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[Cancer Research 66, 4781-4788, May 1, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Inhibition of the Phosphatidylinositol 3'-Kinase Pathway Promotes Autocrine Fas-Induced Death of Phosphatase and Tensin Homologue–Deficient Prostate Cancer Cells

Jerod Bertram1,2, James W. Peacock1,2, Clara Tan3, Alice L-F. Mui2,3, Stephen W. Chung2, Martin E. Gleave1,2, Shoukat Dedhar3, Michael E. Cox1,2 and Christopher J. Ong1,2

1 The Prostate Centre, Vancouver General Hospital; Departments of 2 Surgery and 3 Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada

Requests for reprints: Christopher J. Ong, Department of Surgery, University of British Columbia, 2660 Oak Street, Vancouver, British Columbia, Canada V6H 3Z6. Phone: 604-875-5555, ext. 63120; Fax: 604-875-5654; E-mail: chriso{at}interchange.ubc.ca.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rationally designed therapeutics that target the phosphatidylinositol 3'-kinase (PI3K) cell survival pathway are currently in preclinical and clinical development for cancer therapy. Drugs targeting the PI3K pathway aim to inhibit proliferation, promote apoptosis, and enhance chemosensitivity and radiosensitivity of cancer cells. The phosphatase and tensin homologue (PTEN) phosphatidylinositol 3'-phosphatase is a key negative regulator of the PI3K pathway. Inactivation of the PTEN tumor suppressor results in constitutive activation of the PI3K pathway and is found in ~50% of advanced prostate cancers, which correlates with a high Gleason score and poor prognosis. Inhibition of the PI3K pathway leads to apoptosis of prostate cancer cells; however, the precise mechanism by which this occurs is unknown. Here we report that apoptotic cell death of PTEN-deficient LNCaP and PC3 prostate cancer cells induced by the PI3K inhibitor LY294002 can be abrogated by disrupting Fas/Fas ligand (FasL) interactions with recombinant Fas:Fc fusion protein or FasL neutralizing antibody (Nok-1), or by expressing dominant-negative Fas-associated death domain. Furthermore, we find that apoptosis induced by expression of wild-type PTEN, driven by a tetracycline-inducible expression system in LNCaP cells, can be inhibited by blocking Fas/FasL interaction using Fas:Fc or Nok-1. These data show that apoptosis induced by blockade of the PI3K pathway in prostate tumor cells is mediated by an autocrine Fas/FasL apoptotic mechanism and the Fas apoptotic pathway is both necessary and sufficient to mediate apoptosis by PI3K inhibition. (Cancer Res 2006; 66(9): 4781-8)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Phosphatase and tensin homologue (PTEN), also known as MMAC or TEP1, is among the most commonly mutated genes in a wide spectrum of cancers (1). The PTEN tumor suppressor gene is inactivated in ~30% of primary prostate cancers (2, 3) and 63% of metastatic prostate tumors (4). Loss of PTEN correlates with a high Gleason score, an advanced pathologic stage, and a poor prognosis (5, 6). PTEN is a lipid phosphatase that cleaves the D3 phosphate of the second messenger phosphatidylinositol 3,4,5-trisphosphate and thus negatively regulates the phosphatidylinositol 3'-kinase (PI3K) pathway (7, 8). The PI3K signaling pathway is an important intracellular mediator of cell survival and antiapoptotic signals originating from trophic factors (1). PI3K activation leads to production of 3'-phosphoinositide second messengers, such as phosphatidylinositol 3,4,5-trisphosphate, which activate a variety of downstream cell survival signals. Accumulation of phosphatidylinositol 3,4,5-trisphosphate at the membrane recruits a number of signaling proteins containing pleckstrin homology domains, including protein kinase B (also known as Akt). On recruitment, protein kinase B becomes phosphorylated and activated and exerts its antiapoptotic activity through inactivation of proapoptotic proteins, such as the Bcl-2 family member, BAD, caspase-9, and forkhead family transcription factors, which regulate the expression of proapoptotic genes, such as Fas ligand (FasL). In addition, the PI3K pathway has also been shown to be capable of negatively regulating Fas-induced cell death (9–11).

The lack of negative regulation of PI3K signaling as a consequence of PTEN inactivation results in elevated steady-state levels of phosphatidylinositol 3,4,5-trisphosphate and constitutive activation of protein kinase B, leading to cytoprotection from various apoptotic stimuli as well as accelerated cell cycle progression (1). Blockade of the PI3K pathway leads to apoptosis of PTEN-deficient cancer cells including prostate cancer (12–14). Consequently, the PI3K pathway is currently a major therapeutic target for treatment of cancer and several novel therapies are in late-stage phase II and phase III clinical trials (15–17). However, the manner through which PI3K pathway blockade mediates apoptosis is currently unknown. Thus, a better understanding of mechanism(s) by which these agents mediate their anticancer activity may improve insight into strategies to fully exploit the potential clinical benefits of these new agents.

