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Endocrinology Vol. 143, No. 10 3897-3904
Copyright © 2002 by The Endocrine Society

ARTICLE

Androgen Modulation of Adhesion and Antiadhesion Molecules in PC-3 Prostate Cancer Cells Expressing Androgen Receptor

Andreas Evangelou, Michelle Letarte, Alexander Marks and Theodore J. Brown

Cancer and Blood Research Program (A.E., M.L.), The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8; Division of Reproductive Science (A.E., T.J.B.), The Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada M5G 1X5; The Banting and Best Department of Medical Research (A.M.), University of Toronto, Ontario, Canada M5G 1L6; Department of Zoology (A.E., T.J.B.), University of Toronto, Toronto, Ontario, Canada M5S 3G5; Department of Obstetrics and Gynecology (M.L., T.J.B.), University of Toronto, Toronto, Ontario, Canada M5G 1L4; and Department of Immunology (M.L.), University of Toronto, Toronto, Ontario, Canada M5S 1A8

Address all correspondence and requests for reprints to: Theodore J. Brown, Ph.D., Associate Professor, Department of Obstetrics and Gynecology, Samuel Lunenfeld Research Institute, 600 University Avenue, University of Toronto, Toronto, Canada M5G 1X5. E-mail: brown{at}mshri.on.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The metastatic spread of cancer cells involves a complex process of detachment via antiadhesion molecules and attachment and migration through adhesion. In the prostate, androgens are generally thought to contribute to the development and progression of prostate cancer by promoting cell proliferation and survival through poorly defined mechanisms. We have reported previously that PC-3 prostate cancer cells, which are unresponsive to androgens, show androgen-dependent detachment and ultimately apoptosis when stably transfected with a full-length human androgen receptor (AR) cDNA. We now demonstrate that treatment of these cells with 5{alpha}-dihydrotestosterone (DHT) for 24 or 48 h increased the expression of antiadhesion mucin MUC-1 at the cell surface as detected by flow cytometry with two independent antibodies. This increase in protein was concordant with up-regulation of MUC-1 mRNA in the AR-transfected PC-3 sublines, as determined by quantitative RT-PCR. Treatment with DHT for 48 h also down-regulated the cell surface expression of {alpha}2ß1-integrin but having little effect on the levels of {alpha}3ß1- and {alpha}5ß1-integrins. Androgen also decreased, in a dose-dependent manner, the adhesion of AR-transfected PC-3 cells to collagen type I, which was shown to be specifically inhibited by blocking antibody to {alpha}2ß1-integrin. The present data demonstrate that DHT can modulate expression of adhesion and antiadhesion molecules and suggest that this effect of androgen might contribute to prostate cancer progression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAJORITY OF cancer-related deaths are due to the metastatic spread of malignant tumor cells to distant sites by uncontrolled growth and invasion. Metastatic spread is a complex multistep process involving detachment of cells from primary tumors and their selective uptake from the circulation at distant sites (1, 2). Whereas integrins function in cell-cell and cell-extracellular matrix (ECM) interactions, antiadhesive molecules such as mucin MUC-1 participate in preventing these interactions. The extracellular domain of MUC-1 can extend 200–500 nm beyond the cell surface, up to 100-fold further than most other cell membrane proteins including integrins (3, 4). Because of its extensive hydrated sugar linkages, MUC-1 is thought to form a glycocalyx on the apical surface of epithelial cells that serves as a protective barrier to prevent adhesion (5). Studies on embryo implantation by Aplin (6) and cancer cells by Wesseling et al. (7) have suggested a fine balance between integrin-mediated cell-cell or cell-ECM interactions and antiadhesion by MUC-1. MUC-1 is overexpressed in many human adenocarcinomas (8) and may regulate cancer cell growth by altering cell-cell and cell-ECM interactions (9). Several studies have demonstrated the regulation of MUC-1 expression by steroid hormones, including estrogens, progesterone, glucocorticoids, and androgens (10, 11, 12, 13, 14, 15, 16, 17, 18, 19).

In the prostate, androgens maintain the structural and functional integrity of epithelial cells by stimulating proliferation and survival (20, 21). Prostate adenocarcinoma cells maintain these responses to androgens but eventually become androgen independent (22, 23). Human prostate cancer PC-3 cells, derived from a bony metastasis, do not express androgen receptor (AR) and have been used extensively as a model of androgen-independent disease. These cells metastasize to bone in mouse models and can adhere and spread onto bone matrix via the {alpha}2ß1-integrins (24, 25). We have previously studied the impact of rendering PC-3 cells androgen sensitive by establishing clonally selected sublines of these cells expressing a full-length human AR cDNA. Upon treatment with 5{alpha}-dihydrotestosterone (DHT), these cells detach from the culture dish and ultimately undergo apoptosis (26). This observation raised the possibility that androgens might affect the balance between adhesion and antiadhesion. Whereas detachment in vitro may lead to apoptosis, prostate cancer cells that detach in vivo may survive and form metastases.

To examine the influence of androgens on adhesion, we investigated the regulation of MUC-1 and integrins by DHT in AR-transfected PC-3 cells. We present evidence that DHT up-regulates the expression of MUC-1 and down-regulates the cell surface expression of {alpha}2ß1-integrin in these cells. Furthermore, we show that DHT treatment decreases adhesion of these cells to collagen type I, a major component of bone matrix. These findings thus support a potential role for androgens in modulating cell adhesion and metastasis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
Clonally selected sublines of PC-3 human prostate cancer cells stably transfected with pCEP4 (Invitrogen Canada Inc., Burlington, Ontario, Canada) containing a full-length AR cDNA were previously established in our laboratory (26). Three sublines, PC-3(AR)13, PC-3(AR)6, PC-3(AR)10, expressing androgen-binding levels approximately 30%, 100%, and 180%, respectively, of that measured in LNCaP cells (26), were chosen for this study. PC-3 cells transfected with pCEP4 vector without insert, mock-transfected, PC-3(M) cells were included as controls.

