothiocyanate (Sigma Chemical Co.). Photographs were taken at 60x magnification using a Zeiss MC-63 microscope (Carl Zeiss Canada, Don Mills, Ontario, Canada) (28).

Nuclear runoff assay of gene transcription
Nuclear runoff assays were performed by harvesting cells after treatment with various agents in cold PBS. Cells were collected and lysed in cold NP-40 lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40] for 5 min on ice. Cell nuclei were collected by centrifugation at 4 C and were resuspended in storage buffer [50 mM Tris-HCl (pH 8.3), 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 5 mM dithiothreitol]. The nuclei were either used immediately or frozen in liquid nitrogen for later use. Nuclear runoff assays were carried out by adding 100 µl nuclear suspension (2–4 x 107 nuclei) to 100 µl reaction buffer [50 mM Tris-HCl (pH 7.5); 0.3 M KCl; 5 mM MgCl2; 5 mM dithiothreitol; 0.5 mM each of ATP, cytidine 5'-triphosphate, and GTP; 50–100 µCi [32P]{alpha}-uridine triphosphate, >600 Ci/mmol, ICN Pharmaceuticals, Inc., Costa Mesa, CA] for 60 min at room temperature. After the incubation, deoxyribonuclease I (150 U per reaction) and proteinase K (0.2 mg/ml) were added and incubated for 30 min at 37 C, respectively (27). Newly synthesized RNAs were isolated by spin column and ethanol precipitation and were pelleted by centrifugation. RNAs were hybridized with uPA and 18S cDNAs and with Bluescript vector DNA (Stratagene, La Jolla, CA) previously immobilized on Nytran membranes using a slot blot manifold (Bio-Rad Laboratories, Inc., Richmond, CA). These membranes were incubated in the hybridization solution [6 x SSC (pH 7.4), 50% formamide, 1% SDS, 0.1 mg/ml sonicated salmon sperm DNA] at 42 C for 48 h. After hybridization, membranes were washed in a final wash solution of 0.1 x SSC, 0.1% SDS at 42 C, and exposed to Kodak XAR film with intensifying screens. The intensity of each band was quantitated using laser densitometry.

Boyden chamber matrigel invasion assay
The invasive capacities of PC-3 and PC-3T cells were determined by two-compartment Boyden chambers (Transwell, Costar, Fisher Scientific, Montréal, Québec, Canada) and basement membrane Matrigel (Becton Dickinson and Co., Bedford, MA) invasion assay, as previously described (28, 29). In some experiments, PC-3T cells were cultured in SFBS in the presence or absence of DHT (10-8 M), or with DHT and AR antagonist flutamide (10-6 M), or with rat anti-uPA antibody (100 µg/ml).

Animal protocols
Six-week-old BALB/c nu/nu male normal and castrated mice were obtained from Charles River Laboratories, Inc. (St. Constant, Québec, Canada). Before inoculation, GBF-labeled PC-3 and PC-3T tumor cells (PC-3-GAP and PC-3T-GAP) grown in serum-containing medium were washed with Hanks’ buffer and were trypsinized for 5 min. Cells were then collected in Hanks’ buffer and centrifuged at 1500 rpm for 5 min. Cell pellets (3 x 106 cells) were resuspended in 200 µl of a matrigel and saline mixture (20% matrigel) and injected sc into the flank region of the hinder leg of the mice. All animals were numbered, kept separately, and examined for the development of tumors weekly. The tumor mass was measured in two dimensions by callipers, and the tumor volume was calculated. Animals were killed at timed intervals and were examined and scored for the development of macroscopic metastases in various tissues.

Statistical analysis
Results are expressed as the mean ± SEM of at least triplicate determinations, and statistical comparisons are based on the Student’s t test or ANOVA. P < 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of androgen on prostate cancer cell growth in vitro
The effect of restoration of androgen responsiveness was examined by a cell proliferation assay in PC-3T cells, and comparison was made with cell-doubling time of wild-type PC-3 cells over 5 days. PC-3T cells exhibited a significantly reduced (40–50%) cell-doubling time throughout the course of this study. No significant difference in cell-doubling time was seen between PC-3T and PC-3 cells transfected with empty vector (PC3-V) (Fig. 1Go). After culture of PC-3T cells in a culture medium containing charcoal SFBS to remove androgens, treatment of PC-3T cells with 10-8 M DHT showed a marked increase in PC-3T cell growth. The specificity of AR in mediating this growth-stimulatory effect of androgen was confirmed by coincubation of PC-3T cells with DHT and 10-6 M AR antagonist flutamide (Flu), which completely abolished the increase in PC-3T cell growth seen after DHT treatment (Fig. 1Go). Under similar experimental conditions, no effect on PC-3 cell-doubling time was seen after treatment with DHT (data not shown).