In mammals, programmed cell death can be initiated by two major pathways: the extrinsic and intrinsic death pathways. The extrinsic pathway is triggered by extracellular ligands that induce oligomerization of death receptors, such as Fas or other members of the tumor necrosis factor receptor superfamily, resulting in activation of a caspase cascade leading to apoptosis. The instrinsic pathway, on the other hand, is triggered in response to a variety of apoptotic stimuli that induce damage within the cell, including anticancer agents, oxidative damage, UV irradiation, and growth factor withdrawal, and is mediated through the mitochondria. These stimuli induce the loss of mitochondrial membrane integrity and result in the release of proapoptotic molecules, including cytochrome c, which associates with apoptotic protease-activating factor-1 and caspase-9 to promote caspase activation, and SMAC/Diablo and Omi/HtrA2, which promote caspase activation by eliminating inhibition by inhibitor of apoptosis proteins. PI3K pathway inhibitors have been dogmatically presumed to mediate apoptosis via activation of the intrinsic pathway. However, the role of the extrinsic pathway in PI3K blockade–induced apoptosis has not been examined.

Fas-induced death is the best understood extrinsic apoptotic pathway both in terms of mechanism and its physiologic importance in vivo (18). Multivalent cross-linking of the Fas receptor, as a result of FasL binding to preassociated Fas receptor trimers, triggers the recruitment of a set of effector proteins to the receptor, resulting in the formation of the death-inducing signaling complex. The death-inducing signaling complex is composed of intracellular signaling proteins including Fas-associated death domain (FADD/MORT1), a death domain–containing adaptor protein, and caspase-8 (also known as FLICE/MACH). On recruitment to the death-inducing signaling complex, caspase-8 is autoproteolytically cleaved and activated, which then directly activates caspase-3, leading to execution of apoptosis. Caspase-8 also leads to activation of the mitochondrial amplification loop by proteolytic cleavage of the proapoptotic Bcl-2 member Bid. The truncated Bid then translocates to the mitochondria and promotes cytochrome c release into the cytosol. In association with apoptotic protease-activating factor-1 and procaspase-9, cytochrome c forms the apoptosome complex, leading to the activation of caspase-9, which subsequently cleaves and activates effector caspases.

Fas and FasL are commonly coexpressed in prostate cancer cells (19, 20). Despite expression of both cell-surface Fas and FasL as well as constitutive secretion of biologically active soluble FasL, prostate cancer cells are resistant to Fas-mediated apoptosis (21–26). PTEN inactivation has been shown to result in impairment of Fas-induced apoptosis and Pten heterozygous mice develop a lymphoproliferative syndrome that mimics that observed in Fas and FasL deficient mice (11). We therefore hypothesized that blockade of PI3K pathway may lead to reactivation of apoptotic potential of autocrine Fas signals in prostate cancer cells.

Abrogation of the PI3K/protein kinase B pathway using pharmacologic inhibitors of PI3K, such as LY294002, or reconstitution with PTEN in PTEN-deficient prostate cancer cells induces apoptotic cell death by an unknown mechanism (12, 13). In this study, we examined the role of Fas in mediating apoptosis induced by inhibition of the PI3K pathway in LNCaP and PC3 prostate cancer cells through expression of PTEN or treatment with the potent pharmacologic inhibitor of PI3K, LY294002. Here, we show that apoptosis induced by these agents can be effectively inhibited by blocking Fas signaling. These data suggest that apoptosis, as a result of abrogating PI3K signaling in LNCaP and PC3 cells, is mediated by a Fas-dependent mechanism and occurs in an autocrine manner.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies and chemicals. The following antibodies were used: anti–phospho-protein kinase B (Ser473), anti-PTEN, antihuman specific cleaved poly(ADP-ribose) polymerase (Asp214; Cell Signaling, Inc., Beverly, MA), and anti-Fas (CH-11) antibody (Immunotech, Marseille, France). Rabbit polyclonal antiactin antibody, MOPC-31c immunoglobulin G1 (IgG1), {kappa} antibody, DMSO, and LY294002 were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). Nok-1 antibody, Annexin V-biotin, phycoerythrin-conjugated streptavidin, anti–cleaved caspase-3-phycoerythrin, antihuman Fas antibody DX-2, matrix metalloproteinase inhibitor (KB8301), and BD Cytofix were purchased from BD PharMingen (San Diego, CA). Fas:Fc was purchased from R&D Systems (Minneapolis, MN) and Thy1:Fc from Alexis Biochemicals (Lausen, Switzerland). Anti-FADD antibody (mouse IgG1 isotype-matched control antibody) was purchased from Transduction Laboratories (Lexington, KY) and anti–extracellular signal–regulated kinase-2 antibody from Upstate Biotechnology (Lake Placid, NY). FITC-conjugated affinipure donkey anti-mouse IgG antibody was obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).

