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

  • Marina Cruz-Lozano
  • Adrián González-González
  • Juan A. Marchal
  • Esperanza Muñoz-Muela
  • Maria P. Molina
  • Francisca E. Cara
  • Anthony M. Brown
  • Gerardo García-Rivas
  • Carmen Hernández-Brenes
  • Jose A. Lorente
  • Pedro Sanchez-Rovira
  • Jenny C. Chang
  • Sergio Granados-PrincipalEmail author
Original Contribution



This 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.


BCSCs 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 TGFβ signaling were investigated in SUM159PT, BT549 and MDA-MB-231 cell lines. Wnt/β-catenin signaling was assessed by Western blot in BT549 cells expressing WNT1 and MDA-MB-231 cells. Changes in TGFβ activity was determined by SMAD Binding Element (SBE) reporter assay.


HT reduced BCSCs self-renewal, ALDH+ (aldehyde dehydrogenase) and CD44+/CD24−/low subpopulations, tumor cell migration and invasion. Consistently, HT suppressed Wnt/β-catenin signaling by decreasing p-LRP6, LRP6, β-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 TGFβ activity.


In conclusion, we report for the first time the inhibitory role of HT on BCSCs and tumor cell migration by targeting EMT, Wnt/β-catenin and TGFβ 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.


Hydroxytyrosol Olive oil Triple-negative breast cancer Cancer stem cells Epithelial-to-mesenchymal transition 



Funding was provided by Instituto de Salud Carlos III (CP14/00197, PI15/00336, PIE16/00045), European Regional Development Fund (European Union), and the Chair “Doctors Galera-Requena in Cancer Stem Cell Research”.

Compliance with ethical standards

Conflict of interest

The authors declare no potential conflict of interests.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Marina Cruz-Lozano
    • 1
    • 2
  • Adrián González-González
    • 1
    • 2
  • Juan A. Marchal
    • 3
    • 4
  • Esperanza Muñoz-Muela
    • 1
    • 2
  • Maria P. Molina
    • 1
    • 2
  • Francisca E. Cara
    • 1
    • 2
  • Anthony M. Brown
    • 5
  • Gerardo García-Rivas
    • 6
  • Carmen Hernández-Brenes
    • 6
  • Jose A. Lorente
    • 2
  • Pedro Sanchez-Rovira
    • 1
    • 2
  • Jenny C. Chang
    • 7
  • Sergio Granados-Principal
    • 1
    • 2
    Email author
  1. 1.UGC de Oncología Médica, Complejo Hospitalario de JaénJaénSpain
  2. 2.GENYO. Centre for Genomics and Oncological ResearchPfizer/University of Granada/Andalusian Regional GovernmentGranadaSpain
  3. 3.Instituto de Investigación Biosanitaria de Granada (ibs.GRANADA)Universidad de GranadaGranadaSpain
  4. 4.Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM)University of GranadaGranadaSpain
  5. 5.Department of Cell and Developmental BiologyWeill Cornell Medical CollegeNew YorkUSA
  6. 6.Centro de Investigación Biomédica, Hospital Zambrano HellionTecnológico de MonterreyMonterreyMexico
  7. 7.Houston Methodist Cancer Center, Houston Methodist HospitalHoustonUSA

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