Repression of beta-catenin function in malignant cells by nonsteroidal antiinflammatory drugs

Abstract

Activation of the Wnt/beta-catenin pathway promotes the development of several cancers and is an attractive target for chemopreventive and chemotherapeutic agents. Nonsteroidal antiinflammatory drugs (NSAIDs) have been reported to antagonize beta-catenin function, but their mechanism of action is not known. We demonstrate here that interference with beta-catenin function by NSAIDs does not correlate with cyclooxygenase (COX) inhibition. Instead, NSAID inhibition of beta-catenin requires the high level expression of peroxisome proliferator-activated receptor gamma (PPAR-gamma) and its co-receptor retinoid-X-receptor alpha (RXR-alpha). Immunoprecipitation experiments show that beta-catenin interacts with RXR-alpha and PPAR-gamma in some malignant cells. Repression of beta-catenin-dependent transcription by NSAIDs is thus indirect and depends on the coexpression of other nuclear receptors.

Fig. 1.

NSAIDs inhibit Dsh/PPAR-γ/RXR-α-mediated transcription. HEK293 cells were transfected with a TCF/LEF-dependent reporter gene, expression plasmids for Dsh, RXR-α, and either active PPAR-γ (A) or inactive PPAR-γEA469 (B). After overnight incubation, the cells were treated for 24 h with 250 μM R-etodolac, 125 μM indomethacin, 250 μM sulindac, 200 μM fenoprofen, 300 μM salsalate, and 250 μM naproxen or vehicle alone, after which the reporter gene activity was measured. All cells were also transfected with a β-gal reporter gene to control for transfection efficiency. The results are expressed as % control TCF/LEF-dependent reporter gene activity ± SEM (n = 3).

Fig. 2.

Troglitazone and R-etodolac inhibit Dsh/β-catenin-mediated transcription through a process that requires PPAR-γ and RXR-α. (A) TOPflash reporter was transfected into HEK293 cells with expression plasmids for Dsh, PPAR-γ, and RXR-α, as indicated. After overnight incubation, the cells were treated for 24 h with 1 μM troglitazone, 1 μM9-cis-RA, 10 μM of the PPAR-α activator WY14,643, 250 μM R-etodolac, or vehicle alone. (B) This experiment was performed similar to A, except the inactive PPAR-γEA469 plasmid was used along with the indicated concentrations of troglitazone and R-etodolac. TOPflash reporter gene activity was measured. The results are expressed as fold induction of TCF/LEF-dependent reporter gene activity ± SEM (n = 3). All cells were also transfected with a β-gal reporter gene to control for transfection efficiency.

Fig. 3.

NSAID inhibition of TCF/LEF-dependent transcription is specific and downstream of β-catenin. (A) The TCF/LEF-dependent reporter was transfected into HEK293 cells with expression plasmids for either Dsh (A) or β-catenin (B). Then, the cells were treated with the indicated concentrations of R-etodolac for 24 h. The fold induction values are the ratios of the normalized luciferase activities in cells transfected with both expression and reporter plasmids, compared with the activities in cells receiving the respective reporter plasmids alone. The results are the mean ± SEM of triplicate experiments. (C) Reporter plasmids for TCF/LEF, activator protein 1 (AP-1), or NFAT were transfected into HEK293 cells along with the respective expression plasmids for β-catenin, H-Ras^V12 and NFAT, as indicated. Transfected cells were treated with the indicated concentrations of R-etodolac for 24 h, and the fold increase in luciferase activities was determined. The results are the mean ± SEM of triplicate determinations.

Fig. 4.

Interaction of PPAR-γ and β-catenin. (A) Expression plasmids for Dsh, PPAR-γ, and RXR-α were cotransfected into HEK293 cells as indicated. At 48 h after transfection, cell extracts were prepared for immunoprecipitation (IP) with an anti-β-catenin monoclonal antibody. The immune complexes were analyzed by immunoblotting with anti-β-catenin, anti-PPAR-γ, and anti-RXR-α.(B) HEK293 cells were transfected with expression plasmids for PPAR-γ in the presence or absence of β-catenin. At 48 h after transfection, cell extracts were prepared for IP with the anti-PPAR-γ monoclonal antibody. The immune complexes were analyzed by immunoblotting with anti-β-catenin or anti-PPAR-γ antibodies. (C) LNCaP cells were grown at 37°C for 24–36 h. Cells were lysed, and IP was completed with anti-PPAR-γ (lane 1) and anti-β-catenin (lane 2) monoclonal antibodies. The immune complexes were analyzed by immunoblotting with anti-β-catenin or anti-PPAR-γ antibodies.

Fig. 5.

Interaction of R-etodolac with PPAR-γ. (A) HEK293 cells were transfected with expression plasmids for PPAR-γ and β-gal. After overnight incubation, the cells were treated for 24 h with 5 μM troglitazone, 10 μM WY14,643, 500 μM R-etodolac, and DMSO alone. Cell lysates were prepared and analyzed by SDS/PAGE under reducing conditions, transferred to a membrane, and probed with anti-PPAR-γ and anti-β-gal (β-gal) antibodies. (B) Inhibition by R-etodolac of the ligand-dependent interaction between PBP and PPAR-γ in a mammalian two-hybrid system. The UAS-TK-Luc reporter and expression plasmids for Gal4-PBP, VP16, VP16-PPAR-γ were transfected into HEK293 cells. After 16 h, the cells were treated with the indicated amount of R-etodolac, troglitazone, and vehicle alone for 24 h.