Inhibition of endogenous FOXO1 activity by LPA requires FHL2. (A) DU145 cells were transfected with 4 μg of pcDNA3 (vector) or the indicated siRNA vectors. Cell extracts were immunoblotted with anti-FHL2 or anti-β-actin antibody. (B–D) DU145 (B), JCA1 (C), and 267B1 (D) cells were transfected with 3 × IRSLuc (0.3 μg), pRL-null (0.01 μg), and 1 μg of the indicated siRNA vectors. Luciferase activity was assayed and normalized as in Figure 1D.

FHL2 suppresses FOXO1-induced apoptosis. (A) DU145 cells were transfected with pCMV-CD20 (3 μg), Flag-FOXO1 (2.5 μg), and Myc-FHL2 (2.5 μg) as indicated. At 16 h post-transfection, CD20-positive cells were separated from CD20-negative cells by FACS-based sorting. The apoptotic index of CD-20 positive cells was determined as described in Materials and Methods. Representative profiles are shown in the top graphs. Apoptotic indexes from four independent experiments are shown in the table below the graphs. (B) DU145 cells were transfected with FOXO1 (4 μg) and FHL2 (4 μg) as indicated. The activity of capase-3 was determined as described in Materials and methods.

A model of how FHL2 regulates FOXO1 activity by SIRT1-mediated deacetylation. Active FOXO1 suppresses prostate tumorigenesis by inhibiting cell cycle progression and by inducing apoptosis. FOXO1 activation in prostate cancer cells requires acetylation at yet to be defined sites. These sites are essential for its modulation of gene expression by both direct DNA binding and ‘tethering'. FHL2 promotes prostate tumorigenesis by serving as an adaptor that couples SIRT1 and FOXO1 and thus inhibits FOXO1 action in prostate cells.

FHL2 suppresses FOXO1 action through the mode of direct DNA binding. (A) Diagram depicting FOXO1 action by direct DNA binding. (B) DU145 cells were transfected with 3 × IRSLuc (0.3 μg), pCMVβGal (0.3 μg), and the indicated amounts of FOXO1 and FHL2 vectors. FOXO1 activity is expressed as relative luciferase units (RLU) normalized to β-gal activity. (C) DU145 cells were transfected with Flag-FOXO1 (4 μg) and Myc-FHL2 (4 μg). Cell extracts were immunoblotted with anti-Fas ligand antibody 24 h post-transfection. A β-actin immunoblot is included as a loading control.

FHL2 suppresses the ‘tethering' mode of FOXO1 action. (A) Diagram depicting FOXO1 action through ‘tethering'. (B) LNCaP cells were transfected with the pGL2-CD1 reporter plasmid (0.3 μg), pRL-null (0.01 μg), and the indicated amounts of Flag-FOXO1 and Myc-FHL2. Luciferase activity was assayed and normalized as in Figure 1D. (C) DU145 cells were transfected with Flag-FOXO1 (4 μg) and 4 μg of either Myc-FHL2 or pcDNA3. Amounts of endogenous cyclin D1 protein were determined by immunoblotting with anti-cyclin D1 antibody. A β-actin immunoblot is included as a loading control.

FHL2 decreases FOXO1 acetylation by promoting the interaction of FOXO1 with SIRT1. (A) DU145 cells were transfected with Myc-FHL2 (1 μg) and Flag-SIRT1 (1 μg) as indicated. Cell extracts were immunoprecipitated with anti-Myc or rabbit anti-Flag antibody, and immune complexes were immunoblotted with rabbit anti-Flag, anti-Myc, M2 anti-Flag, or anti-FHL2 antibody. (B) DU145 cells were transfected with Flag-FOXO1 (0.3 μg) and the indicated amounts of Myc-FHL2. Cell extracts were immunoprecipitated and immunoblotted with the indicated antibodies. (C) DU145 cells were transfected with Flag-FOXO1 (3 μg), Myc-FHL2 (3 μg), and 6 μg of siRNA vectors. Cell extracts were immunoprecipitated with the indicated antibodies. (D) DU145 cells were transfected with Flag-FOXO1 (3 μg), Myc-FHL2 (3 μg), and 6 μg of the siRNA vectors. Cells were harvested 24 h after transfection, and amounts of Fas ligand, Bim, and β-actin (loading control) were determined by immunoblotting.

