Androgens repress FKHR activity in prostate cancer cells in an androgen receptor- and dose-dependent manner.
To investigate the possibility that androgen-mediated prostate cancer cell survival involves the suppression of FKHR function, PTEN-intact and androgen receptor-negative DU145 cells were transfected with wild-type and mutant FKHR(AAA), in which all three AKT phosphorylation sites were mutated to alanines (14
). FKHR transcriptional activity was determined by cotransfection with a reporter gene containing copies of the IGFBP-1 insulin response sequence immediately upstream of the minimal thymidine kinase promoter (14
As shown in Fig. , IGF-1 inhibits transcriptional activity of the wild-type FKHR but not the AAA mutant, indicating that phosphorylation of these sites is critical for the ability of growth factors to suppress transactivation by FKHR, consistent with previous reports (37
). In cells transfected with the androgen receptor, treatment with R1881, a synthetic androgen receptor agonist, represses the activity of both the wild-type FKHR and the AAA mutant (Fig. ), indicating that repression of transactivation of FKHR by androgens occurs independently of FKHR phosphorylation by AKT and that androgens are more powerful repressors of FKHR than growth factors. R1881 does not affect the FKHR activity in the absence of the androgen receptor, and the expression of the androgen receptor does not have an effect on the FKHR activity in the absence of R1881, demonstrating that the FKHR inhibition is androgen receptor dependent and only occurs if androgen receptor is activated. Activated androgen receptor also decreases the reporter activity in the absence of ectopic FKHR (Fig. ), suggesting that, when activated, androgen receptor may also inhibit the activity of endogenous forkhead proteins in DU145 cells. Overall, the activity in Fig. is lower than in Fig. , presumably because the vehicle is ethanol for R1881 and medium for IGF-1.
FIG. 1. Inhibition of FKHR reporter activity by activated androgen receptor. (A) Inhibition of wild-type FKHR by IGF-1. DU145 cells were transfected with 0.5 μg of 3×IRS-Luc, 0.2 μg of pCMVβ, 0.1 μg of FKHR:WT (WT) or FKHR:TSS (more ...)
To directly demonstrate the dosage dependency, the activities of wild-type FKHR (Fig. ) and the AAA mutant (Fig. ) were determined in DU145 cells transfected with various amounts of androgen receptor vector. Although in the absence of androgens, the androgen receptor expressed at different levels has little effect on FKHR activity, in the presence of R1881, it decreases the activities of both wild-type FKHR and the AAA mutant in a dosage-dependent manner.
To determine whether the decrease in FKHR activity is due to a decrease in the level of FKHR protein, the level of FKHR and androgen receptor proteins was examined by immunoblotting under the same conditions that the reporter activity was determined. As shown in Fig. , R1881 treatment of DU145 cells expressing different amounts of androgen receptor protein does not decrease the level of FKHR protein, suggesting that activated androgen receptor decreases the specific activity of FKHR.
To make sure that the decreased reporter activity truly reflects inhibition of FKHR, we next tested the effect of activated androgen receptor on the FKHR reporter in LNCaP cells without FKHR cotransfection. LNCaP cells contain no functional PTEN (42
). AKT is constitutively active in these cells, which would phosphorylate endogenous FKHR, resulting in its cytoplasmic localization. As a consequence of the AKT phosphorylation, LNCaP cells contain little transcriptionally active FKHR in the nucleus. As shown in Fig. , activated androgen receptor does not inhibit the basal activity of the FKHR reporter in LNCaP cells, demonstrating that the decreased reporter activity in DU145 cells without FKHR transfection must be due to the repression of endogenous activity by activated androgen receptor.
FIG. 2. Androgen inhibition of FKHR activity mediated through endogenous androgen receptor. (A) Lack of an androgen effect on basal FKHR reporter activity in LNCaP cells. Cells were transfected with 0.1 μg of pCMVhAR, 0.2 μg of pCMVβ and (more ...)
