Abstract
Abstract The Wnt/β-catenin signaling pathway is frequently activated in invasive triple negative breast cancer (TNBC), and it is able to modify epithelial cancer cells towards a more aggressive and metastatic mesenchymal-like phenotype through activation of epithelial-mesenchymal transition (EMT). Hallmarks of EMT are loss of epithelial markers (e.g. E-cadherin) while gaining those of mesenchymal cells associated with metastatic potential (e.g. Snail, Slug, nuclear β-catenin). The antioxidant hydroxytyrosol (HT) (3,4-dihydroxyphenilethanol) is a phenolic compound present in olive oil which exerts anti-proliferative and pro-apoptotic activity on cancer cells. The aim of this study was to investigate whether HT exerts inhibitory features on the Wnt/EMT axis and migration of TNBC cells. BT549 cells were infected with either the retroviral vector pMV/Wnt-1, pMV/DKK1 or pMV7 (parental retrovirus). BT549/Wnt-1 and MDA-MB-231 cells were seeded at a density of 1x106 in DMEM (supplemented with 10% fetal bovine serum and antibiotics) and treated with HT for 48h (0, 0.5, 1, 5, 10, 25, 50, 75, 100μM). BT549/DKK1 and BT549/MV7 were used as controls. Western blotting was employed to evaluate Wnt pathway activation (p-LRP6, LRP6, β-catenin, cyclin D1) and EMT marker expression (E-cadherin, Snail, Slug), with β-actin as a loading control. Cell motility was assessed by “scratch” assays of confluent MDA-MB-231 cells in presence of HT (10 and 25μM) for 72h in low serum conditions (0.1%), followed by recovery in regular media without treatment for 14h. Our results show, for both cell lines, that treatment with HT (at 10, 25, 50, 75, and 100μM) reduces total levels of the Wnt co-receptor LRP6 and reduces its phosphorylation status only at high concentrations (50, 75, and 100μM). The diminished receptor activation is correlated with a dose-dependent decrease of β-catenin and cyclin D1 protein levels. Similar findings were observed with EMT markers, with a significant decrease in both Snail and Slug transcription factors and an increase in protein levels of the epithelial marker E-cadherin. This dramatic reduction of EMT phenotype was further correlated with a clear inhibition of the migration rate of metastatic MDA-MB-231 cells. In conclusion, this is the first report indicating that the small molecule HT acts as a novel Wnt inhibitor, possibly by interference with the onset of the signaling cascade. This natural compound can also reduce the aggressiveness and invasive potential of TNBC cell lines by altering EMT markers and cell motility. Further studies will be needed in order to get insight into the anti-cancer properties of hydroxytyrosol and evaluate its future potential for clinical trials. Citation Format: Sergio Granados-Principal, Dong Soon Choi, Anthony M.C. Brown, Jenny Chang. The natural compound hydroxytyrosol inhibits the Wnt/EMT axis and migration of triple-negative breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2586. doi:10.1158/1538-7445.AM2013-2586
Published Version
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