A Small Peptide Targeting the Ligand-Induced Androgen Receptor/Filamin a Interaction Inhibits the Invasive Phenotype of Prostate Cancer Cells

Abstract

Prostate cancer (PC) is one of the most widespread malignancies among males worldwide. The androgen receptor (AR) plays a major role in prostate cancer development and progression and is the main target of PC therapy. Nonetheless, its action is not yet fully elucidated. We report here that the AR associates with Filamin A (FlnA) promoting migration and invasiveness of various PC-derived cells after androgen challenging. Inhibition of the AR/FlnA complex assembly by a very low concentration of Rh-2025u, an AR-derived peptide specifically interfering with this association, impairs such phenotype in monolayer cells and in 3D models. This study, together with our recent data in cancer-associated fibroblasts (CAFs), indicates that targeting the AR/FlnA complex could improve the clinical management of invasive PC, as the limited number of new drugs reaching the market suggests that we must re-examine the way invasive PC is currently treated. In this context, the synthesis of new biologically active molecules, such as the Rh-2025u peptide, which has been shown to efficiently interfere in the complex assembly in CAFs and PC cells, should overcome the limits of current available therapies, mostly based on hormone antagonists.

Keywords: androgen receptor; androgens; cell migration; prostate cancer; spheroids.

Figure 1

The Rh-2025u peptide effect on the LNCaP-spheroid size. The GFP-LNCaP cells were used. (A) After three days, LNCaP in Matrigel were left unchallenged (basal) or challenged as indicated. Peptide or enzalutamide alone were used as controls. (B) Representative images by phase-contrast (left section), or fluorescence microscopy (middle section) were captured and shown. Merged images are shown in the right section. Scale bar, 100 μm. In (B), the spheroid size was monitored for 15 days and calculated under basal conditions (three days) or in cells left unstimulated (basal) or stimulated for 15 days, in the absence or presence of the indicated compounds. It was expressed as fold increase in the relative organoid size. n represents the number of experiments. Means and SEM are shown. * p  <  0.05, for the indicated experimental point (R1881) versus the corresponding untreated control (basal).

Figure 2

The Rh-2025u peptide does not affect LNCaP cells proliferation and viability. Quiescent LNCaP cells were employed. (A) Cells were left unchallenged or challenged for 18 h with the indicated compounds and pulsed in vivo with 100 μM BrdU, then analyzed by immunofluorescence (IF) and expressed as the percentage of nuclei. (B) Cells were left untreated or treated for 24, 48, and 72 h with the indicated compounds. WST-1 reagent was used for testing cell proliferation and graph represents the ratios of proliferation expressed as the fold increase over the basal absorbance. R1881 induced a significant (p < 0.05) increase in cell proliferation as compared with the untreated (nt) cells. (C) LNCaP-Spheroid viability was analyzed by the MTT assay after 15 days of treatment. Graph represents the ratios of proliferation expressed as the fold increase over the basal absorbance. (D) Cells were transfected as reported in Methods. Luciferase activity was assayed, normalized using β-gal and expressed as fold induction. (E) Cells were left unstimulated or stimulated for 48 h with R1881 in absence or presence of the indicated compounds and conditioned media were collected and analyzed by ELISA. The graph represents the release of PSA expressed in ng/mL and detected by absorbance. In (A–E), n represents the number of experiments. Means and SEM are shown. When indicated, * p  <  0.05, for the indicated experimental point (R1881) versus the corresponding untreated control (basal). In (A–E) only the enzalutamide inhibits significantly the effects triggered by R1881.

Figure 3

The Rh-2025u impairs the migratory behavior in LNCaP cells. Quiescent LNCaP cells were used. (A) Cells, unstimulated or stimulated for 40 min as indicated, were analyzed for F-actin and arrows indicate cells protrusions and ruffles. The Immunofluorescence images (IF) are representative of 3 different experiments, each in duplicate. Scale bar 10 μm. In (B) cells were wounded and unstimulated or stimulated as indicated. Phase-contrast images are representative of three different experiments, each in triplicate. The wound area was calculated and data, expressed as the percentage of decrease in wound width over the control cells (analyzed at time 0; left panel), are reported below each image. In (C,D), cells unchallenged or challenged as indicated, were allowed to migrate (C) or invade (D) for seven h or 24 h, respectively and scored as described in “Methods” section. Means and SEMs are shown. In (C,D), n represents the number of experiments. * p  <  0.05 for the indicated experimental points versus the corresponding untreated control (basal).

Figure 4

The inhibitory effect of Rh-2025u on androgen-induced migration and invasiveness of various PC cells. Quiescent DuCaP (A,B), 22Rv1 (C,D), DU145 and PC3 (F) cells were used. In (A–F), cells, left unstimulated or stimulated as indicated and allowed to migrate (A,C,F) or invade (B,D) for nine h or 24 h, respectively, were scored as described in “Methods” and data were expressed as increase fold. Means and SEMs are shown. n represents the number of experiments. * p  <  0.05 for the indicated experimental points versus the corresponding untreated control (basal). In (C,D), only the Rh-2025u inhibits significantly (*) the cell migration (C) and invasiveness (D) triggered by androgens. In (E), lysates from the indicated cell lines were prepared and analyzed by Western blotting using the antibodies against the indicated proteins. AR fl stands for the AR full length isoform. ARv7 for the AR truncated form.

Figure 5

The androgen triggers AR/FlnA complex assembly and co-localization. Quiescent LNCaP were used. In (A), cells were left unchallenged or challenged for 10 min as indicated. The upper section shows the WB with the indicated antibodies to reveal lysate proteins (loading), which were then immunoprecipitated using the anti-FlnA (filamin A) antibody (middle section) or control IgG (lower section, ctrl IgG). Results are representative of three different experiments. In (B), cells were stained for AR and FlnA. Images captured by confocal microscope, and representative of three independent experiments, show the staining of Fln A (red) and AR (green). On the right merged images are presented. Bar, 5 μm. Co-localization factors are indicated in the right panels.

Figure 6

The expression of Filamin A and the activation of Rac 1 and FAK (Tyr 397) are important for androgen induced LNCaP migration. In (A–E), siRNA Alexa Fluor 488, control siRNA (Ctrl siRNA) or siRNA FlnA (FlnA siRNA) or siRNA AR (AR siRNA) were used as in “Methods” section. (A) After transfection, cellular lysates were obtained and FlnA (left) and AR (right) expression were analyzed by WB using the appropriate antibodies. Transfected and quiescent cells, unstimulated or stimulated as indicated, were used for migration (B–D) or invasiveness assays (C–E) for seven h or 24 h, respectively. Migrating (B–D) or invading (C–E) cells were scored as in “Methods” section and data expressed as fold increase. Means and SEM are shown. In (F), proteins lysates were used for Rac pull down assay using a commercially available kit, as described under “Methods”. The WB with anti-Rac antibody revealed the total amount of Rac expressed in the corresponding lysates (upper panel) and the eluted Rac (Rac-GTP; lower panel). In (G), lysate proteins were analyzed for Fak activation (P-Fak Y397), using the anti-P-Tyr397Fak antibody. The filter was stripped and re-probed using anti-Fak antibody (upper section). The WB for tubulin expression in lysate proteins was finally done, as loading control. In (A–E) results are representative of 3 different experiments and when indicated n represents the number of experiments. In (B–E), in ctrl siRNA transfected cells, R1881 increases significantly the cell migration (B,D) or BrdU incorporation (C,E).