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J. Biol. Chem., Vol. 277, Issue 34, 30935-30941, August 23, 2002

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Phosphatidylinositol 3-Kinase/Akt Stimulates Androgen Pathway through GSK3 Inhibition and Nuclear -Catenin Accumulation^*

Manju Sharma, William W. Chuang, and Zijie Sun

From the Departments of Surgery and Genetics, Stanford University School of Medicine, Stanford, California 94305-5328

Received for publication, February 26, 2002, and in revised form, June 10, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PI3K/Akt plays a critical role in prostate cancer cell growth and survival. Recent studies have shown that the effect of PI3K/Akt in prostate cells is mediated through androgen signaling. The PI3K inhibitor, LY294002, and a tumor suppressor, PTEN, negatively regulate the PI3K/Akt pathway and repress AR activity. However, the molecular mechanisms whereby PI3K/Akt and PTEN regulate the androgen pathway are currently unclear. Here, we demonstrate that blocking the PI3K/Akt pathway reduces the expression of an endogenous AR target gene. Moreover, we show that the repression of AR activity by LY294002 is mediated through phosphorylation and inactivation of GSK3, a downstream substrate of PI3K/Akt, which results in the nuclear accumulation of -catenin. Given the recent evidence that -catenin acts as a coactivator of AR, our findings suggest a novel mechanism by which PI3K/Akt modulates androgen signaling. In a PTEN-null prostate cancer cell line, we show that PTEN expression reduces -catenin-mediated augmentation of AR transactivation. Using the mutants of -catenin, we further demonstrate that the repressive effect of PTEN is mediated by a GSK3-regulated degradation of -catenin. Our results delineate a novel link among the PI3K, wnt, and androgen pathways and provide fresh insights into the mechanisms of prostate tumor development and progression.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Prostate cancer is the most common malignancy in men and the second leading cause of cancer death in the United States (1). The fact that androgen ablation is an effective treatment for the majority of prostate cancers indicates that androgen plays an essential role in regulating the growth of prostate cancer cells (2, 3). The growth-promoting effects of androgen in prostate cells are mediated mostly through the androgen receptor (AR).¹ The AR belongs to the nuclear receptor superfamily and acts as a ligand-dependent transcription factor (4, 5). Recent studies suggest that other signal transduction pathways can modulate AR activity and that they may also contribute to the development and progression of prostate cancer (6, 7).

The phosphatidylinositol 3-kinase (PI3K) consists of regulatory (p85) and catalytic (p110) subunits that participate in multiple cellular processes including cell growth, transformation, differentiation, and survival (8). An oncoprotein, Akt/PKB, has been identified as a key effector of the PI3K signaling pathway (9, 10). The binding of PI3K-generated phospholipids to Akt results in the translocation of Akt from the cytoplasm to the inner surface of the plasma membrane where Akt is phosphorylated by the upstream kinases, PDK-1, PDK-2, and ILK (11, 12). The activation of Akt results in the phosphorylation of a number of downstream substrates such as glycogen synthase kinase (GSK3), Bad, and caspase9 and the forkhead transcription factors, Raf, Ib kinase, and phosphodiesterase 3B (13). As one of the principal physiological substrates of Akt, GSK3 is a ubiquitously expressed protein serine/threonine kinase that was initially identified as an enzyme that regulates glycogen synthesis in response to insulin (14, 15). It has been shown that GSK3 plays an important role in the Wnt pathway by regulating the degradation of -catenin (16, 17).

