doi: 10.1128/MCB.23.1.104-118.2003.
AKT-independent protection of prostate cancer cells from apoptosis mediated through complex formation between the androgen receptor and FKHR
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- PMID:12482965
- PMCID: PMC140652
- DOI: 10.1128/MCB.23.1.104-118.2003
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AKT-independent protection of prostate cancer cells from apoptosis mediated through complex formation between the androgen receptor and FKHR
Pengfei Li et al. Mol Cell Biol.2003 Jan.
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Abstract
Recent studies suggested that the protection of cell apoptosis by AKT involves phosphorylation and inhibition of FKHR and related FOXO forkhead transcription factors and that androgens provide an AKT-independent cell survival signal in prostate cancer cells. Here, we report receptor-dependent repression of FKHR function by androgens in prostate cancer cells. Transcriptional analysis demonstrated that activation of the androgen receptor caused an inhibition of both wild-type FKHR and a mutant in which all three known AKT sites were mutated to alanines, showing that the repression is AKT independent. In vivo and in vitro coprecipitation studies demonstrated that the repression is mediated through protein-protein interaction between FKHR and the androgen receptor. Mapping analysis localized the interacting domains to the carboxyl terminus between amino acids 350 and 655 of FKHR and to the amino-terminal A/B region and the ligand binding domain of the receptor. Further analysis demonstrated that the activated androgen receptor blocked FKHR's DNA binding activity and impaired its ability to induce Fas ligand expression and prostate cancer cell apoptosis and cell cycle arrest. These studies identify a new mechanism for androgen-mediated prostate cancer cell survival that appears to be independent of the activity of the receptor on androgen response element-mediated transcription and establish FKHR and related FOXO forkhead proteins as important nuclear targets for both AKT-dependent and -independent survival signals in prostate cancer cells.
Figures
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 (AAA). Transfected cells were treated for 24 h or not with 50 ng of IGF-1 per ml as indicated. Luciferase activity was determined and normalized to cognate β-galactosidase activity. FKHR activity was expressed as relative luciferase units (RLU) after normalization. Duplicate samples were analyzed for each data point, and the data have been reproduced three times. (B) Androgen receptor (AR)-dependent inhibition of wild-type and AAA mutant FKHR by androgens. DU145 cells were transfected with 0.2 μg of pCMVhAR, 0.2 μg of pCMVβ, 0.5 μg of 3×IRS-Luc, and 0.1 μg of FKHR:WT (WT) or FKHR:TSS (AAA). Transfected cells were treated for 24 h with 10−8 M R1881 or ethanol (EOH) as vehicle controls. FKHR activity was determined and expressed as for panel A. (C) Dosage-dependent inhibition of wild-type FKHR by activated androgen receptor (AR). DU145 cells were transfected with 0.5 μg of 3×IRS-Luc, 0.2 μg of pCMVβ, 0.1 μg of FKHR:WT, and the indicated amounts of pCMVhAR. Transfected cells were treated and FKHR activity was determined as for panel B. (D) Dosage-dependent inhibition of the AAA mutant by activated androgen receptor (AR). DU145 cells were transfected as in panel C except that 0.1 μg of FKHR:TSS was used in place of FKHR:WT. Transfected cells were treated and FKHR activity was determined as for panel B. (E) No decrease at the level of FKHR protein after androgen treatment. DU145 cells were transfected and treated as for panels C and D. Androgen receptor (AR) and FKHR proteins were detected by Western blotting with PG-21 anti-androgen receptor (Upstate) and M2 anti-Flag (Sigma) antibodies, respectively. Equal amounts of protein were loaded. W, wild-type FKHR; M, AAA mutant.
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 0.5 μg of 3×IRS-Luc. Transfected cells were treated and FKHR activity was determined as in Fig. 1B. (B) Androgen inhibition of FKHR activity mediated through endogenous androgen receptor (AR). LNCaP cells were transfected as in A but with 0.1 μg of FKHR:WT (WT) or FKHR:TSS (AAA). Transfected cells were treated and FKHR activity was determined as for Fig. 1B. (C) No decrease at the level of FKHR protein in LNCaP cells after androgen treatment. LNCaP cells were transfected and treated as for panel B. DU145 cells transfected with or without Flag-tagged FKHR were included as controls. Note that there is a background band recognized by the M2 antibody which moves slightly faster than Flag-FKHR in the gel. Androgen receptor (AR), FKHR, and β-actin proteins were detected by Western blotting with the PG-21, M2, and anti-β-actin monoclonal (Sigma) antibodies, respectively. Equal amounts of protein were loaded.