All cells were maintained in RPMI 1640 culture medium (without phenol red) supplemented with 5% (vol/vol) fetal bovine serum (FBS), 50 U/ml penicillin, 50 µg/ml streptomycin, 0.625 µg/ml Amphotericin B (Invitrogen Canada Inc.) and 100 µg/ml Hygromycin B (Calbiochem, La Jolla, CA). All cultures were maintained at 37 C in a humidified 5% CO2 atmosphere. Charcoal-stripped FBS, prepared as described by Jones et al. (27), was substituted for normal FBS 48 h before experiments. DHT (Sigma, St. Louis, MO) was dissolved in ethanol and diluted with medium before addition to cultures. The final ethanol concentration in treated cultures was 0.001%.

Quantitative RT-PCR
Cells were washed in ice-cold PBS or serum-free medium and total RNA was isolated using TRIZOL Reagent (Invitrogen Corp., Carlsbad, CA) according to manufacturer instructions. The RNA was quantified spectrophotometrically and reverse transcribed using an oligo(dT) primer.

Quantitative analysis of PCR products was performed according to Murphy et al. (28) with modifications. The selected primers used for amplification of MUC-1 cDNA were 5'-TGATGTGCCATTTCCTTTCTC-3' (forward primer) and 5'-TACAAGTTGGCAGAAGTGGCT-3' (reverse primer), which produced a 342-bp PCR product. ß-Actin was amplified using primers previously reported (29). PCR amplification conditions were as described previously (29) with an annealing temperature of 58 C and 28 cycles for both MUC-1 and ß-actin. Aliquots (20 µl) of the PCR products were fractionated by gel electrophoresis on 2% agarose gels and detected with SYBRgold (Molecular Probes, Inc., Eugene, OR) staining. The bands from each gel were digitized by computer-assisted densitometry using 1DImage Analysis Software (Kodak Digital Science, Scientific Imaging Systems, Eastman Kodak Co., Rochester, NY). Data were plotted as the log of densitometric units against the log of equivalent RNA concentration to identify a linear range of exponential amplification. Linear regressions were obtained using SigmaPlot Scientific Graphing Software (version 2.00, Jandel Corp., San Rafael, CA). The amount of total MUC-1 mRNA estimated from the linear range of amplification after DHT treatment was expressed as a percentage of that measured in cells treated with ethanol vehicle (control). All measurements were normalized for ß-actin expression.

Flow cytometry
Cells were washed twice with PBS (PBS: 137 mM NaCl, 2.68 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4), detached with 0.25% trypsin EDTA, and resuspended in 0.1% BSA in PBS (0.1% BSA/PBS). Cells were incubated for 45 min at 4 C with saturating amounts of murine monoclonal antibodies (mAb) to human leukocyte antigen class I (44D6) (30), MUC-1 (2G3) (31) and BC-2 (Serotec Ltd., Kidlington, Oxford, UK), integrin subunits {alpha}2 (P1E6, Telios, La Jolla, CA), {alpha}3 (P1B5, Telios), {alpha}5 (JBS5, donated by Dr. John Wilkins, Winnipeg, Manitoba, Canada), and ß1 (4B4, Coulter Clone, Fullerton, CA) or control nonimmune murine IgG1. After two washes with 0.1% BSA/PBS, the cells were incubated with fluorescein isothiocyanate-conjugated affinity-purified goat F(ab')2 antimouse IgG (Biosource Technologies, Inc. International, Camarillo, CA) at 20 ng/ml for 45 min at 4 C in the dark. After washing twice with PBS, the cells were stained with propidium iodide (PI) to allow for gating of viable cells during analysis by FACScan with CellQUEST software (Becton Dickinson and Co., Mountain View, CA). The mean fluorescence intensity is reported for all viable cells.

Adhesion assays
Adhesion of cells to collagen type I was performed as described by Kostenuik et al. (25) with minor modifications. Briefly, cells were grown in medium with or without DHT for 48 h, detached with 0.25% trypsin-EDTA, washed with PBS, and resuspended in serum-free RPMI-1640 medium with 20 mM HEPES. Plastic Microtest Optilux 96-well assay flat-bottom plates (Becton Dickinson and Co. Labware, Franklin Lakes, NJ) were coated overnight at 4 C with varying concentrations of rat-tail collagen type I (Fisher Scientific, Collaborative Biomedical Products, Bedford, MA) diluted with PBS. Control wells were coated with 1% BSA to correct for nonspecific binding in the adhesion assay. All wells were blocked with 1% BSA/PBS for 1–2 h and washed with PBS. Cells were labeled with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF, Molecular Probes, Inc.) as described by Regimbald et al. (32) before distribution to appropriate wells. For experiments involving a blocking antibody to {alpha}2-integrin, cells were incubated for 45 min at 4 C with varying amounts of mAb P1E6 or murine IgG in serum-free RPMI-1640 media with 20 mM HEPES before plating. After incubation at 37 C for 30 min, the wells were washed and the percentage of adherent cells was determined using a SpectraMAX GEMINI Fluorescence Reader and SOFTMAX PRO Version 3.1.1 Microplate Data Acquisition and Analysis Software (Molecular Devices, Sunnyvale, CA). Data were acquired by fluorescence excitation at 485 nm and emission at 530 nm.

