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. 2014 Sep 9;9(9):e107530.
doi: 10.1371/journal.pone.0107530. eCollection 2014.

Regulation of the tumor suppressor FOXO3 by the thromboxane-A2 receptors in urothelial cancer

Philip M Sobolesky  1 , Perry V Halushka  2 , Elizabeth Garrett-Mayer  3 , Michael T Smith  4 , Omar Moussa  1
Affiliations

Regulation of the tumor suppressor FOXO3 by the thromboxane-A2 receptors in urothelial cancer

Philip M Sobolesky et al. PLoS One. .

Abstract

The transcription factor FOXO3 is a well-established tumor suppressor whose activity, stability, and localization are regulated by phosphorylation and acetylation. Previous data by our laboratory demonstrated amplified thromboxane-A2 signaling was associated with poor prognoses in bladder cancer patients and overexpression of the thromboxane-A2 isoform-β receptor (TPβ), but not TPα, induced malignant transformation of immortalized bladder cells in vivo. Here, we describe a mechanism of TP mediated modulation of FOXO3 activity and localization by phosphorylation and deacetylation in a bladder cancer cell model. In vitro gain and loss of function studies performed in non-transformed cell lines, UROsta and SV-HUC, revealed knockdown of FOXO3 expression by shRNA increased cell migration and invasion, while exogenously overexpressing TPβ raised basal phosphorylated (p)FOXO3-S294 levels. Conversely, overexpression of ERK-resistant, mutant FOXO3 reduced increases in UMUC3 cell migration and invasion, including that mediated by TP agonist (U46619). Additionally, stimulation of UMUC3 cells with U46619 increased pFOXO3-S294 expression, which could be attenuated by treatment with a TP antagonist (PTXA2) or ERK inhibitor (U0126). Initially U46619 caused nuclear accumulation of pFOXO3-S294; however, prolonged stimulation increased FOXO3 cytoplasmic localization. U46619 stimulation decreased overall FOXO3 transcriptional activity, but was associated with increased expression of its pro-survival target, manganese superoxide dismutase. The data also shows that TP stimulation increased the expression of the histone deacetylase, SIRT1, and corresponded with decreased acetylated-FOXO3. Collectively, the data suggest a role for TP signaling in the regulation of FOXO3 activity, mediated in part through phosphorylation and deacetylation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Loss of FOXO3 results in increased cell migration and invasion.
(A) Western blot of whole cell lysates from SV-HUC and UROsta cells stably transfected with FOXO3-shRNA or control scrambled (scr−) shRNA immunoblotted for total FOXO3 and its known downstream target MnSOD. α-tubulin was applied as loading control. Blots shown are representative of three individual experiments and numbers represent the averaged ratios of FOXO3 to α-tubulin. Ratios quantified using Image Studio Lite Ver.3.1. SV-HUC and UROsta FOXO3 knockdown and control clones were seeded into inserts coated with fibronectin or matrigel and allowed to migrate (B) or invade (C) for 16 hours. Knockdown of FOXO3 increased SV-HUC and UROsta cell migration (39% and 114%) and invasion (63% and 18%, respectively) compared to scr-shRNA. Data represent three independent assays done in duplicates and expressed as the mean ± SEM. Significance was determined with a two-sided two-sample t-test, (* = P<0.05, n = 3) compared to scr- control. (D) Overexpression of GFP tagged TPβ in UROsta and SVHUC cells increases basal pFOXO3-S294 expression (E) compared to vector control (n = 3). Numbers represent the averaged ratios of pFOXO3-S294 to FOXO3. Ratios quantified using Image Studio Lite Ver.3.1. [AU] = arbitrary units.
Figure 2
Figure 2. TP agonist activates ERK and increases FOXO3 phosphorylation at –S294.