Tumor cell lines and cell culture. Human prostate cancer PC-3 cells were purchased from the American Type Culture Collection (Manassas, VA). LNCaP cells were kindly provided by Dr. Leland W.K. Chung (University of Virginia, Charlottesville, VA). LNCaP and PC3 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) at 37°C, 5% CO2.

Flow cytometry. For Fas labeling, 0.2 x 106 to 1.0 x 106 cells were resuspended in fluorescence-activated cell sorting (FACS) buffer (2% FBS containing Ca2+- and Mg2+-free PBS). Cells were stained with anti-Fas (DX-2) primary antibody at 37°C for 1 hour, then stained with FITC-conjugated antimouse IgG at 37°C for 45 minutes. As controls, cells were stained with secondary antibody alone without addition of primary antibody. For FasL staining, cells were fixed in BD cytofix solution (BD Biosciences PharMingen, San Diego CA) overnight followed by permeabilization in PBS containing 0.2% Triton X-100 (Sigma-Aldrich) for 20 minutes at room temperature. Cells were then washed once with wash buffer [PBS and 0.05% Tween 20, 0.1% fraction V bovine serum albumin (BSA); Roche Diagnostics Corporation, Indianapolis, IN], followed by treatment with pre-block containing 5% normal goat serum (NGS; Sigma-Aldrich). After two washes in wash buffer, cells were stained with anti-FasL (Nok-1) primary antibody (or MOPC-31c isotype-matched primary antibody as control) at 37°C for 1 hour. Cells were then washed and stained with secondary FITC-conjugated antimouse IgG antibody. Annexin V staining was done per instructions of the manufacturer (BD Biosciences, Mississauga, Ontario, Canada) by direct labeling with phycoerythrin-conjugated Annexin V or by staining with Annexin V-biotin followed by streptavidin-phycoerythrin. Intracellular active-caspase-3 assay was done as previously described (27). Briefly, cells were fixed, permeabilized, and blocked as above and stained with anti-caspase-3 phycoerythrin-conjugated antibody in wash buffer containing 1.0% NGS at 37°C for 1 hour. Cells were washed twice in wash buffer, washed once in FACS buffer, and finally resuspended in 500 µL of FACS buffer.

Fluorescently labeled cells were examined by flow cytometry on the Coulter Epics XL-2988 (Beckman Coulter, Burlington, Ontario, Canada). Data were analyzed using the WinMidi version 2.8 software (J. Trotter, Scripps Institute, La Jolla, CA).

Apoptosis induction by LY294002. LNCaP and PC3 cells were cultured for 24 hours under low-serum (0.1% FBS) conditions in RPMI containing 40 µmol/L LY294002 or vehicle alone (0.2% DMSO) in the presence of the indicated concentrations of Fas:Fc, Thy1:Fc, Nok-1, or isotype-matched control antibody before flow cytometric analyses of apoptosis.

Expression constructs and transfection of LNCaP cells. The dominant-negative FADD (DN FADD) expression construct, AU1-tagged DN FADD pcDNA3, was kindly provided by Dr. Claudius Vincenz (University of Michigan, Ann Arbor, MI) and the PTEN expression vector, PTEN-wild-type (WT) pcDNA3, was a gift from Dr. Charles Sawyers (University of California, Los Angeles). The original pcDNA3 vector was purchased from Invitrogen (Carlsbad, CA). The enhanced green fluorescence protein (EGFP) reporter expression vector, pEGFP-C1, was obtained from Clontech (Palo Alto, CA).

Expression vectors were transiently transfected into LNCaP cells using Lipofectin (Invitrogen) in six-well culture dishes as per recommended protocol of the supplier. An equivalent total amount of DNA (8 µg per six-well plate) was transfected. GFP reporter construct (pEGFP-C1; 1.6 µg) was used to tag and monitor the transfected cell population. For DN FADD expression, cells were transfected with 1.6 µg pEGFP-C1 plasmid + 6.4 µg of pcDNA3-DN FADD or pcDNA3 empty vector. Following transfection, cells were cultured in 10% FBS containing RPMI overnight. Cells were then rinsed in PBS, overlaid with 0.1% FBS/RPMI medium containing 40 µmol/L LY294002 or 0.2% DMSO control, and cultured for 24 hours before flow cytometric analysis of apoptosis. For triple transfections, cells were transfected with 1.6 µg pEGFP-C1, 3.2 µg WT PTEN pcDNA, and 3.2 µg DN FADD pcDNA plasmids. As controls, WT PTEN and/or DN FADD expression plasmids were replaced with 3.2 µg pcDNA empty vector. Following transfection, cells were cultured in 10% FBS containing RPMI at 37°C, 5% CO2 overnight before transfer and culture in 0.1% FBS–containing medium for 72 hours. Apoptosis was monitored by flow cytometric analyses of Annexin V and intracellular active caspase-3 staining.