Statistics
Data obtained from cell adhesion assays and flow cytometry experiments were subjected to ANOVA using SPSS, Inc. for Windows statistical software (version 10.0.7, SPSS, Inc., Chicago, IL). Further group comparisons were made using Tukey honestly significant difference (HSD) multiple range test or Student-Newman-Keuls test at P < 0.05 as indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Androgen up-regulates MUC-1 protein in AR-transfected PC-3 cells
Clonally selected sublines of AR-transfected PC-3 cells that express varying levels of androgen binding were established and previously characterized (26). In this study we used several of these sublines to determine whether androgen could influence the cell surface expression of MUC-1. PC-3(AR)13, PC-3(AR)6, and PC-3(AR)10 cells, which express low, moderate, and high levels of androgen binding, respectively, were treated with 10 nM DHT or vehicle 24 or 48 h before harvesting. MUC-1 immunoreactivity was measured on the surface of viable cells by flow cytometry using mAb 2G3, reported to be directed at a carbohydrate epitope (33) of MUC-1 from normal cells (34) and hypoglycosylated MUC-1 of malignant cells (35). PC-3(M) cells were included as a negative control.

Representative profiles of 2G3 immunostaining are shown in Fig. 1Go. Little or no MUC-1 immunoreactivity was measured in PC-3(M) cells or in AR-transfected PC-3 cells in the absence of androgen treatment. DHT shifted the 2G3 profiles in all three AR-transfected PC-3 cell sublines resulting in an increased mean fluorescence intensity of the viable cells measured at both 24 and 48 h after treatment (Fig. 1Go). As expected, DHT treatment did not affect MUC-1 immunoreactivity in AR-negative PC-3(M) cells. A summary of data obtained for cells treated for 48 h is shown in Fig. 2Go. DHT increased 2G3 immunoreactivity 4- to 6-fold relative to vehicle-treated cells in the three AR-transfected PC-3 sublines.



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  		Figure 1. Representative flow cytometry profiles of 2G3 antigen on AR-transfected PC-3 cells 24 h (A) and 48 h (B) after treatment with 10 nM DHT or vehicle. The PC-3(M) mock transfected cells were included as controls. Cells were stained with 1 µg/ml PI to allow for gating of viable cells. M1 indicates the region of positively stained cells, set with isotype control IgG1 such that no more than 5% of cells fell within the M1 gate. Heavy lines represent profiles obtained with DHT-treated cells, and dotted lines represent untreated cells. The mean fluorescence intensity values for all viable cells were as follows: A, PC-3(M): vehicle, 9.3, DHT, 9.0; PC-3(AR)13: vehicle, 5.3, DHT, 34.2; PC-3(AR)6: vehicle, 14.0, DHT, 52.5;PC-3(AR)10: vehicle, 11.6, DHT, 64.9; B, PC-3(M): vehicle, 8.8, DHT, 10.3; PC-3(AR)13: vehicle, 6.1, DHT, 42.1;PC-3(AR)6: vehicle, 19.6, DHT, 81.6; and PC-3(AR)10: vehicle, 18.9, DHT, 84.2.

 


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  		Figure 2. Summary of flow cytometry experiments showing an increase in 2G3 mean fluorescence intensities following treatment of AR-transfected PC-3 sublines with 10 nM DHT for 48 h, relative to vehicle-treated cells. Values represent mean ±SEM of several experiments for PC-(M) (n = 14), PC-3(AR)13 (n = 12), PC-3(AR)6 (n =14), and PC-3(AR)10 (n = 4). b is significantly different from a as determined by ANOVA followed by Tukey’s HSD multiple range test, P < 0.05.

 
To determine whether androgen-induced up-regulation of 2G3 antigen reflected an actual increase in MUC-1 protein rather than an alteration in glycosylation, we analyzed the reactivity of mAb BC-2, which recognizes a five amino acid epitope (APDTR) on the MUC-1 core protein (36). Treatment of PC-3(AR)6 or PC-3(AR)13 cells with 10 nM DHT up- regulated the expression of BC-2 antigen in both AR-transfected sublines but not in PC-3(M) cells (Fig. 3Go). In six independent experiments in which both BC-2 and 2G3 were used, DHT treatment for 48 h increased BC-2 and 2G3 immunoreactivity in PC-3(AR)6 cells by 4.86 ± 0.66- and 5.32 ± 0.65-fold, respectively (mean ± SEM), and in PC-3(AR)13 cells by 2.58 ± 0.49- and 3.79 ± 0.44-fold, respectively.



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  		Figure 3. Representative flow cytometry profiles of 2G3 and BC-2 antigens on PC-3(AR)6, PC-3(AR)13, and PC-3(M) cells 48 h after treatment with 10 nM DHT (heavy lines) or vehicle (dotted lines). Cells were stained with 1 µg/ml PI and live cells gated as in Fig. 1Go. The mean fluorescence intensity values for PC-3(M), PC-3(AR)13, and PC-3(AR)6 when treated with ethanol were 9.1, 12.7, and 10.3 for 2G3 and 4.4, 6.3, and 6.1 for BC-2. For the DHT-treated cells, the mean fluorescence intensity values were 9.6, 72.5, and 30.0 for 2G3 and 4.0, 41.5, and 12.7 for BC-2.

