Melanoma antigen-A11 (MAGE-A11) enhances transcriptional activity by linking androgen receptor dimers

Abstract

Prostate cancer growth and progression depend on androgen receptor (AR) signaling through transcriptional mechanisms that require interactions with coregulatory proteins, one of which is the primate-specific steroid receptor coregulator melanoma antigen-A11 (MAGE-A11). In this report, we provide evidence how increased expression of MAGE-A11 during prostate cancer progression enhances AR signaling and prostate cancer growth. MAGE-A11 protein levels were highest in castration-recurrent prostate cancer. The cyclic AMP-induced increase in androgen-dependent and androgen-independent AR transcriptional activity correlated with an increase in MAGE-A11 and was inhibited by silencing MAGE-A11 expression. MAGE-A11 mediated synergistic AR transcriptional activity in LAPC-4 prostate cancer cells. The ability of MAGE-A11 to rescue transcriptional activity of complementary inactive AR mutants and promote coimmunoprecipitation between unlike forms of AR suggests that MAGE-A11 links transcriptionally active AR dimers. A model for the AR·MAGE-A11 multidimeric complex is proposed in which one AR FXXLF motif of the AR dimer engages in the androgen-dependent AR NH(2)- and carboxyl-terminal interaction, whereas the second FXXLF motif region of the AR dimer interacts with dimeric MAGE-A11. The AR·MAGE-A11 multidimeric complex accounts for the dual functions of the AR FXXLF motif in the androgen-dependent AR NH(2)- and carboxyl-terminal interaction and binding MAGE-A11 and for synergy between reported AR splice variants and full-length AR. We conclude that the increased expression of MAGE-A11 in castration-recurrent prostate cancer, which is enhanced by cyclic AMP signaling, increases AR-dependent growth of prostate cancer by MAGE-A11 forming a molecular bridge between transcriptionally active AR dimers.

FIGURE 1.

MAGE-A11 immunostaining in benign prostate and prostate cancer. MAGE-A11 was immunostained using 3 μg/ml MAGE-(94–108) antibody as described under “Experimental Procedures.” The four samples shown are representative of 17 specimens of androgen-stimulated benign prostate (A–D), 16 specimens of androgen-stimulated prostate cancer (E–H), and 11 specimens of castration-recurrent prostate cancer (I–L). The brown positive reaction product is shown against background staining using hematoxylin. Initial magnification, 400×.

FIGURE 2.

Cyclic AMP-dependent increase in MAGE-A11 correlates with increased AR transactivation of PSA in LAPC-4 cells. A, empty vector pCMV5 (−) or pCMV-AR (0.1 μg) was expressed in LAPC-4 cells (2 × 10⁵/well) in 12-well plates with 0.2 μg of MMTV-Luc/well. The next day, medium was exchanged or unchanged, and 24 h later, it was replaced with serum-free medium or unchanged. Cells were then incubated for 24 h with or without 1 nm DHT, and luciferase activity was measured. B, LAPC-4 cells (4 × 10⁵/well) in 6-well plates were transfected using Lipofectamine 2000 with 0.25 μg of MMTV-Luc. The next day, cells were placed in serum-free medium with or without 0.2, 0.5, 1, 3, or 6 mm dibutyryl-cAMP (dbcAMP) with or without 1 nm DHT, and luciferase activity was measured. C, the day after plating LAPC-4 cells (2 × 10⁶/6-cm dish), cells were transferred to medium containing 10% charcoal-stripped serum, and the next day, cells were treated with or without 0.5, 2, or 8 mm dibutyryl-cAMP for 48 h. RNA was extracted and analyzed using quantitative RT-PCR for MAGE-A11 as described under “Experimental Procedures.” D, the day after plating LAPC-4 cells as in C, cells were transferred to medium containing 5% charcoal-stripped serum and treated with or without 0.5, 2, or 8 mm dibutyryl-cAMP for 48 h. Cells were harvested in immunoblot lysis buffer, and 40 μg of protein/lane was probed using FLAG-MAGE antibody (10 μg/ml). Lane 1 contains 0.1 μg of COS1 cell extract control (C) from cells expressing pSG5-MAGE. E, complementary DNA used in C was analyzed using quantitative RT-PCR for PSA as described under “Experimental Procedures.” PPIA, peptidylprolyl isomerase A. Data in A–C and E are the mean ± S.D. (error bars) and are representative of three independent experiments.