Inhibition of FOXO1 activity by FHL2 requires SIRT1 activity. (A) DU145 cells were transfected with 3 × IRSLuc (0.3 μg) and pCMVβGal (0.3 μg). As indicated, cells were cotransfected with Flag-FOXO1 (0.3 μg) and Myc-FHL2 (0.3 μg). Transfected cells received 5 or 20 mM nicotinamide for 24 h. Luciferase activity was determined and normalized to β-gal activity. Fold activation was calculated by dividing the normalized activity of cells expressing FOXO1 and FHL2 by that of the corresponding vector controls. (B) DU145 cells were transfected with Flag-FOXO1 (3 μg), Myc-FHL2 (3 μg), and 6 μg of SIRT1 or GFP siRNA. Cell extracts were immunoblotted with anti-SIRT1, anti-β-actin, or M2 antibody. (C) DU145 cells were transfected with 3 × IRSLuc (0.3 μg), pRL-null (0.01 μg), Flag-FOXO1 (0.3 μg), and 0.6 μg of the indicated siRNA vectors. Luciferase activity was assayed and normalized as in Figure 1D.

Interaction of endogenous FHL2 and FOXO1 in the nucleus of PTEN-intact prostate cancer cells in the presence or absence of LPA. (A) DU145 cells were starved in serum-free medium for 36 h and treated with or without 20 μM LPA for 10 h. Nuclear and cytosolic extracts were immunoblotted with anti-FHL2, anti-FOXO1, anti-Hsp60, and anti-PARP antibodies as indicated. Hsp60 and PARP are cytosolic and nuclear proteins, respectively, and are used as controls for subfractionation. (B–D) DU145 (B), JCA1 (C), and 267B1 (D) cells were starved and treated with 20 mM LPA as in (A). Nuclear extracts were immunoprecipitated with rabbit anti-FOXO1 or rabbit anti-Flag (as a control) antibody. The immunoprecipitates were immunoblotted with anti-FHL2 or goat anti-FOXO1 antibody as indicated. Immunoblotting with anti-PARP antibody without prior immunoprecipitation was included as a control.

SIRT1 binds to FOXO1 and inhibits FOXO1 by deacetylating FOXO1. (A) DU145 cells were transfected with HA-FOXO1 (1 μg) and Flag-SIRT1 (1 μg). Cell extracts were immunoprecipitated with rabbit anti-Flag or mouse anti-HA antibody, and immunoprecipitates were immunoblotted with rabbit anti-HA or M2 anti-Flag antibody as indicated. (B) DU145 cells were transfected with 4 μg of either Flag-FOXO1, pcDNA-SIRT1, pcDNA-SIRT1HY, or pcDNA3 and treated with or without 5 mM nicotinamide as indicated. Cell extracts were immunoprecipitated with M2 anti-Flag antibody. The immunoprecipitates were immunoblotted with an anti-acetyl lysine antibody (upper panel) or rabbit anti-Flag antibody (lower panel). (C) DU145 cells were transfected with 3 × IRSLuc (0.3 μg), pRL-null (0.01 μg), and the indicated amounts of Flag-FOXO1 and pcDNA3 SIRT1. Luciferase activity was assayed and normalized as in Figure 1D. (D) DU145 cells were transfected with 3 × IRSLuc (0.3 μg), pRL-null (0.01 μg), Flag-FOXO1 (0.3 μg), and 0.3 μg of either pcDNA3 or pcDNA-SIRT1. Transfected cells were treated with or without nicotinamide for 24 h as indicated. Luciferase activity was assayed and normalized as in Figure 1D. FOXO1 activity is expressed as % of control and was calculated by normalizing the activity in cells transfected with SIRT1 to that of the vector controls (as 100%).

FHL2 interacts with FOXO1 and inhibits the transcriptional activity of the amino terminus of FOXO1. (A) DU145 cells were transfected with Myc-FHL2 (1 μg) and Flag-FOXO1 (1 μg). Cell extracts were immunoprecipitated with rabbit anti-Flag or anti-Myc antibody. Immunoprecipitates were immunoblotted with anti-Myc, rabbit anti-Flag, M2 anti-Flag, or goat anti-FHL2 antibody as indicated. (B) DU145 cells in medium containing 5% sFBS were transfected with Myc-FHL2 (1 μg) and Flag-FOXO1 (1 μg). Cells were stained with DAPI and incubated with anti-FHL2 and M2 antibodies. Immunoreactivity was detected with IgG conjugated to Alexa Fluor 594 (red, detects FHL2) or FITC (green, detects FOXO1). Colocalization was visualized by high-resolution imaging with deconvolution microscopy (panel B6). (C) DU145 cells were transfected with 1 μg of His-FOXO1(1–150), and cell extracts were incubated with GST or with GST-FHL2 fusion proteins prebound to glutathione beads. Proteins bound to the beads and the input (50% of extract protein used for precipitations) were immunoblotted with goat anti-FOXO1 antibody. (D) DU145 cells were transfected with Gal4 reporter 3 × 17merLuc (firefly, 0.3 μg) and pRL-null (Renilla, 0.01 μg) and the indicated amounts of Gal4-FOXO1(1–150) and Myc-FHL2. Activity of the firefly luciferase was normalized to that of the Renilla luciferase and expressed as relative luciferase unit (RLU).