-catenin plays a pivotal role in cadherin-based cell adhesion and in the Wnt signaling pathway (18). Corresponding to its dual functions in cells, -catenin is localized to two cellular pools. Most of the -catenin is located in the cell membrane where it is associated with the cytoplasmic region of E-cadherin, a transmembrane protein involved in homotypic cell-cell contacts (19). A smaller pool of -catenin is located in both the nucleus and cytoplasm where it mediates Wnt signaling. In the absence of a Wnt signal, -catenin is constitutively down-regulated by a multicomponent destruction complex containing GSK3, axin, and the tumor suppressor adenomatous polyposis coli. These proteins promote the phosphorylation of serine and threonine residues in the amino-terminal region of -catenin and thereby target it for degradation by the ubiquitin proteasome pathway (20). Wnt signaling inhibits this process, which leads to an accumulation of -catenin in the nucleus and promotes the formation of transcriptionally active complexes with members of the Tcf/LEF family (21) and other transcription factors (22, 23).

The tumor suppressor PTEN is a phosphoprotein/phospholipid dual specificity phosphatase (24). Early studies indicated that somatic mutation of PTEN is a common event in a variety of human tumors including prostate cancer (25). PTEN was found to be mutated in primary prostate tumors, metastatic prostate cancers, and in prostate cancer cell lines (25, 26). In addition, the reduced expression of PTEN protein as well as increased Akt activity has been observed in xenograft models (27). Recently, it has been shown that PTEN inhibits PI3K/Akt-stimulated androgen-promoted cell growth and AR-mediated transcription in prostate cancer cells (28).

PI3K/Akt has been shown to promote prostate cancer cell survival and growth via enhancing AR-mediated transcription. Both PTEN and the PI3K inhibitor LY294002 negatively regulate this process (28, 29). Although several potential mechanisms have been suggested for this cross-talk, the precise molecular basis by which PI3K/AKT and PTEN regulate AR-mediated transcription is currently unclear. Recently, a specific protein-protein interaction between -catenin and AR was identified by us and others (22, 23). Through this interaction, -catenin augments the ligand-dependent activity of AR in prostate cancer cells. Here, we provide multiple lines of evidence showing that the cross-talk between the androgen and PI3K/Akt pathways is mediated through the modulation of the PI3K/Akt downstream effector GSK3. Its inactivation by phosphorylation results in increased nuclear levels of -catenin, which augment AR activity. These findings delineate a novel mechanism by which PI3K/Akt and PTEN regulate the androgen pathway during prostate cell growth and survival.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Cultures and Transfections-- An AR-positive prostate cancer cell line LNCaP was maintained in T-medium (Invitrogen) with 5% fetal calf serum. Transient transfections were carried out in RPMI 1640 medium using LipofectAMINE 2000 (Invitrogen) as described previously (23). In the experiments with the PI3K inhibitor LY294002 (Alexis, San Diego, CA), cells were usually cultured for 16 h and then were treated with different concentrations of the inhibitor in Me₂SO or vehicle only for 20 min to 2 h. For androgen induction experiments, cells were grown in T-medium with charcoal-stripped fetal calf serum (HyClone, Denver, CO) for 14 h and treated with 10 nM DHT in ethanol and different concentrations of LY294002 for 4 h.

Northern Blot Analysis-- Total RNAs were isolated from LNCaP cells treated with LY294002 for 4 h in the presence of 10 nM DHT in ethanol or vehicle alone using an RNAwiz kit (Ambion, Austin, TX). For Northern blotting, 5 µg of total RNA were electrophoresed on a 1% agarose-formaldehyde gel, transferred to Hybond-N nylon membranes (Amersham Biosciences) by capillary blotting in 20× SSC, and hybridized with a DNA fragment (amino acids 1-261) derived from the human prostate-specific antigen (PSA) gene. The blots were stripped and rehybridized with a -actin probe (30).