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 of HA-FKHRL1-WT. Transfected cells were treated and FKHRL1 activity was determined as for Fig. 1B. (B) Inhibition of FKHRL1 activity by androgens through endogenous androgen receptor (AR) in LNCaP cells. Cells were transfected as for panel A except that HA-FKHRL1-MT was used in the place of HA-FKHRL1-WT and no pCMVhAR was included in the transfection. Transfected cells were treated and FKHRL1 activity was determined as for Fig. 1B.
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 of 3×IRS-Luc, 0.1 μg of pSG5L-HA-PTEN:WT (WT) or pSG5L-HA-PTEN:G129R (MT), and 0.1 μg of FKHR:WT (WT). Transfected cells were treated and FKHR activity was determined as for Fig. 1B. (B) Expression of PTEN and androgen receptor (AR) in PC3 cells. Cells were transfected and treated as for panel A. PTEN and androgen receptor proteins were detected by Western blotting with anti-PTEN (Upstate), PG-21 anti-androgen receptor, and anti-β-actin antibodies.
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 receptor (AR). DU145 cells were transfected with 0.2 μg of pCMVhAR or control vector, 0.2 μg of pCMVβ, 0.5 μg of 3×IRS-Luc, 0.1 μg of FKHR:WT (WT), and the indicated amounts of coactivators. Transfected cells were treated and FKHR activity was determined as for Fig. 1B. (B) Enhancement of ERα activity by coactivators. DU145 cells were transfected with 0.1 μg of pLENhERα, 0.5 μg of pLENβgal, 0.5 μg of EREe1bLuc, and the indicated amounts of CBP, SRC1, or AIB1. Transfected cells were treated for 24 h with 10−8 M 17β-estradiol (E2) or ethanol (EOH) as vehicle controls. ERα activity was determined and expressed as for Fig. 1B. (C) Lack of an effect on FKHR activity of activated ERβ. DU145 cells were transfected with 0.5 μg of pLENβgal, 0.5 μg of 3×IRS-Luc, 0.1 μg FKHR:WT (WT) or FKHR:TSS (AAA), and 0.1 or 0.5 μg of pLENhERβ. Transfected cells were treated with 17β-estradiol or ethanol as for panel B, and FKHR activity was determined as for Fig. 1B. (D) Inhibition of FKHR activity by activated ERα. DU145 cells were transfected as for panel C except that 0.1 μg of pLENhERα was used in place of pLENhERβ. Transfected cells were treated with 17β-estradiol or ethanol, and FKHR activity was determined as for panel C. Co-A, coactivator.
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 (WT), and 0.2 μg of pCMVβ. Transfected cells were treated as for Fig. 1B. Immunoprecipitates with M2 anti-Flag antibody were probed with M2 anti-Flag (top panel) or PG-21 anti-androgen receptor (middle panel) antibodies. Androgen receptor (AR) protein expression in cell lysates was detected with the PG-21 anti-androgen receptor antibody (bottom panel). IB, immunoblot. (B) Interaction between FKHR and two separate regions of the androgen receptor (AR).35S-labeled FKHR synthesized in in vitro transcription-translation reactions was incubated with GST-androgen receptor fusion proteins, precipitated with glutathione beads, and visualized by autoradiography (top panel). The amount of GST proteins used in the pulldown assays was visualized by Coomassie blue staining after SDS-PAGE (bottom panel). Note that the amount of reticulocyte lysate used for precipitations was 10 times the input amount. EOH, ethanol. (C) A diagram depicting the FKHR-interacting regions of the androgen receptor. aa, amino acids; DBD, DNA binding domain; LBD, ligand binding domain.