 
Androgen up-regulates MUC-1 mRNA in AR-transfected PC-3 cells
The finding that DHT treatment increased both 2G3 and BC-2 immunoreactivity indicated that androgen up-regulated the expression of MUC-1 protein at the cell surface of AR-transfected PC-3 cells. To determine whether androgen influenced the expression of MUC-1 mRNA in these cells, levels of transcript were compared in vehicle and DHT-treated PC-3(M), PC-3(AR)13, and PC-3(AR)6 cells by quantitative RT-PCR. DHT treatment for 24 h increased the level of MUC-1 mRNA by 3- and 10-fold in PC-3(AR)13 and PC-3(AR)6 cells, respectively (Fig. 4Go). Similar results were obtained after 48 h of DHT treatment (data not shown). MUC-1 transcript levels were not altered at either time point in PC-3(M) cells.



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  		Figure 4. Androgen-induced up-regulation of mucin MUC-1 mRNA expression in PC-3(AR)6 and PC-3(AR)13 cells determined by quantitative RT-PCR. Cells were harvested 24 h after treatment with 10 nM DHT ({bullet}) or ethanol (vehicle) control ({circ}). Total RNA was extracted and serial dilutions of cDNAs subjected to RT-PCR using primers specific for MUC-1 and ß-actin cDNA. A, Representative images of PCR products on 2% agarose gels, stained with SYBRgold. The amounts of RNA equivalents loaded onto the gel are indicated at the top of each lane. B, Graphical representation of MUC-1 and ß-actin mRNA levels.

 
Androgen decreases {alpha}2-integrin in AR-transfected PC-3 cells
We have previously reported that AR-transfected PC-3 cells treated with 10 nM DHT for 48 h detach from the culture plate and undergo apoptotic cell death as determined by flow cytometry and DNA fragmentation (26). Although an up-regulation of MUC-1 expression might be associated with this outcome, the loss of integrin-mediated cell-matrix contacts might also be implicated in detachment from substrate and subsequent apoptosis (37). To determine whether androgen treatment affects integrin expression, PC-3(AR)6 and PC-3(M) cells were harvested 48 h after treatment with 10 nM DHT or vehicle and cell surface expression of {alpha}2-, {alpha}3-, {alpha}5-, and ß1-integrins was determined by flow cytometry. Both PC-3(M) and PC-3(AR)6 cells expressed {alpha}2-, {alpha}3-, and ß1-integrins but very low levels of {alpha}5-integrin (Fig. 5Go). Treatment with DHT for 48 h down-regulated the expression of {alpha}2-integrin in PC-3(AR)6 cells by approximately 2-fold (Fig. 5Go). DHT had no effect on {alpha}3- or ß1-integrin expression in either cell line (Fig. 5Go). However, in PC-3(AR)6 cells treated with DHT (0.1, 1.0, or 10 nM), the low level of {alpha}5-integrin expression had increased from a mean fluorescence intensity of 3 to 10, in an average of four determinations (Fig. 5Go).



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  		Figure 5. Representative flow cytometry profiles of integrins on PC-3(M) and PC-3(AR)6 cells 48 h after treatment with 10 nM DHT or vehicle. Cells were immunostained with mAb to integrin subunits ({alpha}2, {alpha}3, {alpha}5, and ß1) and with 1 µg/ml PI to allow gating on viable cells. Heavy lines represent profiles obtained with DHT-treated cells. The mean fluorescence intensity values for all viable cells were as follows: {alpha}2: PC-3(M), vehicle, 55.7, DHT, 56.1;PC-3(AR)6, vehicle, 56.0, DHT, 29.0; {alpha}3: PC-3(M), vehicle, 48.2, DHT, 49.4; PC-3(AR)6, vehicle, 54.2, DHT, 55.1; {alpha}5: PC-3(M), vehicle, 3.3, DHT 2.7; PC-3(AR)6, vehicle, 3.6, DHT, 7.4; ß1: PC-3(M), vehicle, 89.3, DHT, 90.3;PC-3(AR)6, vehicle, 94.2, DHT, 95.2.

 
Androgen decreases adhesion of AR-transfected PC-3 cells to collagen type I
The {alpha}2ß1-integrin is the main receptor for collagen type I, a major component of bone matrix. Because DHT modulated the expression of this integrin, we examined the ability of DHT to modulate adhesion of PC-3(AR)6 cells to collagen type I. PC-3(AR)6 cells were treated with 0.1–10 nM DHT or vehicle for 48 h before seeding at different cell densities in wells coated with 0.25 µg/ml collagen type I or 1% BSA/PBS. Initial experiments established that a concentration of 0.25 µg/ml collagen type I resulted in a maximal number of adherent cells (Fig. 6AGo). DHT significantly decreased in a dose-dependent manner the number of PC-3(AR)6 cells that adhered to collagen type I at all cell densities examined (Fig. 6BGo), whereas treatment of PC-3(M) cells with 10 nM DHT had no effect on adhesion of these cells (Fig. 6CGo). Essentially no adherent cells were detected on wells coated with BSA.



