• Home
  • Search Results
  • Regulation of the tumor suppressor FOXO3 by the thromboxane-A2 receptors in urothelial cancer.

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

PloS one (2014-09-10)
Philip M Sobolesky, Perry V Halushka, Elizabeth Garrett-Mayer, Michael T Smith, Omar Moussa
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.

MATERIALS
Product Number
Brand
Product Description

Roche
X-tremeGENE HP DNA Transfection Reagent, High-performance polymer reagent for transfecting many cell lines
Sigma-Aldrich
Indomethacin, 98.5-100.5% (in accordance with EP)
Sigma-Aldrich
Methyl acetate, anhydrous, 99.5%
Sigma-Aldrich
L-(−)-Glucose, ≥99%
Sigma-Aldrich
Methyl acetate, ReagentPlus®, 99%
Supelco
Methyl acetate, analytical standard
Sigma-Aldrich
Methyl acetate, ≥98%, FG
Supelco
Indomethacin, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Methyl acetate, suitable for HPLC, ≥99.8%
Sigma-Aldrich
Indomethacin, meets USP testing specifications
USP
Indomethacin, United States Pharmacopeia (USP) Reference Standard
Indomethacin, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Methyl acetate, natural, 98%, FG