Antagonism between PTEN/MMAC1/TEP-1 and Androgen Receptor in Growth and Apoptosis of Prostatic Cancer Cells*

Pengfei Li, Santo V. Nicosia, and Wenlong BaiDagger

From the Department of Pathology, University of South Florida College of Medicine and Program of Molecular Oncology and Drug Discovery, H. Lee Moffitt Cancer Center, Tampa, Florida 33612-4799

Received for publication, November 9, 2000, and in revised form, February 12, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PTEN/MMAC1/TEP-1 (PTEN) tumor suppressor and androgen receptor play important roles in prostatic tumorigenesis by exerting opposite effects on homeostasis of prostatic epithelium. Here, we describe a mutual repression and selective dominance between PTEN and the androgen receptor (AR) in the growth and the apoptosis of prostatic cancer cells. On the one hand, PTEN and an inhibitor of phosphoinositide 3-kinase repressed the transcriptional activity of the AR as well as androgen-induced cell proliferation and production of prostate-specific antigen. On the other hand, androgens protected prostate cancer cells from PTEN-induced apoptosis in an AR-dependent manner. Whereas the repression of the transcriptional activity of the AR by PTEN is likely to involve the down-regulation of AKT, androgens protected prostate cancer cells from PTEN-induced apoptosis without an effect on AKT activity, demonstrating a differential involvement of AKT in the interaction between PTEN and the AR. Our data suggest that the loss of PTEN function may induce tumorigenesis through unopposed activity of the AR as well as contribute to the resistance of prostate cancers to androgen ablation therapy.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Androgens are responsible for the development, maintenance, and regulation of male phenotype and reproductive physiology. These activities are mediated through the AR1 (1-4), which belongs to the steroid/thyroid receptor superfamily, a group of ligand-regulated transcription factors (5). Like other members of the family, the AR protein is modular in nature and composed of an amino-terminal A/B region, a DNA-binding domain and a "hinge" region in the middle, and a hormone-binding domain at the carboxyl terminus. Whereas the amino-terminal A/B region contains the major transcriptional activation function, a weaker activation function in the hormone-binding domain also contributes to the total transcriptional activity. The hormone-binding domain represses the activation functions until androgens bind to it and relieve the repression by inducing the formation of a conformation suitable for the interaction with transcriptional cofactors (6-8). In addition, the polymeric stretches within the amino-terminal region (9) and receptor phosphorylation (10, 11) also contribute to the AR transcriptional activities.

Besides their established physiological functions, androgens are implicated in multiple pathological processes including prostate cancer (PCa), which is the most commonly diagnosed malignancy and second only to lung cancer in the mortality rate of American males (12). Chronically maintained androgen level sustains the total prostate cell number by both stimulating the rate of proliferation and inhibiting the rate of death of prostate epithelial cells (13); both lead to an increase in the cell number. To maintain the homeostasis of prostate epithelium, there must exist a "brake" system that opposes these effects of androgens.

PTEN tumor suppressor is a 403-amino acid phosphoprotein/phospholipid dual-specificity phosphatase (14, 15). Somatic mutation of PTEN is a common event in diverse human cancers including PCa (14, 15), and heterozygous deletion of PTEN in mice leads to neoplasm in multiple tissues including the prostate (16, 17). The significance of the phosphoprotein phosphatase in tumor suppression is largely unknown, although one known substrate is focal adhesion kinase (18). In contrast, multiple findings support a role of the lipid phosphatase activity in PTEN-mediated tumor suppression (19-23). PTEN lipid phosphatase catalyzes the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) (24), resulting in inactivation of the downstream protein kinase B, also named AKT, activity (21, 22). Ectopic PTEN expression in PTEN-null PCa cells induced cell cycle arrest and apoptosis (22, 25); both activities are opposite to those of androgens, suggesting that PTEN may provide the brake to balance the functions of AR in prostate cells.

In the current study, we provide experimental evidence that the activities of PTEN and AR are antagonistic in PCa cells. Interestingly, PTEN and AR do not simply shut down each others activity but differentially antagonize and selectively dominate each other in PCa cell apoptosis and proliferation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- AREe1bLuc (26), pCMVhAR (27), pLENbeta gal (28), PSALuc (29), GalLuc (30), and Gal-VP16 (6), pSG5L-HA-PTEN:WT (22), pSG5L-HA-PTEN:G129R (22), PLNCX-HA-myr-AKT (22), and PLNCX-HA-myr-AKT179 M (22) have already been described. pCMVbeta and pLNCE are from CLONTECH (Palo Alto, CA). pLENhAR was constructed by replacing beta -galactosidase (beta -gal) cDNA of pLENbeta gal with human AR cDNA. Partial human AR cDNA was excised from pCMVhAR with BamHI and EagI. Because EagI cuts the AR cDNA at an internal position close to the initiator ATG, the digestion yield a partial AR cDNA fragment. The partial AR cDNA was ligated to a synthetic linker that extended the AR cDNA sequence from the internal EagI site to a designed BamHI site immediately upstream of the initiator ATG. The full-length AR cDNA was inserted into the BamHI site of pLEN vector in the correct orientation for mammalian expression.