Western blot. A lysate volume corresponding to 20 µg total protein, as determined by the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL), was diluted in protein sample buffer containing 2% SDS, 0.1% bromophenol blue, 10% glycerol, 25 mmol/L Tris (pH 6.8). Samples were heated at 100°C for 10 minutes before SDS-PAGE. The filters were blocked with 5% BSA fraction V (Boehringer Mannheim, Indianapolis, IN) and then incubated overnight at 4°C in TBST containing 1% BSA supplemented with the indicated primary antibody as directed by the manufacturer. After three additional washes in TBST, the filters were incubated for 1 hour in the appropriate horseradish peroxidase–conjugated secondary antibody (1:1,000; Dako, Diagnostics Canada, Inc., Mississauga, Ontario, Canada), followed by three washes in TBST. Proteins were detected using enhanced chemiluminescence (Amersham, Arlington Heights, IL) and exposure to Full Blue Film (Island Scientific, Bainbridge Island, WA).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LNCaP cells express both Fas and FasL. Previous reports have shown that LNCaP cells coexpress both Fas and FasL on its cell surface (19). Moreover, LNCaP cells have been shown to constitutively secrete functional soluble FasL (25). To confirm that both Fas and FasL are expressed by LNCaP cells, we did immunofluorescence flow cytometry and found that LNCaP cells do indeed express both Fas and FasL (Fig. 1 ).


Figure 1
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  		Figure 1. LNCaP cells coexpress both Fas and FasL. A, cell-surface expression of Fas was assessed by flow cytometric analyses of LNCaP cells stained with antihuman Fas DX-2 antibody (solid line; geometric mean fluorescence intensity = 24.80 ± 0.14) or stained with FITC-conjugated secondary antibody alone (dotted line; geometric mean fluorescence intensity = 3.03 ± 0.22). B, FasL expression on LNCaP cells was assessed by intracellular staining with an antihuman FasL antibody (Nok-1) followed by labeling with polyclonal anti-IgG-FITC secondary antibody (solid line; geometric mean fluorescence intensity = 9.60 ± 1.07) or staining with isotype-matched primary antibody followed by FITC-conjugated secondary antibody (dotted line; geometric mean fluorescence intensity = 3.97 ± 0.32). Representative of three independent experiments. Average geometric mean fluorescence intensities and SEs are presented in brackets.

 
Apoptosis induced by treatment of LNCaP cells with PI3K inhibitor LY294002 is FADD-dependent. To determine if the apoptotic response that is triggered by inhibition of the PI3K pathway is dependent on FADD, we examined whether expression of DN FADD could protect LNCaP cells from cell death induced by LY294002 treatment or by expression of WT PTEN. LNCaP cells were transiently cotransfected with a GFP expression vector and either an empty pcDNA control expression vector or pcDNA vector containing DN FADD. Transfected cells were then treated with 40 µmol/L LY294002 under low-serum (0.1%) conditions for a period of 24 hours. Apoptosis was monitored by flow cytometry using Annexin V-phycoerythrin. GFP-positive cells were gated and analyzed for phycoerythrin fluorescence. As shown in Fig. 1A, treatment of LNCaP cells with LY294002 resulted in the significant induction of apoptosis as determined by Annexin V–positive staining. Furthermore, expression of DN FADD was able to significantly inhibit LY294002-induced apoptosis. Next, we considered whether inhibition of PI3K by transfection of WT PTEN in LNCaP cells could be inhibited by expression of DN FADD. As shown in Fig. 2B , transfection of LNCaP cells with WT PTEN resulted in a significant induction of apoptosis as compared with pcDNA3 empty vector– or DN FADD–transfected cells. The degree of apoptosis induced by PTEN expression in LNCaP cells that we observe is consistent with that observed by others in previously published reports (28–31). Furthermore, cotransfection of cells with DN FADD and WT PTEN significantly inhibited PTEN-induced apoptosis. These data suggest that apoptosis induced by PI3K inhibition with LY294002 or WT PTEN expression in LNCaP cells is triggered in a FADD-dependent manner.