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  		Figure 1. Effect of androgen on PC-3 cell growth. Growth curves were compared between the wild-type PC-3 and PC-3 cells transfected with the full-length human AR (PC-3T) and with empty vector (PC-3V). The rate of PC-3T cell-doubling time was also compared after treatment of PC-3T with DHT (PC-3T+DHT) alone or in combination with AR antagonist flutamide (PC-3T+DHT+Flu). Cells from triplicate dishes were trypsinized at each time point and counted as described in Materials and Methods. Each point represents ± SEM of three experiments. Significant difference in the growth of PC-3T cells from the control is represented by asterisks (P < 0.05).

 
Effect of androgen on uPA production
To examine the effect of restoration of androgen responsiveness of PC-3 cells on uPA mRNA expression, Northern blot analysis was performed on PC-3 and PC-3T cells cultured in the presence of 10% FBS or charcoal SFBS to remove androgens. Restoration of androgen responsiveness resulted in a marked decrease in uPA mRNA expression in PC-3T cells, as compared with wild-type PC-3 cells. This inhibition in uPA mRNA expression could be restored in PC-3T cells after removal of androgens from FBS with charcoal treatment (Fig. 2AGo). A similar change in uPA protein production was seen in PC-3T cells, as determined by immunohistochemistry (Fig. 2BGo). These effects of induction of uPA expression in PC-3T cells by stripped serum treatment were time dependent, with a maximal induction of uPA after 24 h of androgen removal (data not shown).



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  		Figure 2. Effect of restoration of hormone responsiveness on uPA production. A, Total cellular RNA was extracted from PC-3 and PC-3T cells cultured in medium containing 10% FBS and in culture medium supplemented with charcoal SFBS; B, uPA protein production was determined in PC-3 cells and PC-3T cells maintained in culture medium containing FBS (PC-3T-FBS) and in charcoal SFBS (PC-3T-SFBS) by indirect immunofluorescence under 60x magnification, as described in Materials and Methods. HuPA, Human uPA.

 
The specificity of the effect of androgen on uPA production was further evaluated by treating PC-3T cells with different concentrations of DHT after 24 h of androgen withdrawal. DHT inhibited uPA mRNA expression in a dose-dependent manner (10-6–10-9 M) (Fig. 3AGo). A similar decrease in uPA protein production was seen in DHT-treated PC-3T cells (Fig. 3BGo). To determine the role of AR in mediating these effects, PC-3T cells were treated with either DHT alone or in combination with the AR antagonist flutamide. Because of the ability of flutamide to compete with androgens for AR binding, coincubation of PC-3T cells with DHT and flutamide resulted in a complete restoration of uPA mRNA expression, as determined by Northern blot analysis (Fig. 4Go). In contrast to this, addition of DHT into wild-type and empty vector-transfected PC-3 cells failed to alter uPA production (data not shown).



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  		Figure 3. Effect of androgen on uPA production in PC-3T cells. A, Total cellular RNA was extracted from vehicle-treated PC-3T cells (CTL) and PC-3T cells treated with different concentrations of DHT (DHT). Twenty micrograms of total cellular RNA from each group were electrophoresed on a 1.1% agarose/formaldehyde gel and blotted to a nylon membrane by capillary action. All blots were hybridized with a 32P-labeled human uPA cDNA or with a 32P-labeled 18S cDNA, as described in Materials and Methods. Blots were scanned by laser densitometric scanning, and changes in uPA mRNA expression were determined by plotting the ratio of uPA and 18S mRNA. Results are representative of at least four different experiments. Significant difference from control cells is represented by asterisks (P < 0.05). B, uPA protein production was determined in CTL and PC-3T cells receiving androgen treatment (DHT) by indirect immunofluorescence under 60x magnification, as described in Materials and Methods.

 


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  		Figure 4. Role of AR in mediating the inhibitory effect of androgen on uPA mRNA expression. The role of the AR in mediating the inhibitory effect of androgen on uPA mRNA expression was determined by Northern blot analysis. Total cellular RNA was extracted from CTL, PC-3T cells treated with 10-8 M of DHT (DHT), and PC-3T cells treated with both 10-8 M DHT and 10-6 M flutamide (DHT+Flu). Twenty micrograms of total cellular RNA from each group were electrophoresed on a 1.1% agarose/formaldehyde gel and blotted to a nylon membrane by capillary action. All blots were hybridized with a 32P-labeled human uPA cDNA or with a 32P-labeled 18S cDNA, as described in Materials and Methods. Blots were scanned by laser densitometric scanning, and the changes in uPA mRNA expression were determined by plotting the ratio of uPA and 18S mRNA. Results are representative of at least four different experiments. Significant difference from control cells is represented by asterisks (P < 0.05).

