Hydroxytyrosol inhibits cancer stem cells and the metastatic capacity of triple-negative breast cancer cell lines by the simultaneous targeting of epithelial-to-mesenchymal transition, Wnt/β-catenin and TGFβ signaling pathways

PurposeThis study was aimed to determine the impact of hydroxytyrosol (HT), a minor compound found in olive oil, on breast cancer stem cells (BCSCs) and the migration capacity of triple-negative breast cancer (TNBC) cell lines through the alteration of epithelial-to-mesenchymal transition (EMT) and embryonic signaling pathways.MethodsBCSCs self-renewal was determined by the mammosphere-forming efficiency in SUM159PT, BT549, MDA-MB-231 and Hs578T TNBC cell lines. Flow cytometric analysis of CD44(+)/CD24(-/low) and aldehyde dehydrogenase positive (ALDH(+)) subpopulations, migration by the "wound healing assay", invasion and Western blot of EMT markers and TGF beta signaling were investigated in SUM159PT, BT549 and MDA-MB-231 cell lines. Wnt/beta -catenin signaling was assessed by Western blot in BT549 cells expressing WNT1 and MDA-MB-231 cells. Changes in TGF beta activity was determined by SMAD Binding Element (SBE) reporter assay.ResultsHT reduced BCSCs self-renewal, ALDH(+) (aldehyde dehydrogenase) and CD44(+)/CD24(-/low) subpopulations, tumor cell migration and invasion. Consistently, HT suppressed Wnt/beta -catenin signaling by decreasing p-LRP6, LRP6, beta -catenin and cyclin D1 protein expression and the EMT markers SLUG, ZEB1, SNAIL and VIMENTIN. Finally, HT inhibited p-SMAD2/3 and SMAD2/3 in SUM159PT, BT549 and MDA-MB-231 cells, what was correlated with a less TGF beta activity.ConclusionIn conclusion, we report for the first time the inhibitory role of HT on BCSCs and tumor cell migration by targeting EMT, Wnt/beta -catenin and TGF beta signaling pathways. Our findings highlight the importance of the chemopreventive compound HT as a novel candidate to be investigated as an alternative targeted therapy for TNBC.