Preparation of Whole Cell and Nuclear Extracts-- LNCaP cells were cultured in duplicate flasks to collect both whole cell lysates and nuclear extracts. To make the whole cell lysates, cells were washed with phosphate-buffered saline and were resuspended in RIPA buffer (1% Nonidet P-40, 0.1% SDS, 50 mM NaF, 0.2 mM Na₃VO₄, 0.5 mM dithiothreitol, 150 mM NaCl, 2 mM EDTA, 10 mM sodium phosphate buffer, pH 7.2). Nuclear extracts were prepared from LNCaP cells essentially according to the method of Dignam et al. (31) with minor modifications. The cells were washed with phosphate-buffered saline and mechanically disrupted by scraping into homogenization buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) and incubated on ice for 10 min. Cells were further disrupted by 10 strokes of a homogenizer and centrifuged at 15,000 rpm for 20 min. The pellet was resuspended in buffer containing 20 mM Hepes, pH 7.9, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 25% glycerol and then homogenized with 10 strokes. The lysate was incubated on ice for 30 min and centrifuged for 10 min at 15,000 rpm. The supernatant was saved and analyzed as the nuclear fraction.

To prepare the cytosolic fraction, LNCaP cells treated with LY294002 were lysed in digitonin lysis buffer (1% digitonin, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂). The lysates were centrifuged at 13,000 rpm for 10 min, and the supernatants were saved as cytosolic components. The pellets representing cytoskeletal and nuclear components were lysed in RIPA buffer.

SDS-PAGE and Immunoblotting-- Protein fractions for immunoblotting were boiled in SDS sample buffer and then resolved on a 10% SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane and probed with appropriate antibodies including an anti-human Akt (provided by Dr. Richard Roth, Stanford University, Stanford, CA), phospho-Akt-(Ser-473) (catalog number 9271, Cell Signaling Technology, Beverly, MA), phospho-GSK3/-(Ser-21/Ser-9) (catalog number 9331, Cell Signaling Technology), AR (catalog number sc-816, Santa Cruz Biotechnology, Santa Cruz, CA), Sin3A (catalog number sc-996, Santa Cruz Biotechnology), tubulin (catalog number MS-581-P, Neomarker, Fremont, CA), -catenin (catalog number C19220, Transduction Laboratories, Lexington, KY), and GSK3 (catalog number G22320, Transduction Laboratories). Proteins were detected using the ECL kit (Amersham Biosciences). The nuclear fractions were analyzed by SDS-PAGE. Equal loading of the nuclear proteins was ascertained by reversible staining with the Ponceau S solution (Catalog number P7767, Sigma).

Plasmid Construction-- The pcDNA3-AR expression vector was generated in the laboratory and used for the transient transfection experiments. Expression constructs of human PTEN were generously provided by Dr. William Sellers (Dana-Farber Cancer Institute, Boston, MA) and used for subcloning into the pCMV5 vector. PLNCX-HA-myr-AKT and PLNCX-HA-myr-AKT179M were also kindly provided by Dr. Sellers (32). The reporter plasmid pPSA7kb-luc with the luciferase gene under the control of promoter fragments of the human prostate-specific antigen was provided by Dr. Jan Trapman (33). The mutants of -catenin with a single point mutation in the GSK3 phosphorylation sites were generated by a PCR-based mutagenesis scheme. The key serine amino acid residues were mutagenized by using sets of primers containing two or three nucleotide changes in conjunction with upstream and downstream primers. The appropriate fragments with in-frame restriction enzyme sites were generated by PCR, cleaved with restriction enzymes, and cloned into the pcDNA3 vector (Invitrogen). All of the constructs were sequenced from both ends of the inserts to confirm that no extraneous mutations were introduced by PCR.