 
Effect of androgen on uPA gene transcription
To explore the underlying molecular mechanisms involved in the regulation of uPA gene expression by androgen in PC-3T cells, the effect of DHT on uPA gene transcription was examined by nuclear runoff assays. Comparison of uPA gene transcription in wild-type PC-3 and PC-3T cells showed a marked reduction in uPA gene transcription in PC-3T cells. This reduction in uPA gene transcription could be restored after SFBS treatment (Fig. 5Go). Treatment of PC-3T cells grown in SFBS with 10-8 M DHT showed a marked decrease in uPA gene transcription, and such a reduction was effectively prevented by coincubation of PC-3T cells with DHT (10-8 M) and AR antagonist flutamide (10-6 M) (Fig. 5Go). Collectively, these results demonstrate that DHT can decrease uPA gene transcription, and these actions of DHT are mediated via the AR.



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  		Figure 5. Effect of androgen on uPA gene transcription. Nuclear runoff assays were performed as described in Materials and Methods. 32P-labeled runoff transcripts were prepared from PC-3 cells and PC-3T cells after treatment with 10% FBS, SFBS, 10-8 M DHT alone (DHT), or 10-8 M DHT and 10-6 M flutamide (DHT+Flu). All blots were probed with human uPA and 18S cDNA, scanned by laser densitometric scanning, and fold stimulation of uPA gene transcription (relative to that of 18S) was determined. Results are representative of three different experiments. Significant difference from control cells is represented by asterisks (P < 0.05).

 
Effect of androgen on PC-3T cell invasion
The effect of restoration of hormone sensitivity on the invasive capacity of PC-3 cells was determined by Boyden Chamber Matrigel invasion assays. Androgen-sensitive PC-3T cells exhibited a significantly lower invasive capacity through Matrigel, as compared with wild-type PC-3 cells, which are hormone insensitive (Fig. 6Go). However, the number of PC-3T cells invading through the Matrigel was higher when these cells were incubated in the presence of SFBS. In contrast to this, after DHT treatment, a marked decrease in PC-3T cell invasion was observed. The inhibitory effect of androgen on PC-3T cell invasion could be blocked by addition of AR antagonist flutamide or antihuman uPA IgG (Fig. 6Go). Collectively, these results demonstrated that the inhibitory effect of androgen on PC-3T cell invasion was attributable to its ability to inhibit uPA production, and these actions of androgen were mediated by the AR.



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  		Figure 6. Effect of androgen on PC-3 and PC-3T cell invasion. Both PC-3 and PC-3T cells were grown in culture, as described in Materials and Methods. Number of PC-3T cells migrating to the lower aspect of the Boyden chamber filter after treatment with SFBS, or with 10-8 M of DHT, or with 10-8 M DHT and 10-6 M flutamide (DHT+Flu) and 50 µg/ml of anti-uPA antibody (SFBS+uPA IgG) were counted and compared with vehicle-treated controls (FBS). Results represent ± SEM of four different experiments. Significant difference in cell invasion from the wild-type control cells is represented by asterisks (P < 0.05).

 
Results from these in vitro studies clearly demonstrated that restoration of androgen responsiveness rendered PC-3 cells in a less virulent phenotype, which is characterized by lower rate for proliferation, decreased uPA production, and the resultant decreased capability of tumor cells to invade through the Matrigel.

Effect of androgen on tumor growth, invasion, and metastasis in vivo
After in vitro characterization of the effect of restoration of androgen responsiveness of PC-3T cells on cell growth, invasion, and uPA production, we compared the growth and metastatic characteristics of PC-3 and PC-3T cells in vivo. sc inoculation of the wild-type PC-3 cells into the right flank of 6-week-old male BALB/c nu/nu mice resulted in the development of palpable tumors by 6-week posttumor cell inoculation (Fig. 7Go). In contrast to this, development of primary tumors was significantly delayed when the animals were inoculated with PC-3T cells, where tumors could be palpated only at 12 weeks after tumor cell inoculation. Once developed, both PC-3 and PC-3T tumors showed a linear growth rate up to week 16 (Fig. 7Go). To evaluate the effect of androgen ablation on PC-3T tumor development, PC-3T cells were inoculated into castrated animals. Castrated nude mice developed palpable tumors, by 10-week posttumor cell inoculation, which was at least 2 weeks earlier, compared with noncastrated PC-3T tumor-bearing animals. Although the tumor volume of castrated animals was significantly smaller, compared with PC-3 tumors, it was markedly larger than that of noncastrated animals (Fig. 7Go).