Wild-type FKHR introduced into LNCaP cells contains little activity (Fig. ), which is expected because the activity of wild-type FKHR, similar to the endogenous FKHR activity, should be suppressed by the AKT phosphorylation. Different from wild-type FKHR, the AAA mutant is active in the cells and its activity is inhibited by R1881 through endogenous androgen receptor (Fig. ), showing that FKHR inhibition is not limited to ectopic androgen receptor introduced into androgen receptor-negative cells. The transfection of additional androgen receptor enhances the androgen inhibition, confirming the data from DU145 cells that the degree of inhibition depends on the amount of androgen receptor in the cells. Similar to the data in Fig. , androgen treatment does not decrease the level of FKHR protein expression (Fig. ), demonstrating that R1881, acting through the endogenous androgen receptor, decreased the specific activity of the AAA mutant.
To determine whether androgens also repress the activity of other mammalian homologues of Daf-16, the effect of activated androgen receptor on FKHRL1 was determined in DU145 cells. As shown in Fig. , activated androgen receptor inhibits the activity of FKHRL1 on the reporter constructed with the promoter of the Fas ligand gene (4
), albeit to a lesser degree than FKHR (Fig. ). To test whether FKHRL1 activity is also inhibited by androgens through endogenous androgen receptor, the activity of a mutant FKHRL1, similar to the AAA of FKHR, was analyzed in LNCaP cells treated or not with R1881, As shown in Fig. , the mutant FKHRL1 is again inhibited by R1881 treatment. The data suggest that androgen inhibition is not limited to FKHR and that androgens might be general suppressors of FOXO forkhead factors.
FIG. 3. Inhibition of FKHRL1 activity by activated androgen receptor. (A) Inhibition of FKHRL1 activity by androgens in DU145 cells. Cells were transfected with 0.2 μg of pCMVhAR, 0.2 μg of pCMVβ, 0.5 μg of FHRE-Luc, and 0.1 μg (more ...) PTEN reexpression in PTEN-null prostate cancer cells increases FKHR activity that is suppressed by activated androgen receptor.
As shown in our earlier studies (30
), PTEN represses the transcriptional activity of the androgen receptor but is unable to override the protective effect of androgens on PTEN-induced apoptosis. It is thus possible that androgens protect prostate cancer cells from PTEN-induced death through inhibition of FKHR. If so, androgen repression of FKHR should occur despite the PTEN repression of androgen receptor transcriptional activity. Thus, we assayed the androgen repression of FKHR in PTEN-null PC3 cells transfected with wild-type PTEN.
As shown in Fig. , FKHR activity in PC3 cells is increased by wild-type PTEN in comparison to an inactive PTEN mutant, and the increase is repressed by R1881. As expected, PTEN and androgen receptor vectors express the corresponding proteins in the cells (Fig. ). The data suggest that the repression of FKHR by activated androgen receptor may contribute to the androgen receptor-mediated androgen protection of prostate cancer cells from PTEN-induced apoptosis.
FIG. 4. Activated androgen receptor repressed PTEN-induced FKHR activity. (A) Inhibition of PTEN-induced FKHR activity by activated androgen receptor (AR). PC3 cells were transfected with 0.2 μg of pCMVhAR, 0.2 μg of pCMVβ, 0.5 μg (more ...) Androgen repression of FKHR is not due to competition for transcriptional coactivators.
Competition for the limited amounts of transcriptional coactivators in cells has been held accountable for the inhibition between AP-1 and steroid receptors (24
) as well as among steroid receptors (40
). To test whether the FKHR repression by activated androgen receptor is due to competition for coactivators, DU145 cells were transfected with various amounts of coactivators, and the repression of FKHR by activated androgen receptor was determined. As shown in Fig. , expression of the coactivators at different dosages does not relieve the FKHR repression. As controls, the transcriptional activity of ERα (Fig. ) is increased by the increased dosages of the coactivators. In addition, different amounts of activated ERβ do not affect FKHR activity (Fig. ). Since steroid receptors share coactivators, activated ERβ would also be expected to repress FKHR activity if the repression were due to the competition for coactivators. Overall, the data in Fig. indicate that the FKHR repression by androgens is not due to competition between the activated androgen receptor and FKHR for coactivators.
FIG. 5. Lack of effect of coactivator expression on inhibition of FKHR by androgens and differential effect of activated ERα and ERβ on FKHR activity. (A) Lack of an effect of coactivator expression on the inhibition of FKHR by activated androgen (more ...)
Different from ERβ but similar to androgen receptor, ERα represses FKHR in the presence but not in the absence of 17β-estradiol (Fig. ), suggesting that FKHR repression is not limited to the androgen receptor. The data are consistent with two recent studies that identified FKHR as an ERα-interacting protein in yeast two-hybrid screening and in cells (43
Androgen receptor forms a complex with FKHR.