Figure 2
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  		Figure 2. DN FADD protects LNCaP cells from apoptosis triggered by PI3K blockade. A, LNCaP cells were transiently transfected using Lipofectin with AU1-tagged DN FADD or control empty pcDNA vector (gift from Claudius Vincenz) and treated in the presence or absence of 40 µmol/L LY294002 for 24 hours. Under all conditions, cells were cotransfected with a GFP reporter plasmid (pEGFP-C1, Clontech) to mark transfected cells. Apoptosis was determined by staining with phycoerythrin-conjugated Annexin V and analyzed by flow cytometry. GFP-positive cells were gated and analyzed for phycoerythrin fluorescence. Representative of at least three independent experiments. B, LNCaP cells were transfected with WT PTEN and DN FADD and cultured in 0.1% FBS containing serum for 72 hours before flow cytometric analyses of Annexin V staining. Cells were transfected with either empty pcDNA3, pcDNA3-DN FADD, or pcDNA3-WT PTEN expression vector alone as control. Average of triplicate measurements and representative of no fewer than three independent experiments showing similar results.

 
Apoptosis induced by LY294002 treatment in LNCaP cells is mediated by an autocrine Fas signal. To determine if apoptosis that is induced by blocking PI3K signaling in LNCaP cells is initiated by Fas/FasL signaling in an autocrine fashion, we treated LNCaP cells with 40 µmol/L LY294002 for 24 hours in 0.1% FBS–containing medium in the presence or absence of Fas:Fc (1 µg/mL) or Nok-1 (0.1, 1, and 2 µg/mL). Fas:Fc is a recombinant protein encoding the extracellular portion of Fas fused to the Fc portion of human IgG. Nok-1 is an antihuman FasL neutralizing antibody. Both of these reagents inhibit interactions between the Fas and FasL by binding and sequestering FasL. To control for the use of the Fas:Fc fusion protein and Nok-1 antibody, we used Thy1:Fc protein, which encodes the extracellular domain of the mouse Thy1 protein fused to the Fc portion of human IgG, and an isotype-matched antibody, respectively. Apoptosis was monitored by labeling with either Annexin V-biotin followed by streptavidin-phycoerythrin staining or by direct labeling with anti–active caspase-3-phycoerythrin and analyzed by flow cytometry.

LY294002 induced a significant increase in the proportion of Annexin V–positive cells (Fig. 3A ) and the percentage of cells exhibiting intracellular anti–active caspase-3 staining (Fig. 3B). There was no significant difference in the apoptotic response between cells treated with LY294002 alone and cells treated with LY294002 in the presence of either control molecule, Thy1:Fc, or the mouse IgG1 isotype-matched control antibody. Both Fas:Fc and Nok-1 were able to significantly suppress apoptosis caused by PI3K blockade. Furthermore, Nok-1 was able to suppress apoptosis in a dose-dependent manner.


Figure 3
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  		Figure 3. LY294002-induced apoptosis in LNCaP cells is inhibited by blocking Fas/FasL interactions. LNCaP cells were culture in 0.2% DMSO alone or in 40 µmol/L LY294002 in the presence of Thy1:Fc or Fas:Fc at 2.0 µg/mL each, or Nok-1 or isotype-matched control antibody at the indicated concentrations. Cell death was determined by flow cytometric analyses of cell-surface Annexin V-biotin/streptavidin-phycoerythrin labeling (A) and phycoerythrin-conjugated intracellular active caspase-3 staining (B). Average of triplicate measurements and representative of no fewer than three independent experiments showing similar results. **, P < 0.01; *, P < 0.05; n.s., not significant (P > 0.05), compared with LY294002 alone treatment (Student's t test). C, Fas and FasL expression in LNCaP cells grown in serum-free (SF), 10% charcoal-stripped serum (CSS), and 10% FBS conditions, and grown in 0.1% FBS in the presence of the indicated treatment conditions (LY, LY294002; No Tx, no treatment).

 
To determine whether the induction of apoptosis by LY294002 was due to changes in levels of expression of either the Fas or FasL, we examined the protein levels of both Fas and FasL in LNCaP cells (Fig. 3C) treated with LY294002 in the presence of Fas:Fc, Nok-1, and their respective control. Immunoblots show no marked change in Fas and FasL levels in the various medium conditions or in response to PI3K blockade and Fas:Fc or Nok-1 treatment. These data suggest that LY294002-induced apoptosis is likely not primarily caused by marked changes in Fas and FasL expression under the defined experimental conditions used.