(A) UMUC3 cells were treated with 1 µM U46619 for the indicated times. Cell lysates were analyzed by immunoblotting for both total and phosphorylated ERK and FOXO3. GAPDH served as loading control. Blots were quantified and ratios for (B) pFOXO3-S294 to FOXO3 and (C) pERKT202/Y204 to ERK1/2 were graphed using GraphPad Prism 5. UMUC3 cells pretreated for 15 minutes with (D) 2 µM TP antagonist Pinane (PTXA2) or (G) 10 µM ERK inhibitor (U0126) attenuated the activation of ERK and phosphorylation of FOXO3 following treatment with 1 µM U46619 for 30 minutes. Cell lysates were immunoblotted for both total and phosphorylated ERK and FOXO3. α-Tubulin served as loading control. Graphs represent quantified ratios for (E and I) pFOXO3-S294 to FOXO3 and (F and G) pERKT202/Y204 to ERK1/2 were graphed. Significance was determined for all quantified blots using a repeated measure One-way ANOVA with Tukey post-hoc test. P<0.05.
Figure 3
Figure 3. Decreased FOXO3 transcriptional activity and nuclear localization following TP agonist treatment.
(A) UMUC3 cells transiently transfected with 3× forkhead response element (FHRE) reporter construct and Renilla luciferase control vector were treated with 1 µM U46619 for 30 or 120 minutes and resulted in decreased FOXO3 transcriptional activity. The average Firefly luciferase expression was adjusted to total protein and control vector Renilla luciferase expression before normalization to vehicle control. Results represent the average percent luciferase activity of three independent experiments conducted in duplicates. *P<0.05, One-way ANOVA with Tukey post-hoc test. (B) UMUC3 cells were serum starved overnight and pretreated for 15 min. with 1 µM Indomethacin prior to treatment with either vehicle or 1 µM U46619 for 5, 30, or 120 minutes. The cells were subjected to nuclear and cytoplasmic fractionation and proteins were analyzed by Western blot. α-Tubulin and Lamin-B1 served as loading controls for cytoplasmic and nuclear fractions, respectively. (C) Densitometry of blots showing prolonged agonist stimulation increased cytoplasmic FOXO3 protein and increased nuclear pFOXO3 proteins compared to vehicle controls. Significance was determined for all quantified blots using a repeated measure One-way ANOVA with Tukey post-hoc test. P<0.05.
Figure 4
Figure 4. Effect of TP agonist on FOXO3 target proteins expressions and acetylation status.
(A) UMUC3 cells treated with vehicle or 1 µM U46619 for 30, 120, and 240 minutes. Cell lysates were collected and analyzed by Western blot for total FOXO3 expression and downstream targets p27Kip1, MnSOD, and Bim. (B) Protein expression of p300 and SIRT1 in same cell lysates. α-tubulin served as loading control. (C) Quantified values from blots of three independent experiments are graphed. (*) indicates p<0.05, One-way ANOVA, compared to time 0 for each Target ratio. (D) Increased SIRT1 expression mediated by U46619 in UMUC3 cells corresponded with decreased acetylated FOXO3. Total FOXO3 was immunoprecipitated from serum starved UMUC3 cells treated with vehicle or 1 µM U46619 for 30, 120, or 240 minutes. Eluted proteins were analyzed by Western blot with antibodies against acetylated lysine residues and FOXO3. Blots are representative of three independent experiments. Blots were quantified and ratios for (E) acetylated-lysine (acetyl-K) to FOXO3 were graphed. Significance was determined using repeated measures One-way ANOVA with Tukey post-hoc test. P<0.05.
Figure 5
Figure 5. Expression of mutant FOXO33A attenuated TP agonist mediated migration of UMUC3 cells.
(A) GFP-FOXO33A or EGFP-C2 (Vector) was stably transfected into UMUC3 cells and mutant FOXO3 expression was confirmed by immunoblotting for GFP. α-tubulin served as loading control. Cells were seeded into inserts coated with fibronectin for migration or matrigel for invasion in serum free media containing 1 µM U46619 and placed in wells containing complete media with 1 µM U46619. Cells were allowed to migrate (B) for 8 hours or invade (C) for 16 hours. Significance was determined with a two-sided two-sample t-test, (* = P<0.05, n = 3) compared to GFP-vector control. (D) UMUC3 cells were pretreated with 10 µM U0126 for 15 minutes before stimulation with 1 µM U46619 and allowed to migrate for 16 hours. Data represent three independent assays done in duplicates and expressed as the mean ± SEM. Significance was determined with a One-way ANOVA with Tukey post-hoc test. P<0.05.
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