Luciferase and -Galactosidase Assay-- Luciferase activity was measured in relative light units as described previously (30). 50 µl of cell lysate was used for luciferase assays. The light output is measured after a 5-s delay following injection of 50 µl of luciferase buffer and 50 µl of Luciferin by the dual injector luminometer according to manufacturer's instruction (Analytical Luminescence Laboratories, San Diego, CA). The relative light units from individual transfections were normalized by the measurement of -galactosidase activity expressed from a cotransfected plasmid in the same samples. Individual transfection experiments were done in triplicate, and the results are reported as the luciferase/-galactosidase mean ± S.D. from representative experiments.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inhibition of the PI3K/AKT Pathway Represses AR-mediated Transcription-- PI3K/Akt enhances the activity of AR-regulated reporter genes in transient transfection experiments (28, 29). To evaluate the effect of PI3K/Akt on AR-mediated transcription in a physiologically relevant cellular context, we examined the expression of the endogenous PSA gene in an AR-positive prostate cancer cell line LNCaP treated with the PI3K inhibitor LY294002. In the presence of 10 nM DHT, PSA expression was increased ~4-fold in LNCaP cells over that found in cells not treated with DHT (Fig. 1A). At concentrations of LY294002 from 25-100 µM, the expression of PSA was significantly reduced. An ~4-fold reduction of PSA transcripts was found in the cells treated with 100 µM LY294002 using the level of -actin transcripts as an internal control (Fig. 1A). Low concentrations (5 µM) of LY294002 induced only a slight reduction of PSA expression during a 4-h treatment but showed a significant reduction of PSA expression after 16 h (data not shown). To ensure that this repression was not the result of LY294002-induced changes in the intracellular steady-state levels of AR protein, we examined both the AR and tubulin protein levels in the cell samples used for the Northern blotting. We found that there was no significant change in protein expression (Fig. 1B). This result provided the first line of evidence that inhibition of PI3K/Akt could suppress endogenous AR-mediated transcription in prostate cancer cells.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   The PI3K inhibitor represses AR-mediated transcription. A, total RNAs were isolated from LNCaP cells cultured in T-medium with or without 10 nM DHT, treated for 4 h with the PI3K inhibitor LY294002 or vehicle, and analyzed by Northern blotting. Expression of the endogenous PSA gene was detected by a cDNA probe derived from the human PSA gene. A -actin probe was used to confirm equal RNA loading. Densitometry of the membrane blot was performed, and the relative numbers were reported as optical density units of -actin (PSA/-actin). B, whole cell lysates were isolated from LNCaP cells treated as described above and analyzed by Western blotting to detect the expression of AR and tubulin proteins.

Repression of the PI3K/AKT Pathway Inhibits Phosphorylation of GSK3 and Nuclear Accumulation of -catenin in Prostate Cancer Cells-- To further elucidate the mechanism by which LY294002 inhibits endogenous AR transactivation in LNCaP cells, we first assessed the phosphorylation state of Akt. It has been reported that PDK-1 phosphorylation of threonine 308 in the activation loop of the catalytic domain of Akt allows autophosphorylation of serine 473 (a hydrophobic phosphorylation site) in the carboxyl terminus (34). To demonstrate that the effect of LY294002 on PSA transcription was attributed to inhibition of Akt, we evaluated Akt activation using a phosphorylation-specific antibody for Ser-473. As shown in Fig. 2A, the phosphorylation of Akt proteins was significantly inhibited by LY294002 in LNCaP cells, even after a very short pulse (20 min). In contrast, the total amount of Akt protein showed no differences in the presence or absence of LY294002.

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Inhibition of Akt and GSK3 phosphorylation by LY294002 in prostate cancer cells. Whole cell lysates were isolated from LNCaP cells that were treated as indicated in Fig. 1 and "Experimental Procedures," and were analyzed by Western blotting. Both total and phosphorylated Akt (A) and GSK3 (B) were detected by specific antibodies as indicated in the figure.

Because GSK3 is one of the major downstream targets of Akt, we next assessed whether LY294002 also affected the phosphorylation of GSK3. Using specific antibodies, we examined both the total and phosphorylated GSK3 proteins in the same cell samples used for detecting Akt. As expected, the phosphorylation of GSK3 proteins was also significantly impaired by treatment with LY294002, whereas almost equal amounts of total GSK3 proteins were found in both treated and untreated cells (Fig. 2B). At either 5 or 20 µM LY294002, we observed a similar inhibitory effect on the phosphorylation of both Akt and GSK3 in cells treated for 12 h (data not shown). Taken together, the results demonstrate that the suppression of the PI3K pathway by the PI3K inhibitor LY294002 blocks the phosphorylation of both Akt and GSK3 proteins in LNCaP cells.