Generation of PTEN Tet-ON inducible LNCaP cells. Expression of WT PTEN in LNCaP cells potently induces cell death (28–31). Generation of stable LNCaP cell lines that constitutively express PTEN is not possible without selecting for genetic alterations that confer resistance to PTEN-induced apoptosis (data not shown). Therefore, to determine the role of Fas in PTEN expression-induced apoptosis of LNCaP cells, we generated LNCaP cells that express WT PTEN under the control of a tetracycline inducible promoter (Clontech). To verify the expression and activity of PTEN, we analyzed the levels of PTEN protein, phosphorylation status of protein kinase B, as well as poly(ADP-ribose) polymerase cleavage in the presence and absence of 1 µg/mL of doxycycline by immunoblotting of total protein lysates derived from cells grown in three different medium conditions: serum-free, 10% charcoal-stripped serum, and 10% FBS (Fig. 4 ). A marked increase in PTEN expression was observed in all doxycycline-treated conditions. This was accompanied by a decrease in phospho-protein kinase B (Ser473) levels with the greatest decline occurring in serum-free medium. A large increase in cleaved poly(ADP-ribose) polymerase was observed in cells cultured in the serum-free medium in the presence of doxycycline. These data validate that the expression of PTEN is regulated in a tetracycline inducible manner in PTEN-Tet-ON LNCaP cells and show that, on induction with doxycycline, PTEN expression is able to cause apoptosis in these cells under low-serum (0.1% FBS) or serum-free conditions.


Figure 4
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  		Figure 4. Generation of PTEN Tet-ON inducible LNCaP cells. Cells were grown in serum-free, charcoal-stripped serum, and FBS conditions in the presence or absence of 1 µg/mL doxycycline. Changes in PTEN, Akt-P473, and cleaved poly(ADP-ribose) polymerase (PARP) were examined by Western blot analyses. Mitogen-activated protein kinase (MAPK) levels are shown as protein loading controls.

 
Apoptosis induced by expression of WT PTEN in LNCaP cells is mediated by an autocrine Fas death signal. PTEN Tet-ON LNCaP cells were grown for 24 hours in full serum with 1 µg/mL doxycycline to induce PTEN protein expression. Then, PTEN Tet-ON LNCaP cells were transferred to 0.1% FBS medium and cultured in the presence of 1 µg/mL doxycycline for 24 hours in the presence or absence of Fas:Fc (1 µg/mL) or Nok-1 (0.1, 1, and 2 µg/mL). Apoptosis was monitored by flow cytometric analyses of cell-surface Annexin V, or intracellular anti–cleaved caspase-3 antibody staining.

From the analyses of the apoptotic response induced by WT PTEN expression as detected by Annexin V staining (Fig. 5A ), we saw roughly a 25% increase in the proportion of cells that were apoptotic and a 12% to 15% increase using intracellular anti-caspase-3 staining (Fig. 5B). There was no difference between cells treated with doxycycline alone and cells cultured with doxycycline in the presence of either control molecule, Thy1:Fc, or the isotype-matched control antibody. Both Fas:Fc and Nok-1 were able to suppress apoptosis caused by PTEN expression. Moreover, Nok-1 was able to suppress apoptosis in a dose-dependent manner. Both Fas:Fc and Nok-1 completely suppressed apoptosis in the PTEN Tet-ON LNCaP cells to levels below those seen in untreated cells.


Figure 5
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  		Figure 5. PTEN-induced apoptosis of LNCaP cells is inhibited by abrogating Fas/FasL interactions. PTEN Tet-ON LNCaP cells were cultured for 72 hours in the presence or absence of 1 µg/mL doxycycline and 2.0 µg/mL Fas:Fc, 2.0 µg/mL Thy1:Fc, or Nok-1 or isotype-matched control antibodies at the indicated concentrations, and cell death was determined by flow cytometric analyses of cell-surface Annexin V-biotin/streptavidin-phycoerythrin labeling (A) and phycoerythrin-conjugated intracellular active caspase-3 staining (B). Average of triplicate measurements and representative of no fewer than three independent experiments showing similar results. **, P < 0.01; n.s., not significant (P > 0.05), compared with treatment with doxycycline alone (Student's t test). C, Fas and FasL expression in PTEN Tet-ON cells grown in serum-free, 10% charcoal-stripped serum, and 10% FBS conditions, and grown in 0.1% FBS in the presence or absence of 1 µg/mL doxycycline under the indicated conditions.

 
As shown in Fig. 5C, no overt changes in Fas and FasL levels were observed in Tet-ON PTEN LNCaP cells cultured in varying medium conditions or in response to doxycycline-induced PTEN expression and Fas:Fc or Nok-1 treatment, suggesting that changes in Fas or FasL expression likely do not play a major role in PTEN expression–induced apoptosis of LNCaP cells under these experimental conditions.