FIGURE 3.

Requirement for MAGE-A11 in cyclic AMP-dependent AR transcriptional activity and LAPC-4 prostate cancer cell growth. A, top, LAPC-4 cells (3 × 10⁵/well) in 6-well plates were transfected with 0.25 μg of MMTV-Luc and 5 nm nonspecific (NS) or MAGE-A11 siRNA-2 or siRNA-3 using Lipofectamine 2000. The next day, cells were incubated for 24 h with or without 1 nm DHT and 0.2 mm dibutyryl-cAMP (dbcAMP) before assaying for luciferase. Bottom, empty vector pSG5 (−) (lane 1) or pSG5-MAGE (1 μg; lanes 2–5) was expressed in COS1 cells using Lipofectamine 2000 with or without a 5 nm concentration of two nonspecific siRNAs (NS), MAGE-A11 siRNA-2 (M2), or MAGE-A11 siRNA-3 (M3). Cell extracts (10 μg of protein/lane) were probed on the transblot using 10 μg/ml MAGE-(94–108) antibody incubated for 72 h at 4 °C. B, LAPC-4 cells were transfected with 0.5 μg of MMTV-Luc and 5 nm nonspecific siRNA (NS), MAGE-A11 siRNA-2, or MAGE-A11 siRNA-3. Cells were incubated with or without 1 nm DHT and 10 ng/ml EGF before assaying for luciferase. C, LAPC-4 cells (3 × 10⁶/well) in 6-well plates were transduced with 150 μl of lentivirus for MAGE-A11 shRNA-169 (M169), shRNA-827 (M827), shRNA-947 (M947), and shRNA-964 (M964), AR shRNA-5, empty vector, or 18-bp nonspecific (NS) shRNA. Cells were selected using puromycin as described under “Experimental Procedures.” Cell extracts (100 μg of protein/lane) were analyzed on immunoblots using AR32 (1 μg/ml) and AR52 (10 μg/ml) antibodies, 10 μg/ml of FLAG-MAGE antibody, 5 μg/ml MAGE-(94–108) antibody, and a 1:5000 dilution of β-actin antibody. Lane 8 contains extracts of COS1 cells expressing pCMV-AR (5 μg of protein/lane) or pSG5-MAGE (0.1 μg of protein/lane) as control (C). D, LAPC-4 cells transduced with lentivirus and selected as in C using puromycin were grown in the presence of 0.1 nm DHT and 10 ng/ml EGF in 24-well plates. Shown also is the no-hormone control of LAPC-4 cells transduced with nonspecific lentivirus shRNA and grown in the absence of DHT and EGF (−). Cell growth was assayed as described under “Experimental Procedures.” NV, no virus. Data in A, B, and D are the mean ± S.D. (error bars) representative of at least three independent experiments.

FIGURE 4.

AR requirements for transcriptional activity in LAPC-4 cells. A, schematic diagram of AR deletion mutants expressed in LAPC-4 cells. LBD, ligand binding domain. B, top, pCMV5 (−) or pCMV-AR wild-type (WT) or mutants (0.1 μg) were expressed in LAPC-4 cells with 25 ng of MMTV-Luc. Cells were incubated with or without 1 nm DHT. Bottom, pCMV5 (−) or pCMV-AR WT or mutants (5 μg) were expressed in COS1 cells. The transblot of cell extracts (15 μg of protein/lane) was probed using AR32 antibody. C, top, pCMV5 (−) or pCMV-AR WT or mutants (0.1 μg) were expressed in LAPC-4 cells with 25 ng of MMTV-Luc. Cells were incubated with or without 1 nm DHT. Bottom, pCMV5 (−) or pCMV-AR WT or mutants (5 μg) were expressed in COS1 cells as in B. The transblot of cell extracts (15 μg of protein/lane) was probed using AR32 antibody. D, top, pCMV5 (−) or pCMV-AR WT or AR deletion mutants (0.1 μg) were expressed in LAPC-4 cells with 25 ng of MMTV-Luc. Cells were incubated with or without 1 nm DHT. Bottom, pCMV5 (−) or pCMV-AR WT or AR deletion mutants (6 μg) were expressed in COS1 cells. The transblot of cell extracts (20 μg of protein/lane) was probed using AR32 antibody. E, pCMV5 (−), pCMV-AR WT, or pCMV-ARΔDBD (0.1 μg) was expressed in LAPC-4 cells with 0.1 μg of PSA-Enh-Luc, PSA-Enh-SIII-Luc, or pGL3-E4-Luc. Cells were incubated with or without 1 nm DHT. Shown in B–E are the mean ± S.D. (error bars) representative of three independent experiments.