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 (IP) of anti-Gal4 antibody were probed with PG-21 anti-androgen receptor (AR) antibody (upper panel) or the same anti-Gal4 antibody (lower panel). Note that Gal4-FKHR(1-150), Gal4-FKHR(350-655), and IgG light chain run close to one another in the gel due to their similar molecular weights. After quantifying the intensity of the corresponding bands, the ratio of androgen receptor to Gal4-FKHR in the immunoprecipitates was plotted (bottom panel). IB, immunoblot; H-chain, heavy chain.
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 μg of pCMVhAR and treated with R1881 or ethanol (EOH). Then 50 μg of nuclear extracts of transfected cells was subjected to immunoblotting analysis with M2 anti-Flag antibody. Lane 1, transfected with control vectors; lane 2, transfected with Flag-FKHR; lane 3, transfected with both Flag-FKHR and androgen receptor; lane 4, transfected with Flag-FKHR and androgen receptor and treated with 10−8 M R1881. (B) Inhibition of FKHR DNA binding by activated androgen receptor. We used 10 μg of nuclear extracts for the electrophoretic mobility shift assay with radiolabeled insulin response sequence (IRS) probe as described in the text. The arrow points to the DNA complex containing Flag-FKHR protein. Lanes 1 to 4, as in panel A; lane 2b, as in lane 2, but the nuclear extract was incubated with M2 antibody. (C) Characterization of the FKHR-DNA complexes. DU145 cells were transfected as for panel A, and electrophoretic mobility shift assays performed as for panel B. Cotreatment with antibodies and an excess amount of nonradiolabeled wild-type (WT) and mutant (MT) insulin response sequence oligonucleotides was performed as indicated.
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 FKHR:TSS (AAA) with or without 2.5 μg of pCMVhAR. Transfected cells were treated with 10−8 M R1881 or vehicle for 36 h. CD20-positive cells were separated from CD20-negative cells, and Fas ligand (FasL) expression on the surface of CD20-positive cells was determined. Similar data were obtained in multiple experiments, and a representative experiment is shown. Cy3, indocarbocyanine. (B) A bar graph showing the inhibition of FKHR-induced Fas ligand expression by activated androgen receptor (AR). (C) Inhibition of Fas ligand expression by androgens mediated through the endogenous androgen receptor in LNCaP cells. LNCaP cells were transfected with CD20 with or without AAA. Transfected cells were treated with R1881 or ethanol (EOH) as for panel A. The data from two independent experiments are presented as in panel B.
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 treated with R1881 or ethanol (EOH). The viability of transfected cells in each well was determined by counting the total number of green cells. Triplicate samples were analyzed for each data point, and the data were reproduced three times. (B) Representative micrographs of LNCaP cells transfected with FKHR:TSS. Cells were transfected with FKHR:TSS and GFP vectors as for panel A, fixed, and stained with DAPI. Representative micrographs were captured by a charge-coupled device camera attached to the fluorescence microscope. (C) Androgen effect on FKHR-induced increase in apoptotic index of LNCaP cells. Cells were transfected as for panel A and processed as for panel B. Apoptotic index of GFP-positive cells was determined by scoring 300 GFP-positive cells for chromatin condensation and nuclear fragmentation. Triplicate samples were analyzed per data point, and the graph represents three independent experiments. (D) Androgen effect on the viability of FKHR-transfected DU145 cells. Cells were transfected as for panel A but with or without 0.2 μg of pCMVhAR. Transfected cells were treated and determined for cell viability as for panel A. (E) Dosage-dependent protection of DU145 cells from FKHR-induced cell death by activated androgen receptor (AR). Cells were transfected as for panel D except that different amount of pCMVhAR were used, as indicated. Transfected cells were treated and cell viability was determined as for panel A.
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 pCMVhAR. Transfected cells were treated with 10−8 M R1881 for 36 h, fixed, and stained with propidium iodide. CD20-positive cells were separated from CD20-negative cells and subjected to flow cytometry analysis. Data from a representative experiment are shown. (B) A bar graph showing the inhibition of FKHR-induced cell cycle arrest by activated androgen receptor in DU145 cells. Each data point was analyzed in duplicate.
References
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