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  		Figure 7. Effect of restoration of androgen sensitivity on PC-3 tumor growth. Tumor volumes of noncastrated BALB/c nu/nu male nude mice receiving PC-3 or PC-3T cells, and of castrated animals inoculated with PC-3T cells were determined at timed intervals, as described in Materials and Methods. Results represent the mean ± SEM of six starting animals in each group in three different experiments. Significant difference from control PC-3 and PC-3T tumor-bearing animals is marked by asterisks (P < 0.05).

 
To assess the effect of restoration of androgen sensitivity on PC-3 and PC-3T tumor metastases, we used tumor cells that had previously been transfected with the plasmid containing GFP. These green fluorescent cells showed similar growth characteristics in vitro and in vivo, compared with the wild-type PC-3 and PC-3T cells (data not shown). However, the presence of green cells allowed us to easily visualize and characterize metastatic tumor foci in the distant organs of host animals. At the time of development of palpable PC-3 tumors (week 6), tumor metastases were seen in the lymph nodes, livers, and lungs. In contrast to this, at the time of PC-3T tumor development (week 12), no evident tumor metastases were detected in the lymph nodes and livers; only a few small metastatic foci were seen in the lungs of these animals. However, when PC-3T cells were inoculated into castrated hosts, tumor metastases were observed in the livers and lungs by week 10 (Table 1Go). Removal of the primary tumors and lung metastases, after Northern blot analysis, showed significantly lower levels of uPA mRNA expression in animals inoculated with PC-3T cells, compared with animals bearing PC-3 tumors. Moreover, the tumoral level of uPA mRNA was significantly higher in castrated PC-3T tumor-bearing animals, compared with that of noncastrated controls (Fig. 8Go).


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  		Table 1. Effect of restoration of hormone sensitivity and androgen on PC-3 tumor metastases

 


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  		Figure 8. Effect of androgen sensitivity on tumoral uPA mRNA expression. Total cellular RNA was extracted from PC-3, PC-3T, and PC-3T tumors of castrated animals (PC-3T-Cast). Twenty micrograms of RNA from each group were electrophoresed on a 1.1% agarose-formaldehyde gel and blotted to a nylon membrane. Twenty micrograms of total cellular RNA from each group of cells were electrophoresed on a 1.1% agarose/formaldehyde gel and blotted to a nylon membrane by capillary action. All blots were hybridized with a 32P-labeled human uPA cDNA or with a 32P-labeled 18S cDNA, as described in Materials and Methods. Blots were scanned by laser densitometric scanning, and change in uPA mRNA expression was determined by plotting the ratio of uPA and 18S mRNA. Results are representative of at least four different experiments. Significant difference from control cells is represented by asterisks (P < 0.05).

 
These results collectively, for the first time, demonstrated that restoration of hormone responsiveness in PC-3 cells resulted in a decrease in tumor growth, invasion, and metastases in vivo. These results also demonstrated that this change in growth and metastasis of tumor cells, after induction of their androgen responsiveness, was caused by inhibition of uPA production, which plays a key role in tumor growth and metastasis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Patients with prostate cancer initially respond to hormone therapies, including androgen ablation and antiandrogen treatment. However, because of a slow transition into androgen-independent tumor, currently available hormonal and chemotherapeutic strategies have limited success in controlling prostate cancer progression (30, 31). In the present study, we have investigated the mechanisms of high invasiveness and poor responsiveness to anticancer therapies of hormone-insensitive prostate cancer cells. For these studies, we have used androgen-insensitive human prostate cancer cell line PC-3 and compared its growth, invasion, and metastatic behavior with those of PC-3 cells transfected with a functional AR (PC-3T) (31). Restoration of PC-3T cells into a hormone-sensitive state significantly reduced the cell-doubling time and their ability to invade through the Matrigel. The observed decrease in cell-doubling time is consistent with reports that establishment of androgen responsiveness in PC-3 cells leads to a lower malignant state of cells characterized by the decreased rate of proliferation, impaired ability of colony formation, increased epithelial cell differentiation, and increased production of insulin-like growth factor binding proteins (32, 33). In addition to altered growth role, we observed a significant reduction in the production of uPA, a key protease, by androgen-responsive PC-3T cells at the levels of mRNA expression, gene transcription, and protein production. We and others (34, 35, 36, 37, 38) have previously shown that uPA has mitogenic effects on several cell lines. Therefore, decreased uPA production in PC-3T cells could also contribute to their slower rate of cell proliferation, compared with nontransfected PC-3 cells, which produce abundant amounts of uPA. Increased uPA production is associated with increased tumor growth, invasion, and metastasis of various human malignancies, including prostate cancer. Results presented in this study indicate that the decrease in the invasive capacity of PC-3T cells is caused by the reduced production of uPA in response to androgen.