Since the repression of FKHR by androgens does not appear to be due to competition for coactivators, we next tested the possibility that activated androgen receptor represses FKHR through protein-protein interaction. DU145 cells were transfected with Flag-FKHR and androgen receptor vectors, and complex formation was determined by coimmunoprecipitation with M2 anti-Flag antibody. As shown in Fig. , the androgen receptor is coimmunoprecipitated with FKHR only in cells transfected with both FKHR and the androgen receptor, and the coprecipitation is enhanced by R1881 (Fig. , lanes 1 and 2). Without Flag-FKHR, the M2 anti-Flag antibody does not bring down androgen receptor in either the presence (Fig. , lane 3) or the absence of R1881 (Fig. , lane 4), demonstrating that there is no cross-reactivity between androgen receptor and M2 antibody.
FIG. 6. Complex formation between FKHR and the androgen receptor in prostate cancer cells and in vitro. (A) Coimmunoprecipitation (IP) of FKHR and the androgen receptor (AR). DU145 cells were transfected with 0.5 μg of pCMVhAR, 0.5 μg of FKHR:WT (more ...)
In cells transfected only with Flag-FKHR, no androgen receptor is detected in the precipitates (Fig. , lanes 5 and 6), showing that the band detected in lane 1 is indeed androgen receptor. Since androgens are known to increase the stability of androgen receptor protein, which is confirmed under our conditions for R1881 (Fig. , lanes 1 and 2 of the bottom panel), the increased amount of androgen receptor protein in the immunoprecipitates may be due simply to the higher amount of total androgen receptor protein in R1881-treated cells. Based on our estimation of the enhanced chemiluminescence signals on the immunoblots, the amount of the androgen receptor in the immunoprecipitates, after normalization to the total androgen receptor protein, is induced by R1881 about fourfold. This experiment shows that the androgen receptor and FKHR coexist in a protein complex in prostate cancer cells and that complex formation is increased by R1881.
To determine whether the androgen receptor and FKHR interact in vitro, GST fusions with the androgen receptor amino-terminal A/B region (GST-AR-NT), the DNA binding domain (GST-AR-DBD), or the ligand binding domain (GST-AR-LBD) were produced in and purified from bacteria. These fusion proteins were used to precipitate 35S-labeled FKHR protein produced by in vitro transcription-translation reactions. In these so-called GST pulldown assays, FKHR coprecipitates with GST-AR-NT (Fig. , lane 2) but not with GST-DBD (Fig. , lane 5). In addition, the coprecipitation could be detected with GST-AR-LBD in the presence (Fig, 6B, lane 4) but not in the absence of R1881 (Fig. , lane 3). This demonstrates that androgen receptor interacts with FKHR through both the NT region and the ligand binding domain (Fig. ). No FKHR was detected in GST precipitates (Fig. , lane 6), showing the specificity of FKHR coprecipitation with the androgen receptor fragments.
Compared to the input (Fig. , lane 1), the NT region showed a much stronger interaction with FKHR than the liganded ligand binding domain. Coomassie blue staining shows that the amounts of GST and GST-AR-DBD used in the pulldown experiment are not lower than the amounts of the GST-AR-NT or GST-AR-LBD constructs and that the amount of GST-AR-LBD used for the pulldown assays performed in the presence or absence of R1881 is comparable (Fig. , lower panel), eliminating the possibility that the difference in the FKHR signal in the pulldown assays is due to different amounts of GST protein used for the precipitations. Multiple bands smaller than the predicted size of the fusion protein are present in the GST-AR-NT and GST-AR-LBD lanes. Presumably, these are the products of partial degradation of the full-length GST fusion proteins. It is known that androgen receptor expressed in bacteria as GST fusion proteins is not stable, and so far nobody has succeeded in generating a GST fusion protein with a full-length androgen receptor.