Transfections and Transcriptional Assays-- PC3 cells were plated in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS) at 2 × 105 cells/well in 6-well plates and LNCaP cells were plated in RPMI 1640 at 4 × 105 cells/well. One day after plating, cells were transfected with a LipofectAMINE plus-mediated transfection procedure following the protocol from Life Technologies, Inc. Transfected cells were treated with synthetic androgens or phosphoinositide 3-kinase (PI3K) inhibitors in medium containing 1% charcoal-stripped FBS for 24 h and washed with phosphate-buffered saline (PBS). Cell lysate was prepared by directly adding lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N',N',N',N'-tetraacetic acid, 10% glycerol, 0.2% Triton X-100) to the cells on ice. Luciferase activity was determined using luciferase assay systems from Promega Corporation (Madison, WI) following the company's protocol. beta -gal activity was determined as previously described (31).

ELISA and Colorimetric MTT Assay-- LNCaP cells were plated in RPMI 1640 containing 10% FBS at 1 × 105 cells/well in 96-well plates for MTT assays. After cells attached, they were starved in RPMI 1640 with 1% charcoal-stripped FBS for 48 h and treated with synthetic androgens or PI3K inhibitors. The medium was collected and PSA levels were determined using the Tandem-E PSA ImmunoEnzyMetric Assay Kit (Hybritech Inc., San Diego, CA) following manufacturer's protocols. The absorbance at 405 nm was measured using a UV spectrophotometer. PSA concentration (ng/ml) was determined based on a standard curve generated with PSA controls of known concentrations provided by the kit. MTT assays were performed as described (32). The plates were read on a MRX microplate reader (DYNEX Technologies, Chantilly, VA) using a test wavelength of 595 nm.

Apoptotic Assays-- Transfected cells were washed with PBS and fixed in 3.7% formaldehyde/PBS at room temperature for 10 min. The viability of transfected cells in each well was determined by counting the total number of green cells in each well under a fluorescence microscope. For the demonstration of apoptotic cells, fixed cells were stained at room temperature for 15 min with 4',6'-diamidino-2-phenylindole (DAPI) at a concentration of 1.5 mg/ml in the VECTASHIELD Mounting Medium (Vector laboratories, Burlingame, CA). Green and blue fluorescence were observed with a Leitz Orthoplan 2 Microscope. Representative micrographs were captured by a CCD camera with the Smart Capture Program (Vysis, Downers Grove, IL). The apoptotic index of GFP-positive cells was determined by scoring 300 GFP-positive cells for chromatin condensation and apoptotic body formation.

In Vitro Immunocomplex Kinase Assays-- LNCaP cells were transfected as described above for transcriptional assays, washed with ice-cold PBS, and lysed in 20 mM Tris, pH. 7.6, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 5 mM beta -glycerophosphate, 100 µM Na3VO4, 0.5% Nonidet P-40, and protease inhibitor mixture (1 tablet/10 ml). After centrifugation, half of the supernatant was immunoprecipitated with the 12CA5 anti-HA monoclonal antibody (Roche Molecular Biochemicals Corp., Indianapolis, IN). Kinase reactions were performed at 30 °C in 30 µl of buffer containing 20 mM HEPES, pH 7.4, 10 mM p-nitrophenyl phenylphosphonate, 20 mM MgCl2, 2 mM EDTA, 2 mM EGTA, 100 µM Na3VO4, 10 µM ATP, 10 µCi [gamma -32P]ATP with 3 µg of H2B as substrate. After incubation for 30 min, reactions were terminated by adding 2× SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, analyzed by SDS-PAGE, and visualized by autoradiography.