The above data demonstrate that the treatment of LNCaP cells with LY294002 results in a decreased level of expression of the endogenous PSA gene and an inhibition of the phosphorylation of Akt and GSK3. It has been shown that GSK3 regulates the cellular levels of -catenin by targeting it to the ubiquitin proteasome pathway via the destruction complex (20). Previous studies have shown that inactivation of GSK3 by phosphorylation can induce the nuclear accumulation of -catenin because of decreased degradation (17, 35). To evaluate the downstream effect of GSK3 in LNCaP cells, we next examined the nuclear levels of -catenin. Nuclear extracts and whole cell lysates were prepared from cells that were treated with LY294002 or with vehicle only. As shown in Fig. 3A, there was no significant change in the amount of total -catenin protein in the treated compared with the untreated cells. However, there was a 2-3-fold reduction in nuclear -catenin in the cells treated with LY294002 (Fig. 3, A and B). In contrast, the controls, total nuclear protein, and the transcriptional repressor Sin3A showed no change (Fig. 3A). To confirm these findings, we examined the level of free cytosolic -catenin protein in LNCaP cells treated with LY294002 (36). As shown in Fig. 3C, after LY294002 treatment, free -catenin in the cytosolic compartment (Digi) was significantly reduced, whereas -catenin in the cytoskeletal compartment (RIPA) remained unchanged. Taken together, these results demonstrate that blocking PI3K signaling results in a decrease in both the free cytosolic and nuclear -catenin in prostate cells.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Inhibition of PI3K signaling results in decreased nuclear accumulation of -catenin in prostate cancer cells. A, both nuclear extracts and whole cell lysates were isolated from LNCaP cells treated with LY294002 and DHT and resolved by SDS-PAGE. The -catenin and Sin3A antibodies were used for the detection of protein expression. The same membrane used for the Western blotting was also stained with Ponceau S stain solution for measuring equal protein loading. B, densitometry of nuclear -catenin proteins is shown as relative -catenin density (optical density units of nuclear proteins/optical density units of total proteins). C, both cytosolic fraction (Digi) and cytoskeletal fraction (RIPA) were prepared from LNCaP cells as described under "Experimental Procedures" and were analyzed by Western blotting. Both -catenin and tubulin were detected using specific antibodies.

Repression of AR Activity by LY294002 Is Mediated through the Downstream Effectors of PI3K, Akt, and GSK3-- To further study the repressive effect of LY294002 on AR-mediated transcription, we next used an inactive and a dominantly active mutant of Akt to directly examine the involvement of Akt in LY294002-induced AR repression. Transient transfection assays were performed in LNCaP cells. In the presence of 10 nM DHT, the overexpression of AR induces approximately a 10-fold induction of the PSA promoters. Cotransfection with the wild type -catenin expression vector augments AR activity to nearly 20-fold above base line (Fig. 4A). The addition of LY294002 to the cells results in a large reduction in AR activity. At 5 µM LY294002, AR activity was reduced by ~60%. Coexpression of the dominantly active Akt reversed the inhibition of AR activity by LY294002, whereas an inactive mutant of Akt used as a control showed no effect (Fig. 4A). These data directly demonstrate that repression of AR activity by LY294002 is mediated through the down-regulation of PI3K and the subsequent inactivation of Akt activity.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Inhibition of AR activity by LY294002 is mediated through Akt and GSK3. A, transient transfections were performed in LNCaP cells with 100 ng of PSA7kb-luc reporter, 5 ng of pcDNA3-AR, 25 ng of pcDNA3--galactosidase, and 50 ng of wild type pcDNA3-FLAG--catenin in the presence or absence of 50 ng of an inactive mutant (IA) or a dominantly active mutant (DA) of Akt. The cells were incubated in RPMI 1640 medium with 5% charcoal-stripped fetal calf serum for 12 h and then were treated with different concentrations of LY294002 in the presence or absence of 10 nM DHT for 18 h. Cell lysates were measured for luciferase and -galactosidase activities. The data represent the mean ± S.D. of three independent samples. B, LNCaP cells were cotransfected with 50 ng of wild type -catenin or the mutants of -catenin containing a point mutation within the GSK3 binding site as well as with the other plasmids indicated in the figure. The cells were treated with DHT and LY294002 as described above.