To determine which region of FKHR interacts with the androgen receptor, we transfected DU145 cells with androgen receptor and FKHR fragments fused to the Gal4 DNA binding domain and determined the binding in coimmunoprecipitation assays with an anti-Gal4 antibody. As shown in Fig. , no interaction with the androgen receptor is detected with Gal4-FKHR(1-150) in either the absence or the presence of R1881, whereas interaction with the androgen receptor is detected with both Gal4-FKHR(211-655) and Gal4-FKHR(350-655) in the presence but not the absence of R1881. As controls, Gal4-FKHR proteins are detected in the immunoprecipitates (Fig. , middle panel), demonstrating that the lack of androgen receptor interaction with Gal4-FKHR fusions in the absence of R1881 as well as with Gal4-FKHR(1-150) in the presence of R1881 is not due to insufficient amounts of Gal4-FKHR protein expression.
FIG. 7. Mapping the androgen receptor-interacting domain of FKHR by coimmunoprecipitation. DU145 cells were transfected with 0.5 μg of pCMVhAR, 0.5 μg of Gal4-FKHR, and 0.2 μg of pCMVβ and treated with R1881 or ethanol (EOH). Immunoprecipitates (more ...)
Based on the estimation of the enhanced chemiluminescence signals on the immunoblots, the amount of the androgen receptor in the immunoprecipitates, after normalization to the amount of Gal4-FKHR fusion proteins, is increased by R1881 more than 10-fold (Fig. , bottom panel). Although the quantitative data shown in Fig. , bottom panel, suggest an androgen induction of complex formation between the androgen receptor and FKHR, the data do not exclude the possibility that the increased complex formation detected in the presence of androgens is the result of androgen-induced accumulation of both androgen receptor and FKHR to a higher level in the cells.
Activated androgen receptor inhibits the ability of FKHR to bind DNA.
Since the androgen receptor and FKHR exist in the same protein complex and activated androgen receptor represses FKHR activity, we performed electrophoretic mobility shift assays to test whether activated androgen receptor inhibits the ability of FKHR to bind DNA. A synthetic oligonucleotide probe (8
) containing an insulin response sequence from the IGFBP-1 promoter was used in the electrophoretic mobility shift assay. As shown in Fig. , neither androgen receptor expression nor t treatment with R1881 decreased the level of FKHR expression, consistent with data presented earlier (Fig. and ). Compared to the control (Fig. , lane 2), androgen receptor coexpression does not decrease FKHR DNA binding in the absence of R1881 (Fig. , lane 3, and 8C, lane 9), whereas, after R1881 treatment, the androgen receptor blocks the formation of FKHR-DNA complex (Fig. , lane 4, and 8C, lane 10).
FIG. 8. Androgen receptor-dependent inhibition of FKHR DNA binding by androgens. (A) Detection of transfected Flag-FKHR by immunoblotting. DU145 cells in 100-mm dishes were transfected with 2.5 μg of Flag-tagged FKHR or empty vector with or without 2.5 (more ...)
Interestingly, the unliganded androgen receptor appears to enhance the FKHR binding (Fig. , lane 3, and 8C, lane 9), for which the mechanism is unclear. The specificity of the FKHR DNA complex is demonstrated by the inhibition of complex formation with the M2 anti-Flag antibody (Fig. , lane 2b, and 8C, lane 2), but not with 12CA5 antihemagglutinin (anti-HA) antibody (Fig. , lane 1). Further analysis shows that excess amounts of nonradiolabeled wild-type insulin response sequence (Fig. , lanes 5 and 6), but not that of a mutant insulin response sequence (Fig. , lanes 7 and 8), inhibit the complex formation, confirming the specificity of the FKHR complex. Although the activity of endogenous FKHR is detected in DU145 cells (Fig. ), no specific DNA binding is observed with nuclear extracts from DU145 cells transfected with control vector (Fig. , lane 1, and Fig. , lane 3), presumably due to the limited sensitivity of electrophoretic mobility shift assays.
Activated androgen receptor suppresses FKHR-induced Fas ligand expression.
Since activated androgen receptor inhibited the transcriptional activity of FKHR as well as its ability to bind DNA, it is reasonable to expect that the activated androgen receptor would suppress the expression of FKHR target genes such as Fas ligand. To test this possibility, DU145 cells were transfected with the mutant FKHR and an expression vector for a cell surface marker, CD20, with or without the androgen receptor. CD20-positive cells were separated from CD20-negative cells by flow cytometry-based sorting, and Fas ligand expression on the surface of the CD20-positive cells was analyzed. As shown in Fig. and B, the expression of AAA in DU145 cells increases Fas ligand expression on the cell surface about fourfold, and the increase is suppressed by R1881 in the presence of androgen receptor. In the absence of AAA expression, androgens and androgen receptor, alone or in combination, have little effect on the basal level of Fas ligand on the cell surface. Similar analysis in LNCaP cells shows that the endogenous androgen receptor is sufficient to mediate the androgen inhibition of FKHR-induced Fas ligand expression (Fig. ).