Because higher levels of uPA production are observed in prostate cancer (17, 18, 21), the inhibitory effect of androgens on uPA production may explain the noninvasive and low-virulent phenotypes of tumor cells at the initial hormone-sensitive state of prostate cancer. This may also provide a mechanism for a role of androgens in the acquisition of a more malignant phenotype, during hormone treatment, to promote the growth of hormone-insensitive tumor cells. Decreased availability of androgens to tumor cells in the presence of competing antiandrogens may promote a subset of hormone-responsive prostate cancer cells to produce higher levels of uPA (which, in turn, endows tumor cells with a higher invasive and metastatic potential). On the other hand, increased uPA production may have a direct effect on promoting tumor progression via its stimulatory effect on angiogenesis, cell adhesion, and migration (39, 40). It is not yet known whether the effect of androgen on uPA gene expression is direct; however, the presence of a putative AR-responsive element within the uPA promoter region (23) strongly suggests that AR can directly down-regulate the expression of uPA mRNA in hormone-sensitive PC-3T cells.

When tested in vivo, athymic nude mice, inoculated with hormone-responsive PC-3T cells, developed significantly smaller tumors and exhibited a later onset of tumor development, as compared with animals receiving wild-type hormone-insensitive PC-3 cells. Moreover, PC-3T tumor-bearing animals developed limited micrometastases at their livers, as compared with the development of extensive microscopic and macroscopic tumor metastases in the livers, lungs, and auxiliary lymph nodes of animals inoculated with PC-3 cells. Detection of micrometastase was made possible by using GFP-labeled PC-3 and PC-3T cells (41). Previous in vivo studies by Chishima et al. (42) have demonstrated the effectiveness, simplicity, and sensitivity of the GFP gene as a marker to visualize micrometastases in fresh viable target organs, such as the livers, lungs, and draining and regional lymph nodes at the single-cell level. To examine the effect of uPA induction by androgen ablation to alter tumor growth, invasion, and metastases, PC-3T cells were implanted into the castrated nude mice. The early onset of PC-3T tumor development in castrated host further underscored the role of androgens in tumor development. Decreased production of uPA in primary tumors and at various metastatic sites of PC-3T tumor-bearing animals strongly points to the role of uPA in altering the growth and metastatic ability of tumor cells in vivo. Recent clinical studies have shown that, in castrated human males, intraprostatic DHT concentrations range as high as 20–50% of that measured before castration (43), illustrating the significant contribution of extragonadal sources of androgen. Results from our studies demonstrate that the extragonadal sources of androgen are sufficient to provide the required amount of androgen for the initiation and progression of prostate cancer, a finding with significant clinical implications. It points to the importance of complete deprivation of any androgenic source to prevent the growth of early stage, hormone-responsive prostate tumors, which can be achieved by performing both surgical and medical castrations. A common approach to medical castration is via drug-induced suppression of the hypothalamic-pituitary-testicular axis by administration of GnRH analogs. If complete androgen ablation is not achieved, the residual amount of androgen could not only stimulate tumor growth but also promote a subset of hormone-sensitive tumor cells, to acquire a more malignant phenotype via increased production of tumor progression factors, like uPA, to cause a continued high rate of morbidity and mortality.

In summary, these results clearly indicate that androgens could help to maintain androgen-responsive prostate cancer cells in a low invasive and metastatic state, by down-regulating uPA production. They also provide a potential mechanism for the emergence of hormone-independent and more malignant phenotype during hormonal therapy, because of an induction of uPA production by hormone-responsive prostate cancer cells, in response to androgen suppression. These observations have significant clinical implications. They point to the importance of careful designing of treatment regimen in which the timing of hormonal therapy and the procedures of androgen suppression are carefully chosen and determined. These could help to limit the undesired effects of current therapies, thus improving the outcome of treatment.


   Footnotes
 
1 This work was supported by the Medical Research Council of Canada Grants MT-12609 and MT-10603 (to S.A.R.). Back

Received September 28, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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