FIGURE 5.

AR requirements for transactivation in CV1 cells. A, pCMV-AR WT, ΔDBD, C576A, or ΔTR (25 ng) was expressed in CV1 cells with 0.25 μg of PSA-Enh-Luc with or without 0.1 μg of pSG5 (−) or 50 ng of pSG5-MAGE (M) and 50 ng of pSG5-HA-p300 (P). Cells were incubated with or without 1 nm DHT. B, pCMV-AR (50 ng) was expressed in CV1 cells (3 × 10⁴/well) in 12-well plates with 0.1 μg of PSA-Enh-Luc and 0.1 of μg pSG5 (−) or 50 ng of pSG5-MAGE (M) and/or 50 ng of pSG5-HA-p300 (P). Cells were incubated with or without 1 nm DHT. C, CV1 cells were plated in phenol red-free medium containing 5% charcoal-stripped fetal calf serum, and the medium was not changed. pCMV-AR (25 ng) was expressed with 0.25 μg of PSA-Enh-Luc and 0.1 μg of pSG5 (−) or 50 ng of pSG5-MAGE (M) and 50 ng of pSG5-HA-p300 (P). Twenty-four hours before harvest, DHT was added to 0.1 or 1 nm with or without 0.5 μm hydroxyflutamide (HF) or 1 μm bicalutamide (BIC). Data shown are mean ± S.D. (error bars) representative of three independent experiments.

FIGURE 6.

Dependence of ARΔDBD activity on endogenous AR and MAGE-A11 in LAPC-4 cells. A, schematic diagram of human AR (hAR) with the ²³FXXLFXXVXXV³³ interaction site for MAGE-A11, AF1, DBD, ligand binding domain (LBD), and AF2. The target position is indicated for AR siRNA-3 in the ligand binding domain and for AR siRNA-4 in the DBD. B, pCMV5 (−) (lanes 1 and 6), pCMV-AR (lanes 2–5), or pCMV-ARΔDBD (lanes 7–10) (0.5 μg) was expressed in COS1 cells using Lipofectamine 2000 with or without 10 nm AR siRNA-3, AR siRNA-4, or nonspecific (NS) siRNA. Cells were incubated without DHT, and cell extracts (15 μg of protein/lane) were probed on transblots using AR32 antibody. C, pCMV5 (−) or pCMV-AR WT or ΔDBD (25 ng) was expressed in LAPC-4 cells (4 × 10⁵/well) in 6-well plates using Lipofectamine with 25 ng of MMTV-Luc with or without 10 nm AR siRNA-3 (AR3), AR siRNA-4 (AR4), MAGE-A11 siRNA-2 (MAG2), or nonspecific (NS) siRNA. Cells were incubated with or without 1 nm DHT. Data are the mean ± S.D. (error bars) and are representative of three independent experiments.

FIGURE 7.

