Abstract 3894: Regulation of VDUP1 expression in breast cancer cells by cyclopamine

Abstract

Abstract VDUP1 (TXNIP) is a tumor suppressor protein that contributes to the regulation of cell proliferation through its role as an inhibitor of thioredoxin (TRX). VDUP1 expression is decreased in a variety of tumor cells relative to non-tumor cells and enforced expression of VDUP1 slows tumor cell growth. 1,25-Dihydroxyvitamin D3 (VD3) has long been known to increase VDUP1 expression in tumor cells, although the full extent of VDUP1 regulation is not known. Preliminary experiments with Drosophila mutants lacking Hedgehog (Hh) secretion suggested that VDUP1 expression could be regulated by the Hh signaling pathway. Experiments were conducted to determine the ability of cyclopamine (CYCLO), an inhibitor of Hh signaling, to regulate VDUP1 expression in breast cancer cells. MDA-231 and MCF-7 cells, grown in RPMI 1640 medium containing 10% fetal calf serum, were treated with CYCLO (5-20 μM) or vehicle (methanol) for 24-72 hrs. Using an anti-VDUP1 antibody, VDUP1 localization was determined by immunofluorescence staining of cells grown on gelatin-coated coverslips following fixation in 4% paraformaldehyde. VDUP1 expression in methanol-treated MDA-231 cells showed a greater expression of VDUP1 in nuclei, indicated by overlapping expression with the nuclear marker histone deacetylase, as compared to cytoplasm. Treatment with 20 μM CYCLO for 72 hours resulted in a decrease in nuclear VDUP1 expression, coupled with a dramatic increase in cytoplasmic VDUP1. This redistribution of VDUP1 from the nucleus to the cytoplasm was detected as early as 24 hours after initiation of treatment with 20 μM CYCLO and was also detected following treatment with 10 μM CYCLO for 72 hours. The CYCLO-induced decrease in nuclear VDUP1 was confirmed with Western blotting. In contrast, no such changes were detected in CYCLO-treated MCF-7 cells. To investigate the direct effect of CYCLO treatment on vdup1 promoter activity, a 1.7 kb fragment of the human vdup1 promoter was inserted upstream of a luciferase gene to generate a vdup1/pLuc plasmid. vdup1/pLuc was co-transfected into CYCLO-treated cells (20 μM), along with a β-galactosidase plasmid and luciferase activity was determined. CYCLO treatment of MDA-231 cells resulted in a significant decrease in luciferase activity, as compared to methanol-treated cells. No changes in luciferase activity were seen following similar experiments conducted with MCF-7 cells. These results demonstrate the novel finding that inhibition of Hh signaling alters both the level of VDUP1 expression and the intracellular distribution of VDUP1 in MDA-231 cells. Future experiments will be directed at determining the mechanisms responsible for the change in the subcellular distribution of VDUP1 seen following CYCLO treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3894.