We next performed the transient transfection experiments using either wild type or -catenin mutants containing a point mutation within the NH₂-terminal GSK3 binding site. Because these mutants are resistant to GSK3-mediated degradation, we further assessed whether the repression of AR by LY294002 is mediated through GSK3. As shown in Fig. 4B, an ~40% reduction in expression was induced by 5 µM LY294002 in the cells that were cotransfected with wild type -catenin but not in the cells cotransfected with the -catenin mutants. As mentioned above, because the -catenin mutants used in these experiments are impervious to the effects of the destructive complex attributed to point mutations within the GSK3 phosphorylation sites (20), the results from these experiments suggest that GSK3 is involved in the regulation of -catenin-mediated augmentation of AR activity.

Expression of PTEN in LNCaP Cells Represses -Catenin-mediated Augmentation of AR Activity-- Recent data have shown that the tumor suppressor PTEN appears to negatively control the PI3K signaling pathway by blocking the activation of the downstream target Akt (24). The mutations in the PTEN gene were found in prostate cancer tissues and cell lines (25). In a previous report, Li et al. (28) showed that the transfection of the wild type PTEN repressed an AR-regulated reporter gene in PTEN-null prostate cancer cells. The results from our experiments indicate that the inhibition of PI3K/Akt signaling represses the expression of an endogenous AR target gene and reduces the levels of nuclear -catenin. To further examine whether repression of AR activity by PTEN is also mediated by PI3K/Akt modulation of nuclear -catenin, we performed transient transfections using either the wild type -catenin or the -catenin mutants described above. As shown in Fig. 5A, in the absence of PTEN vector, both the wild type and -catenin mutants augment AR-mediated transcription ~1.5-fold using a 7-kilobase PSA promoter in the PTEN-null cells, LNCaP. However, when a wild type PTEN vector was cotransfected into the cells, the wild type -catenin showed less enhancement of AR activity than the mutants, indicating a repressive effect of PTEN on wild type -catenin (p < 0.05). The results with the mutants of -catenin demonstrate that the effect of PTEN on AR-mediated transcription is regulated through GSK3 via degradation of nuclear -catenin. To further confirm this finding, we examined the phosphorylation status of Akt and GSK3 proteins as well as the levels of nuclear -catenin protein in LNCaP cells, which were transfected with either wild type or the loss-of-function PTEN expression vector. As shown in Fig. 5B, both the phosphorylation of Akt and GSK3 proteins was significantly reduced in the cells transfected with wild type PTEN vector. Moreover, a reduction of nuclear -catenin protein was observed only in the nuclear extracts isolated from cells transfected with the wild type PTEN vector, although the total -catenin protein detected was almost equal in all of the samples (Fig. 5C).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   PTEN represses -catenin-mediated augmentation of AR activity by reducing nuclear -catenin protein. A, LNCaP cells were transfected with a PSA7kb-luc reporter (100 ng), pcDNA3--galactosidase (25 ng), pcDNA3-AR (5 ng), and the wild type or mutants of pcDNA3-FLAG--catenin (50 ng) as indicated. Either an empty pCMV5 vector or pCMV5-PTEN was cotransfected with the above plasmids. Ten hours after transfection, the cells were treated with 10 nM DHT or with vehicle only for 18 h. Cell lysates were measured for luciferase and -galactosidase activities. The data represent the mean ± S.D. of three independent samples. (B and C) The PTEN expression constructs were transfected into LNCaP cells. Nuclear extracts and whole cell lysates were prepared from the cells 30 h after transfection and analyzed by Western-blotting. D, transient transfections were performed with the plasmids as labeled in the figure. After a 10 h transfection, 10 nM DHT and 50 mM LiCl were added to the cells. Whole cell lysates were prepared after another 18 h of incubation and were used to measure luciferase and -galactosidase activities.