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  		Figure 6. Adhesion of PC-3(AR)6 and PC-3(M) cells to collagen type I. A, Effect of collagen type I concentration on PC-3 cell adhesion. Ninety-six-microwell plates were coated with varying concentrations of collagen type I. PC-3(M) cells labeled with BCECF were added at a concentration of 100,000 cells/well for 30 min at 37 C. Adherent cell number was estimated as described in Materials and Methods. Data are shown as the mean ± SEM of three replicates per group. B, Effect of 0.1–10 nM DHT treatment for 48 h on the adhesion of PC-3(AR)6 cells to collagen type I. Wells were coated with 0.25 µg/ml collagen type I, and a varying number of BCECF-labeled cells were plated. Data are shown as the mean ± SEM of six replicates per group. Curves with different letters are significantly different from one another as determined by two-way ANOVA. All points within each plating density except 50,000 cells/well were significantly different from one another as determined by Tukey’s HSD multiple range test. C, Lack of effect of DHT on adhesion of PC-3(M) cells to collagen type I. Wells were coated with 0.25 µg/ml collagen type I, and a varying number of BCECF-labeled PC-3(M) cells treated with 10 nM DHT or vehicle for 48 h were plated. Data are shown as the mean ± SEM of three replicates per group. D, Effect of {alpha}2-integrin-blocking mAb on the adhesion of PC-3(AR)6 cells to collagen type I. BCECF-labeled cells were incubated with 0.06–1.0 µg mAb P1E6/ml or 1.0 µg/ml control murine IgG before plating in 96-microwell plates coated with 0.25 µg/ml collagen type I. Results are expressed as percent of adherent untreated control cells. Bars represent the mean ± SEM of six replicates per group. Bars with the same letter are not statistically different from one another as determined by ANOVA on ranks followed by Student-Newman-Keuls multiple range test.

 
Because the binding of PC-3 cells to collagen type I was previously shown to be mediated by {alpha}2ß1-integrin (24, 25), a blocking antibody to this integrin was tested for the ability to inhibit adhesion of AR-transfected PC-3 cells to collagen type I. PC-3(AR)6 cells were treated with varying levels of the blocking mAb P1E6 immediately before the adhesion assay. This antibody decreased adhesion to collagen in a dose-dependent manner with concentrations of 0.5 µg/ml or 1.0 µg/ml blocking adhesion by 96% and 97%, respectively, whereas a control murine IgG (1.0 µg/ml) had minimal effects (Fig. 6DGo). Thus, adhesion of PC-3(AR)6 cells to collagen is specifically mediated by {alpha}2ß1-integrin.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrate that androgens can modulate the expression of MUC-1 and {alpha}2ß1-integrin, thereby affecting the balance between adhesion and antiadhesion in AR-transfected PC-3 cells. Mitchell et al. (19) also recently reported androgen-induced up-regulation of cell surface MUC-1 AR-transfected DU-145 cells. In contrast to its polarized distribution in normal epithelial cells, MUC-1 is found throughout the entire surface of cancer cells in which it is frequently overexpressed in a hypoglycosylated state (3, 38). This reflects changes in the activity of glycosylating enzymes (39) and is thought to reveal previously masked epitopes that might be exploited as unique targets for immunotherapy (38, 40). Because of this possibility, numerous antibodies to MUC-1 have been generated that recognize hypoglycosylated epitopes, carbohydrate groups, or the MUC-1 core peptide. In the present study, we determined that androgen up-regulated the cell surface expression of MUC-1 protein using mAb recognizing either a carbohydrate epitope (2G3) or the APDTR peptide of the core protein (BC-2). These data thus indicate that the effect of androgen is on MUC-1 protein production rather than on altered activity of enzymes involved in glycosylation. This is further supported by our demonstration that DHT increased MUC-1 mRNA levels, likely via effects upon transcription or transcript stability. Although a potential hormone receptor response element compatible with the glucocorticoid, progesterone, and AR has been identified in the MUC-1 promoter region (16), AR interaction with this site has not yet been demonstrated.

Increased expression of MUC-1 has been associated with decreased patient survival or advanced tumor grade in breast and prostate cancer (41, 42). In addition, transfection of MUC-1 into myeloma cells increases their metastatic potential as determined in a nude mouse model (43, 44), indicating a role in cancer progression. However, the precise actions of MUC-1 in cancer or how overexpression may lead to tumor progression are not well understood. There is some evidence that hypoglycosylated MUC-1 recovered from ascites of patients with adenocarcinoma inhibits T cell proliferation and may thus be immunosuppressive (45). Other studies have implicated a role for MUC-1 in modulating cell adhesion. Wesseling et al. (7) have shown that transfection of MUC-1 cDNA into a human melanoma cell line reduced integrin-mediated adhesion to fibronectin, laminin, and collagen types I and IV. Treatment of the cells with a ß1-integrin-activating antibody restored adhesion to all substrates except fibronectin, leading the authors to propose that MUC-1 may modulate the balance between cell adhesion and detachment. Capping of MUC-1 using a cross-linking antibody directed to the extracellular repeat domain of MUC-1 restored adhesion to all ECM components tested (7). This suggests that the antiadhesive action of MUC-1 requires its uniform expression at the cell surface and may cause steric hindrance of integrin-ECM binding. This interpretation was further strengthened by studies of overexpression of repeat-deletion mutants of MUC-1 in murine L929 fibroblasts, which led to blocking of E-cadherin-mediated cell-cell adhesion that was directly correlated with the number of repeats present in the extracellular domain (46).

In the present study, DHT treatment of AR transfected PC-3 cells led to an increase in MUC-1 and a 50% decrease in cell surface expression of {alpha}2-integrin. Confirming the findings of Kostenuik et al. (24, 25), we demonstrated that adhesion of PC-3 cells to collagen type I is mediated by the {alpha}2ß1-integrin receptor. Our finding that DHT treatment decreased adhesion to collagen type I is thus consistent with its effect on {alpha}2ß1-integrin expression. Bonaccorsi et al. (47) reported that androgen treatment reduced {alpha}6ß4-integrin mRNA levels in AR-transfected PC-3 cells and their adhesion to laminin. However, androgen treatment did not decrease the level of {alpha}6ß4 integrin at the cell surface. Furthermore, the AR-transfected cells had a lower level of {alpha}6ß4-integrin at the cell surface than parental cells and a reduced adhesion to laminin before androgen treatment, suggesting mechanisms different from the ones observed in our studies.