Immunoblotting Analysis-- To determine the expression of AR, duplicate wells of PC3 cells transfected in parallel to the cells prepared for transcriptional assays were pooled and lysed in the same buffer as described above for kinase assays. The lysate was mixed with one-sixth of 6× SDS-PAGE sample buffer, heated for 10 min at 100 °C, and separated on 8% SDS-PAGE. To determine the expression of HA-AKT, half of the cell lysate prepared for kinase assays was processed similarly as for AR and separated on 10% SDS-PAGE. After separation on SDS-PAGE, samples were transferred to a nitrocellulose membrane, probed with the PG-21 anti-AR (Upstate Biotechnology, Lake Placid, NY) or the 12CA5 anti-HA antibodies, and visualized using ECL as described (28).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Repression of AR Transcriptional Activity by PTEN and a PI3K Inhibitor and Relief of the Repression by a Dominantly Active AKT-- To test whether PTEN inhibits AR transcriptional activity, we transfected into AR-negative, PTEN-null PC3 cells (22) an AR expression vector, pLENhAR, together with a PTEN expression vector or a control vector expressing a phosphatase-inactive PTEN. The transcriptional activity of AR was measured by assaying the luciferase activity from a co-transfected AR reporter, AREe1bLuc, in which luciferase expression is under the control of synthetic androgen response elements (AREs) placed in front of the simple adenovirus E1b promoter. The effect of PTEN on the AR activity was measured by comparing the AR activity in cells transfected with PTEN to those transfected with the control vector. Because of the concern that co-transfected PTEN may alter the expression of AR by affecting the activity of the promoter or the enhancer of the AR expression vector, the reporter activity was normalized with beta -gal activity from co-transfected pLENbeta gal vector in which beta -gal expression is under the control of human metallothionin-II promoter and SV40 enhancer, the same regulatory sequences used by the AR vector. As shown in Fig. 1A, AR basal activity was low but the activity was significantly induced by treatment with R1881, a stable synthetic androgen agonist. Whereas the basal activity was not affected, R1881-induced AR activity was repressed, but not abolished, by PTEN in a dosage-dependent manner.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 1.   PTEN repressed the transcriptional activity of the AR. A, dosage-dependent inhibition of AR activity by PTEN in PC3 cells. Cells were transfected with 0.1 µg of pLENhAR, 0.5 µg of pLENbeta gal, 0.5 µg of AREe1bLuc, and the indicated amounts of pSG5L-HA-PTEN:WT (WT) or pSG5L-HA-PTEN:G129R (MT). Transfected cells were treated for 24 h with 10-8 M R1881 or ethanol (EOH) as vehicle controls. All samples were normalized with beta -gal activity and expressed as relative luciferase unit (RLU). Duplicate samples were analyzed for each single data point, and the data have been reproduced three times. B, inhibition of AR activity by PTEN was not caused by decreased AR protein levels. PC3 cells were transfected with 0.1 µg of pCMVhAR, 0.5 µg of pCMVbeta , 0.5 µg of AREe1bLuc, and 0.1 µg of pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R. The level of AR protein was detected by Western blotting.

Because both AR and beta -gal were expressed using the same vector, the way we normalize the reporter activity should have eliminated the potential interference caused by either variation in transfection efficiency or PTEN-induced alterations in the promoter activity of the AR expression vector. However, the lower AR activity in the presence of PTEN could arguably be because of lower level of AR protein as a result of decreased protein stability or reduced efficiency of protein translation. To test whether the level of AR protein was altered by PTEN, PC3 cells were transfected with pCMVhAR and pCMVbeta , which express the receptor and beta -gal, respectively, under the control of the stronger cytomegalovirus (CMV) promoter, permitting the analysis of AR protein level and transcriptional activity in the same experiment. The AR activity from pCMVhAR was decreased by PTEN to the same degree as demonstrated for pLENhAR (Fig. 1A). Parallel Western blotting demonstrated that PTEN, in the presence of R1881, did not decrease the AR protein level (Fig. 1B). These data demonstrate that PTEN repressed the transcriptional activity of the AR per molecule. In the absence of R1881, PTEN expression caused a decrease in the level of AR protein. The decrease, based on later studies, was evidently because of the death of transfected cells that expressed PTEN to high levels.

To further substantiate the AR repression by PTEN, we performed a time course study as shown in Fig. 2. In cells transfected with mutant PTEN, R1881-induced a dramatic AR activation at times as early as 12 h post-transfection and the continuous treatment proportionally increased the AR activity over time. Whereas the basal AR activity in the absence of R1881 was not affected, the proportional increase in AR activity as a result of prolonged R1881 treatment was almost eliminated by PTEN.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2.   PTEN repressed the androgen-induced increase in AR activity over time. PC3 cells were transfected with 0.1 µg of pCMVhAR, 0.5 µg of pCMVbeta , 0.5 µg of AREe1bLuc, and 0.1 µg of pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R. Transfected cells were treated for indicated times with 10-8 M R1881 or ethanol (EOH) as vehicle controls. AR activity was assayed and expressed as described in the legend to Fig. 1.

In theory, the AR repression could be mediated through either the protein or lipid phosphatase activity of PTEN. Because the lipid phosphatase activity has a well established role in tumor suppression, we decided to first investigate the role of the lipid phosphatase activity in the AR repression. PTEN lipid phosphatase is known to decrease the level of PI(3,4,5)P3 by catalyzing its dephosphorylation. Thus, we examined whether the decrease of PI(3,4,5)P3 by other means such as inhibition of its synthesis will have the same effect on AR activity as PTEN expression. AR was transfected into PC3 cells and treated with either R1881 alone or co-treated with LY294002, a synthetic inhibitor for PI3K. As shown in Fig. 3A, LY294002 repressed the AR activity in a dosage-dependent manner. A time course study showed that the percentage of AR repression by LY294002 was best observed at the shortest time point (Fig. 3B). This is different from the PTEN data (Fig. 2) and probably reflects the fact that LY294002 exerts its effect through PI3K-mediated signaling pathways (which usually takes just minutes), and the effectiveness of synthetic drugs decreases over time because of the issue of stability and active clearance by cells. These studies indicate that the PTEN-induced AR repression is most likely mediated through the PI(3,4,5)P3 phosphatase activity of PTEN.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3.   A chemical inhibitor of PI3K also repressed AR activity. A, dosage-dependent inhibition of AR by LY294002. PC3 cells were transfected with 0.1 µg of pLENhAR, 0.5 of µg AREe1bLuc, 0.5 µg of pLENbeta Gal and treated for 24 h with or without 10-8 M R1881 and LY294002 (LY) at indicated concentrations. B, repression of AR activity by LY294002 over time. PC3 cells were transfected as described in A and treated for indicated times with or without 10-8 M R1881 and 20 µM LY294002. LY294002 was dissolved in Me2SO, and the absolute amount of Me2SO was the same for all samples.