We next examined whether the inhibition of GSK3 can directly affect -catenin-mediated augmentation of AR activity. As lithium chloride has been shown to inhibit GSK3 through a mechanism independent of serine 9 phosphorylation (37), we examined whether -catenin-mediated AR augmentation is affected in cells treated with LiCl. As shown in Fig. 5D, in the presence of PTEN, the transfection of wild type -catenin showed less stimulation of AR-mediated PSA promoter activity than that of the mutant -catenin (black bars 2 and 4). However, the inhibition of GSK3 by LiCl treatment increases AR activity in the presence of wild type -catenin (black bar 3), whereas there is little change in the PSA promoter activity in the mutant -catenin-transfected cells treated with LiCl (black bar 5). These data are consistent with previous reports on other human cell lines (36, 38). Taken together, our results demonstrate that PTEN negatively regulates the augmentation of AR activity by -catenin through targeting of the -catenin degradation pathway mediated by GSK3.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The PI3/Akt pathway plays a critical role in prostate cell proliferation and survival (24). PTEN, which is frequently mutated in prostate cancer cells, negatively regulates this process by blocking the PI3K/Akt pathway. Recently, several lines of evidence showed that PI3K/Akt and PTEN can modulate androgen-induced cell growth and AR-mediated transcription in prostate cancer cells (28, 29), suggesting a potential link between the PI3K/Akt and androgen pathways. In this study, we demonstrated that -catenin acts as the point of convergence for the cross-talk between the PI3K/Akt and androgen signaling pathways. The data presented here are consistent with what is known regarding the degradation of -catenin by GSK3, a downstream effector of PI3K/Akt, and fit very well with our recent finding that -catenin interacts with AR and augments its ligand-dependent transcription (23).

The dysregulation of -catenin expression and Wnt-mediated signaling is now recognized as important events in the pathogenesis of variety of human malignancies including prostate cancer (18, 39). Tumor cells contain high levels of free cellular -catenin by acquiring loss-of-function mutations in the components of the destruction complex or by altering regulatory sequences in -catenin itself. Besides Wnt signaling, other signaling pathways are also involved in regulating cellular -catenin levels (36, 38, 40). In this study, we showed that PI3K/Akt increases the stability of nuclear -catenin by phosphorylation and inactivation of the downstream substrate GSK3 in prostate cancer cells. Given that -catenin acts as a transcriptional coactivator of AR, these data provide evidence to suggest a new mechanism whereby PI3K/Akt can affect prostate cell proliferation and survival through androgen signaling.

Earlier studies showed that PTEN negatively regulates the PI3K/Akt pathway in prostate cancer cells (28). The expression of PTEN in LNCaP, a PTEN-null prostate cancer cell line, blocks androgen-induced cell growth and AR-mediated transcription. In this study, we demonstrated that the overexpression of PTEN in LNCaP reduces -catenin-mediated augmentation of AR activity; however, PTEN showed no effect in cells transfected with -catenin mutants containing a single point mutation within the GSK3 phosphorylation sites. The results from our biochemical experiments further demonstrated that PTEN reduces the nuclear accumulation of -catenin proteins in prostate cells. Because the -catenin mutants used in our experiments are impervious to degradation by the destruction complex, we conclude that the regulation of -catenin by PTEN is mediated through GSK3. Our results are consistent with a recent study showing that nuclear -catenin protein is constitutively elevated in PTEN null cells, and this elevated expression can be reduced upon the reexpression of PTEN (41). The data presented here also confirm that PTEN negatively regulates the PI3K pathway by inhibiting phosphorylation of Akt. In addition, the experiments using PTEN as a natural PI3K inhibitor are consistent with our data showing the important effects mediated by the synthetic PI3K inhibitor LY294002.