AR-transfected PC-3 cells exhibit normal responses to DHT such as increased production and secretion of prostate specific antigen and human kallikrein 2 (Kollara A., E. Diamandis, and T. Brown, manuscript submitted). These cells also undergo cell cycle arrest and ultimately cell death following DHT treatment. We previously demonstrated DNA fragmentation characteristic of programed cell death in floating (nonadherent) cells collected from DHT-treated cultures but not in adherent cells (26), suggesting that cell death occurs following detachment from the monolayer. Flow cytometry studies of PC-3(AR) cells stained with Annexin-V (AnV) and PI are consistent with this interpretation. On viable (PI negative) cells, AnV detects phosphatidylserine transposition on the cell surface, which is an early event in programed cell death. AnV-positive/PI-negative cells are not detected in adherent cells treated with DHT but are present among nonadherent cells (our unpublished observations). The altered expression of MUC-1 and {alpha}2-integrin reported in this study was determined in viable adherent cells. Thus, the observed changes in expression were not the result of cells undergoing apoptosis. Rather, it is likely that the cells detach from the monolayer after DHT treatment as a result of a decrease in {alpha}2ß1-integrin receptor and an up-regulation of MUC-1 and subsequently undergo apoptosis (anoikis).

Androgen-induced up-regulation of MUC-1 and decreased {alpha}2ß1-integrin expression would likely favor increased tumor progression. Loss of this integrin in breast cancer has been associated with a more aggressive phenotype and its reexpression in a poorly differentiated breast carcinoma resulted in more differentiated and less malignant cells (48, 49). The altered expression of these cell surface molecules would favor detachment and migration and would thus promote metastasis. Metastasizing prostate and breast cancer cells have a predilection for bone, which contains high concentrations of collagen type I that is thought to mediate the retention of cancer cells within the bone marrow stroma (24). Intriguingly, in the case of parental PC-3 cells, which were originally derived from a bony metastasis, the loss of androgen responsiveness may have allowed increased levels of {alpha}2ß1-integrin and decreased MUC-1, allowing these cells to establish in bone. However, this is not true for most bony metastases, which retain AR expression (22). Factors produced by bone cells may influence the balance between adhesive and antiadhesive molecules. For example, TGF-ß1, which is present at high concentrations in bone, increases {alpha}2ß1-integrin expression and collagen type I binding of PC-3 cells (25). The action of such factors could reverse or compete with the effects of androgen on {alpha}2ß1 expression to promote bone retention.

To date, 25 distinct combinations of integrin subunits have been identified in mammals, each having its own binding specificity and signaling properties (50). Furthermore, each integrin binds to several ECM ligands, and each ECM component can bind to several integrins. Although the decreased expression of a single integrin can result in decreased affinity for particular ECM ligands, concomitant changes in other integrins might increase binding to other substrates. Interestingly, we consistently observed an increase in {alpha}5ß1-integrin, the fibronectin receptor, in cells treated with DHT. Such treatment of PC-3(AR) cells might favor their local migration from a collagen type I-rich to a fibronectin-rich microenvironment. Because the PC-3 cells are negative for the {alpha}5ß1-integrin, the induction of low levels of this integrin by androgen might be significant in terms of altering their migration pattern. Binding of integrins to their substrates is known to activate several signaling transduction pathways to regulate not only cell migration but also cell differentiation, growth, and survival. Thus, the effects of androgen on modulation of MUC-1 and integrins observed in PC-3(AR) cells, if occurring in tumor cells in vivo, might contribute to androgen regulation of tumor cell growth.

By demonstrating androgen regulation of MUC-1 and {alpha}2ß1-integrin, this article presents a novel way androgens might influence prostate cancer progression and raises the possibility that the expression of other cell surface molecules that regulate cell adhesion might also be altered. Further studies are required to understand the mechanisms by which androgens alter cell surface proteins directly implicated in cellular adhesion, such as integrins, or those modulating antiadhesion, such as MUC-1. Such studies will help to clarify the role of AR in the progression of prostate cancer.


   Footnotes
 
This work was supported by a grant from the Canadian Institute of Health Research (MOP-42437).

Abbreviations: AR, Androgen receptor; BCECF, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester; DHT, dihydrotestosterone; ECM, extracellular matrix; FBS, fetal bovine serum; HSD, honestly significant difference; mAb, monoclonal antibody; PI, propidium iodide.

Received February 7, 2002.