Because AKT is the major downstream target of PI(3,4,5)P3, we next examined the involvement of AKT in PTEN-induced AR repression. PC3 cells were transfected with PTEN, AR, and a dominantly active AKT of which the activity cannot be repressed by PTEN or a kinase-inactive AKT. The AR repression by PTEN was analyzed as shown in Fig. 4. Whereas PTEN-induced AR repression occurred in the presence of the kinase-inactive AKT, co-expression of the dominantly active AKT blocked the AR repression by PTEN. These data suggest that the down-regulation of PI(3,4,5)P3 by PTEN lipid phosphatase, and the subsequent inactivation of AKT kinase mediated the AR repression, establishing the role of the PTEN lipid phosphatase in AR repression.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4.   A dominantly active AKT blocked PTEN-induced AR repression. PC3 cells were transfected with 0.1 µg of pLENhAR, 0.5 µg of AREe1bLuc, 0.5 µg of pLENbeta Gal, 0.1 µg of pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R and 0.1 µg of PLNCX-HA-myr-Akt (Active) or PLNCX-HA-myr-AKTK179M (Inactive). AR activity was assayed and expressed as described in the legend to Fig. 1.

Impairment of the Biological Activities of Endogenous AR by PTEN and the PI3K Inhibitor-- So far, we have demonstrated an inhibitory effect of PTEN and the PI3K inhibitor on the activity of transiently transfected AR with a reporter constructed with synthetic AREs. To determine whether PTEN also represses endogenous AR activity with natural promoters, we transfected PSALuc into PTEN-null but AR-positive LNCaP cells (22) and examined the effect of PTEN and the PI3K inhibitor on endogenous AR activity. PSALuc is an AR reporter in which luciferase expression is under the control of the promoter of human PSA, a PCa marker, which is transcriptionally regulated by AR through complex AREs (29). As shown in Fig. 5A, the transcriptional activity of the endogenous AR in cells transfected with the PSALuc was increased by R1881, and R1881 induction was blocked by PTEN. This demonstrates that PTEN represses endogenous AR activity on natural promoters and thus is not limited to ectopic AR or synthetic AREs.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   PTEN repressed the transcriptional activity of endogenous AR in LNCaP cells. A, PTEN repression of endogenous AR activity on PSA promoter in LNCaP cells. Cells were transfected with 0.5 µg of PSALuc, 0.5 µg of pLENbeta Gal, and 0.1 µg of pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R. Transfected cells were treated with or without 10-8 M R1881, and AR activity was assayed and expressed as described in the legend to Fig. 1. B, lack of Gal-VP16 repression by PTEN in LNCaP cells. Cells were transfected with 0.5 µg of GalLuc, 0.5 µg of pCMVbeta , 0.1 µg of pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R and with or without 0.1 µg of Gal-VP16. Transfected cells were treated with 10-8 M R1881. Transcriptional activity of VP16 was assayed and expressed as described in the legend to Fig. 1.

To rule out the possibility that the AR repression was caused by nonspecific repression of LNCaP cell transcription by PTEN, we transfected into LNCaP cells a Gal-VP16 expression vector and a Gal4 reporter and measured the activity of the fusion activator in the presence or absence of PTEN. As shown in Fig. 5B, the reporter was inactive in the absence of Gal-VP16, and the co-expression of the fusion activator induced the activity dramatically. More importantly, the Gal-VP16 activity was not inhibited by PTEN. These data demonstrate that the AR repression was not caused by a nonspecific effect of PTEN on LNCaP cell transcription.

To determine the biological consequence of the observed repression of AR transcriptional activity, the effect of PI3K inhibitor on androgen-induced PSA production and cell proliferation was examined in LNCaP cells. LNCaP cells were treated with either R1881 alone or co-treated with LY294002 for indicated times. Culture medium from the treated cells was collected and analyzed for PSA level by ELISA. As shown in Fig. 6A, R1881 treatment stimulated PSA production and the stimulation was inhibited by the co-treatment with LY294002. Similar to PSA production, R1881 induced an increase in cell number as measured by MTT assays, and this increase was blocked by co-treatment with LY294002 (Fig. 6B). LY294002 did not consistently decrease the basal PSA level or LNCaP cell numbers in the absence of R1881. These analyses demonstrate that the repression of AR transcriptional activity by the PI3K inhibitor impaired the biological functions of endogenous AR.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6.   PI3K inhibitor impaired the biological activities of the AR. A, repression of androgen-induced PSA production in LNCaP cells by LY294002. LNCaP cells were starved and treated with vehicle, 10-8 M R1881 or 10-8 M R1881 plus 20 µM LY294002 for the indicated times. Absolute amounts of Me2SO and ethanol were the same for all samples. PSA levels were quantified by ELISA. Duplicate samples were analyzed for each data points, and the experiment was repeated three times. B, repression of androgen-induced LNCaP cell proliferation by LY294002. LNCaP cells were starved and treated as in A. Cell numbers were determined by the MTT colorimetric assays. Eight samples were analyzed for each data point, and the data were reproduced three times.