FIG. 9. Inhibition of FKHR-induced cell surface Fas ligand expression by activated androgen receptor. (A) Determination of cell surface Fas ligand expression by flow cytometry. DU145 cells were transfected with 3.0 μg of pCMVCD20 and 2.5 μg of (more ...) Activated androgen receptor impairs the ability of FKHR to induce prostate cancer cell apoptosis and cell cycle arrest.
Up to this point, our investigation has provided strong evidence that activated androgen receptor binds and inhibits FKHR transcriptional activity in prostate cancer cells as well as the ability of FKHR to bind DNA and induce target gene expression. To determine the effect of the inhibition on the biological activity of FKHR in prostate cancer cells, FKHR-induced cell apoptosis was analyzed in the presence and absence of R1881. LNCaP cells were transfected with wild-type FKHR or the AAA mutant together with a GFP expression vector, and the survival of transfected cells was examined. As shown in Fig. , expression of the AAA mutant decreases LNCaP cell survival by fourfold, whereas the expression of wild-type FKHR has little effect. The decrease in cell survival induced by the AAA mutant is prevented by R1881, showing that the endogenous androgen receptor is sufficient to mediate the androgen protection.
FIG. 10. Androgen protection of prostate cancer cells from FKHR-induced cell death. (A) Androgen effect on the viability of FKHR-transfected LNCaP cells. Cells were transfected with 0.5 μg of pLNCE and 0.1 μg of FKHR:WT (WT) or FKHR:TSS (AAA) and (more ...)
To show that the decreased prostate cancer cell survival induced by FKHR is due to apoptosis, LNCaP cells transfected with the AAA mutant and GFP vectors were stained with DAPI and examined for features of cell apoptosis under a fluorescence microscope. As shown in representative micrographs in Fig. , two of eight of the transfected cells are going through apoptosis, while none of the surrounding nontransfected cells are dying of apoptosis. After scoring 300 cells, the results suggest that the AAA mutant increases the apoptotic index of LNCaP cells by fourfold and that R1881 treatment restores the index to the basal level (Fig. ). In a parallel analysis, wild-type FKHR causes only a marginal increase in the apoptotic index, which is nevertheless also restored to the basal level by R1881. Consistent with the fact that PTEN is intact in DU145 cells, wild-type FKHR, like the AAA mutant, decreased the survival of DU145 cells (Fig. ). R1881 treatment restores the cell survival to the control level in cells cotransfected with the androgen receptor but showed no effect in the absence of androgen receptor, suggesting that the protection of prostate cancer cells from FKHR-induced death by androgens is androgen receptor dependent. By varying the amount of androgen receptor introduced into the cells, it is obvious that the ability of the androgen receptor to mediate the protection is dose dependent (Fig. ).
To extend the functional analysis beyond cell death, the effect of activated androgen receptor on the regulation of DU145 cell cycle progression by FKHR was determined with a strategy similar to what we have used for the Fas ligand study. DU145 cells were transfected with AAA, CD20, and androgen receptor, and cell cycle distribution of CD20-positive cells was analyzed by flow cytometry. As shown in Fig. , the expression of AAA in DU145 cells decreases the percentage of cells in S phase and increases the percentage in G1 phase about 15%. The effect of FKHR on both G1/G0 and S phases is suppressed by R1881 treatment. Similar analysis in LNCaP cells shows that the endogenous androgen receptor is sufficient to mediate the androgen suppression of FKHR-induced alteration in cell cycle progression, with the exceptions that FKHR induces the accumulation of LNCaP cells in G2/M instead of G1/G0 phase and that the effect of FKHR on LNCaP cell cycle progression is relatively minor (about 5%) (data not shown).
FIG. 11. Inhibition of FKHR-induced cell cycle arrest by activated androgen receptor. (A) Flow cytometry profile of CD20-positive DU145 cells. DU145 cells were transfected with 3.0 μg of pCMVCD20, 2.5 μg of FKHR TSS (AAA), and 2.5 μg of (more ...)