Accepted for publication June 27, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tuszynski GP, Wang TN, Berger D 1997 Adhesive proteins and the hematogenous spread of cancer. Acta Haematol 97:29–39[Medline]
  2. Kawaguchi T 1996 Adhesion molecules and carbohydrates in cancer metastasis. Rinsho Byori 44:1138–1146[Medline]
  3. Hilkens J, Ligtenberg MJ, Vos HL, Litvinov SV 1992 Cell membrane-associated mucins and their adhesion-modulating property. Trends Biochem Sci 17:359–363[CrossRef][Medline]
  4. Hilkens J, Vos HL, Wesseling J, Boer M, Storm J, van der Valk S, Calafat J, Patriarca C 1995 Is episialin/MUC1 involved in breast cancer progression? Cancer Lett 90:27–33[CrossRef][Medline]
  5. Gendler SJ 2001 MUC1, the renaissance molecule. J Mammary Gland Biol Neoplasia 6:339–353[CrossRef][Medline]
  6. Aplin JD 1997 Adhesion molecules in implantation. Rev Reprod 2:84–93[Abstract]
  7. Wesseling J, van der Valk SW, Vos HL, Sonnenberg A, Hilkens J 1995 Episialin (MUC1) overexpression inhibits integrin-mediated cell adhesion to extracellular matrix components. J Cell Biol 129:255–265[Abstract/Free Full Text]
  8. Ho SB, Niehans GA, Lyftogt C, Yan PS, Cherwitz DL, Gum ET, Dahiya R, Kim YS 1993 Heterogeneity of mucin gene expression in normal and neoplastic tissues. Cancer Res 53:641–651[Abstract/Free Full Text]
  9. Makiguchi Y, Hinoda Y, Imai K 1996 Effect of MUC1 mucin, an anti-adhesion molecule, on tumor cell growth. Jpn J Cancer Res 87:505–511[CrossRef][Medline]
  10. Gollub EG, Waksman H, Goswami S, Marom Z 1995 Mucin genes are regulated by estrogen and dexamethasone. Biochem Biophys Res Commun 217:1006–1014[CrossRef][Medline]
  11. Treon SP, Mollick JA, Urashima M, Teoh G, Chauhan D, Ogata A, Raje N, Hilgers JM, Nadler L, Belch AR, Pilarski LM, Anderson KC 1999 Muc-1 core protein is expressed on multiple myeloma cells and is induced by dexamethasone. Blood 93:1287–1298[Abstract/Free Full Text]
  12. Hey NA, Graham RA, Seif MW, Aplin JD 1994 The polymorphic epithelial mucin MUC1 in human endometrium is regulated with maximal expression in the implantation phase. J Clin Endocrinol Metab 78:337–342[Abstract]
  13. Aplin JD, Seif MW, Graham RA, Hey NA, Behzad F, Campbell S 1994 The endometrial cell surface and implantation: expression of the polymorphic mucin MUC-1 and adhesion molecules during the endometrial cycle. Ann NY Acad Sci 734:103–121[Medline]
  14. Braga VM, Gendler SJ 1993 Modulation of Muc-1 mucin expression in the mouse uterus during the estrus cycle, early pregnancy and placentation. J Cell Sci 105:397–405[Abstract]
  15. Surveyor GA, Gendler SJ, Pemberton L, Das SK, Chakraborty I, Julian J, Pimental RA, Wegner CC, Dey SK, Carson DD 1995 Expression and steroid hormonal control of Muc-1 in the mouse uterus. Endocrinology 136:3639–3647[Abstract]
  16. Lancaster CA, Peat N, Duhig T, Wilson D, Taylor-Papadimitriou J, Gendler SJ 1990 Structure and expression of the human polymorphic epithelial mucin gene: an expressed VNTR unit. Biochem Biophys Res Commun 173:1019–1029[CrossRef][Medline]
  17. Seregni E, Botti C, Bajetta E, Ferrari L, Martinetti A, Nerini-Molteni S, Bombardieri E 1999 Hormonal regulation of MUC1 expression. Int J Biol Markers 14:29–35[Medline]
  18. McGuckin MA, Quin RJ, Ward BG 1998 Progesterone stimulates production and secretion of MUC1 epithelial mucin in steroid-responsive breast cancer cell lines. Int J Oncol 12:939–945[Medline]
  19. Mitchell S, Abel P, Madaan S, Jeffs J, Chaudhary K, Stamp G, Lalani E-N 2002 Androgen-dependent regulation of human MUC1 mucin expression. Neoplasia 4:9–18[CrossRef][Medline]
  20. Buttyan R, Ghafar MA, Shabsigh A 2000 The effects of androgen deprivation on the prostate gland: cell death mediated by vascular regression. Curr Opin Urol 10:415–420[CrossRef][Medline]
  21. Buttyan R, Shabsigh A, Perlman H, Colombel M 1999 Regulation of apoptosis in the prostate gland by androgenic steroids. Trends Endocrinol Metab 10:47–54[CrossRef][Medline]
  22. McLeod DG, Crawford ED, DeAntoni EP 1997 Combined androgen blockade: the gold standard for metastatic prostate cancer. Eur Urol 32:70–77
  23. Newling DW 1996 The management of hormone refractory prostate cancer. Eur Urol 29:69–74
  24. Kostenuik PJ, Sanchez-Sweatman O, Orr FW, Singh G 1996 Bone cell matrix promotes the adhesion of human prostatic carcinoma cells via the {alpha}2ß1 integrin. Clin Exp Metastasis 14:19–26[CrossRef][Medline]
  25. Kostenuik PJ, Singh G, Orr FW 1997 Transforming growth factor ß upregulates the integrin-mediated adhesion of human prostatic carcinoma cells to type I collagen. Clin Exp Metastasis 15:41–52[CrossRef][Medline]
  26. Heisler LE, Evangelou A, Lew AM, Trachtenberg J, Elsholtz HP, Brown TJ 1997 Androgen-dependent cell cycle arrest and apoptotic death in PC-3 prostatic cell cultures expressing a full-length human androgen receptor. Mol Cell Endocrinol 126:59–73[CrossRef][Medline]