Modification of the AR protein such as by phosphorylation or acetylation has been suggested to be an important mechanism for modulating AR activity in prostate cancer cells (42-44). The putative consensus sequences for Akt phosphorylation were identified in both the transactivation and the ligand binding domains of AR (29). Those authors showed that Akt can directly bind to and phosphorylate AR (29). However, using both biochemical and functional approaches, we were not able to show a physical protein-protein interaction between Akt and AR or the phosphorylation of AR by Akt in vitro (data not shown). Results similar to ours were also reported by Li et al. (28). These conflicting results may be attributed to the use of different reagents and experimental conditions, but they also suggest that other alternative pathways may be involved in this regulation (Fig. 6). As presented in this study, we propose a novel molecular mechanism for PI3K/Akt and PTEN regulation of androgen signaling in prostate cancer cells.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   -catenin acts as a mediator in the cross-talk between PI3K and androgen signaling. A model summarizes PI3K/Akt signaling in prostate cells and the pathways for PTEN and the PI3K inhibitor LY294002 in the regulation of AR activity.

The major role of -catenin in tumorigenesis has been implicated via its interaction with the Tcf/LEF transcription factors (45). Interestingly, as we and others have reported recently (23, 46), -catenin is shown to have no effect on the activation of Tcf/LEF-mediated transcription in prostate cancer cells despite the expression of Tcf/LEF. A similar observation was also reported recently in breast cancer cells (47). In this study, using Tcf/LEF reporters, we were also not able to demonstrate an effect of PTEN on the regulation by -catenin of Tcf/LEF-mediated transcription in LNCaP cells (data not shown). This raises the question as to whether the growth-promoting effect of -catenin is mediated through partners outside of the Tcf/LEF pathway in prostate cancer and/or other tumor cells.

In this study, we demonstrate that -catenin mediates the cross-talk between PI3K/Akt and androgen pathways. Based on these results and previous studies by others, we summarize our findings in Fig. 6. The PI3K/Akt signal induces phosphorylation and inactivation of GSK3, resulting in increased nuclear levels of -catenin. Consequently, increased -catenin elevates AR activity to stimulate prostate cell growth and survival. Both the PI3K inhibitor LY294002 and PTEN negatively regulate these processes. A loss-of-expression or mutational inactivation of PTEN has been frequently observed in human tumors, which induce the suppression of apoptosis and accelerates cell cycle progression (24, 25). Additionally, the mutation or aberrant expression of the destruction complex and the reduction of E-cadherin, which results in increased nuclear -catenin, also occurs during prostate cancer progression (39). Our data showing that PTEN reduces nuclear -catenin in prostate cancer cells suggest a novel role of PTEN in down-regulating androgen-induced cell growth and survival. A further study of the regulation of the interaction among PI3K, Wnt, and the androgen signaling pathways in prostate cancer cells should provide fresh insight into the pathogenesis of prostate cancer that may help us to identify new pathways that can be targeted for prostate cancer treatment.

	ACKNOWLEDGEMENTS

We are especially grateful to Drs. Jan Trapman, Richard Roth, and William Sellers for the various reagents. We thank Homer Abaya for administrative assistance and help in preparing this paper.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants CA70297 and CA87767 and the Department of Army Prostate Cancer Grant PC01-0690.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.

To whom correspondence should be addressed: Departments of Surgery and Genetics, R135, Edwards Bldg., Stanford University School of Medicine, Stanford, CA 94305-5328. E-mail: zsun@stanford.edu.

Published, JBC Papers in Press, June 12, 2002, DOI 10.1074/jbc.M201919200

	ABBREVIATIONS

The abbreviations used are: AR, androgen receptor; PI3K, phosphatidylinositol 3,4,5-trisphosphate; GSK3, glycogen synthase kinase 3; PTEN, phosphatase and tensin homolog deleted on chromosome 10; DHT, dihydrotestosterone; PSA, prostate-specific antigen.

	REFERENCES

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