Cooperativity between endogenous AR and AR-(1–660) in LAPC-4 cells. A, top, pCMV5 (−) or pCMV-AR-(1–660) WT, C576A, or L26A,F27A (LFAA)-C576A (0.1 μg) was expressed with 25 ng of MMTV-Luc in LAPC-4 cells. Cells were incubated with or without 1 nm DHT. Bottom, pCMV5 (−) or pCMV-AR-(1–660) WT, C576A, or LFAA-C576A (5 μg) was expressed in COS1 cells. Cell extracts (20 μg of protein/lane) were probed on transblots using AR32 antibody. B, pCMV-AR-(1–660)-C576A (25 ng) was expressed in LAPC-4 cells using Lipofectamine 2000 with 25 ng of MMTV-Luc and 5 nm nonspecific (NS) siRNA, AR siRNA-3 (AR3), AR siRNA-4 (AR4), MAGE-A11 siRNA-2 (M2), or MAGE-A11 siRNA-3 (M3). Cells were incubated with or without 1 nm DHT. C, top, pCMV5 (−) or pCMV-AR-(1–503) WT, LFAA, W433A,L436A,F437A (AX), or LFAA-AX combined mutant (0.1 μg) was expressed in LAPC-4 cells with 25 ng of MMTV-Luc. Cells were incubated with or without 1 nm DHT. Bottom, pCMV5 (−) or pCMV-AR-(1–503) WT, LFAA, AXXAA (AX), or combined mutant (5 μg) was expressed in COS1 cells. The transblot of cell extracts (10 μg of protein/lane) was probed using AR32 antibody. D, pCMV5 (−) or pCMV-AR-(1–660)-C576A or -AR-(1–660)-LFAA-C576A (10 ng) was expressed with 0.25 μg of PSA-Enh-Luc with or without 25 ng of pCMV-ARΔTR or ARΔTR-V716R with 50 ng of pSG5 (−) or pSG5-MAGE (M). Cells were incubated with or without 1 nm DHT. E, 10 ng of pCMV5 (−) or 2 ng of pCMV-AR-(1–660) was expressed in CV1 cells with 0.25 μg of PSA-Enh-Luc with or without 10 ng of pCMV-AR and 50 ng of pSG5 (−) or pSG5-MAGE (M). Cells were incubated with or without 1 nm DHT. Data in A–E are the mean ± S.D. (error bars) and are representative of three independent experiments.

FIGURE 8.

MAGE-A11 rescues complementary inactive AR mutants. A, pCMV5 (−), pCMV-ARΔTR, -C576A, and/or -ΔDBD (25 ng) were expressed in CV1 cells with 0.25 μg of PSA-Enh-Luc and 0.1 μg of pSG5 (−) or 50 ng of pSG5-MAGE (M) and 50 ng of pSG5-HA-p300 (P). Cells were incubated with or without 1 nm DHT. Inset, pCMV5 (−) or pCMV-AR WT, ΔTR, or ΔTR-C576A (8 μg) was expressed in COS1 cells. Cells were incubated with 10 nm DHT. The transblot of cell extracts (20 μg of protein/lane) was probed using AR32 antibody. B, pCMV-ARΔTR and -AR-C576A alone (first dash, 25 ng; second dash, 50 ng) or ARΔTR and AR-C576A (25 ng each) were expressed in CV1 cells with 0.25 μg of PSA-Enh-Luc with or without 0.1 μg of pSG5 or 50 ng of pSG5-MAGE (M) and/or 50 ng of pSG5-HA-p300 (P). Cells were incubated with or without 1 nm DHT. C, pCMV-ARΔTR and -ARΔDBD (25 ng) were expressed in CV1 cells with 0.25 μg of PSA-Enh-Luc and 50 ng of pSG5 (−), pSG5-MAGE (MAG), pSG5-HA-p300, and/or pSG5-TIF2. Cells were incubated with or without 1 nm DHT. D, pCMV-ARΔTR and/or -ARΔDBD (25 ng) were expressed in CV1 cells with 0.25 μg of PSA-Enh-Luc and 100 ng of pSG5 (−) or 25, 50, or 100 ng of pSG5-MAGE. Cells were incubated with or without 1 nm DHT. Data in A–D are the mean ± S.D. (error bars) and are representative of three independent experiments.

FIGURE 9.