AR-dependent Protection of PTEN-induced Apoptosis by Androgens in PCa Cells-- Our data so far have established a functional relationship between PTEN and AR by showing that PTEN or a PI3K inhibitor opposed AR function in PCa cells. We next investigated whether androgen also opposes the function of PTEN. Because the expression of PTEN in LNCaP cells induced apoptosis (25), we examined the effect of androgens on PTEN-induced apoptosis. LNCaP cells were transfected with a green fluorescence protein (GFP) expression vector and the PTEN expression vector or the control vector expressing the mutant PTEN. The transfected cells were then treated with or without R1881, and the viability of transfected cells was determined. As shown in Fig. 7A, the viability of PTEN-transfected cells in the absence of R1881 was only about 15% of the cells transfected with the control vector. Treatment with R1881 restored the viability of PTEN-transfected cells to the level of controls. This decrease in the viability of PTEN-transfected cells as well as its blockage by R1881 was more directly shown by the number of GFP-positive cells in representative micrographic fields (Fig. 7B, panels 1-3).


View larger version (56K):
[in this window]
[in a new window]
 
Fig. 7.   Androgen protected LNCaP cells from PTEN-induced apoptosis. A, androgen effect on the viability of PTEN-transfected LNCaP cells. LNCaP cells were transfected with 0.5 µg of pLNCE, a GFP expression vector and 0.1 µg of pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R. Transfection cells were treated with 10-8 M R1881 or ethanol for 24 h, and the viability of transfected cells in each well was determined by counting the total number of green cells. Triplicate samples were analyzed for each data point, and the data were reproduced three times. B, representative micrographs of PTEN-transfected cells in the presence or absence of androgen. Cells were transfected and fixed as in A. Fixed cells were stained with DAPI, and representative micrographs were captured by a CCD camera attached to the fluorescence microscope using objective lens of × 10 (panels 1-3) or × 100 (panels 4-6). C, androgen effect on PTEN-induced increase in apoptotic index of LNCaP cells. Cells were processed as in B, and the apoptotic index of GFP-positive cells was determined by scoring 300 GFP-positive cells for chromatin condensation. Triplicate samples were analyzed per data point, and the graph represents three independent experiments.

To confirm that the decreased viability of PTEN-transfected cells is the result of cell apoptosis, cells were fixed after transfection and stained with DAPI, and the nuclear morphology of transfected cells was examined for features of apoptosis under a fluorescence microscope that permits the simultaneous visualization of both blue and green fluorescence. As shown in Fig. 7B, panels 4-6, as representative micrographs, cells transfected with the control vector displayed a normal morphology similar to surrounding non-transfected cells (Fig. 7B, panel 4). Cells transfected with PTEN without R1881 treatment frequently displayed an apoptotic morphology (Fig. 7B, panel 5). Similar to controls, most cells transfected with PTEN but treated with R1881 showed a normal morphology (Fig. 7B, panel 6). Apoptotic index, as determined by counting apoptotic cells in 300 green cells per sample 24 h after treatment, was 5% for controls, 20% for cells transfected with PTEN without R1881 treatment, and 5% for cells transfected with PTEN but treated with R1881 (Fig. 7C). These analyses show that PTEN induced apoptosis in LNCaP cells, and this PTEN function was blocked by androgen treatment.

Because of the presence of endogenous AR in LNCaP cells, LNCaP cell experiments did not show that the androgen protection of PTEN-induced apoptosis is mediated through the AR. To determine whether the androgen effect on apoptosis is AR-dependent, AR-negative PC3 cells were transfected with or without AR, and the effect of androgen on PTEN-induced apoptosis was examined as in LNCaP cells. As shown in Fig. 8A, R1881 had no effect on the viability of PTEN-transfected cells in the absence of ectopic AR expression. After co-transfection with AR, both PTEN-induced decreases in cell viability (Fig. 8A) and increases in apoptotic index (Fig. 8B) were blocked by R1881, demonstrating that the androgen protection of PTEN-induced apoptosis was mediated through the AR.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 8.   Androgen effect on PTEN-induced apoptosis was AR-dependent. A, AR-dependent androgen effect on PTEN-induced decrease in PC3 cell viability. PC3 cells were transfected with 0.5 µg of pLNCE, 0.1 µg of pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R and with or without 0.1 µg of pLENhAR. Transfected cells were treated and processed for cell viability determination as described in the legend to Fig. 7. B, effect of androgen on PTEN-induced increase in apoptotic index in AR-transfected PC3 cells. Cells were transfected with AR and PTEN expression vectors and processed as in A. Apoptotic index was scored as described in the legend to Fig. 7. C, androgen protection of PTEN-induced PC3 cell death over time. Cells were transfected with AR and PTEN expression vectors as in A and treated with 10-8 M R1881 or ethanol for indicated times. The viability of transfected cells in each well was determined as described in the legend to Fig. 7.

