Sulindac Reverses Aberrant Expression and Localization of β-Catenin in Papillary Thyroid Cancer Cells with the BRAF V600E Mutation

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Abstract
Background: Activation of the Wnt/β-catenin signaling pathway is implicated in thyroid tumorigenesis, and up to 90% of papillary thyroid cancer (PTC) demonstrate aberrant expression of β-catenin. Nonsteroidal antiinflammatory drugs reverse aberrant β-catenin expression and localization in colon cancer. In this study, we tested the hypothesis that the nonsteroidal antiinflammatory drug sulindac would reverse aberrant β-catenin activity in thyroid cancer cells. Methods: β-catenin protein levels were determined in thyroidectomy specimens from six consecutive patients and in three different thyroid cancer cells lines (8505-C, KTC-1, and TPC-1) by immunoblotting. Cells of 8505-C and KTC-1 harbor the BRAFV600E mutation, and TPC-1 has the RET/PTC rearrangement. All cell lines were treated with sulindac (100 μM for up to 72 hours). Protein levels of c-myc and cyclin D1 were detected by immunoblotting, and β-catenin localization was determined by immunocytochemistry in the PTC cell lines. PCCL3 rat thyroid cells that conditionally overexpress either BRAFV600E or RET/PTC were also treated with sulindac. Results: All PTC specimens and cell lines expressed high levels of β-catenin protein and displayed aberrant nuclear and cytoplasmic localization of β-catenin. Exposure to sulindac for 48 hours reduced β-catenin expression in 8505-C and KTC-1 cells, but not in TPC-1 cells. Further, sulindac treatment reduced c-myc and cyclin D1 levels in 8505-C and KTC-1 cells, but had no effect in TPC-1 cells. Immunocytochemistry demonstrated that sulindac treatment redistributed β-catenin from the nucleus to the membrane in 8505-C and KTC-1 cells. However, sulindac did not affect β-catenin localization in TPC-1 cells. Finally, sulindac was effective in decreasing β-catenin expression and cellular proliferation in BRAFV600E-overexpressing cells, but not in RET/PTC3-overexpressing cells. Conclusions: Taken together, our findings demonstrate that sulindac treatment reverses β-catenin activity in 8505-C and KTC-1 cell lines with the BRAFV600E, but not in TPC-1 cells with the RET/PTC mutation. Future studies should investigate the potential for β-catenin-directed therapy for patients with advanced thyroid cancers.

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