  27. Jones SA, Brooks AN, Challis JRG 1989 Steroids modulate corticotropin-releasing hormone production in human fetal membranes and placenta. J Clin Endocrinol Metab 68:825–830[Abstract/Free Full Text]
  28. Murphy LD, Herzog CE, Rudick JB, Fojo AT, Bates SE 1990 Use of the polymerase chain reaction in the quantitation of mdr-1 gene expression. Biochemistry 29:10351–10356[CrossRef][Medline]
  29. Jindal SK, Ishii E, Letarte M, Vera S, Teerds KJ, Dorrington JH 1995 Regulation of transforming growth factor-{alpha}gene expression in an ovarian surface epithelial cell line derived from a human carcinoma. Biol Reprod 52:1027–1037[Abstract]
  30. Quackenbush EJ, Letarte M 1985 Identification of several cell surface proteins of non-T, non-B acute lymphoblastic leukemia by using monoclonal antibodies. J Immunol 134:1276–1285[Abstract]
  31. Linsley PS, Brown JP, Magnani JL, Horn D 1988 Monoclonal antibodies reactive with mucin glycoproteins found in sera from breast cancer patients. Cancer Res 48:2138–2148[Abstract/Free Full Text]
  32. Regimbald LH, Pilarski LM, Longenecker BM, Reddish MA, Zimmermann G, Hugh JC 1996 The breast mucin MUC-1 as a novel adhesion ligand for endothelial intercellular adhesion molecule-1 in breast cancer. Cancer Res 56:4244–4249[Abstract/Free Full Text]
  33. Linsley PS, Ochs V, Horn D, Ring DB, Frankel AE 1986 Heritable variation in expression of multiple tumor associated epitopes on a high molecular weight mucin-like antigen. Cancer Res 46:6380–6386[Abstract/Free Full Text]
  34. Auersperg N, Maines-Bandiera SL, Dyck HG, Kruk PA 1994 Characterization of cultured human ovarian surface epithelial cells: phenotypic plasticity and premalignant changes. Lab Invest 71:510–518[Medline]
  35. Auersperg N, Maines-Bandiera S, Booth JH, Lynch HT, Godwin AK, Hamilton TC 1995 Expression of two mucin antigens in cultured human ovarian surface epithelium: influence of a family history of ovarian cancer. Am J Obstet Gynecol 173:558–565[CrossRef][Medline]
  36. Xing PX, Prenzoska J, McKenzie IF 1992 Epitope mapping of anti-breast and anti-ovarian mucin monoclonal antibodies. Mol Immunol 29:641–650[CrossRef][Medline]
  37. Frisch SM, Ruoslahti E 1997 Integrins and anoikis. Curr Opin Cell Biol 9: 701–706
  38. Finn OJ, Jerome KR, Henderson RA, Pecher G, Domenech N, Magarian-Blander J, Barratt-Boyes SM 1995 MUC-1 epithelial tumor mucin-based immunity and cancer vaccines. Immunol Rev 145:61–89[CrossRef][Medline]
  39. Brockhausen I, Yang JM, Burchell J, Whitehouse C, Taylor-Papadimitriou J 1995 Mechanisms underlying aberrant glycosylation of MUC1 mucin in breast cancer cells. Eur J Biochem 233:607–617[Medline]
  40. Barratt-Boyes SM 1996 Making the most of mucin: a novel target for tumor immunotherapy. Cancer Immunol Immunother 43:142–151[CrossRef][Medline]
  41. McGuckin MA, Walsh MD, Hohn BG, Ward BG, Wright RG 1995 Prognostic significance of MUC1 epithelial mucin expression in breast cancer. Hum Pathol 26:432–439[CrossRef][Medline]
  42. Kirschenbaum A, Itzkowitz SH, Wang JP, Yao S, Eliashvili M, Levine AC 1999 MUC1 expression in prostate carcinoma: correlation with grade and stage. Mol Urol 3:163–168[Medline]
  43. Van de Wiel-van Kemenade E, Ligtenberg MJ, de Boer AJ, Buijs F, Vos HL, Melief CJ, Hilkens J, Figdor CG 1993 Episialin (MUC1) inhibits cytotoxic lymphocyte-target cell interaction. J Immunol 151:767–776[Abstract]
  44. Rowse GJ, Tempero RM, VanLith ML, Hollingsworth MA, Gendler SJ 1998 Tolerance and immunity to MUC1 in a human MUC1 transgenic murine model. Cancer Res 58:315–321[Abstract/Free Full Text]
  45. Agrawal B, Krantz MJ, Reddish MA, Longenecker BM 1998 Cancer-associated MUC1 mucin inhibits human T-cell proliferation, which is reversible by IL-2. Nat Med 4:43–49[CrossRef][Medline]
  46. Wesseling J, van der Valk SW, Hilkens J 1996 A mechanism for inhibition of E-cadherin-mediated cell-cell adhesion by the membrane-associated mucin episialin/MUC1. Mol Biol Cell 7:565–577[Abstract]
  47. Bonaccorsi L, Carloni V, Muratori M, Salvadori A, Giannini A, Carini M, Serio M, Forti G, Baldi E 2000 Androgen receptor expression in prostate carcinoma cells suppresses {alpha}6ß4 integrin-mediated invasive phenotype. Endocrinology 141:3172–3182[Abstract/Free Full Text]
  48. Zutter MM, Sun H, Santoro SA 1998 Altered integrin expression and the malignant phenotype: the contribution of multiple integrated integrin receptors. J Mammary Gland Biol Neoplasia 3:191–200[CrossRef][Medline]
  49. Zutter MM, Santoro SA, Staatz WD, Tsung YL 1995 Re-expression of the {alpha}2ß1 integrin abrogates the malignant phenotype of breast carcinoma cells. Proc Natl Acad Sci USA 92:7411–7415[Abstract/Free Full Text]
  50. Humphries MJ 2000 Integrin structure. Biochem Soc Trans 28:311–339[Medline]



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