Apoptosis induced by blockade of PI3K signaling in PC3 cells is mediated by an autocrine Fas signal. To determine if blockade of PI3K signaling in a second PTEN-deficient prostate cancer cell line, PC3, also induced autocrine Fas-mediated cell death, PC3 cells were treated with LY294002 in the presence of Fas:Fc (2 µg/mL) or Thy1:Fc as above. Cells were treated for 48 hours in serum-free medium. Apoptosis was monitored by staining with Annexin V-biotin followed by phycoerythrin-streptavidin. Cells were then analyzed by flow cytometry. We saw a 15% increase in the proportion of cells that were apoptotic (Fig. 6A ). There was no difference between cells treated with LY294002 alone and those also given the control molecule Thy1:Fc. Fas:Fc was able to completely suppress apoptosis induced by PI3K signal blockade. Consistent with previous reports (20), Fig. 6B and C shows that PC3 cells express both Fas and FasL.


Figure 6
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  		Figure 6. PI3K inhibitor LY294002-induced apoptosis of PTEN-deficient PC3 prostate cancer cells is mediated by an autocrine Fas death signal. A, PC3 cells were cultured in the presence of 0.2% DMSO alone or 40 µmol/L LY294002 plus Thy1:Fc or Fas:Fc at 2 µg/mL each. Apoptosis was determined by flow cytometric analyses of Annexin V-biotin/streptavidin-phycoerythrin staining. Average of triplicate measurements and representative of no fewer than three independent experiments showing similar results. **, P < 0.01; n.s., not significant (P > 0.05), compared with LY294002 alone treatment (Student's t test). B, cell-surface expression of Fas was assessed by staining with antihuman Fas (DX-2) followed by labeling with FITC-conjugated antimouse IgG secondary antibody (solid line; geometric mean fluorescence intensity = 26.28 ± 0.16) or by staining with secondary antibody alone (dotted line; geometric mean fluorescence intensity = 2.86 ± 0.09). C, FasL expression on PC3 cells was assessed by intracellular staining with antihuman FasL antibody (Nok-1) followed by labeling with anti-IgG-FITC secondary antibody (solid line; geometric mean fluorescence intensity = 6.57 ± 0.28) or by staining with secondary antibody alone (dotted line; geometric mean fluorescence intensity = 2.33 ± 0.05). Representative of three independent experiments. Average geometric mean fluorescence intensities and SEs are presented in brackets.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The PI3K pathway has long been known to be important in promoting cancer cell survival and proliferation (1). PTEN-deficient tumors are exquisitely dependent on continuous PI3K signaling for their growth and survival and exhibit enhanced susceptibility to inhibition of PI3K pathway compared with cells expressing WT PTEN (32–35). Hence, considerable attention has been devoted toward development of novel therapeutics targeting this pathway in treatment of a wide variety of cancers including prostate cancer (15–17). Inhibitors of the PI3K pathway show particular promise for treatment of PTEN-deficient cancers. An understanding of the mechanism by which these agents induce cell death will provide better appreciation for strategies by which these drugs might be most effectively used in the clinic as single-agent therapies or in combination with other therapeutic agents.

Attenuation of signaling through the PI3K pathway in PTEN-deficient prostate cancer cells by pharmacologic PI3K inhibitors triggers a rapid and extensive apoptotic response when grown without androgens or growth factors under low-serum or serum-free conditions (13, 14). It is commonly accepted that this apoptotic response is mediated by loss of mitochondrial membrane integrity, release of cytochrome c, and apoptosome-mediated activation of caspase-3 (12, 13). Whereas the precise mechanism initiating this event has not been directly addressed, these observations have implied that PI3K inhibition–induced apoptosis occurs via activation of the intrinsic mitochondrial death pathway. However, the relative contribution of the extrinsic death pathway in mediating apoptosis has previously been unappreciated.

Here we show that the extrinsic death pathway is a significant contributor in PI3K blockade–induced death. Consistent with previous reports (19, 20), we have confirmed that LNCaP and PC3 indeed coexpress both Fas and FasL. Moreover, it has also been shown that PC3 and LNCaP constitutively secrete biologically active soluble FasL in vitro. We have found that cell death induced by PI3K inhibition or by expression of WT PTEN can be significantly attenuated by expression of DN FADD or by exogenous treatment with Fas:Fc protein or neutralizing FasL antibody. We now show herein that cytotoxicity due to PI3K inhibition is mediated through activation of the extrinsic death pathway via Fas-induced apoptosis.

The term autocrine denotes a mode of hormone action in which a hormone/factor binds to receptors and affects the function of the cell type that produced it. The ability of exogenously applied Fas:Fc and neutralizing FasL antibody to block PI3K inhibition–induced cell death suggests that apoptosis occurs in an autocrine fashion. Thus, collectively, our data imply that PI3K blockade–induced cell death occurs in an autocrine manner through the action of sFasL secreted by the cell (25) acting on itself and neighboring like cells and/or through the action of Fas/FasL interactions on the cell surface of same cell or between like cells.