Site-specific requirements for synergistic effects of AR and MAGE-A11. A, pCMV-ARΔTR, -ΔDBD, -LFAAΔDBD, and/or -LFAAΔTR (25 ng) were expressed in CV1 cells with 0.25 μg of PSA-Enh-Luc and 50 ng of pSG5 (−) or pSG5-MAGE. Cells were incubated with or without 1 nm DHT. Inset, pCMV5 (−) or pCMV-AR WT, ΔTR, LFAAΔTR, ΔDBD, or LFAAΔDBD (5 μg) was expressed in COS1 cells. Cells were incubated with 10 nm DHT. Cell extracts (20 μg of protein/lane) were probed on the transblot using AR32 antibody. B, pCMV-ARΔDBD, -ΔTR, -ΔTRm4, and/or -ΔTR-C576A (25 ng) was expressed in CV1 cells with 0.25 μg of PSA-Enh-Luc and 50 ng of pSG5 (−) or pSG5-MAGE (MAG). Cells were incubated with or without 1 nm DHT. Inset, pCMV5 (−) or pCMV-AR WT or mutants (5 μg) were expressed in COS1 cells. Cells were incubated with 2 nm DHT, and extracts (20 μg of protein/lane) were probed on transblots using AR32 antibody. C, top, pCMV-ARΔTR and -ARΔDBD (25 ng) were expressed alone or together with 0.25 μg of PSA-Enh-Luc and 50 of ng pSG5 (−) or pSG5-MAGE WT or mutants. Cells were incubated with or without 1 nm DHT. Bottom, pSG5 (−) or pSG5-MAGE WT or mutants (8 μg) were expressed in COS1 cells. Cell extracts (20 μg of protein/lane) were probed on the transblot using 0.5 μg/ml FLAG-MAGE antibody. D, top left and right, pCMV-ARΔTR and -ARΔDBD (25 ng) were expressed in CV1 cells with 0.25 μg of PSA-Enh-Luc and 50 ng of pSG5 (−), pSG5-MAGE WT or mutants (left), or pSG5-HA-MAGE WT or mutants (right). Cells were incubated with or without 1 nm DHT. Bottom, pSG5 (−) or pSG5-HA-MAGE WT or mutants (8 μg) were expressed in COS1 cells. Cell extracts (20 μg of protein/lane) were probed on transblots using HA antibody. Data in A–D are the mean ± S.D. (error bars) and are representative of three independent experiments.

FIGURE 10.

MAGE-A11 links AR dimers. FLAG empty vector or FLAG-AR (2 μg) was expressed in COS1 cells with 2 μg of pCMV-ARΔDBD (A), pCMV-ARΔ14–83ΔDBD (B), or pCMV-AR-LFAAΔDBD (C) with or without 2 μg of pSG5-MAGE and 4 μg of pSG5-HA-p300. Cells were incubated in the presence of 0.1 μg/ml EGF with or without 10 nm DHT. The cell extract (20 μg of protein/lane) and immunoprecipitate (IP) transblots were probed using AR32, FLAG-MAGE, and p300 antibodies.

FIGURE 11.

Synergy between AR and MAGE-A11 on multiple AREs. pCMV-ARΔTR and/or -ARΔDBD (25 ng) were expressed in CV1 cells with 50 ng of pSG5 (−) or pSG5-MAGE (M) and 0.25 μg of pGL3-E4-Luc, PSA-Enh-Luc, PSA-ARE4-Luc, and PSA-Enh-SIII-Luc (A) or 0.25 μg of PSCA-TATA-Luc or PSCA-ARE4-Luc (B). Cells were incubated with or without 1 nm DHT. Data are the mean ± S.D. (error bars) and are representative of three independent experiments.

FIGURE 12.

Model of synergistic effects of MAGE-A11 bridging AR dimers. The AR dimer undergoes an androgen-dependent interaction between the NH₂-terminal ²³FXXLF²⁷ motif and AF2 in the ligand binding domain. The AR N/C interaction is important for AR transactivation of androgen-dependent genes (67). MAGE-A11 forms a dimer (14) and interacts through its F box-like region with the extended ²³FXXLFXXVXXV³³ motif region in the AR dimer. The ability of MAGE-A11 to rescue transcriptional activity of complementary inactive AR mutants that lack activation function 1 or the DNA binding domain provides evidence that MAGE-A11 links AR dimers. This function of MAGE-A11 was supported by coimmunoprecipitation of unlike AR dimers that required MAGE-A11.