To further analyze the anti-apoptotic function of AR, a time course study was performed (Fig. 8C). In this study, the number of transfected (green) cells peaked at 24 h post-transfection, and R1881 protected PC3 cells from PTEN-induced death similarly at all three tested time points, presumably because of the synchronized expression of AR and PTEN proteins in co-transfected cells.

Lack of an Androgen Effect on AKT Activities in PCa Cells-- Our data in Fig. 1 showed that the PTEN repression of AR depended on the down-regulation of AKT activity. In addition, co-expression of the dominantly active AKT blocked PTEN-induced apoptosis in LNCaP cells (Fig. 9A), demonstrating that PTEN-induced apoptosis was mediated through AKT down-regulation. So we investigated the possibility that androgens might protect apoptosis by regulating AKT activity. Western blotting with anti-[phospho-Ser437]AKT antibody did not detect an androgen effect on endogenous AKT activity in LNCaP cells (data not shown).


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 9.   Androgen protection of PTEN-induced apoptosis in LNCaP cells occurred independent of AKT activity. A, inhibition of PTEN-induced apoptosis by dominantly active AKT. PC3 cells were transfected with 0.5 µg of pLNCE, 0.1 µg of pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R and 0.5 µg of PLNCX-HA-myr-AKT or PLNCX-HA-myr-AKTK179M. The viability of transfected cells was determined as described in the legend to Fig. 7. B, lack of androgen effect on PTEN-induced down-regulation of AKT activity. LNCaP cells were transfected with 0.5 µg of PLNCX-HA-AKT (wtAKT) and 0.1 µg of pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R. Transfected cells were treated with 10-8 M R1881 or vehicle for 24 h. Both AKT kinase activity (top panel) and level of expression (bottom panel) were analyzed by parallel immunocomplex kinase assays and Western blotting using the 12CA5 anti-HA monoclonal antibody. Phosphorylated histone H2b (P-H2b) was resolved on a 15% SDS-PAGE gel and visualized by autoradiography.

LNCaP cells are PTEN-null and contain high levels of endogenous AKT activity. Although androgens do not have a direct effect on endogenous AKT activity, it may affect the negative regulation of AKT by PTEN. So we next examined whether androgen treatment blocked PTEN-induced AKT down-regulation. We transfected a HA-tagged wild-type AKT into LNCaP cells with the PTEN expression vector and analyzed the AKT activity by in vitro immunocomplex kinase assays. As shown in Fig. 9B, in either the presence or absence of R1881, the kinase activity of AKT was dramatically decreased by PTEN expression whereas the level of AKT protein was slightly reduced (Fig. 9B). The data demonstrate that R1881 did not block the down-regulation of AKT activity by PTEN in LNCaP cells.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our studies demonstrated an antagonistic interaction and selective dominance between PTEN and AR in the proliferation and apoptosis of PCa cells as well as a differential involvement of AKT in the interaction. The lack of an androgen effect on both AKT activity and its down-regulation by PTEN in PCa cells suggests that androgens protect PTEN-induced apoptosis either by activating a survival pathway that is independent of AKT as suggested by a previous study (33) or by activating the same survival pathway at steps downstream of AKT.

It appears paradoxical that PTEN-or LY294002-induced repression of AR transcriptional activity was sufficient to block androgen-induced proliferation and PSA production but unable to override the protective effect of androgens on apoptosis. One possibility is that AR-dependent protection of apoptosis by androgens might be mediated through non-genomic effects of the receptor. Our data clearly showed that the androgen effect on PTEN-induced apoptosis is AR-dependent. Consistent with our data, reported studies demonstrated that the "decoy" of ARE triggered apoptosis in LNCaP cells (34), suggesting that the anti-apoptotic effect of androgens is mediated through the genomic effect of the AR. Because the AR transcriptional activity was repressed but not abolished by PTEN or LY294002 in our experiments, it is likely that the androgen induction of genes involved in promoting cell proliferation, and those in apoptosis protection require different amounts of AR transcriptional activity.

Because our studies indicate that androgen target genes involved in proliferation and apoptosis protection may have a differential sensitivity to cellular status of PI(3,4,5)P3 signal pathway, it would be interesting to determine whether AR could still mediate the induction of anti-apoptotic genes under conditions when androgen-induced cell proliferation is blocked by the suppression of PI(3,4,5)P3 signaling. Unfortunately, genes mediating the anti-apoptotic effect of androgens in PCa cells remain to be identified. Under the same conditions when PTEN-induced apoptosis of LNCaP cells was blocked by androgens, we did not detect any androgen effect on Bcl-2 expression (data not shown), although Bcl-2 up-regulation by androgens in LNCaP cells had been described in previous studies (35).