Normal prostatic epithelial cells express both Fas and FasL, and this feature has been proposed to contribute to the immune privileged status of the normal prostate (36, 37). In prostate cancer, the coexpression of both Fas and FasL is commonly maintained, and yet prostate cancer cells remain resistant to autocrine Fas-induced cell death (25, 38, 39). The mechanism by which these cells maintain resistance to Fas is unclear. A plethora of signaling molecules have been shown to regulate Fas-induced cell death and have thus been implicated in potentially mediating Fas resistance including PTEN, PI3K, and protein kinase B, among many others (10, 11, 18, 40). Our findings suggest that inhibition of the PI3K pathway results in reactivation of the apoptotic potential of Fas.

The PI3K/PTEN pathway has been shown to be capable of regulating Fas-induced apoptosis (10, 11). Pten haploinsufficiency leads to impaired Fas-induced cell death and results in autoimmune lymphoproliferative disease that mimics that seen in Fas- and FasL-deficient mice (11). Because PI3K blockade is able to reactivate the death potential of Fas in prostate cancer cells, our data suggest that Fas resistance in PTEN-deficient prostate cancer cells is primarily imparted by activation of the PI3K pathway. Furthermore, our data imply that prostate cancer cells retain the intrinsic capacity to undergo cell death in an autocrine Fas-mediated manner and that prostate cancer cells are chronically attempting to commit suicide through an autocrine Fas-induced apoptotic mechanism and sustained PI3K signals, as a result of PTEN deficiency, are required to protect cells from autocrine cell death.

Previous studies have shown that LNCaP cells are resistant to Fas-mediated apoptosis in response to stimulation with exogenously added FasL or agonistic anti-Fas antibodies (21–23, 26). However, overexpression of high levels of endogenous FasL in LNCaP cells was able to overcome protection from Fas-induced apoptosis (26), suggesting that Fas-mediated apoptosis can be triggered beyond a set threshold of stimulation. Based on our data, blockade of PI3K is capable of enhancing the sensitivity of LNCaP and PC3 cells to Fas-induced apoptosis and effectively lower the threshold of Fas stimulation that is required to induce apoptosis, such that the low levels of endogenous FasL expression in LNCaP and PC3 cells are sufficient to trigger Fas-induced apoptosis.

Under our experimental conditions, we have found that low-serum or serum-free conditions are necessary to permit autocrine Fas-mediated apoptosis induced by PI3K inhibition or WT PTEN expression to occur (data not shown) presumably because additional signals that are activated by serum derived factors may attenuate Fas-induced apoptosis in a PI3K-independent manner. Consistent with this hypothesis, PI3K-independent signals have been shown to protect from LY294002-induced cell death of LNCaP cells (12, 13). Thus, these data suggest that other autocrine, paracrine, and/or endocrine growth factor– or hormone-derived PI3K-independent signals may regulate apoptotic responsiveness of PTEN-deficient prostate cancer cells in vivo. Thus, from a clinical perspective, our findings suggest that PI3K blockade may be potentially effective antineoplastic agents in treating PTEN-deficient prostate cancer but these therapeutic strategies that incorporate PI3K blockade may require combination therapy to simultaneously inhibit growth factor– (such as insulin-like growth factor or epidermal growth factor) and/or hormone-derived PI3K-independent signals that mediate cytoprotection from Fas-induced cell death.

The coexpression of Fas and FasL is a common feature of a wide variety of cancers (41). In Hodgkin/Reed-Sternberg cells, c-FLIP has been shown to mediate resistance to autocrine Fas-induced apoptosis (42, 43). Intriguingly, PI3K/Akt has been shown to regulate c-FLIP expression in cancer cells including prostate cancer cells (44), and PI3K-dependent protein kinase B activation has been implicated in impairing Fas signaling by suppressing death-inducing signaling complex formation and caspase-8 activation (9, 10, 45–47). Thus, the constitutively activated PI3K pathway in PTEN-deficient cancer cells may confer resistance to autocrine Fas-induced apoptosis by the ability of protein kinase B to impair death-inducing signaling complex formation and caspase-8 activation via, in part, the regulation of FLIP expression. However, a complete understanding of the mechanism(s) by which prostate cancer cells might mediate protection from autocrine Fas-induced apoptosis through the direct or indirect actions of PI3K/PTEN regulated pathways remains to be established.

In summary, blockade of the PI3K pathway causes an apoptotic response in PTEN-deficient prostate cancer cells by some previously unknown mechanism. Here, we show that the mechanism of action of these agents is to trigger an apoptotic response via reactivation of a latent autocrine Fas death pathway.


   Acknowledgments
 
Grant support: Health Canada.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: J. Bertram and J.W. Peacock contributed equally to this work.

Received 9/21/05. Revised 1/20/06. Accepted 3/ 3/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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