Phosphorylation is known to regulate the transcriptional activity of steroid receptors including AR (10, 11, 36). Based on the consensus sequence for AKT phosphorylation (37), there are two potential AKT phosphorylation sites in AR: Ser212 in the A/B region and Ser782 in the ligand-binding domain. Therefore, it is conceivable that AR activation may need the phosphorylation of these sites by AKT and that AR repression by PTEN might be caused by the inhibition of the AKT phosphorylation. However, transfection of the dominantly active AKT into DU145 cells that contain wild type PTEN and low endogenous AKT did not cause either ligand-independent activation nor enhanced the androgen-induced activity of co-transfected AR (data not shown), suggesting that PTEN repression of AR activity may involve more complex processes than a simple inhibition of AR phosphorylation at the potential AKT sites.

Our data are the first to clearly demonstrate a mutual antagonism between PTEN and AR in PCa cells. The mutual antagonism implies that the balance between the function of PTEN and AR may maintain the homeostasis of prostate epithelium in adult males. The demonstration that AR is more active in the absence of functional PTEN suggests that the loss of PTEN function may induce prostatic tumorigenesis by exposing prostatic epithelial cells to unopposed AR activity. Similarly, excessive androgens may induce prostatic tumorigeneis by blocking the apoptosis-promoting function of PTEN. The induction of apoptosis by the restored PTEN expression in LNCaP cells only occurred in the absence of androgen, implying that PTEN mutation or decreased expression may contribute to the resistance of PCa to androgen ablation and that the combinational inhibition of both PI3K/AKT and androgen signals could be an effective approach for the treatment of AR-positive PCa.

    ACKNOWLEDGEMENTS

We thank Dr. W. R. Sellers for PTEN:WT, pSG5L-HA-PTEN:G129R, PLNCX-HA-AKT, PLNCX-HA-myr-AKT, and PLNCX-HA-myr-AKTK179M, P. S. Rennie for PSALuc, Dr. Z. Nawaz for Gal-VP16, Dr. B. Su for GalLuc, and Dr. C. L. Smith for AREe1bLuc. We also thank Drs. S. A. Enkemann, F. Jiang, and B. W. O'Malley for reading the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant R29 CA79530 (to W. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pathology, University of South Florida College of Medicine, 12901 Bruce B. Downs Blvd., MDC 11, Tampa, FL 33612-4799. Tel.: 813-974-0563; Fax: 813-974-5536; E-mail: wbai@com1.med.usf.edu.

Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M010226200

    ABBREVIATIONS

The abbreviations used are: AR, androgen receptor; PCa, prostate cancer; beta -gal, beta -galactosidase; FBS, fetal bovine serum; PI3K, phosphatidylinositol 3-kinase; PI(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PAGE, polyacrylamide gel electrophoresis; PSA, prostate serum antigen; PBS, phosphate-buffered saline; GFP, green fluorescence protein; HA, hemagglutinin; ELISA, enzyme-linked immunosorbent assay; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PTEN, phosphatase and tensin homologue deleted from chromosome 10.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Chang, C., Kokontis, J., and Liao, S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7211-7215[Abstract]
2. Lubahn, D. B., Joseph, D. R., Sullivan, P. M., Willard, H. F., French, F. S., and Wilson, E. M. (1988) Science 240, 327-330[Medline] [Order article via Infotrieve]
3. Trapman, J., Klaassen, P., Kuiper, G. G., van der Korput, J. A., Faber, P. W., van Rooij, H. C., Geurts van Kessel, A., Voorhorst, M. M., Mulder, E., and Brinkmann, A. O. (1988) Biochem. Biophys. Res. Commun. 153, 241-248[Medline] [Order article via Infotrieve]
4. Tilley, W. D., Marcelli, M., Wilson, J. D., and McPhaul, M. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 86, 327-331
5. Tsai, M. J., and O'Malley, B. W. (1994) Annu. Rev. Biochem. 63, 451-486[CrossRef][Medline] [Order article via Infotrieve]
6. Onate, S. A., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1995) Science 270, 1354-1357[Abstract]
7. Ikonen, T., Palvimo, J. J., and Janne, O. A. (1997) J. Biol. Chem. 272, 29821-29828[Abstract/Free Full Text]
8. Yeh, S., and Chang, C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5517-5521[Abstract/Free Full Text]
9. Mhatre, A. N., Trifiro, M. A., Kaufman, M., Kazemi-Esfarjani, P., Figlewicz, D., Rouleau, G., and Pinsky, L. (1993) Nat. Genet. 5, 184-188[Medline] [Order article via Infotrieve]
10. Zhou, Z. X., Kemppainen, J. A., and Wilson, E. M. (1995) Mol. Endocrinol. 9, 605-615[Abstract]
11. Yeh, S., Lin, H. K., Kang, H. Y., Thin, T. H., Lin, M. F., and Chang, C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 5458-5463[Abstract/Free Full Text]
12. Boring, C. C., Squires, T. S., and Tong, T. (1993) Cancer J. Clin. 43, 7-26[Free Full Text]
13. Isaacs, J. T. (1984) Prostate 5, 545-557[Medline] [Order article via Infotrieve]
14. Li, J., Yen, C., Liaw, D., Podsypanina, K., Bose, S., Wang, S. I., Puc, J., Miliaresis, C., Rodgers, L., McCombie, R., Bigner, S. H., Giovanella, B. C., Ittmann, M., Tycko, B., Hibshoosh, H., Wigler, M. H., and Parsons, R. (1997) Science 275, 1943-1947[Abstract/Free Full Text]
15. Steck, P. A., Pershouse, M. A., Jasser, S. A., Yung, W. K., Lin, H., Ligon, A. H., Langford, L. A., Baumgard, M. L., Hattier, T., Davis, T., Frye, C., Hu, R., Swedlund, B., Teng, D. H., and Tavtigian, S. V. (1997) Nat. Genet. 15, 356-362[Medline] [Order article via Infotrieve]
16. Di Cristofano, A., Pesce, B., Cordon-Cardo, C., and Pandolfi, P. P. (1998) Nat. Genet. 19, 348-355[CrossRef][Medline] [Order article via Infotrieve]
17. Suzuki, A., de la Pompa, J. L., Stambolic, V., Elia, A. J., Sasaki, T., del Barco Barrantes, I., Ho, A., Wakeham, A., Itie, A., Khoo, W., Fukumoto, M., and Mak, T. W. (1998) Curr. Biol. 8, 1169-1178[Medline] [Order article via Infotrieve]
18. Tamura, M., Gu, J., Matsumoto, K., Aota, S., Parsons, R., and Yamada, K. M. (1998) Science 280, 1614-1617[Abstract/Free Full Text]
19. Myers, M. P., Pass, I., Batty, I. H., Van der Kaay, J., Stolarov, J. P., Hemmings, B. A., Wigler, M. H., Downes, C. P., and Tonks, N. K. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13513-13518[Abstract/Free Full Text]
20. Sun, H., Lesche, R., Li, D. M., Liliental, J., Zhang, H., Gao, J., Gavrilova, N., Mueller, B., Liu, X., and Wu, H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6199-6204[Abstract/Free Full Text]
21. Stambolic, V., Suzuki, A., de la Pompa, J. L., Brothers, G. M., Mirtsos, C., Sasaki, T., Ruland, J., Penninger, J. M., Siderovski, D. P., and Mak, T. W. (1998) Cell 95, 29-39[Medline] [Order article via Infotrieve]
22. Ramaswamy, S., Nakamura, N., Vazquez, F., Batt, D. B., Perera, S., Roberts, T. M., and Sellers, W. R. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2110-2115[Abstract/Free Full Text]
23. Lee, J. O., Yang, H., Georgescu, M. M., Di Cristofano, A., Maehama, T., Shi, Y., Dixon, J. E., Pandolfi, P., and Pavletich, N. P. (1999) Cell 99, 323-334[Medline] [Order article via Infotrieve]
24. Maehama, T., and Dixon, J. E. (1998) J. Biol. Chem. 273, 13375-13378[Abstract/Free Full Text]
25. Davies, M. A., Koul, D., Dhesi, H., Berman, R., McDonnell, T. J., McConkey, D., Yung, W. K., and Steck, P. A. (1999) Cancer Res. 59, 2551-2556[Abstract/Free Full Text]
26. Nawaz, Z., Lonard, D. M., Dennis, A. P., Smith, C. L., and O'Malley, B. W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1858-1862[Abstract/Free Full Text]
27. Nazareth, L. V., and Weigel, N. L. (1996) J. Biol. Chem. 271, 19900-19907[Abstract/Free Full Text]
28. Lee, H., Jiang, F., Wang, Q., Nicosia, S. V., Yang, J., Su, B., and Bai, W. (2000) Mol. Endocrinology 14, 1882-1896[Abstract/Free Full Text]
29. Snoek, R., Bruchovsky, N., Kasper, S., Matusik, R. J., Gleave, M., Sato, N., Mawji, N. R., and Rennie, P. S. (1998) Prostate 36, 256-263[CrossRef][Medline] [Order article via Infotrieve]
30. Yang, J., New, L., Jiang, Y., Han, J., and Su, B. (1998) Gene (Amst.) 212, 95-102[CrossRef][Medline] [Order article via Infotrieve]
31. Bai, W., and Weigel, N. L. (1996) J. Biol. Chem. 271, 12801-12806[Abstract/Free Full Text]
32. Mosmann, T. (1983) J. Immunol. Methods 65, 55-63[CrossRef][Medline] [Order article via Infotrieve]
33. Carson, J. P., Kulik, G., and Weber, M. J. (1999) Cancer Res. 59, 1449-1453[Abstract/Free Full Text]
34. Kuratsukuri, K., Sugimura, K., Harimoto, K., Kawashima, H., and Kishimoto, T. (1999) Prostate 41, 121-126[CrossRef][Medline] [Order article via Infotrieve]
35. Berchem, G. J., Bosseler, M., Sugars, L. Y., Voeller, H. J., Zeitlin, S., and Gelmann, E. P. (1995) Cancer Res. 55, 735-738[Abstract]
36. Bai, W., and Weigel, N. L. (1995) Vitam. Horm. 51, 289-313[Medline] [Order article via Infotrieve]
37. Alessi, D. R., Caudwell, F. B., Andjelkovic, M., Hemmings, B. A., and Cohen, P. (1996) FEBS Lett. 399, 333-338[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.